bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒12‒25
78 papers selected by
Viktor Korolchuk, Newcastle University
  1. Orchestration of selective autophagy by cargo receptors.
  2. mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species.
  3. Autophagic degradation of membrane-bound organelles in plants.
  4. mTOR Signaling Network in Cell Biology and Human Disease.
  5. Mitochondrial Autophagy in Ischemic Aged Livers.
  6. Role of autophagy in aging: The good, the bad, and the ugly.
  7. Toosendanin Induces Hepatocyte Damage by Inhibiting Autophagic Flux via TFEB-Mediated Lysosomal Dysfunction.
  8. The endo-lysosomal regulatory protein BLOC1S1 modulates hepatic lysosomal content and lysosomal lipolysis.
  9. PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS.
  10. The role of autophagy in acute myeloid leukemia development.
  11. Secretory autophagy: a turn key for understanding AMD pathology and developing new therapeutic targets?
  12. mTORC1 takes control of lysosomal lipid breakdown.
  13. SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells.
  14. Network analysis of the autophagy biochemical network in relation to various autophagy-targeted proteins found among SARS-CoV-2 variants of concern.
  15. Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome.
  16. PLEKHM2 Loss of Function Impairs the Activity of iPSC-Derived Neurons via Regulation of Autophagic Flux.
  17. Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.
  18. Role of autophagy in lung diseases and ageing.
  19. Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM).
  20. A conserved MTMR lipid phosphatase increasingly suppresses autophagy in brain neurons during aging.
  21. The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
  22. Ring domains are essential for GATOR2-dependent mTORC1 activation.
  23. Intersections of Ubiquitin-Proteosome System and Autophagy in Promoting Growth of Glioblastoma Multiforme: Challenges and Opportunities.
  24. Potential Diets to Improve Mitochondrial Activity in Amyotrophic Lateral Sclerosis.
  25. Endoplasmic reticulum stress regulates autophagic response that is involved in Saikosaponin a-induced liver cell damage.
  26. Highly expressed SERCA2 triggers tumor cell autophagy and is a druggable vulnerability in triple-negative breast cancer.
  27. Fluid Shear Stress Regulates Osteogenic Differentiation via AnnexinA6-Mediated Autophagy in MC3T3-E1 Cells.
  28. Targeting the Lysosomal Degradation of Rab22a-NeoF1 Fusion Protein for Osteosarcoma Lung Metastasis.
  29. Therapeutic role of mTOR inhibitors in control of SARS-CoV-2 viral replication.
  30. Autophagy Is a Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel.
  31. Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.
  32. The role of mTORC1 in the regulation of skeletal muscle mass.
  33. Miro1 R272Q disrupts mitochondrial calcium handling and neurotransmitter uptake in dopaminergic neurons.
  34. The multifaced role and therapeutic regulation of autophagy in ovarian cancer.
  35. Object location learning in mice requires hippocampal somatostatin interneuron activity and is facilitated by mTORC1-mediated long-term potentiation of their excitatory synapses.
  36. 3'-epi-12β-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells.
  37. Autophagy-Related ncRNAs in Pancreatic Cancer.
  38. Inhibition of autophagy reduces the rate of fluoride-induced LS8 apoptosis via regulating ATG5 and ATG7.
  39. Changes in AMPK activity induces cellular senescence in human dental follicle cells.
  40. Prunus spinosa Extract Sensitized HCT116 Spheroids to 5-Fluorouracil Toxicity, Inhibiting Autophagy.
  41. Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease.
  42. HDAC6 Inhibition Alleviates Ischemia- and Cisplatin-Induced Acute Kidney Injury by Promoting Autophagy.
  43. Blockage of Nrf2 and autophagy by L-selenocystine induces selective death in Nrf2-addicted colorectal cancer cells through p62-Keap-1-Nrf2 axis.
  44. Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion.
  45. The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain.
  46. The Sag-Shoc2 axis regulates conversion of mPanINs to cystic lesions in Kras pancreatic tumor model.
  47. Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway.
  48. New insights into the interplay between autophagy and cartilage degeneration in osteoarthritis.
  49. Sulforaphane attenuates microglia-mediated neuronal damage by down-regulating the ROS/autophagy/NLRP3 signal axis in fibrillar Aβ-activated microglia.
  50. CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment.
  51. mTOR Signaling Regulates Zika Virus Replication Bidirectionally through Autophagy and Protein Translation.
  52. Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.
  53. Stevia and Stevioside Attenuate Liver Steatosis through PPARα-Mediated Lipophagy in db/db Mice Hepatocytes.
  54. Co-Targeting the EGFR and PI3K/Akt Pathway to Overcome Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma: What about Autophagy?
  55. Limonin, a Component of Immature Citrus Fruits, Activates Anagen Signaling in Dermal Papilla Cells.
  56. FOXO3a-dependent PARKIN negatively regulates cardiac hypertrophy by restoring mitophagy.
  57. Lysine crotonylation regulates leucine-deprivation-induced autophagy by a 14-3-3ε-PPM1B axis.
  58. The SsAtg1 Activating Autophagy Is Required for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum.
  59. The UBE2C/CDH1/DEPTOR axis is an oncogene-tumor suppressor cascade in lung cancer cells.
  60. Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.
  61. Detection and modulation of neurodegenerative processes using graphene-based nanomaterials: Nanoarchitectonics and applications.
  62. Pro-prion, as a membrane adaptor protein for E3 ligase c-Cbl, facilitates the ubiquitination of IGF-1R, promoting melanoma metastasis.
  63. N6-methyladenosine-modified USP13 induces pro-survival autophagy and imatinib resistance via regulating the stabilization of autophagy-related protein 5 in gastrointestinal stromal tumors.
  64. DEPTOR loss impairs brown adipocyte development in vitro but has limited impacts in mice.
  65. Perspectives on mitochondrial relevance in cardiac ischemia/reperfusion injury.
  66. Mechanisms of RNA and Protein Quality Control and Their Roles in Cellular Senescence and Age-Related Diseases.
  67. Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery.
  68. Host autophagy limits Toxoplasma gondii proliferation in the absence of IFN-γ by affecting the hijack of Rab11A-positive vesicles.
  69. Zinc-finger Protein CXXC5 Promotes Breast Carcinogenesis by Regulating the TSC1/mTOR Signaling Pathway.
  70. ASIC1a involves the acid-mediated activation of pancreatic stellate cells associated with autophagy induction.
  71. Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.
  72. Hepatic PLIN5 Deficiency Impairs Lipogenesis through Mitochondrial Dysfunction.
  73. Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation.
  74. ALDH1-Mediated Autophagy Sensitizes Glioblastoma Cells to Ferroptosis.
  75. Targeting mTOR Signaling by Dietary Polysaccharides in Cancer Prevention: Advances and Challenges.
  76. BAG3 promotes autophagy and suppresses NLRP3 inflammasome activation in Parkinson's disease.
  77. Sestrin2 Overexpression Ameliorates Endoplasmic Reticulum Stress-Induced Apoptosis via Inhibiting mTOR Pathway in HepG2 Cells.
  78. Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration.
  1. Curr Biol. 2022 Dec 19. pii: S0960-9822(22)01758-4. [Epub ahead of print]32(24): R1357-R1371
    Orchestration of selective autophagy by cargo receptors.
    Elias Adriaenssens, Luca Ferrari, Sascha Martens.
      Cellular homeostasis requires the swift and specific removal of damaged material. Selective autophagy represents a major pathway for the degradation of such cargo material. This is achieved by the sequestration of the cargo within double-membrane vesicles termed autophagosomes, which form de novo around the cargo and subsequently deliver their content to lysosomes for degradation. The importance of selective autophagy is exemplified by the various neurodegenerative diseases associated with defects in this pathway, including Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. It has become evident that cargo receptors are acting as Swiss army knives in selective autophagy by recognizing the cargo, orchestrating the recruitment of the machinery for autophagosome biogenesis, and closely aligning the membrane with the cargo. Furthermore, cargo receptors sequester ubiquitinated proteins into larger condensates upstream of autophagy induction. Here, we review recent insights into the mechanisms of action of cargo receptors in selective autophagy by focusing on the roles of sequestosome-like cargo receptors in the degradation of misfolded, ubiquitinated proteins and damaged mitochondria. We also highlight at which steps defects in their function result in the accumulation of harmful material and how this knowledge may guide the design of future therapies.
    DOI:  https://doi.org/10.1016/j.cub.2022.11.002
  2. Nat Metab. 2022 Dec;4(12): 1792-1811
    mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species.
    Aaron M Hosios, Meghan E Wilkinson, Molly C McNamara, Krystle C Kalafut, Margaret E Torrence, John M Asara, Brendan D Manning.
      The mechanistic target of rapamycin complex 1 (mTORC1) senses and relays environmental signals from growth factors and nutrients to metabolic networks and adaptive cellular systems to control the synthesis and breakdown of macromolecules; however, beyond inducing de novo lipid synthesis, the role of mTORC1 in controlling cellular lipid content remains poorly understood. Here we show that inhibition of mTORC1 via small molecule inhibitors or nutrient deprivation leads to the accumulation of intracellular triglycerides in both cultured cells and a mouse tumor model. The elevated triglyceride pool following mTORC1 inhibition stems from the lysosome-dependent, but autophagy-independent, hydrolysis of phospholipid fatty acids. The liberated fatty acids are available for either triglyceride synthesis or β-oxidation. Distinct from the established role of mTORC1 activation in promoting de novo lipid synthesis, our data indicate that mTORC1 inhibition triggers membrane phospholipid trafficking to the lysosome for catabolism and an adaptive shift in the use of constituent fatty acids for storage or energy production.
    DOI:  https://doi.org/10.1038/s42255-022-00706-6
  3. Biosci Rep. 2022 Dec 23. pii: BSR20221204. [Epub ahead of print]
    Autophagic degradation of membrane-bound organelles in plants.
    Jiaojiao Wang, Qian Zhang, Yan Bao, Diane Bassham.
      Eukaryotic cells have evolved membrane-bound organelles, including the endoplasmic reticulum (ER), Golgi, mitochondria, peroxisomes, chloroplasts (in plants and green algae) and lysosomes/vacuoles, for specialized functions. Organelle quality control and their proper interactions are crucial both for normal cell homeostasis and function and for environmental adaption. Dynamic turnover of organelles is tightly controlled, with autophagy playing an essential role. Autophagy is a programmed process for efficient clearing of unwanted or damaged macromolecules or organelles, transporting them to vacuoles for degradation and recycling and thereby enhancing plant environmental plasticity. The specific autophagic engulfment of organelles requires activation of a selective autophagy pathway, recognition of the organelle by a receptor, and selective incorporation of the organelle into autophagosomes. While some of the autophagy machinery and mechanisms for autophagic removal of organelles is conserved across eukaryotes, plants have also developed unique mechanisms and machinery for these pathways. In this review, we discuss recent progress in understanding autophagy regulation in plants, with a focus on autophagic degradation of membrane-bound organelles. We also raise some important outstanding questions to be addressed in the future.
    Keywords:  Autophagosome; degradation; organelle; receptor; selective autophagy; stress
    DOI:  https://doi.org/10.1042/BSR20221204
  4. Int J Mol Sci. 2022 Dec 18. pii: 16142. [Epub ahead of print]23(24):
    mTOR Signaling Network in Cell Biology and Human Disease.
    Jane J Yu, Elena A Goncharova.
      The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple processes, including gene transcription, protein synthesis, ribosome biogenesis, autophagy, cell metabolism, and cell growth [...].
    DOI:  https://doi.org/10.3390/ijms232416142
  5. Cells. 2022 Dec 16. pii: 4083. [Epub ahead of print]11(24):
    Mitochondrial Autophagy in Ischemic Aged Livers.
    Jae-Sung Kim, William C Chapman, Yiing Lin.
      Mitochondrial autophagy (mitophagy) is a central catabolic event for mitochondrial quality control. Defective or insufficient mitophagy, thus, can result in mitochondrial dysfunction, and ultimately cell death. There is a strong causal relationship between ischemia/reperfusion (I/R) injury and mitochondrial dysfunction following liver resection and transplantation. Compared to young patients, elderly patients poorly tolerate I/R injury. Accumulation of abnormal mitochondria after I/R is more prominent in aged livers than in young counterparts. This review highlights how altered autophagy is mechanistically involved in age-dependent hypersensitivity to reperfusion injury.
    Keywords:  autophagy; ischemia/reperfusion; liver; mitochondria; mitochondrial dynamics
    DOI:  https://doi.org/10.3390/cells11244083
  6. Aging Cell. 2022 Dec 20. e13753
    Role of autophagy in aging: The good, the bad, and the ugly.
    Siamak Tabibzadeh.
      Autophagy (self-eating) is a conserved catabolic homeostatic process required for cellular metabolic demands by removal of the damaged molecules and organelles and for alleviation of stress initiated by pathology and infection. By such actions, autophagy is essential for the prevention of aging, disease, and cancer. Genetic defects of autophagy genes lead to a host of developmental, metabolic, and pathological aberrations. Similarly, the age-induced decline in autophagy leads to the loss of cellular homeostatic control. Paradoxically, such a valuable mechanism is hijacked by diseases, during tumor progression and by senescence, presumably due to high levels of metabolic demand. Here, we review both the role of autophagy in preventing cellular decline in aging by fulfillment of cellular bioenergetic demands and its contribution to the maintenance of the senescent state and SASP by acting on energy and nutritional sensors and diverse signaling pathways.
    Keywords:  AMPK; SASP; aging; cancer; mTOR; senescence
    DOI:  https://doi.org/10.1111/acel.13753
  7. Pharmaceuticals (Basel). 2022 Dec 03. pii: 1509. [Epub ahead of print]15(12):
    Toosendanin Induces Hepatocyte Damage by Inhibiting Autophagic Flux via TFEB-Mediated Lysosomal Dysfunction.
    Li Luo, Yonghong Liang, Yuanyuan Fu, Zhiyuan Liang, Jinfen Zheng, Jie Lan, Feihai Shen, Zhiying Huang.
      Toosendanin (TSN) is a triterpenoid from the fruit or bark of Melia toosendan Sieb et Zucc, which has clear antitumor and insecticidal activities, but it possesses limiting hepatotoxicity in clinical application. Autophagy is a degradation and recycling mechanism to maintain cellular homeostasis, and it also plays an essential role in TSN-induced hepatotoxicity. Nevertheless, the specific mechanism of TSN on autophagy-related hepatotoxicity is still unknown. The hepatotoxicity of TSN in vivo and in vitro was explored in this study. It was found that TSN induced the upregulation of the autophagy-marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62, the accumulation of autolysosomes, and the inhibition of autophagic flux. The middle and late stages of autophagy were mainly studied. The data showed that TSN did not affect the fusion of autophagosomes and lysosomes but significantly inhibited the acidity, the degradation capacity of lysosomes, and the expression of hydrolase cathepsin B (CTSB). The activation of autophagy could alleviate TSN-induced hepatocyte damage. TSN inhibited the expression of transcription factor EB (TFEB), which is a key transcription factor for many genes of autophagy and lysosomes, such as CTSB, and overexpression of TFEB alleviated the autophagic flux blockade caused by TSN. In summary, TSN caused hepatotoxicity by inhibiting TFEB-lysosome-mediated autophagic flux and activating autophagy by rapamycin (Rapa), which could effectively alleviate TSN-induced hepatotoxicity, indicating that targeting autophagy is a new strategy to intervene in the hepatotoxicity of TSN.
    Keywords:  TFEB; autophagic flux; autophagy; hepatotoxicity; lysosome; toosendanin (TSN)
    DOI:  https://doi.org/10.3390/ph15121509
  8. Biochem Biophys Res Commun. 2022 Dec 14. pii: S0006-291X(22)01703-X. [Epub ahead of print]642 1-10
    The endo-lysosomal regulatory protein BLOC1S1 modulates hepatic lysosomal content and lysosomal lipolysis.
    Kaiyuan Wu, Jizhong Zou, Michael N Sack.
      BLOC1S1 is a common component of BLOC and BORC multiprotein complexes which play distinct roles in endosome and lysosome biology. Recent human mutations in BLOC1S1 associate with juvenile leukodystrophy. As leukodystrophy is linked to perturbed lysosomal lipid storage we explored whether BLOC1S1 itself modulates this biology. Given the central role of the liver in lipid storage, our investigations were performed in hepatocyte specific liver bloc1s1 knockout (LKO) mice and in human hepatocyte-like lines (HLCs) derived from inducible pluripotential stem cells (iPSCs) from a juvenile leukodystrophy subject's with bloc1s1 mutations and from isogenic corrected iPSCs. Here we show that hepatocyte lipid stores are diminished in parallel with increased lysosomal content, increased lysosomal lipid uptake and lipolysis in LKO mice. The lysosomal lipolysis program was independent of macro- and chaperone-mediated lipophagy but dependent on cellular lysosome content. In parallel, genetic induction of lysosomal biogenesis in a transformed hepatocyte cell line replicated depletion of intracellular lipid stores. Interestingly bloc1s1 mutant and isogenic corrected HLCs both showed normal lysosomal enzyme activity. However, relative to the isogenic corrected HLCs, mutant bloc1s1 HLCs showed reduced lysosomal content and increased lipid storage. Together these data show distinct phenotypes in human mutant HLCs compared to murine knockout cells. At the same time, human blcs1s1 mutation and murine hepatocyte bloc1s1 depletion disrupt lysosome content and the cellular lipid storage. These data support that BLOC1S1 modulates lysosome content and lipid handling independent of autophagy and show that lysosomal lipolysis is dependent on the cellular content of functional lysosomes.
    Keywords:  BLOC1S1; Hepatic lipid droplets; Lysosomal lipolysis; Lysosome
    DOI:  https://doi.org/10.1016/j.bbrc.2022.12.038
  9. Autophagy. 2022 Dec 21.
    PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS.
    Nicholas D Demers, Victoria Riccio, Doo Sin Jo, Sushil Bhandari, Kelsey B Law, Weifang Liao, Choy Kim, G Angus McQuibban, Seong-Kyu Choe, Dong-Hyung Cho, Peter K Kim.
      Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.
    Keywords:  PEX13; PEX5; Peroxisomes; pexophagy; reactive oxygen species; selective autophagy; ubiquitin; zebrafish
    DOI:  https://doi.org/10.1080/15548627.2022.2160566
  10. Expert Rev Anticancer Ther. 2022 Dec 23.
    The role of autophagy in acute myeloid leukemia development.
    Martyna Bednarczyk, Karolina Kociszewska, Olga Grosicka, Sebastian Grosicki.
      INTRODUCTION: Autophagy is a highly conservative self-degradative process. It aims at elimination-impaired proteins and cellular organelles. Previous research confirmed the autophagy role in cancer pathogenesis.AREAS COVERED: This article discusses the role of autophagy in the development of AML. Autophagy seems to be a 'double-sword' mechanism, hence, either its suppression or induction could promote neoplasm growth. This mechanism could also be the aim of the 'molecular targeted therapy'. Chemo- and radiotherapy induce cellular stress in neoplasm cells with subsequent autophagy suppression. Simultaneously, it is claimed that the autophagy suppression increases chemosensitivity 'in neoplastic cells. Some agents, like bortezomib, in turn could promote autophagy process, e.g., in AML (acute myeloid leukemia). However, currently there are not many studies focusing on the role of autophagy in patients suffering for AML. In this review, we summarize the research done so far on the role of autophagy in the development of AML.
    EXPERT OPINION: The analysis of autophagy genes expression profiling in AML could be a relevant factor in the diagnostic process and treatment 'individualization'. Autophagy modulation seems to be a relevant target in the oncological therapy - it could limit disease progression and increase the effectiveness of treatment.
    Keywords:  acute myeloid leukemia; autophagy; cancer; hematology; lysosome; mitophagy; myleloid; proteasome
    DOI:  https://doi.org/10.1080/14737140.2023.2161518
  11. Expert Opin Ther Targets. 2022 Dec 18. 1-13
    Secretory autophagy: a turn key for understanding AMD pathology and developing new therapeutic targets?
    Janusz Blasiak, Kai Kaarniranta.
      INTRODUCTION: Age-related macular degeneration (AMD) is an eye disease leading to vision loss with poorly known pathogenesis and limited therapeutic options. Degradative autophagy (DA) is impaired in AMD, but emerging evidence points to secretary autophagy (SA) as a key element in AMD pathogenesis.AREAS COVERED: SA may cause the release of proteins and protein aggregates, lipofuscin, beta amyloid, faulty mitochondria, pro-inflammatory and pro-angiogenic factors from the retinal pigment epithelium (RPE) that may contribute to drusen formation and choroidal neovascularization. SA may replace DA, when formation of autolysosome is impaired, and then a harmful cargo, instead of being degraded, is extruded from the RPE contributing to drusen and/or angiogenic environment. Therefore, the interplay between DA and SA may be critical for drusen formation and choroidal neovascularization, so it can be a turn key to understand AMD pathogenesis.
    EXPERT OPINION: Although SA fulfills some beneficial functions, it is detrimental for the retina in many cases. Therefore, inhibiting SA may be a therapeutic strategy in AMD, but it is challenged by the development of selective SA inhibitors that would not affect DA. The TRIM16, SEC22B and RAB8A proteins, specific for secretory autophagosome, may be primary candidates as therapeutic targets, but their action is not limited to autophagy and therefore requires further studies.
    Keywords:  Age-related macular degeneration; amyloid beta; autophagy; drusen; lipofuscin; secretory autophagy
    DOI:  https://doi.org/10.1080/14728222.2022.2157260
  12. Nat Metab. 2022 Dec;4(12): 1620-1622
    mTORC1 takes control of lysosomal lipid breakdown.
    Laura Tribouillard, Mathieu Laplante.
      
    DOI:  https://doi.org/10.1038/s42255-022-00702-w
  13. Pathogens. 2022 Dec 14. pii: 1535. [Epub ahead of print]11(12):
    SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells.
    Juraj Koči, Marta Novotová, Monika Sláviková, Boris Klempa, Ivan Zahradník.
      The coronavirus transforms the cytoplasm of susceptible cells to support virus replication. It also activates autophagy-like processes, the role of which is not well understood. Here, we studied SARS-CoV-2-infected Vero E6 cells using transmission electron microscopy and autophagy PCR array. After 6-24 h post-infection (hpi), the cytoplasm of infected cells only contained double-membrane vesicles, phagophores, and phagosomes engulfing virus particles and cytoplasmic debris, including damaged mitochondria. The phagosomes interacted with the viral nucleoprotein complex, virus particles, mitochondria, and lipid droplets. The phagosomes transformed into egress vacuoles, which broke through the plasmalemma and discharged the virus particles. The Vero E6 cells exhibited pronounced virus replication at 6 hpi, which stabilized at 18-24 hpi at a high level. The autophagy PCR array tests revealed a significant upregulation of 10 and downregulation of 8 autophagic gene markers out of 84. Altogether, these results underline the importance of autophagy-like processes for SARS-CoV-2 maturation and egress, and point to deviations from a canonical autophagy response.
    Keywords:  SARS-CoV-2; autophagy; gene regulation; mitophagy; virus egress; virus maturation
    DOI:  https://doi.org/10.3390/pathogens11121535
  14. J Mol Graph Model. 2022 Dec 14. pii: S1093-3263(22)00275-3. [Epub ahead of print]119 108396
    Network analysis of the autophagy biochemical network in relation to various autophagy-targeted proteins found among SARS-CoV-2 variants of concern.
    Marni E Cueno, Keiichi Taketsuna, Mitsuki Saito, Sara Inoue, Kenichi Imai.
      Autophagy is an important cellular process that triggers a coordinated action involving multiple individual proteins and protein complexes while SARS-CoV-2 (SARS2) was found to both hinder autophagy to evade host defense and utilize autophagy for viral replication. Interestingly, the possible significant stages of the autophagy biochemical network in relation to the corresponding autophagy-targeted SARS2 proteins from the different variants of concern (VOC) were never established. In this study, we performed the following: autophagy biochemical network design and centrality analyses; generated autophagy-targeted SARS2 protein models; and superimposed protein models for structural comparison. We identified 2 significant biochemical pathways (one starts from the ULK complex and the other starts from the PI3P complex) within the autophagy biochemical network. Similarly, we determined that the autophagy-targeted SARS2 proteins (Nsp15, M, ORF7a, ORF3a, and E) are structurally conserved throughout the different SARS2 VOC suggesting that the function of each protein is preserved during SARS2 evolution. Interestingly, among the autophagy-targeted SARS2 proteins, the M protein coincides with the 2 significant biochemical pathways we identified within the autophagy biochemical network. In this regard, we propose that the SARS2 M protein is the main determinant that would influence autophagy outcome in regard to SARS2 infection.
    Keywords:  Centrality measurements; Membrane protein; Network analysis; SARS-CoV-2 (SARS2); Variants of concern
    DOI:  https://doi.org/10.1016/j.jmgm.2022.108396
  15. J Vis Exp. 2022 11 30.
    Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome.
    Bilin Liu, Anqi Li, Yuan Qin, Lei Chen, Meng Gao, Guohua Gong.
      Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.
    DOI:  https://doi.org/10.3791/64647
  16. Int J Mol Sci. 2022 Dec 17. pii: 16092. [Epub ahead of print]23(24):
    PLEKHM2 Loss of Function Impairs the Activity of iPSC-Derived Neurons via Regulation of Autophagic Flux.
    Hadas Ben-Zvi, Tatiana Rabinski, Rivka Ofir, Smadar Cohen, Gad D Vatine.
      Pleckstrin Homology And RUN Domain Containing M2 (PLEKHM2) [delAG] mutation causes dilated cardiomyopathy with left ventricular non-compaction (DCM-LVNC), resulting in a premature death of PLEKHM2[delAG] individuals due to heart failure. PLEKHM2 is a factor involved in autophagy, a master regulator of cellular homeostasis, decomposing pathogens, proteins and other cellular components. Autophagy is mainly carried out by the lysosome, containing degradation enzymes, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 promotes lysosomal movement toward the cell periphery. Autophagic dysregulation is associated with neurodegenerative diseases' pathogenesis. Thus, modulation of autophagy holds considerable potential as a therapeutic target for such disorders. We hypothesized that PLEKHM2 is involved in neuronal development and function, and that mutated PLEKHM2 (PLEKHM2[delAG]) neurons will present impaired functions. Here, we studied PLEKHM2-related abnormalities in induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) as a neuronal model. PLEKHM2[delAG] iMN cultures had healthy control-like differentiation potential but exhibited reduced autophagic activity. Electrophysiological measurements revealed that PLEKHM2[delAG] iMN cultures displayed delayed functional maturation and more frequent and unsynchronized activity. This was associated with increased size and a more perinuclear lysosome cellular distribution. Thus, our results suggest that PLEKHM2 is involved in the functional development of neurons through the regulation of autophagic flux.
    Keywords:  DCM-LVNC; PLEKHM2; autophagosomes; autophagy; disease model; iPSCs; lysosomes; motor neurons; neurodegeneration; neurons
    DOI:  https://doi.org/10.3390/ijms232416092
  17. Cancers (Basel). 2022 Dec 15. pii: 6195. [Epub ahead of print]14(24):
    Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.
    Jesse D Champion, Kayleigh M Dodd, Hilaire C Lam, Mohammad A M Alzahrani, Sara Seifan, Ellie Rad, David Oliver Scourfield, Melissa L Fishel, Brian L Calver, Ann Ager, Elizabeth P Henske, David Mark Davies, Mark R Kelley, Andrew R Tee.
      Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.
    Keywords:  APE1; HIF-1α; NF-kB; Ref-1; STAT3; TSC; angiogenesis; hypoxia; mTOR; redox
    DOI:  https://doi.org/10.3390/cancers14246195
  18. Eur Respir Rev. 2022 Dec 31. pii: 220134. [Epub ahead of print]31(166):
    Role of autophagy in lung diseases and ageing.
    Yan Zhang, Jin Zhang, Zhiling Fu.
      The lungs face ongoing chemical, mechanical, biological, immunological and xenobiotic stresses over a lifetime. Advancing age progressively impairs lung function. Autophagy is a "housekeeping" survival strategy involved in numerous physiological and pathological processes in all eukaryotic cells. Autophagic activity decreases with age in several species, whereas its basic activity extends throughout the lifespan of most animals. Dysregulation of autophagy has been proven to be closely related to the pathogenesis of several ageing-related pulmonary diseases. This review summarises the role of autophagy in the pathogenesis of pulmonary diseases associated with or occurring in the context of ageing, including acute lung injury, chronic obstructive pulmonary disease, asthma and pulmonary fibrosis, and describes its potential as a therapeutic target.
    DOI:  https://doi.org/10.1183/16000617.0134-2022
  19. Cell Death Dis. 2022 Dec 21. 13(12): 1065
    Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM).
    Fei Li, Yifan Zhang, Zhoujun Lin, Lizhong Yan, Qiao Liu, Yin Li, Xiaolin Pei, Ya Feng, Xiao Han, Juan Yang, Fangxu Zheng, Tianjiao Li, Yupeng Zhang, Zhenkun Fu, Di Shao, Jane Yu, Chenggang Li.
      Lymphangioleiomyomatosis (LAM), a progressive pulmonary disease exclusively affecting females, is caused by defects or mutations in the coding gene tuberous sclerosis complex 1 (TSC1) or TSC2, causing the mammalian target of rapamycin complex 1 (mTORC1) activation and autophagy inhibition. Clinically, rapamycin shows limited cytocidal effects, and LAM recurs after drug withdrawal. In this study, we demonstrated that TSC2 negatively regulated the sphingolipid metabolism pathway and the expressions of sphingosine kinase 1 (SPHK1) and sphingosine-1-phosphate receptor 3 (S1PR3) were significantly elevated in LAM patient-derived TSC2-deficient cells compared to TSC2-addback cells, insensitive to rapamycin treatment and estrogen stimulation. Knockdown of SPHK1 showed reduced viability, migration and invasion in TSC2-deficient cells. Selective SPHK1 antagonist PF543 potently suppressed the viability of TSC2-deficient cells and induced autophagy-mediated cell death. Meanwhile, the cognate receptor S1PR3 was identified to mediating the tumorigenic effects of sphingosine-1-phosphate (S1P). Treatment with TY52156, a selective antagonist for S1PR3, or genetic silencing using S1PR3-siRNA suppressed the viability of TSC2-deficient cells. Both SPHK1 and S1PR3 inhibitors markedly exhibited antitumor effect in a xenograft model of TSC2-null cells, restored autophagy level, and triggered cell death. Together, we identified novel rapamycin-insensitive sphingosine metabolic signatures in TSC2-null LAM cells. Therapeutic targeting of aberrant SPHK1/S1P/S1PR3 signaling may have potent therapeutic benefit for patients with TSC/LAM or other hyperactive mTOR neoplasms with autophagy inhibition.
    DOI:  https://doi.org/10.1038/s41419-022-05511-3
  20. Sci Rep. 2022 Dec 17. 12(1): 21817
    A conserved MTMR lipid phosphatase increasingly suppresses autophagy in brain neurons during aging.
    Tibor Kovács, Janka Szinyákovics, Viktor Billes, Gábor Murányi, Virginia B Varga, Annamária Bjelik, Ádám Légrádi, Melinda Szabó, Sára Sándor, Enikő Kubinyi, Cecília Szekeres-Paracky, Péter Szocsics, János Lőke, Jun Mulder, Balázs Gulyás, Éva Renner, Miklós Palkovits, Károly Gulya, Zsófia Maglóczky, Tibor Vellai.
      Ageing is driven by the progressive, lifelong accumulation of cellular damage. Autophagy (cellular self-eating) functions as a major cell clearance mechanism to degrade such damages, and its capacity declines with age. Despite its physiological and medical significance, it remains largely unknown why autophagy becomes incapable of effectively eliminating harmful cellular materials in many cells at advanced ages. Here we show that age-associated defects in autophagic degradation occur at both the early and late stages of the process. Furthermore, in the fruit fly Drosophila melanogaster, the myotubularin-related (MTMR) lipid phosphatase egg-derived tyrosine phosphatase (EDTP) known as an autophagy repressor gradually accumulates in brain neurons during the adult lifespan. The age-related increase in EDTP activity is associated with a growing DNA N6-adenine methylation at EDTP locus. MTMR14, the human counterpart of EDTP, also tends to accumulate with age in brain neurons. Thus, EDTP, and presumably MTMR14, promotes brain ageing by increasingly suppressing autophagy throughout adulthood. We propose that EDTP and MTMR14 phosphatases operate as endogenous pro-ageing factors setting the rate at which neurons age largely independently of environmental factors, and that autophagy is influenced by DNA N6-methyladenine levels in insects.
    DOI:  https://doi.org/10.1038/s41598-022-24843-w
  21. Nat Commun. 2022 Dec 21. 13(1): 7857
    The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
    Ryosuke Ishimura, Afnan H El-Gowily, Daisuke Noshiro, Satoko Komatsu-Hirota, Yasuko Ono, Mayumi Shindo, Tomohisa Hatta, Manabu Abe, Takefumi Uemura, Hyeon-Cheol Lee-Okada, Tarek M Mohamed, Takehiko Yokomizo, Takashi Ueno, Kenji Sakimura, Tohru Natsume, Hiroyuki Sorimachi, Toshifumi Inada, Satoshi Waguri, Nobuo N Noda, Masaaki Komatsu.
      Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
    DOI:  https://doi.org/10.1038/s41467-022-35501-0
  22. Mol Cell. 2022 Dec 08. pii: S1097-2765(22)01134-0. [Epub ahead of print]
    Ring domains are essential for GATOR2-dependent mTORC1 activation.
    Cong Jiang, Xiaoming Dai, Shaohui He, Hongfei Zhou, Lan Fang, Jianping Guo, Songlei Liu, Tao Zhang, Weijuan Pan, Haihong Yu, Tianmin Fu, Dali Li, Hiroyuki Inuzuka, Ping Wang, Jianru Xiao, Wenyi Wei.
      The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.
    Keywords:  Gator1; Gator2; NPRL2; Sestrin; WDR24; amino acid sensing; mTOR; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2022.11.021
  23. Cells. 2022 Dec 15. pii: 4063. [Epub ahead of print]11(24):
    Intersections of Ubiquitin-Proteosome System and Autophagy in Promoting Growth of Glioblastoma Multiforme: Challenges and Opportunities.
    Rhett Visintin, Swapan K Ray.
      Glioblastoma multiforme (GBM) is a brain tumor notorious for its propensity to recur after the standard treatments of surgical resection, ionizing radiation (IR), and temozolomide (TMZ). Combined with the acquired resistance to standard treatments and recurrence, GBM is an especially deadly malignancy with hardly any worthwhile treatment options. The treatment resistance of GBM is influenced, in large part, by the contributions from two main degradative pathways in eukaryotic cells: ubiquitin-proteasome system (UPS) and autophagy. These two systems influence GBM cell survival by removing and recycling cellular components that have been damaged by treatments, as well as by modulating metabolism and selective degradation of components of cell survival or cell death pathways. There has recently been a large amount of interest in potential cancer therapies involving modulation of UPS or autophagy pathways. There is significant crosstalk between the two systems that pose therapeutic challenges, including utilization of ubiquitin signaling, the degradation of components of one system by the other, and compensatory activation of autophagy in the case of proteasome inhibition for GBM cell survival and proliferation. There are several important regulatory nodes which have functions affecting both systems. There are various molecular components at the intersections of UPS and autophagy pathways that pose challenges but also show some new therapeutic opportunities for GBM. This review article aims to provide an overview of the recent advancements in research regarding the intersections of UPS and autophagy with relevance to finding novel GBM treatment opportunities, especially for combating GBM treatment resistance.
    Keywords:  GBM cell survival and proliferation; autophagy; crosstalk between the UPS and autophagy; glioblastoma multiforme (GBM); induction of apoptosis in GBM; new therapeutic opportunities; treatment resistance; ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.3390/cells11244063
  24. Diseases. 2022 Dec 01. pii: 117. [Epub ahead of print]10(4):
    Potential Diets to Improve Mitochondrial Activity in Amyotrophic Lateral Sclerosis.
    Sayuri Yoshikawa, Kurumi Taniguchi, Haruka Sawamura, Yuka Ikeda, Ai Tsuji, Satoru Matsuda.
      Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, the pathogenesis of which is based on alternations in the mitochondria of motor neurons, causing their progressive death. A growing body of evidence shows that more efficient mitophagy could prevent and/or treat this disorder by suppressing mitochondrial dysfunction-induced oxidative stress and inflammation. Mitophagy has been considered one of the main mechanisms responsible for mitochondrial quality control. Since ALS is characterized by enormous oxidative stress, several edible phytochemicals that can activate mitophagy to remove damaged mitochondria could be considered a promising option to treat ALS by providing neuroprotection. Therefore, it is of great significance to explore the mechanisms of mitophagy in ALS and to understand the effects and/or molecular mechanisms of phytochemical action, which could translate into a treatment for neurodegenerative diseases, including ALS.
    Keywords:  ALS; AMPK; ROS; mTOR; mTORC1; mitophagy; natural product
    DOI:  https://doi.org/10.3390/diseases10040117
  25. Toxicol In Vitro. 2022 Dec 17. pii: S0887-2333(22)00232-6. [Epub ahead of print] 105534
    Endoplasmic reticulum stress regulates autophagic response that is involved in Saikosaponin a-induced liver cell damage.
    Ye-Feng Wang, Rui-Xia Ma, Bin Zou, Jia Li, Yao Yao, Juan Li.
      Saikosaponin a (Ssa) is an active ingredient of the Chinese herbal plant Radix Bupleuri (RB) and has severe hepatotoxicity. However, biomolecular mechanisms involved in Ssa-induced hepatotoxicity are not yet entirely clear. Previous studies reported that Ssd (an isomer of Ssa) as a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor can induce autophagy in apoptotic defective cells, leading to autophagy-dependent cell death. Therefore, we speculate that endoplasmic reticulum (ER) stress and autophagy may also play an important role in Ssa-induced hepatocyte death. This study aimed to explore the connection between ER stress and autophagy and Ssa-induced hepatotoxicity. Experiments in vitro showed that the cell viability of L-02 cells in the Ssa treatment group decreased, the level of autophagy marker LC3-II/LC3-I and Beclin1 increased, the level of p62 decreased, the colocalization of autophagosome and lysosome increased, and the cell viability was significantly increased after the application of autophagy inhibitors 3-MA. In addition, SSa can induce ER stress in L-02 cells in vitro. Further studies demonstrated that SSa activated the PERK/eIF2α/ATF4/CHOP pathway, IRE1-TRAF2 pathway, ATF6 pathway, and AMPK/mTOR pathway associated with ER stress. Application of ER stress inhibitors 4-PBA can significantly down-regulate the level of autophagy and improve cell viability. Results of in vivo experiments showed that treatment with 150 and 300 mg/kg Ssa significantly elevated the liver/body weight ratio and caused histological injury in mice liver. Furthermore, Ssa treatment induced significantly downregulated p62 expression but upregulated LC3-II, CHOP, and GRP78 expression in mice livers. Taken together, our results showed that SSa can activate endoplasmic reticulum stress, promote toxic autophagy, and then induce cell death. We revealed an alternative mechanism involving autophagy and ERs, by which Ssa induced hepatotoxicity.
    Keywords:  Autophagy; Endoplasmic reticulum stress; Hepatotoxicity; Saikosaponin a
    DOI:  https://doi.org/10.1016/j.tiv.2022.105534
  26. Acta Pharm Sin B. 2022 Dec;12(12): 4407-4423
    Highly expressed SERCA2 triggers tumor cell autophagy and is a druggable vulnerability in triple-negative breast cancer.
    Minmin Fan, Jian Gao, Lin Zhou, Wenwen Xue, Yixuan Wang, Jingwei Chen, Wuhao Li, Ying Yu, Bo Liu, Yan Shen, Qiang Xu.
      Chemoresistance remains a major obstacle to successful treatment of triple negative breast cancer (TNBC). Identification of druggable vulnerabilities is an important aim for TNBC therapy. Here, we report that SERCA2 expression correlates with TNBC progression in human patients, which promotes TNBC cell proliferation, migration and chemoresistance. Mechanistically, SERCA2 interacts with LC3B via LIR motif, facilitating WIPI2-independent autophagosome formation to induce autophagy. Autophagy-mediated SERCA2 degradation induces SERCA2 transactivation through a Ca2+/CaMKK/CREB-1 feedback. Moreover, we found that SERCA2-targeting small molecule RL71 enhances SERCA2-LC3B interaction and induces excessive autophagic cell death. The increase in SERCA2 expression predisposes TNBC cells to RL71-induced autophagic cell death in vitro and in vivo. This study elucidates a mechanism by which TNBC cells maintain their high autophagy activity to induce chemoresistance, and suggests increased SERCA2 expression as a druggable vulnerability for TNBC.
    Keywords:  Autophagy; Chemoresistance; Druggable; LC3B; SERCA2; TNBC; Vulnerability
    DOI:  https://doi.org/10.1016/j.apsb.2022.05.009
  27. Int J Mol Sci. 2022 Dec 11. pii: 15702. [Epub ahead of print]23(24):
    Fluid Shear Stress Regulates Osteogenic Differentiation via AnnexinA6-Mediated Autophagy in MC3T3-E1 Cells.
    Tong Pei, Guanyue Su, Jie Yang, Wenbo Gao, Xinrui Yang, Yaojia Zhang, Jie Ren, Yang Shen, Xiaoheng Liu.
      Fluid shear stress (FSS) facilitates bone remodeling by regulating osteogenic differentiation, and extracellular matrix maturation and mineralization. However, the underlying molecular mechanisms of how mechanical stimuli from FSS are converted into osteogenesis remain largely unexplored. Here, we exposed MC3T3-E1 cells to FSS with different intensities (1 h FSS with 0, 5, 10, and 20 dyn/cm2 intensities) and treatment durations (10 dyn/cm2 FSS with 0, 0.5, 1, 2 and 4 h treatment). The results demonstrate that the 1 h of 10 dyn/cm2 FSS treatment greatly upregulated the expression of osteogenic markers (Runx2, ALP, Col I), accompanied by AnxA6 activation. The genetic ablation of AnxA6 suppressed the autophagic process, demonstrating lowered autophagy markers (Beclin1, ATG5, ATG7, LC3) and decreased autophagosome formation, and strongly reduced osteogenic differentiation induced by FSS. Furthermore, the addition of autophagic activator rapamycin to AnxA6 knockdown cells stimulated autophagy process, and coincided with more expressions of osteogenic proteins ALP and Col I under both static and FSS conditions. In conclusion, the findings in this study reveal a hitherto unidentified relationship between FSS-induced osteogenic differentiation and autophagy, and point to AnxA6 as a key mediator of autophagy in response to FSS, which may provide a new target for the treatment of osteoporosis and other diseases.
    Keywords:  annexinA6; autophagy; fluid shear stress; mineralization; osteogenic differentiation
    DOI:  https://doi.org/10.3390/ijms232415702
  28. Adv Sci (Weinh). 2022 Dec 18. e2205483
    Targeting the Lysosomal Degradation of Rab22a-NeoF1 Fusion Protein for Osteosarcoma Lung Metastasis.
    Cuiling Zeng, Li Zhong, Wenqiang Liu, Yu Zhang, Xinhao Yu, Xin Wang, Ruhua Zhang, Tiebang Kang, Dan Liao.
      Rab22a-NeoF fusion protein has recently been reported as a promising target for osteosarcoma lung metastasis. However, how this fusion protein is regulated in cells remains unknown. Here, using multiple screenings, it is reported that Rab22a-NeoF1 fusion protein is degraded by an E3 ligase STUB1 via the autophagy receptor NDP52-mediated lysosome pathway, which is facilitated by PINK1 kinase. Mechanistically, STUB1 catalyzes the K63-linked ubiquitin chains on lysine112 of Rab22a-NeoF1, which is responsible for the binding of Rab22a-NeoF1 to NDP52, resulting in lysosomal degradation of Rab22a-NeoF1. PINK1 is able to phosphorylate Rab22a-NeoF1 at serine120, which promotes ubiquitination and degradation of Rab22a-NeoF1. Consistently, by upregulating PINK1, Sorafenib and Regorafenib can inhibit osteosarcoma lung metastasis induced by Rab22a-NeoF1. These findings reveal that the lysosomal degradation of Rab22a-NeoF1 fusion protein is targetable for osteosarcoma lung metastasis, proposing that Sorafenib and Regorafenib may benefit cancer patients who are positive for the RAB22A-NeoF1 fusion gene.
    Keywords:  degradation; fusion protein; metastasis; osteosarcoma; phosphorylation
    DOI:  https://doi.org/10.1002/advs.202205483
  29. Mol Biol Rep. 2022 Dec 20.
    Therapeutic role of mTOR inhibitors in control of SARS-CoV-2 viral replication.
    Tuba Khalid, Adria Hasan, Jamal E Fatima, Soban Ahmad Faridi, Ahamad Faiz Khan, Snober S Mir.
      By the end of 2019, COVID-19 was reported in Wuhan city of China, and through human-human transmission, this virus spread worldwide and became a pandemic. Initial symptoms of the disease include fever, cough, loss of smell, taste, and shortness of breath, but a decrease in the oxygen levels in the body leads, and pneumonia may ultimately lead to the patient's death. However, the symptoms vary from patient to patient. To understand COVID-19 disease pathogenesis, researchers have tried to understand the cellular pathways that could be targeted to suppress viral replication. Thus, this article reviews the markers that could be targeted to inhibit viral replication by inhibiting the translational initiation complex/regulatory kinases and upregulating host autophagic flux that may lead to a reduction in the viral load. The article also highlights that mTOR inhibitors may act as potential inhibitors of viral replication. mTOR inhibitors such as metformin may inhibit the interaction of SARS-CoV-2 Nsp's and ORFs with mTORC1, LARP1, and 4E-BP. They may also increase autophagic flux by decreasing protein degradation via inhibition of Skp2, further promoting viral cell death. These events result in cell cycle arrest at G1 by p27, ultimately causing cell death.
    Keywords:  Autophagy; Coronavirus; Metformin; Skp2; mTOR inhibition
    DOI:  https://doi.org/10.1007/s11033-022-08188-1
  30. Gels. 2022 Nov 24. pii: 766. [Epub ahead of print]8(12):
    Autophagy Is a Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel.
    Elena Gabusi, Enrico Lenzi, Cristina Manferdini, Paolo Dolzani, Marta Columbaro, Yasmin Saleh, Gina Lisignoli.
      Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.
    Keywords:  adipose-derived mesenchymal stromal cells; autophagy; chondrogenesis; hydrogel
    DOI:  https://doi.org/10.3390/gels8120766
  31. J Med Chem. 2022 Dec 20.
    Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.
    Edita Poláchová, Kathrin Bach, Elena Heuten, Stancho Stanchev, Anežka Tichá, Philipp Lampe, Pavel Majer, Thomas Langer, Marius K Lemberg, Kvido Stříšovský.
      The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.
    DOI:  https://doi.org/10.1021/acs.jmedchem.2c01092
  32. Fac Rev. 2022 ;11 32
    The role of mTORC1 in the regulation of skeletal muscle mass.
    Sue C Bodine.
      Skeletal muscle mass is a very plastic characteristic of skeletal muscle and is regulated by signaling pathways that control the balance between anabolic and catabolic processes. The serine/threonine kinase mechanistic/mammalian target of rapamycin (mTOR) has been shown to be critically important in the regulation of skeletal muscle mass through its regulation of protein synthesis and degradation pathways. In this commentary, recent advances in the understanding of the role of mTORC1 in the regulation of muscle mass under conditions that induce hypertrophy and atrophy will be highlighted.
    Keywords:  aging; atrophy; hypertrophy; protein synthesis
    DOI:  https://doi.org/10.12703/r/11-32
  33. Front Mol Neurosci. 2022 ;15 966209
    Miro1 R272Q disrupts mitochondrial calcium handling and neurotransmitter uptake in dopaminergic neurons.
    Lisa Schwarz, Karan Sharma, Lorenzo D Dodi, Lara-Sophie Rieder, Petra Fallier-Becker, Nicolas Casadei, Julia C Fitzgerald.
      The Rho GTPase Miro1, located at the mitochondrial outer membrane is known to properly distribute mitochondria to synapses, aid calcium buffering and initiate PINK1-Parkin mediated mitophagy. Several heterozygous RHOT1/Miro1 variants were identified in sporadic Parkinson's disease patients. Miro1 R272Q is located within a calcium binding domain, but the functional outcome of this point mutation and its contribution to the development of disease are unclear. To address this, we introduced a heterozygous RHOT1/Miro1 R272Q point mutation in healthy induced pluripotent stem cells. In dopaminergic neurons, Miro1 R272Q does not affect Miro1 protein levels, CCCP-induced mitophagy, nor mitochondrial movement yet causes the fragmentation of mitochondria with reduction of cristae and ATP5A. Inhibition of the mitochondrial calcium uniporter phenocopied Miro1 R272Q cytosolic calcium response to Thapsigargin in active neurons, a similar effect was observed during the calcium buffering phase in Miro1 knockdown neuroblastoma cells. Altered mitochondrial calcium regulation is associated with reduced mitochondrial respiration and reduced catecholamine neurotransmitter uptake. Synaptic changes are not coupled to dopamine distribution or dopamine transporters but are linked to Miro1 R272Q-related calcium handling via the mitochondria concomitant with defective dopamine regulation at the mitochondrial surface by monoamine oxidase. We conclude that the Miro1 R272Q heterozygous point mutation dampens mitochondrial-calcium regulation and mitochondrial capacity via events at the outer membrane that are sufficient to disrupt dopaminergic function.
    Keywords:  Miro1; Parkinson’s disease; calcium; dopaminergic neuron; mitochondria
    DOI:  https://doi.org/10.3389/fnmol.2022.966209
  34. Clin Transl Oncol. 2022 Dec 19.
    The multifaced role and therapeutic regulation of autophagy in ovarian cancer.
    Fahimeh Nokhostin, Mahboobeh Azadehrah, Malihe Azadehrah.
      Ovarian cancer (OC) is one of the tumors that occurs most frequently in women. Autophagy is involved in cell homeostasis, biomolecule recycling, and survival, making it a potential target for anti-tumor drugs. It is worth noting that growing evidence reveals a close link between autophagy and OC. In the context of OC, autophagy demonstrates activity as both a tumor suppressor and a tumor promoter, depending on the context. Autophagy's exact function in OC is greatly reliant on the tumor microenvironment (TME) and other conditions, such as hypoxia, nutritional deficiency, chemotherapy, and so on. However, what can be concluded from different studies is that autophagy-related signaling pathways, especially PI3K/AKT/mTOR axis, increase in advanced stages and malignant phenotype of the disease reduces autophagy and ultimately leads to tumor progression. This study sought to present a thorough understanding of the role of autophagy-related signaling pathways in OC and existing therapies targeting these signaling pathways.
    Keywords:  Autophagy; Cancer therapy; Ovarian cancer; Signaling pathway
    DOI:  https://doi.org/10.1007/s12094-022-03045-w
  35. Mol Brain. 2022 Dec 21. 15(1): 101
    Object location learning in mice requires hippocampal somatostatin interneuron activity and is facilitated by mTORC1-mediated long-term potentiation of their excitatory synapses.
    Eve Honoré, Jean-Claude Lacaille.
      Hippocampus-dependent learning and memory originate from long-term synaptic changes in hippocampal networks. The activity of CA1 somatostatin interneurons (SOM-INs) during aversive stimulation is necessary for contextual fear memory formation. In addition, mTORC1-dependent long-term potentiation (LTP) of SOM-IN excitatory input synapses from local pyramidal cells (PC-SOM synapses) contributes to the consolidation of fear motivated spatial and contextual memories. Although, it remains unknown if SOM-IN activity and LTP are necessary and sufficient for novelty motivated spatial episodic memory such as the object location memory, and if so when it is required. Here we use optogenetics to examine whether dorsal CA1 SOM-IN activity and LTP are sufficient to regulate object location memory. First, we found that silencing SOM-INs during object location learning impaired memory. Second, optogenetic induction of PC-SOM synapse LTP (TBSopto) given 30 min before object location training, resulted in facilitation of memory. However, in mice with mTORC1 pathway genetically inactivated in SOM-INs, which blocks PC-SOM synapse LTP, TBSopto failed to facilitate object location memory. Our results indicate that SOM-IN activity is necessary during object location learning and that optogenetic induction of PC-SOM synapse LTP is sufficient to facilitate consolidation of object location memory. Thus, hippocampal somatostatin interneuron activity is required for object location learning, a hippocampus-dependent form of novelty motivated spatial learning that is facilitated by plasticity at PC-SOM synapses.
    Keywords:  Dorsal CA1 hippocampus; Long-term potentiation; Object location memory; Optogenetic silencing; Optogenetic synaptic plasticity; Somatostatin interneurons; mTORC1
    DOI:  https://doi.org/10.1186/s13041-022-00988-7
  36. Pharmacol Res. 2022 Dec 16. pii: S1043-6618(22)00559-X. [Epub ahead of print]187 106613
    3'-epi-12β-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells.
    Feng-Ying Huang, Shu-Zhen Dai, Wen-Tian Xu, Wei Xiong, Yan Sun, Yong-Hao Huang, Jin-Yan Wang, Ying-Ying Lin, Hengyu Chen, Guang-Hong Tan, Wu-Ping Zheng.
      Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12β-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.
    Keywords:  3′-epi-12β-hydroxyfroside; Autophagy; Cardiac glycosides; Immunogenic cell death; Necroptosis
    DOI:  https://doi.org/10.1016/j.phrs.2022.106613
  37. Pharmaceuticals (Basel). 2022 Dec 13. pii: 1547. [Epub ahead of print]15(12):
    Autophagy-Related ncRNAs in Pancreatic Cancer.
    Simone Donati, Cinzia Aurilia, Gaia Palmini, Irene Falsetti, Teresa Iantomasi, Maria Luisa Brandi.
      Pancreatic cancer (PC) is a malignancy accounting for only 3% of total cancers, but with a low 5-year relative survival rate. Approximately 80% of PC patients are diagnosed at a late stage when the disease has already spread from the primary site. Despite advances in PC treatment, there is an urgently needed for the identification of novel therapeutic strategies for PC, particularly for patients who cannot undergo classical surgery. Autophagy is an evolutionarily conserved process used by cells to adapt to metabolic stress via the degrading or recycling of damaged or unnecessary organelles and cellular components. This process is elevated in PC and, thus, it contributes to the onset, progression, and cancer cell resistance to chemotherapy in pancreatic tumors. Autophagy inhibition has been shown to lead to cancer regression and to increase the sensitivity of pancreatic cells to radiation and chemotherapy. Emerging studies have focused on the roles of non-coding RNAs (ncRNAs), such as miRNAs, long non-coding RNAs, and circular RNAs, in PC development and progression. Furthermore, ncRNAs have been reported as crucial regulators of many biological processes, including autophagy, suggesting that ncRNA-based autophagy targeting methods could be promising novel molecular approaches for specifically reducing autophagic flux, thus improving the management of PC patients. In this review, we briefly summarize the existing studies regarding the role and the regulatory mechanisms of autophagy-related ncRNAs in the context of this cancer.
    Keywords:  autophagy; circular RNAs; long non-coding RNAs; miRNAs; non-coding RNAs; pancreatic tumors
    DOI:  https://doi.org/10.3390/ph15121547
  38. J Biochem Mol Toxicol. 2022 Dec 19. e23280
    Inhibition of autophagy reduces the rate of fluoride-induced LS8 apoptosis via regulating ATG5 and ATG7.
    Lin Zhao, Han Wang, Sijia Liu, Tao Xi, Liyuan Wang, Yang Li, Lu Chen, Ruan Jianping, Kristina Xiao Liang.
      Excessive fluoride affects ameloblast differentiation and tooth development. The fate of fluorinated ameloblasts is determined by multiple signaling pathways in response to a range of stimuli. Both autophagy and apoptosis are involved in the regulation of dental fluorosis as well as in protein synthesis and enamel mineralization. Emerging evidence suggests that autophagy and apoptosis are interconnected and that their interaction greatly influences cell death. However, the effect of autophagy on apoptosis in fluoride-treated ameloblasts is unclear. Here, we employed an in vitro cellular model of fluorosis in mouse ameloblast-like LS8 cells and induced autophagy using sodium fluoride (NaF). Our findings suggest that NaF treatment induces autophagy in LS8 cells, and ATG5 and ATG7 are important molecules involved in this process. We also showed that NaF treatment reduced cell viability in Atg5/7 siRNA and autophagy inhibitor-treated LS8 cells. More importantly, NaF-induced apoptosis can be reversed by inhibiting early stage of autophagy. In conclusion, our study shows that autophagy is closely related to dental fluorosis, and inhibition of autophagy, especially ATG5/7, reduces fluoride-induced cell death and apoptosis.
    Keywords:  Apoptosis; LS8; NaF; autophagy
    DOI:  https://doi.org/10.1002/jbt.23280
  39. Exp Gerontol. 2022 Dec 21. pii: S0531-5565(22)00380-1. [Epub ahead of print]172 112071
    Changes in AMPK activity induces cellular senescence in human dental follicle cells.
    Christian Morsczeck, Anja Reck, Torsten E Reichert.
      Dental Follicle Cells (DFCs) are somatic stem cells with a limited lifespan, but little is known about a possible mechanism of cellular senescence. Previous studies have shown that cellular senescence is associated with increased demand of glycolsis or the "glycolytic metabotype", which can be induced by activation of 5' adenosine monophosphate-activated protein kinase (AMPK), and decreased autophagy. This study examined the role of AMPK in inducing senescence in DFCs. During the induction of cellular senescence, AMPK activity was impaired, suggesting a negative impact on senescence induction. In line with this assumption, cellular senescence was induced upon inhibition of AMPK with a specific siRNA. In addition, after this inhibition, autophagy was also inhibited. Moreover, specific inhibition of autophagy promoted cellular senescence. However, inducers of AMPK such as metformin or AICAR surprisingly increased senescence in DFCs. Interestingly, autophagy was impaired after long-term induction of AMPK with AICAR and metformin. Moreover, activation of AMPK induces the consumption of glucose but decreases NAD/NADH ratio in DFCs that suggest not only "glycolytic metabotype" of DFCs but also Mitochondrial Dysfunction Associated Senescence (MiDAS). Both changes are highly associated with the induction of cellular senescence. Hence, both AMPK activation and inhibition promote the induction of cellular senecence of DFCs.
    Keywords:  AMP-kinase; Autophagy; Cellular senescence; Dental follicle cells; Metformin
    DOI:  https://doi.org/10.1016/j.exger.2022.112071
  40. Int J Mol Sci. 2022 Dec 17. pii: 16098. [Epub ahead of print]23(24):
    Prunus spinosa Extract Sensitized HCT116 Spheroids to 5-Fluorouracil Toxicity, Inhibiting Autophagy.
    Maria Condello, Rosa Vona, Stefania Meschini.
      Autophagy is a lysosomal degradation and recycling process involved in tumor progression and drug resistance. The aim of this work was to inhibit autophagy and increase apoptosis in a 3D model of human colorectal cancer by combined treatment with our patented natural product Prunus spinosa + nutraceutical activator complex (PsT + NAC®) and 5-fluorouracil (5-FU). By means of cytotoxic evaluation (MTT assay), cytofluorimetric analysis, light and fluorescence microscopy investigation and Western blotting evaluation of the molecular pathway PI3/AKT/mTOR, Caspase-9, Caspase-3, Beclin1, p62 and LC3, we demonstrated that the combination PsT + NAC® and 5-FU significantly reduces autophagy by increasing the apoptotic phenomenon. These results demonstrate the importance of using non-toxic natural compounds to improve the therapeutic efficacy and reduce the side effects induced by conventional drugs in human colon cancer.
    Keywords:  5-fluorouracil; Prunus spinosa; apoptosis; autophagy; colorectal cancer; drug resistance; spheroids
    DOI:  https://doi.org/10.3390/ijms232416098
  41. Nat Commun. 2022 Dec 22. 13(1): 7868
    Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease.
    Houfu Leng, Hanlin Zhang, Linsen Li, Shuhao Zhang, Yanping Wang, Selina J Chavda, Daria Galas-Filipowicz, Hantao Lou, Adel Ersek, Emma V Morris, Erdinc Sezgin, Yi-Hsuan Lee, Yunsen Li, Ana Victoria Lechuga-Vieco, Mei Tian, Jian-Qing Mi, Kwee Yong, Qing Zhong, Claire M Edwards, Anna Katharina Simon, Nicole J Horwood.
      Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.
    DOI:  https://doi.org/10.1038/s41467-022-35358-3
  42. Cells. 2022 Dec 07. pii: 3951. [Epub ahead of print]11(24):
    HDAC6 Inhibition Alleviates Ischemia- and Cisplatin-Induced Acute Kidney Injury by Promoting Autophagy.
    Lang Shi, Zhixia Song, Chenglong Li, Fangjing Deng, Yao Xia, Jing Huang, Xiongfei Wu, Jiefu Zhu.
      Histone deacetylase (HDAC) 6 exists exclusively in cytoplasm and deacetylates cytoplasmic proteins such as α-tubulin. HDAC6 dysfunction is associated with several pathological conditions in renal disorders, including UUO-induced fibrotic kidneys and rhabdomyolysis-induced nephropathy. However, the role of HDAC6 in ischemic acute kidney injury (AKI) and the mechanism by which HDAC6 inhibition protects tubular cells after AKI remain unclear. In the present study, we observed that HDAC6 was markedly activated in kidneys subjected to ischemia- and cisplatin (cis)-induced AKI treatment. Pharmacological inhibition of HDAC6 alleviated renal impairment and renal tubular damage after ischemia and cisplatin treatment. HDAC6 dysfunction was associated with decreased acetylation of α-tubulin at the residue of lysine 40 and autophagy. HDAC6 inhibition preserved acetyl-α-tubulin-enhanced autophagy flux in AKI and cultured tubular cells. Genetic ablation of the renal tubular (RT) Atg7 gene or pharmacological inhibition of autophagy suppressed the protective effects of HDAC6. Taken together, our study indicates that HDAC6 contributes to ischemia- and cisplatin-induced AKI by inhibiting autophagy and the acetylation of α-tubulin. These results suggest that HDAC6 could be a potential target for ischemic and nephrotoxic AKI.
    Keywords:  HDAC6; acute kidney injury; autophagy; cisplatin; renal ischemia/reperfusion
    DOI:  https://doi.org/10.3390/cells11243951
  43. Cell Death Dis. 2022 Dec 20. 13(12): 1060
    Blockage of Nrf2 and autophagy by L-selenocystine induces selective death in Nrf2-addicted colorectal cancer cells through p62-Keap-1-Nrf2 axis.
    Wei-Lun Hsu, Chieh-Min Wang, Chao-Ling Yao, Ssu-Ching Chen, Chung-Yi Nien, Yang-Ho Sun, Tsung-Yu Tseng, Yueh-Hsia Luo.
      Persistent Nrf2 activation is typically noted in many cancers, including colorectal cancer (CRC), aiding cancer cells in overcoming growth stress and promoting cancer progression. Sustained Nrf2 activation, which is beneficial for cancer cells, is called "Nrf2 addiction"; it is closely associated with malignancy and poor prognosis in patients with cancer. However, Nrf2 inhibitors may have adverse effects on normal cells. Here, we found that the selenocompound L-selenocystine (SeC) is selectively cytotoxic in the Nrf2-addicted CRC cell line WiDr cells, but not in non-Nrf2-addicted mesenchymal stem cells (MSCs) and normal human colon cells. Another CRC cell line, C2BBe1, which harbored lower levels of Nrf2 and its downstream proteins were less sensitive to SeC, compared with the WiDr cells. We further demonstrated that SeC inhibited Nrf2 and autophagy activation in the CRC cells. Antioxidant GSH pretreatment partially rescued the CRC cells from SeC-induced cytotoxicity and Nrf2 and autophagy pathway inhibition. By contrast, SeC activated Nrf2 and autophagy pathway in non-Nrf2-addicted MSCs. Transfecting WiDr cells with Nrf2-targeting siRNA decreased persistent Nrf2 activation and alleviated SeC cytotoxicity. In KEAP1-knockdown C2BBe1 cells, Nrf2 pathway activation increased SeC sensitivity and cytotoxicity. In conclusion, SeC selectively attacks cancer cells with constitutively activated Nrf2 by reducing Nrf2 and autophagy pathway protein expression through the P62-Nrf2-antioxidant response element axis and eventually trigger cell death.
    DOI:  https://doi.org/10.1038/s41419-022-05512-2
  44. Mol Biol Cell. 2022 Dec 21. mbcE22060236
    Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion.
    Shannon E Collins, Mollie E Wiegand, Alyssa N Werner, Isabella N Brown, Mary I Mundo, Douglas J Swango, Ghassan Mouneimne, Pascale G Charest.
      We previously identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.
    DOI:  https://doi.org/10.1091/mbc.E22-06-0236
  45. Int J Mol Sci. 2022 Dec 14. pii: 15873. [Epub ahead of print]23(24):
    The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain.
    Kuang-I Cheng, Yu-Chin Chang, Li-Wen Chu, Su-Ling Hsieh, Li-Mei An, Zen-Kong Dai, Bin-Nan Wu.
      Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic-lysosomal pathway in the rat CCI model. Sprague-Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.
    Keywords:  apoptosis; autophagy; loganin; neuropathic pain; peripheral nerve injury
    DOI:  https://doi.org/10.3390/ijms232415873
  46. Cell Rep. 2022 Dec 20. pii: S2211-1247(22)01729-6. [Epub ahead of print]41(12): 111837
    The Sag-Shoc2 axis regulates conversion of mPanINs to cystic lesions in Kras pancreatic tumor model.
    Mingjia Tan, Yu Chang, Xiaoqiang Liu, Hua Li, Zaiming Tang, Mukesh K Nyati, Yi Sun.
      SAG/RBX2 is an E3 ligase, whereas SHOC2 is a RAS-RAF positive regulator. In this study, we address how Sag-Shoc2 crosstalk regulates pancreatic tumorigenesis induced by KrasG12D. Sag deletion increases the size of pancreas and causes the conversion of murine pancreatic intraepithelial neoplasms (mPanINs) to neoplastic cystic lesions with a mechanism involving Shoc2 accumulation, suggesting that Sag determines the pathological process via targeting Shoc2. Shoc2 deletion significantly inhibits pancreas growth, mPanIN formation, and acinar cell transdifferentiation, indicating that Shoc2 is essential for KrasG12D-induced pancreatic tumorigenesis. Likewise, in a primary acinar 3D culture, Sag deletion inhibits acinar-to-ductal transdifferentiation, while Shoc2 deletion significantly reduces the duct-like structures. Mechanistically, SAG is an E3 ligase that targets SHOC2 for degradation to affect both Mapk and mTorc1 pathways. Shoc2 deletion completely rescues the phenotype of neoplastic cystic lesions induced by Sag deletion, indicating physiological relevance of the Sag-Shoc2 crosstalk. Thus, the Sag-Shoc2 axis specifies the pancreatic tumor types induced by KrasG12D.
    Keywords:  CP: Cancer; Deptor; KRAS; MAPK and mTORC1 signals; Sag/Rbx2 E3 ligase; Shoc2; cystic lesions; cystogenesis; pancreatic tumorigenesis
    DOI:  https://doi.org/10.1016/j.celrep.2022.111837
  47. Asian J Androl. 2022 Dec 13.
    Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway.
    Hao-Yu Liang, Fan Peng, Min-Jia Pan, Sen-Lin Liao, Cun Wei, Guan-Yang Wei, Xiao Xie, Kang-Yi Xue, Ming-Kun Chen, Jian-Kun Yang, Wen-Bin Guo, Cun-Dong Liu, Qi-Zhao Zhou.
      Cyclophosphamide-induced testosterone deficiency (CPTD) during the treatment of cancers and autoimmune disorders severely influences the quality of life of patients. Currently, several guidelines recommend patients suffering from CPTD receive testosterone replacement therapy (TRT). However, TRT has many disadvantages underscoring the requirement for alternative, nontoxic treatment strategies. We previously reported bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exos) could alleviate cyclophosphamide (CP)-induced spermatogenesis dysfunction, highlighting their role in the treatment of male reproductive disorders. Therefore, we further investigated whether BMSCs-exos affect autophagy and testosterone synthesis in Leydig cells (LCs). Here, we examined the effects and probed the molecular mechanisms of BMSCs-exos on CPTD in vivo and in vitro by detecting the expression levels of genes and proteins related to autophagy and testosterone synthesis. Furthermore, the testosterone concentration in serum and cell-conditioned medium, and the photophosphorylation protein levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were measured. Our results suggest that BMSCs-exos could be absorbed by LCs through the blood-testis barrier in mice, promoting autophagy in LCs and improving the CP-induced low serum testosterone levels. BMSCs-exos inhibited cell death in CP-exposed LCs, regulated the AMPK-mTOR signaling pathway to promote autophagy in LCs, and then improved the low testosterone synthesis ability of CP-induced LCs. Moreover, the autophagy inhibitor, 3-methyladenine (3-MA), significantly reversed the therapeutic effects of BMSCs-exos. These findings suggest that BMSCs-exos promote LC autophagy by regulating the AMPK-mTOR signaling pathway, thereby ameliorating CPTD. This study provides novel evidence for the clinical improvement of CPTD using BMSCs-exos.
    Keywords:  Leydig cells; autophagy; bone marrow mesenchymal stem cells; cyclophosphamide; exosomes; testosterone deficiency
    DOI:  https://doi.org/10.4103/aja202286
  48. Front Cell Dev Biol. 2022 ;10 1089668
    New insights into the interplay between autophagy and cartilage degeneration in osteoarthritis.
    Xiaoman Lv, Ting Zhao, Youwu Dai, Mingqin Shi, Xiaoyi Huang, Yuanyuan Wei, Jiayan Shen, Xiaoyu Zhang, Zhaohu Xie, Qi Wang, Zhaofu Li, Dongdong Qin.
      Autophagy is an intracellular degradation system that maintains the stable state of cell energy metabolism. Some recent findings have indicated that autophagy dysfunction is an important driving factor for the occurrence and development of osteoarthritis (OA). The decrease of autophagy leads to the accumulation of damaged organelles and macromolecules in chondrocytes, which affects the survival of chondrocytes and ultimately leads to OA. An appropriate level of autophagic activation may be a new method to prevent articular cartilage degeneration in OA. This minireview discussed the mechanism of autophagy and OA, key autophagy targets regulating OA progression, and evaluated therapeutic applications of drugs targeting autophagy in preclinical and clinical research. Some critical issues worth paying attention to were also raised to guide future research efforts.
    Keywords:  autophagy; mechanism; osteoarthritis; preclinical and clinical research; therapeutic applications
    DOI:  https://doi.org/10.3389/fcell.2022.1089668
  49. Brain Res. 2022 Dec 17. pii: S0006-8993(22)00430-9. [Epub ahead of print]1801 148206
    Sulforaphane attenuates microglia-mediated neuronal damage by down-regulating the ROS/autophagy/NLRP3 signal axis in fibrillar Aβ-activated microglia.
    Yunzhu Yang, Jiafa Zhang, Canhong Yang, Bo Dong, Yanhong Fu, Yuanyuan Wang, Ming Gong, Tao Liu, Pingming Qiu, Weibing Xie, Tianming Lü.
      The neuroinflammatory hypothesis of Alzheimer's disease (AD) posits that amyloid-beta (Aβ) phagocytosis along with subsequent lysosomal damage and NLRP3 inflammasome activation plays important roles in Aβ-induced microglia activation and microglia-induced neurotoxicity. Sulforaphane (SFN) has neuroprotective effects for AD. However, whether SFN can inhibit its cytotoxic autophagy and NLRP3 inflammasome activation in microglia remain unknown. In this study, results showed SFN played an indirect, protective role on neurons via a series of impacts on Aβ-activated microglia, including inhibition of autophagy initiation as well as autophagic lysosomal membrane permeability and subsequent NLRP3/caspase-1 inflammasomes activation. M1 phenotype polarization was also inhibited. Our results demonstrated that SFN could inhibit the cytostatic autophagy-induced NLRP3 signaling pathway in Aβ-activated microglia by decreasing reactive oxygen species (ROS) production. These results provide novel insight into the potential role of SFN in AD therapy.
    Keywords:  Alzheimer’s disease; Amyloid-beta; Autophagy; Microglia; Reactive oxygen species; Sulforaphane
    DOI:  https://doi.org/10.1016/j.brainres.2022.148206
  50. Hum Genet. 2022 Dec 20.
    CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment.
    Daphne J Smits, Jordy Dekker, Rachel Schot, Brahim Tabarki, Amal Alhashem, Jeroen A A Demmers, Dick H W Dekkers, Antonio Romito, Peter J van der Spek, Tjakko J van Ham, Aida M Bertoli-Avella, Grazia M S Mancini.
      CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.
    DOI:  https://doi.org/10.1007/s00439-022-02511-3
  51. J Med Virol. 2022 Dec 22.
    mTOR Signaling Regulates Zika Virus Replication Bidirectionally through Autophagy and Protein Translation.
    Bin Liu, Yahui Zhang, Hao Ren, Qiufeng Yao, Jianbo Ba, Jie Luan, Ping Zhao, Zhaoling Qin, Zhongtian Qi.
      Zika virus (ZIKV) reemerged in 2016 and attracted much more attention worldwide. To date, the limited knowledge of ZIKV interactions with host cells in the early stages of infection impedes the prevention of viral epidemics and the treatment of ZIKV disease. The Mammalian target of rapamycin (mTOR) signaling pathway plays an essential role in the regulation of autophagy and protein synthesis during multiple viral infections. This study aimed to investigate the functional role of mTOR signaling in ZIKV replication in human umbilical vein endothelial cells (HUVECs). Immunoblotting demonstrated that ZIKV infection inhibited mTORC1 signaling, enhancing autophagy but obstructing protein translation. Drugs or siRNA for interfering with mTOR signaling molecules were utilized to demonstrate that AKT/TSC2/mTORC1 signaling was involved in ZIKV infection and that autophagy promoted ZIKV production, but viral protein expression was regulated by mTORC1 signaling. Moreover, confocal microscopy indicated a robust correlation between autophagy and viral RNA transcription. This study clarifies the dual functions of mTOR signaling during ZIKV infection and provides theoretical support for developing potential anti-ZIKV drugs based on mTOR signaling molecules and deeper insights to better understand the mechanism between ZIKV and host cells. This article is protected by copyright. All rights reserved.
    Keywords:  Zika virus; autophagy; mTOR; viral replication
    DOI:  https://doi.org/10.1002/jmv.28422
  52. Bio Protoc. 2022 Nov 20. pii: e4554. [Epub ahead of print]12(22):
    Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.
    Omar Peña-Ramos, Zheng Zhou.
      During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.72466.
    Keywords:   Apoptosis ; Apoptotic cell clearance ; Autophagosomes ; Autophagy ; Caenorhabditis elegans ; Engulfment ; Fluorescence ; GFP ; LC3 ; LGG-1 ; LGG-2 ; Membrane fusions ; Phagocytosis ; Phagosomes ; Time-lapse imaging ; mCherry ; mNeonGreen (mNG)
    DOI:  https://doi.org/10.21769/BioProtoc.4554
  53. Antioxidants (Basel). 2022 Dec 19. pii: 2496. [Epub ahead of print]11(12):
    Stevia and Stevioside Attenuate Liver Steatosis through PPARα-Mediated Lipophagy in db/db Mice Hepatocytes.
    Miey Park, Anshul Sharma, Hana Baek, Jin-Young Han, Junho Yu, Hae-Jeung Lee.
      Lipophagy, a type of autophagy that breaks down lipid droplets, is essential in the regulation of intracellular lipid accumulation and intracellular free fatty acid levels in numerous organisms and metabolic conditions. We investigated the effects of Stevia rebaudiana Bertoni (S), a low-calorie sweetener, and stevioside (SS) on hepatic steatosis and autophagy in hepatocytes, as well as in db/db mice. S and SS reduced the body and liver weight and levels of serum triglyceride, total cholesterol, and hepatic lipogenic proteins. In addition, S and SS increased the levels of fatty acid oxidase, peroxisome proliferator-activated receptor alpha (PPARα), and microtubule-associated protein light chain 3 B but decreased that of sequestosome 1 (p62) in the liver of db/db mice. Additionally, Beclin 1, lysosomal associated membrane protein 1, and phosphorylated adenosine monophosphate-activated protein kinase protein expression was augmented following S and SS treatment of db/db mice. Furthermore, the knockdown of PPARα blocked lipophagy in response to SS treatment in HepG2 cells. These outcomes indicate that PPARα-dependent lipophagy is involved in hepatic steatosis in the db/db mouse model and that SS, a PPARα agonist, represents a new therapeutic option for managing associated diseases.
    Keywords:  PPARα; lipophagy; non-alcoholic fatty liver disease; stevia; stevioside
    DOI:  https://doi.org/10.3390/antiox11122496
  54. Cancers (Basel). 2022 Dec 12. pii: 6128. [Epub ahead of print]14(24):
    Co-Targeting the EGFR and PI3K/Akt Pathway to Overcome Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma: What about Autophagy?
    Hannah Zaryouh, Jinthe Van Loenhout, Marc Peeters, Jan Baptist Vermorken, Filip Lardon, An Wouters.
      Resistance to EGFR-targeted therapy is a major obstacle on the road to effective treatment options for head and neck cancers. During the search for underlying mechanisms and regulators of this resistance, there were several indications that EGFR-targeted therapy resistance is (partially) mediated by aberrant signaling of the PI3K/Akt pathway. Genomic alterations in and/or overexpression of major components of the PI3K/Akt pathway are common in HNSCC tumors. Therefore, downstream effectors of the PI3K/Akt pathway serve as promising targets in the search for novel therapeutic strategies overcoming resistance to EGFR inhibitors. As both the EGFR/Ras/Raf/MAPK and the PI3K/Akt pathway are involved in autophagy, combinations of EGFR and PI3K/Akt pathway inhibitors can induce an autophagic response in tumor cells. This activation of autophagy can be seen as a "double-edge sword", depending on the cellular context. Autophagy is largely known as a cytoprotective mechanism, but it can also be a mechanism of programmed (autophagic) cell death. The activation of autophagy during anti-cancer treatment is, therefore, not necessarily a bad sign. However, in HNSCC, the role of therapy-induced autophagy as an anti-tumor mechanism is still largely unclear. Further research is warranted to understand the potential of combination treatments targeting both the EGFR and PI3K/Akt pathway.
    Keywords:  EGFR; HNSCC; PI3K/Akt; autophagy; cetuximab; combination therapy; resistance
    DOI:  https://doi.org/10.3390/cancers14246128
  55. Nutrients. 2022 Dec 16. pii: 5358. [Epub ahead of print]14(24):
    Limonin, a Component of Immature Citrus Fruits, Activates Anagen Signaling in Dermal Papilla Cells.
    Jung-Il Kang, Youn Kyoung Choi, Sang-Chul Han, Hyeon Gyu Kim, Seok Won Hong, Jungeun Kim, Jae Hoon Kim, Jin Won Hyun, Eun-Sook Yoo, Hee-Kyoung Kang.
      Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss. We evaluated the hair anagen activation effects of limonin, which is abundant in immature citrus fruits. Limonin increased the proliferation of rat dermal papilla cells (rDPC) by changing the levels of cyclin D1 and p27, and increasing the number of BrdU-positive cells. Limonin increased autophagy by decreasing phosphorylated mammalian target of rapamycin levels and increasing the phospho-Raptor, ATG7 and LC3B. Limonin also activated the Wnt/β-catenin pathway by increasing phospho-β-catenin levels. XAV939, a Wnt/β-catenin inhibitor, inhibited these limonin-induced changes, including induced autophagy, BrdU-positive cells, and cell proliferation. Limonin increased the phosphorylated AKT levels in both two-dimensional cultured rDPC and three-dimensional spheroids. Treatment with the PI3K inhibitor wortmannin inhibited limonin-induced proliferation, and disrupted other limonin-mediated changes, including decreased p27, increased BrdU-positive cells, induced autophagy, and increased ATG7 and LC3B levels. Wortmannin also inhibited limonin-induced cyclin D1 and LC3 expression in spheroids. Collectively, these results indicate that limonin can enhance anagen signaling by activating autophagy via targeting the Wnt/β-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.
    Keywords:  PI3K/AKT; Wnt/β-Catenin; anagen signaling; autophagy; dermal papilla cells; limonin
    DOI:  https://doi.org/10.3390/nu14245358
  56. Cell Biosci. 2022 Dec 19. 12(1): 204
    FOXO3a-dependent PARKIN negatively regulates cardiac hypertrophy by restoring mitophagy.
    Teng Sun, Yu Han, Jia-Lei Li, Xiang-Ying Jiao, Lin Zuo, Jin Wang, Hai-Xiong Wang, Jun-Li Yang, Ji-Min Cao, Jian-Xun Wang.
      BACKGROUND: Sustained cardiac hypertrophy often develops maladaptive myocardial remodeling, and eventually progresses to heart failure and sudden death. Therefore, maladaptive hypertrophy is considered as a critical therapeutic target for many heart diseases. Mitophagy, a crucial mechanism in mitochondria quality control and cellular homeostasis, has been implicated in diverse cardiac disorders such as myocardial infarction, diabetic cardiomyopathy, cardiac hypertrophy and heart failure. However, what role mitophagy plays in heart diseases remains an enigma. PARKIN functions as an E3 ubiquitin protein ligase and mediates mitophagy cascades. It is still unclear whether PARKIN participates in the regulation of cardiac hypertrophy.RESULTS: PARKIN was downregulated in cardiomyocytes and hearts under hypertrophic stress. Enforced expression of PARKIN inhibited Ang II-induced cardiomyocyte hypertrophy. Compared to wide-type mice with Ang II-induced cardiac hypertrophy, Parkin transgenic mice subjected to Ang II administration showed attenuated cardiac hypertrophy and improved cardiac function. In addition, mitophagy machinery was impaired in response to Ang II, which was rescued by overexpression of PARKIN. PARKIN exerted the anti-hypertrophy effect through restoring mitophagy. In further exploring the underlying mechanisms, we found that PARKIN was transcriptionally activated by FOXO3a. FOXO3a promoted mitophagy and suppressed cardiac hypertrophy by targeting Parkin.
    CONCLUSIONS: The present study reveals a novel cardiac hypertrophy regulating model composed of FOXO3a, PARKIN and mitophagy program. Modulation of their levels may provide a new approach for preventing cardiac hypertrophy and heart failure.
    Keywords:  Cardiac hypertrophy; FOXO3a; Mitophagy; PARKIN
    DOI:  https://doi.org/10.1186/s13578-022-00935-y
  57. Cell Rep. 2022 Dec 20. pii: S2211-1247(22)01742-9. [Epub ahead of print]41(12): 111850
    Lysine crotonylation regulates leucine-deprivation-induced autophagy by a 14-3-3ε-PPM1B axis.
    Zilong Zheng, Guokai Yan, Xiuzhi Li, Yuke Fei, Lingling Sun, Haonan Yu, Yaorong Niu, Weihua Gao, Qing Zhong, Xianghua Yan.
      Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy.
    Keywords:  14-3-3ε; CP: Molecular biology; HDAC7; PPM1B; autophagy; crotonylation; leucine deprivation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111850
  58. J Fungi (Basel). 2022 Dec 17. pii: 1314. [Epub ahead of print]8(12):
    The SsAtg1 Activating Autophagy Is Required for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum.
    Wenli Jiao, Huilin Yu, Xueting Chen, Kunqin Xiao, Dongmei Jia, Fengting Wang, Yanhua Zhang, Hongyu Pan.
      Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 down-regulated and 1040 up-regulated, were identified. Transcriptomes data provide the possibility to precisely comprehend the sclerotia development. We further analyzed the differentially expressed genes (DEGs) in sclerotia to explore the molecular mechanism of sclerotia development, which include ribosome biogenesis and translation, melanin biosynthesis, autophagy and reactivate oxygen metabolism. Among these, the autophagy-related gene SsAtg1 was up-regulated in sclerotia. Atg1 homologs play critical roles in autophagy, a ubiquitous and evolutionarily highly conserved cellular mechanism for turnover of intracellular materials in eukaryotes. Therefore, we investigated the function of SsAtg1 to explore the function of the autophagy pathway in S. sclerotiorum. Deficiency of SsAtg1 inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. Notably, ΔSsAtg1 was unable to form sclerotia and displayed defects in vegetative growth under conditions of nutrient restriction. Furthermore, the development and penetration of the compound appressoria in ΔSsAtg1 was abnormal. Pathogenicity analysis showed that SsAtg1 was required for full virulence of S. sclerotiorum. Taken together, these results indicate that SsAtg1 is a core autophagy-related gene that has vital functions in nutrient utilization, sclerotia development and pathogenicity in S. sclerotiorum.
    Keywords:  Sclerotinia sclerotiorum; SsAtg1; autophagy; pathogenicity; sclerotia; transcriptome
    DOI:  https://doi.org/10.3390/jof8121314
  59. J Clin Invest. 2022 Dec 22. pii: e162434. [Epub ahead of print]
    The UBE2C/CDH1/DEPTOR axis is an oncogene-tumor suppressor cascade in lung cancer cells.
    Shizhen Zhang, Xiahong You, Yawen Zheng, Yanwen Shen, Xiufang Xiong, Yi Sun.
      Ubiquitin-conjugating enzyme E2C (UBE2C) mediates the ubiquitylation chain formation via the K11 linkage. While previous in vitro studies showed that UBE2C plays a growth-promoting role in cancer cell lines, the underlying mechanism remains elusive. Still unknown is whether and how UBE2C plays a promoting role in vivo. Here we reported that UBE2C is indeed essential for growth and survival of lung cancer cells harboring Kras mutations, and UBE2C is required for KrasG12D-induced lung tumorigenesis, since Ube2c deletion significantly inhibits tumor formation and extends the life-span of mice. Mechanistically, KrasG12D induces expression of UBE2C, which couples with APC/CCDH1 E3 ligase to promote ubiquitylation and degradation of DEPTOR, leading to activation of the mTORC signals. Importantly, DEPTOR levels are fluctuated during cell cycle progression in a manner dependent of UBE2C and CDH1, indicating their physiological connection. Finally, Deptor deletion fully rescues the tumor inhibitory effect of Ube2c deletion in the KrasG12D lung tumor model, indicating a causal role of Deptor. Taken together, our study shows that the UBE2C/CDH1/DEPTOR axis forms an oncogene-tumor suppressor cascade that regulates cell cycle progression and autophagy and validates that UBE2C is an attractive target for lung cancer associated with Kras mutations.
    Keywords:  Cell Biology; Lung cancer; Oncology; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI162434
  60. Cells. 2022 Dec 16. pii: 4079. [Epub ahead of print]11(24):
    Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.
    Ditte L Fogde, Cristina P R Xavier, Kristina Balnytė, Lya K K Holland, Kamilla Stahl-Meyer, Christoffel Dinant, Elisabeth Corcelle-Termeau, Cristina Pereira-Wilson, Kenji Maeda, Marja Jäättelä.
      Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.
    Keywords:  autophagy; cancer; cationic amphiphilic drugs; cell death; lysosomal membrane permeabilization; ursolic acid
    DOI:  https://doi.org/10.3390/cells11244079
  61. Adv Colloid Interface Sci. 2022 Dec 11. pii: S0001-8686(22)00226-3. [Epub ahead of print]311 102824
    Detection and modulation of neurodegenerative processes using graphene-based nanomaterials: Nanoarchitectonics and applications.
    Priyanka Tiwari, Sanjay Tiwari.
      Neurodegenerative disorders (NDDs) are caused by progressive loss of functional neurons following the aggregation and fibrillation of proteins in the central nervous system. The incidence rate continues to rise alarmingly worldwide, particularly in aged population, and the success of treatment remains limited to symptomatic relief. Graphene nanomaterials (GNs) have attracted immense interest on the account of their unique physicochemical and optoelectronic properties. The research over the past two decades has recognized their ability to interact with aggregation-prone neuronal proteins, regulate autophagy and modulate the electrophysiology of neuronal cells. Graphene can prevent the formation of higher order protein aggregates and facilitate the clearance of such deposits. In this review, after highlighting the role of protein fibrillation in neurodegeneration, we have discussed how GN-protein interactions can be exploited for preventing neurodegeneration. A comprehensive understanding of such interactions would contribute to the exploration of novel modalities for controlling neurodegenerative processes.
    Keywords:  Autophagy; Biomarker detection; Defibrillation; Neurotoxicity; Protein deposits; Scaffolds
    DOI:  https://doi.org/10.1016/j.cis.2022.102824
  62. Cell Rep. 2022 Dec 20. pii: S2211-1247(22)01726-0. [Epub ahead of print]41(12): 111834
    Pro-prion, as a membrane adaptor protein for E3 ligase c-Cbl, facilitates the ubiquitination of IGF-1R, promoting melanoma metastasis.
    Huan Li, Jie Zhang, Jing-Ru Ke, Ze Yu, Run Shi, Shan-Shan Gao, Jing-Feng Li, Zhen-Xing Gao, Chang-Shu Ke, Hui-Xia Han, Jiang Xu, Qibin Leng, Gui-Ru Wu, Yingqiu Li, Lin Tao, Xianghui Zhang, Man-Sun Sy, Chaoyang Li.
      Aberrant activation of receptor tyrosine kinase (RTK) is usually a result of mutation and plays important roles in tumorigenesis. How RTK without mutation affects tumorigenesis remains incompletely understood. Here we show that in human melanomas pro-prion (pro-PrP) is an adaptor protein for an E3 ligase c-Cbl, enabling it to polyubiquitinate activated insulin-like growth factor-1 receptor (IGF-1R), leading to enhanced melanoma metastasis. All human melanoma cell lines studied here express pro-PrP, retaining its glycosylphosphatidylinositol-peptide signal sequence (GPI-PSS). The sequence, PVILLISFLI in the GPI-PSS of pro-PrP, binds c-Cbl, docking c-Cbl to the inner cell membrane, forming a pro-PrP/c-Cbl/IGF-1R trimeric complex. Subsequently, IGF-1R polyubiquitination and degradation are augmented, which increases autophagy and tumor metastasis. Importantly, the synthetic peptide PVILLISFLI disrupts the pro-PrP/c-Cbl/IGF-1R complex, reducing cancer cell autophagy and mitigating tumor aggressiveness in vitro and in vivo. Targeting cancer-associated GPI-PSS may provide a therapeutic approach for treating human cancers expressing pro-PrP.
    Keywords:  CP: Cell biology; CP: Molecular biology; adaptor protein; autophagy; melanoma; metastasis; prion protein; receptor tyrosine kinase; treatment; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2022.111834
  63. Cell Death Differ. 2022 Dec 17.
    N6-methyladenosine-modified USP13 induces pro-survival autophagy and imatinib resistance via regulating the stabilization of autophagy-related protein 5 in gastrointestinal stromal tumors.
    Zhishuang Gao, Chao Li, Haoyu Sun, Yibo Bian, Zhiwei Cui, Nuofan Wang, Zhangjie Wang, Yang Yang, Zonghang Liu, Zhongyuan He, Bowen Li, Fengyuan Li, Zheng Li, Linjun Wang, Diancai Zhang, Li Yang, Zekuan Xu, Xueming Li, Hao Xu.
      Secondary resistance to imatinib (IM) represents a major challenge for therapy of gastrointestinal stromal tumors (GISTs). Aberrations in oncogenic pathways, including autophagy, correlate with IM resistance. Regulation of autophagy-related protein 5 (ATG5) by the ubiquitin-proteasome system is critical for autophagic activity, although the molecular mechanisms that underpin reversible deubiquitination of ATG5 have not been deciphered fully. Here, we identified USP13 as an essential deubiquitinase that stabilizes ATG5 in a process that depends on the PAK1 serine/threonine-protein kinase and which enhances autophagy and promotes IM resistance in GIST cells. USP13 preferentially is induced in GIST cells by IM and interacts with ATG5, which leads to stabilization of ATG5 through deubiquitination. Activation of PAK1 promoted phosphorylation of ATG5 thereby enhancing the interaction of ATG5 with USP13. Furthermore, N6-methyladenosine methyltransferase-like 3 (METTL3) mediated stabilization of USP13 mRNA that required the m6A reader IGF2BP2. Moreover, an inhibitor of USP13 caused ATG5 decay and co-administration of this inhibitor with 3-methyladenine boosted treatment efficacy of IM in murine xenograft models derived from GIST cells. Our findings highlight USP13 as an essential regulator of autophagy and IM resistance in GIST cells and reveal USP13 as a novel potential therapeutic target for GIST treatment.
    DOI:  https://doi.org/10.1038/s41418-022-01107-8
  64. Mol Metab. 2022 Dec 16. pii: S2212-8778(22)00229-0. [Epub ahead of print] 101660
    DEPTOR loss impairs brown adipocyte development in vitro but has limited impacts in mice.
    Charles Colas, Mathilde Mouchiroud, Manal Al Dow, Alona Kolnohuz, Yves Gélinas, Alexandre Caron, Mathieu Laplante.
      OBJECTIVES: The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates growth and metabolism. In mice, activation of mTOR controls cold adaptation by promoting the recruitment and the activation of brown adipose tissue (BAT). DEP-domain containing mTOR-interacting protein (DEPTOR) interacts with mTOR to modulate its activity. Whether DEPTOR levels are modulated by cold in BAT and whether this protein regulates brown adipocyte development and thermogenic activation has never been tested.METHODS: DEPTOR levels were measured in mouse tissues upon cold exposure and in brown preadipocytes following the induction of adipogenesis. Lentiviruses expressing short-hairpin RNA were used to repress DEPTOR expression in brown preadipocytes in vitro. Conditional deletion of DEPTOR in brown preadipocytes and in mature brown fat cells was achieved by crossing DEPTOR floxed mice with either Myf5-Cre or Ucp1-CreERT2 mice. These animals were exposed to cold and extensively phenotyped.
    RESULTS: DEPTOR is highly expressed in BAT and its levels are induced by chronic cold exposure, a condition that triggers BAT expansion and activation. Supporting a role for DEPTOR in brown fat cell recruitment, we found that DEPTOR is induced during brown adipocyte development and that its depletion impairs adipogenesis in vitro. This adipogenic lesion was associated with defects in both Akt activation and the expression of key adipogenic regulators. Conditional deletion of DEPTOR in brown preadipocytes or mature brown fat cells did not impact BAT recruitment and thermogenesis in mice but slightly reduced the expression of adipogenic and lipogenic genes.
    CONCLUSIONS: DEPTOR is highly expressed in BAT and its levels are dynamically regulated during brown fat cell development and upon cold exposure. Although DEPTOR depletion severely represses brown fat adipogenesis in vitro, its deletion is dispensable for BAT development, recruitment, and thermogenic activation in mice.
    Keywords:  Adipogenesis; Cold exposure; DEPTOR; Thermogenesis; brown adipocyte; brown adipose tissue; mTOR
    DOI:  https://doi.org/10.1016/j.molmet.2022.101660
  65. Front Cell Dev Biol. 2022 ;10 1082095
    Perspectives on mitochondrial relevance in cardiac ischemia/reperfusion injury.
    Gaia Pedriali, Daniela Ramaccini, Esmaa Bouhamida, Mariusz R Wieckowski, Carlotta Giorgi, Elena Tremoli, Paolo Pinton.
      Cardiovascular disease is the most common cause of death worldwide and in particular, ischemic heart disease holds the most considerable position. Even if it has been deeply studied, myocardial ischemia-reperfusion injury (IRI) is still a side-effect of the clinical treatment for several heart diseases: ischemia process itself leads to temporary damage to heart tissue and obviously the recovery of blood flow is promptly required even if it worsens the ischemic injury. There is no doubt that mitochondria play a key role in pathogenesis of IRI: dysfunctions of these important organelles alter cell homeostasis and survival. It has been demonstrated that during IRI the system of mitochondrial quality control undergoes alterations with the disruption of the complex balance between the processes of mitochondrial fusion, fission, biogenesis and mitophagy. The fundamental role of mitochondria is carried out thanks to the finely regulated connection to other organelles such as plasma membrane, endoplasmic reticulum and nucleus, therefore impairments of these inter-organelle communications exacerbate IRI. This review pointed to enhance the importance of the mitochondrial network in the pathogenesis of IRI with the aim to focus on potential mitochondria-targeting therapies as new approach to control heart tissue damage after ischemia and reperfusion process.
    Keywords:  PTP; cardiac ischemia/reperfusion; mitochondria; mitochondrial biogenesis; mitochondrial dynamics; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2022.1082095
  66. Cells. 2022 Dec 15. pii: 4062. [Epub ahead of print]11(24):
    Mechanisms of RNA and Protein Quality Control and Their Roles in Cellular Senescence and Age-Related Diseases.
    Donghee Kang, Yurim Baek, Jae-Seon Lee.
      Cellular senescence, a hallmark of aging, is defined as irreversible cell cycle arrest in response to various stimuli. It plays both beneficial and detrimental roles in cellular homeostasis and diseases. Quality control (QC) is important for the proper maintenance of cellular homeostasis. The QC machineries regulate the integrity of RNA and protein by repairing or degrading them, and are dysregulated during cellular senescence. QC dysfunction also contributes to multiple age-related diseases, including cancers and neurodegenerative, muscle, and cardiovascular diseases. In this review, we describe the characters of cellular senescence, discuss the major mechanisms of RNA and protein QC in cellular senescence and aging, and comprehensively describe the involvement of these QC machineries in age-related diseases. There are many open questions regarding RNA and protein QC in cellular senescence and aging. We believe that a better understanding of these topics could propel the development of new strategies for addressing age-related diseases.
    Keywords:  RNA quality control; age-related diseases; cellular senescence; protein quality control
    DOI:  https://doi.org/10.3390/cells11244062
  67. Cells. 2022 Dec 17. pii: 4109. [Epub ahead of print]11(24):
    Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery.
    Yu Zhang, Ofri Karmon, Koyeli Das, Reuven Wiener, Norbert Lehming, Ophry Pines.
      Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research.
    Keywords:  E2; Rad6; dual targeting; eclipsed distribution; mitochondrial matrix; ubiquitination
    DOI:  https://doi.org/10.3390/cells11244109
  68. Front Microbiol. 2022 ;13 1052779
    Host autophagy limits Toxoplasma gondii proliferation in the absence of IFN-γ by affecting the hijack of Rab11A-positive vesicles.
    Lingtao Pan, Yimin Yang, Xueqiu Chen, Mingxiu Zhao, Chaoqun Yao, Kaiyin Sheng, Yi Yang, Guangxu Ma, Aifang Du.
      Introduction: Autophagy has been recognized as a bona fide immunological process. Evidence has shown that this process in IFN-γ stimulated cells controls Toxoplasma gondii proliferation or eliminates its infection. However, little is known about the effect of T. gondii infection on the host cell autophagy in the absence of IFN-γ.Methods: Multiple autophagy detection methods and CRISPR/CAS9 technology were used to study T. gondii-induced autophagy in HeLa and several other mammalian cell lines.
    Results: Here, we report increased LC3 II, autophagosome-like membrane structures, enhanced autophagic flux, and decreased lysosomes in a range of mammalian cell lines without IFN-γ treatment after T. gondii infection. Specifically, disruption of host atg5 (a necessary gene for autophagy) in HeLa cells promoted the intracellular replication of T. gondii, with the transcript level of rab11a increased, compared with that in wild-type cells. Further, after T. gondii infection, the abundance of Rab11A remained stable in wild-type HeLa cells but decreased in atg5 -/- mutant. Disruption of rab11a in the HeLa cells compromised the proliferation of T. gondii, and increased the transcription of gra2 in the parasite. Compared to the T. gondii wild-type RH∆ku80 strain, the ∆gra2 mutant induces enhanced host autophagy in HeLa cells, and results in slower replication of the parasite.
    Discussion: Collectively, these results indicate that host cell autophagy can limit T. gondii proliferation in an IFN-γ independent manner, possibly by affecting the hijack of host Rab11A-positive vesicles by the parasite which involved TgGRA2. The findings provide novel insights into T. gondii infection in host cells and toxoplasmosis research.
    Keywords:  Rab11A; TgGRA2; Toxoplasma gondii; autophagy; proliferation
    DOI:  https://doi.org/10.3389/fmicb.2022.1052779
  69. J Biol Chem. 2022 Dec 17. pii: S0021-9258(22)01255-8. [Epub ahead of print] 102812
    Zinc-finger Protein CXXC5 Promotes Breast Carcinogenesis by Regulating the TSC1/mTOR Signaling Pathway.
    Wenjuan Wang, Zhaohan Zhang, Minghui Zhao, Yu Wang, Yuze Ge, Lin Shan.
      CXXC5, a member of the CXXC family of zinc-finger proteins, is associated with numerous pathological processes. However, the pathophysiological function of CXXC5 has not been clearly established. Herein, we found that CXXC5 interacts with the CRL4B and NuRD complexes. Screening of transcriptional targets downstream of the CXXC5-CRL4B-NuRD complex by next-generation sequencing (ChIP-sequencing) revealed that the complex regulates the transcriptional repression process of a cohort of genes, including TSC1, that play important roles in cell growth and mTOR signaling pathway regulation, and whose abnormal regulation results in the activation of programmed cell death-ligand protein 1 (PD-L1). Intriguingly, CXXC5 expression increased after stimulation with vitamin B2, but decreased after vitamin D treatment. We also found that the CXXC5-CRL4B-NuRD complex promotes the proliferation of tumor cells in vitro and accelerates the growth of breast cancer in vivo. The expression of CXXC5, CUL4B, and MTA1 increased during the occurrence and development of breast cancer, and correspondingly, TSC1 expression decreased. Meanwhile, a high expression of CXXC5 was positively correlated with the histological grade of high malignancy and poor survival of patients. In conclusion, our study revealed that CXXC5-mediated TSC1 suppression activates the mTOR pathway, reduces autophagic cell death, induces PD-L1-mediated immune suppression, and results in tumor development, shedding light on the mechanism of the pathophysiological function of CXXC5.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102812
  70. Physiol Res. 2022 Dec 22.
    ASIC1a involves the acid-mediated activation of pancreatic stellate cells associated with autophagy induction.
    T Wang, Q Wang, G Pan, G Jia, X Li, C Wang, L Zhang, C Zuo.
      The acidic tumor microenvironment (TME) of pancreatic cancer affects the physiological function of pancreatic stellate cells (PSCs), which in turn promotes cancer progression. Acid-sensing ion channel 1a (ASIC1a) is responsible for acidosis-related physiopathological processes. In this study, we investigated the effect of acid exposure on the activation and autophagy of PSCs, and the role of ASIC1a in these events. The results showed that acidic medium upregulated the expression of ASIC1a, induced PSCs activation and autophagy, which can be suppressed by inhibiting ASIC1a using PcTx1 or ASIC1a knockdown, suggesting that ASIC1a involves these two processes. In addition, the acid-induced activation of PSCs was impaired after the application of autophagy inhibitor alone or in combination with ASIC1a siRNA, meaning a connection between autophagy and activation. Collectively, our study provides evidence for the involvement of ASIC1a in the acid-caused PSCs activation, which may be associated with autophagy induction.
  71. J Med Chem. 2022 Dec 19.
    Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.
    G Leslie Burnett, Yu C Yang, James B Aggen, Jennifer Pitzen, Micah K Gliedt, Chris M Semko, Abby Marquez, James W Evans, Gang Wang, Walter S Won, Aidan C A Tomlinson, Gert Kiss, Christos Tzitzilonis, Arun P Thottumkara, James Cregg, Kevin T Mellem, Jong S Choi, Julie C Lee, Yongyuan Zhao, Bianca J Lee, Justin G Meyerowitz, John E Knox, Jingjing Jiang, Zhican Wang, David Wildes, Zhengping Wang, Mallika Singh, Jacqueline A M Smith, Adrian L Gill.
      Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.
    DOI:  https://doi.org/10.1021/acs.jmedchem.2c01658
  72. Int J Mol Sci. 2022 Dec 09. pii: 15598. [Epub ahead of print]23(24):
    Hepatic PLIN5 Deficiency Impairs Lipogenesis through Mitochondrial Dysfunction.
    Enxiang Zhang.
      Regulation of lipid droplets (LDs) metabolism is the core of controlling intracellular fatty acids (FAs) fluxes, and perilipin 5 (PLIN5) plays a key role in this process. Our previous studies have found that hepatic PLIN5 deficiency reduces LDs accumulation, but the trafficking of FAs produced from this pathway and the interaction between mitochondria and LDs in this process are largely unknown. Here, we found that the deficiency of PLIN5 decreases LDs accumulation by increasing FAs efflux. In addition, the decreased lipogenesis of PLIN5-deficient hepatocytes is accompanied by mitochondrial dysfunction, suggesting that PLIN5 plays an important role in mediating the interaction between LDs and mitochondria. Importantly, PLIN5 ablation negates oxidative capacity differences of peri-droplet and cytosolic mitochondria. In summary, these data indicate that PLIN5 plays a vital role in maintaining mitochondrial-mediated lipogenesis, which provides an important new perspective on the regulation of liver lipid storage and the relationship between PLIN5 and mitochondria.
    Keywords:  LDs-mitochondria interaction; PLIN5; fatty acids; lipid droplets; lipogenesis
    DOI:  https://doi.org/10.3390/ijms232415598
  73. J Biol Chem. 2022 Dec 20. pii: S0021-9258(22)01264-9. [Epub ahead of print] 102821
    Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation.
    Goonho Park, Ke Xu, Leon Chea, Kyle Kim, Lance Safarta, Keon-Hyoung Song, Jian Wu, Soyoung Park, Hyejung Min, Nobuhiko Hiramatsu, Jaeseok Han, Jonathan H Lin.
      Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms - Unfolded Protein Response (UPR) and Integrated Stress Response (ISR). Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation while inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the ER luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.
    Keywords:  EIF2AK3; ER stress; Integrated Stress Response; Neurodegeneration; PERK; Tau aggregation; Tauopathy; Unfolded Protein Response; eIF2α phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2022.102821
  74. Cells. 2022 Dec 12. pii: 4015. [Epub ahead of print]11(24):
    ALDH1-Mediated Autophagy Sensitizes Glioblastoma Cells to Ferroptosis.
    Yang Wu, Helena Kram, Jens Gempt, Friederike Liesche-Starnecker, Wei Wu, Jürgen Schlegel.
      The fatal clinical course of human glioblastoma (GBM) despite aggressive adjuvant therapies is due to high rates of recurrent tumor growth driven by tumor cells with stem-cell characteristics (glioma stem cells, GSCs). The aldehyde dehydrogenase 1 (ALDH1) family of enzymes has been shown to be a biomarker for GSCs, and ALDH1 seems to be involved in the biological processes causing therapy resistance. Ferroptosis is a recently discovered cell death mechanism, that depends on iron overload and lipid peroxidation, and it could, therefore, be a potential therapeutic target in various cancer types. Since both ALDH1 and ferroptosis interact with lipid peroxidation (LPO), we aimed to investigate a possible connection between ALDH1 and ferroptosis. Here, we show that RSL3-induced LPO and ferroptotic cell death revealed RSL3-sensitive and -resistant malignant glioma cell lines. Most interestingly, RSL3 sensitivity correlates with ALDH1a3 expression; only high ALDH1a3-expressing cells seem to be sensitive to ferroptosis induction. In accordance, inhibition of ALDH1a3 enzymatic activity by chemical inhibition or genetic knockout protects tumor cells from RSL3-induced ferroptotic cell death. Both RSL-3-dependent binding of ALDH1a3 to LC3B and autophagic downregulation of ferritin could be completely blocked by ALDH inhibition. Therefore, ALDH1a3 seems to be involved in ferroptosis through the essential release of iron by ferritinophagy. Our results also indicate that ferroptosis induction might be a particularly interesting clinical approach for targeting the highly aggressive cell population of GSC.
    Keywords:  autophagy; cancer stem cells; ferroptosis; glioblastoma; therapy
    DOI:  https://doi.org/10.3390/cells11244015
  75. J Agric Food Chem. 2022 Dec 21.
    Targeting mTOR Signaling by Dietary Polysaccharides in Cancer Prevention: Advances and Challenges.
    Mengyuan Li, Zuomin Hu, Tianyi Guo, Tiantian Xie, Yanqin Tang, Xiuxiu Wu, Feijun Luo.
      Cancer is the most serious problem for public health. Traditional treatments often come with unavoidable side effects. Therefore, the therapeutic effects of natural products with wide sources and low toxicity are attracting more and more attention. Polysaccharides have been shown to have cancer-fighting potential, but the molecular mechanisms remain unclear. The mammalian target of rapamycin (mTOR) pathway has become an attractive target of antitumor therapy research in recent years. The regulation of mTOR pathway not only affects cell proliferation and growth but also has an important effect in tumor metabolism. Recent studies indicate that dietary polysaccharides play a vital role in cancer prevention and treatment by regulating mTOR pathway. Here, the progress in targeting mTOR signaling by dietary polysaccharides in cancer prevention and their molecular mechanisms are systemically summarized. It will promote the understanding of the anticancer effects of polysaccharides and provide reference to investigators of this cutting edge field.
    Keywords:  cancer; dietary polysaccharides; mTOR; signaling pathway
    DOI:  https://doi.org/10.1021/acs.jafc.2c06780
  76. Ann Transl Med. 2022 Nov;10(22): 1218
    BAG3 promotes autophagy and suppresses NLRP3 inflammasome activation in Parkinson's disease.
    Zhong-Ming Ying, Qian-Kun Lv, Xiao-Yu Yao, An-Qi Dong, Ya-Ping Yang, Yu-Lan Cao, Fen Wang, Ai-Ping Gong, Chun-Feng Liu.
      Background: Neuroinflammation mediated by microglia plays a key role in the pathogenesis of Parkinson's disease (PD), and our previous studies showed this was significantly inhibited by enhanced autophagy. In the autophagy pathway, Bcl2-associated athanogene (BAG)3 is a prominent co-chaperone, and we have shown BAG3 can regulate autophagy to clear the PD pathogenic protein α-synuclein. However, the connection between BAG3 and microglia mediated neuroinflammation is not clear.Methods: In this study, we explored whether BAG3 regulated related neuroinflammation and its original mechanism in PD. An inflammatory model of PD was established by injecting adeno-associated virus (AAV)-BAG3 into the bilateral striatum of C57BL/6 male mice to induce overexpression of BAG3, followed by injection of lipopolysaccharide (LPS). The striatum was extracted at 3 days after injection of LPS for Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and immunohistochemical staining was performed at 21 days after injection. At the same time, LPS was used to induce activation of BV2 cells to verify the effect of BAG3 in vitro.
    Results: Overexpression of BAG3 reduced LPS-induced pyroptosis by reducing activation of caspase-1, the NOD-like receptor family, and the pyrin domain-containing 3 (NLRP3) inflammasome, and by release of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. The LPS-induced inflammatory environment inhibits autophagy, and overexpression of BAG3 can restore autophagy, which may be the mechanism by which BAG3 reduces neuronal inflammation in PD.
    Conclusions: Our results demonstrate BAG3 promotes autophagy and suppresses NLRP3 inflammasome formation in PD.
    Keywords:  Bcl2-associated athanogene 3 (BAG3); NOD-like receptor family, and the pyrin domain-containing 3 inflammasome (NLRP3 inflammasome); Parkinson’s disease (PD); autophagy; microglia
    DOI:  https://doi.org/10.21037/atm-22-5159
  77. Int J Endocrinol. 2022 ;2022 2009753
    Sestrin2 Overexpression Ameliorates Endoplasmic Reticulum Stress-Induced Apoptosis via Inhibiting mTOR Pathway in HepG2 Cells.
    Huiling Hu, Zhijun Luo, Xiuli Liu, Lisi Huang, Xiaoxia Lu, Rui Ding, Chaohui Duan, Yuqing He.
      Sestrin2 is a highly conserved stress-inducible protein, acting as a crucial part in regulating homeostasis in response to various stress conditions in the cell. However, the role of Sestrin2 in regulating cell apoptosis related to endoplasmic reticulum (ER) has not been fully investigated. Our study presented here aims to reveal the effect of Sestrin2 in tunicamycin (TM)-induced cell apoptosis related to ER stress and its underlying molecular mechanisms. The results demonstrated that Sestrin2 expression was significantly upregulated correlated with ER stress responses in TM treated HepG2 cells. Sestrin2 overexpression obviously alleviated ER stress with the determination of ER stress-related proteins expression. In addition, Sestrin2 overexpression inhibited cell apoptosis with the examination of apoptosis-related proteins and TUNEL assay. However, Sestrin2 knockdown further promoted the ER stress-mediated cell apoptosis. The further mechanistic study revealed that Sestrin2 overexpression inhibited TM-induced mTOR pathway activation. Taken together, our current study indicated that Sestrin2 overexpression ameliorates ER stress-induced apoptosis via inhibiting mTOR pathway in HepG2 cells.
    DOI:  https://doi.org/10.1155/2022/2009753
  78. Nat Neurosci. 2022 Dec 19.
    Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration.
    Sean K Ryan, Matija Zelic, Yingnan Han, Erin Teeple, Luoman Chen, Mahdiar Sadeghi, Srinivas Shankara, Lilu Guo, Cong Li, Fabrizio Pontarelli, Elizabeth H Jensen, Ashley L Comer, Dinesh Kumar, Mindy Zhang, Joseph Gans, Bailin Zhang, Jonathan D Proto, Jacqueline Saleh, James C Dodge, Virginia Savova, Deepak Rajpal, Dimitry Ofengeim, Timothy R Hammond.
      Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson's disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.
    DOI:  https://doi.org/10.1038/s41593-022-01221-3
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