bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒07‒23
47 papers selected by
Viktor Korolchuk, Newcastle University
  1. Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy.
  2. Mitophagy in the retina: Viewing mitochondrial homeostasis through a new lens.
  3. P62/SQSTM1 binds with claudin-2 to target for selective autophagy in stressed intestinal epithelium.
  4. Expression of mTOR in normal and pathological conditions.
  5. The mitophagy receptor NIX induces vIRF-1 oligomerization and interaction with GABARAPL1 for the promotion of HHV-8 reactivation-induced mitophagy.
  6. Aging Differentially Affects Axonal Autophagosome Formation and Maturation.
  7. Driving arginine catabolism to activate systemic autophagy.
  8. Noncanonical autophagy is a new strategy to inhibit HSV-1 through STING1 activation.
  9. Autophagy orchestrates the crosstalk between cells and organs.
  10. Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson's disease.
  11. The human acetylcholinesterase C-terminal T30 peptide activates neuronal growth through alpha 7 nicotinic acetylcholine receptors and the mTOR pathway.
  12. Therapeutic strategies targeting AMPK-dependent autophagy in cancer cells.
  13. LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles.
  14. LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner.
  15. Spartin is a receptor for the autophagy of lipid droplets.
  16. Editorial: Mitophagy in health and disease, volume II.
  17. Structure of the Schizosaccharomyces pombe Gtr-Lam complex reveals evolutionary divergence of mTORC1-dependent amino acid sensing.
  18. Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.
  19. Development of small fluorescent probes for the analysis of autophagy kinetics.
  20. Urolithin A alleviates neuropathic pain and activates mitophagy.
  21. Plant reoviruses hijack autophagy in insect vectors.
  22. Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase.
  23. Autophagy protein 5 controls flow-dependent endothelial functions.
  24. Application and value of hydrogen sulfide modulated autophagy in sepsis.
  25. Analysis of ATG4C function in vivo.
  26. Homocysteine Metabolites Inhibit Autophagy, Elevate Amyloid Beta, and Induce Neuropathy by Impairing Phf8/H4K20me1-dependent Epigenetic Regulation of mTOR in Cystathionine β-Synthase-Deficient Mice.
  27. Qizhi Jiangtang capsule activates podocyte autophagy in diabetic kidney disease by inhibiting phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways.
  28. Autophagy mediates cementoblast mineralization under compression through periostin/β-catenin axis.
  29. Design of negative-regulating proteins of Rheb/mTORC1 with much reduced sizes of the tuberous sclerosis protein complex.
  30. Structural and functional understanding of disease-associated mutations in V-ATPase subunit a1 and other isoforms.
  31. Protrudin-mediated ER-endosome contact sites promote phagocytosis.
  32. APOE ε4 allele, along with G206D-PSEN1 mutation, alters mitochondrial networks and their degradation in Alzheimer's disease.
  33. Lysosomal enzyme trafficking: from molecular mechanisms to human diseases.
  34. Autophagy in sarcopenia: Possible mechanisms and novel therapies.
  35. Dietary restriction to optimize T cell immunity is an ancient survival strategy conserved in vertebrate evolution.
  36. Autophagy signaling in hypertrophied muscles of diabetic and control rats.
  37. The C-terminal tail of CSNAP attenuates the CSN complex.
  38. Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer's disease patient lymphocytes.
  39. Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells.
  40. Mitochondrial reactive oxygen species: double agents in Mycobacterium tuberculosis infection.
  41. CERS6-derived ceramides aggravate kidney fibrosis by inhibiting PINK1-mediated mitophagy in diabetic kidney disease.
  42. Increased apoptosis of gingival epithelium is associated with impaired autophagic flux in medication-related osteonecrosis of the jaw.
  43. MdHB7-like positively modulates apple salt tolerance by promoting autophagic activity and Na+ efflux.
  44. Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis.
  45. Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.
  46. Fluorescence-On Imaging of Reticulophagy Enabled by an Acidity-Reporting Solvatochromic Probe.
  47. The Organization and Function of the Phagophore-ER Membrane Contact Sites.
  1. Autophagy. 2023 Jul 16.
    Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy.
    Tomoyuki Fukuda, Kentaro Furukawa, Tatsuro Maruyama, Nobuo N Noda, Tomotake Kanki.
      Mitophagy is a selective form of autophagy that targets dysfunctional or superfluous mitochondria for degradation. During mitophagy, specific selective autophagy receptors (SARs) mark a portion of mitochondria to recruit the autophagy-related (Atg) machinery and nucleate a phagophore. The phagophore expands and surrounds the mitochondrial cargo, forming an autophagosome. Fission plays a crucial role in separating the targeted portion of mitochondria from the main body to sequester it within the autophagosome. Our recent study, utilizing fission and budding yeasts as model systems, has identified Atg44 as a mitochondrial fission factor that generates mitochondrial fragments suitable for phagophore engulfment. Atg44 resides in the mitochondrial intermembrane space (IMS) and interacts with lipid membranes, with the capacity of mediating membrane fragility and fission. Based on our findings, we propose the term mitofissin to refer to Atg44 and its homologous proteins, which might participate in diverse cellular processes requiring membrane remodeling across various species.
    Keywords:  Atg44; autophagy; mitochondria; mitochondrial fission; mitofissin; mitophagy; yeast
    DOI:  https://doi.org/10.1080/15548627.2023.2237343
  2. Prog Retin Eye Res. 2023 Jul 15. pii: S1350-9462(23)00044-7. [Epub ahead of print]96 101205
    Mitophagy in the retina: Viewing mitochondrial homeostasis through a new lens.
    Juan Ignacio Jiménez-Loygorri, Rocío Benítez-Fernández, Álvaro Viedma-Poyatos, Juan Zapata-Muñoz, Beatriz Villarejo-Zori, Raquel Gómez-Sintes, Patricia Boya.
      Mitochondrial function is key to support metabolism and homeostasis in the retina, an organ that has one of the highest metabolic rates body-wide and is constantly exposed to photooxidative damage and external stressors. Mitophagy is the selective autophagic degradation of mitochondria within lysosomes, and can be triggered by distinct stimuli such as mitochondrial damage or hypoxia. Here, we review the importance of mitophagy in retinal physiology and pathology. In the developing retina, mitophagy is essential for metabolic reprogramming and differentiation of retina ganglion cells (RGCs). In basal conditions, mitophagy acts as a quality control mechanism, maintaining a healthy mitochondrial pool to meet cellular demands. We summarize the different autophagy- and mitophagy-deficient mouse models described in the literature, and discuss the potential role of mitophagy dysregulation in retinal diseases such as glaucoma, diabetic retinopathy, retinitis pigmentosa, and age-related macular degeneration. Finally, we provide an overview of methods used to monitor mitophagy in vitro, ex vivo, and in vivo. This review highlights the important role of mitophagy in sustaining visual function, and its potential as a putative therapeutic target for retinal and other diseases.
    Keywords:  Age-related macular degeneration; Autophagy; Diabetic retinopathy; Glaucoma; Metabolism; Mitochondria; Mitophagy; Retinal development; Retinal homeostasis; Retinitis pigmentosa
    DOI:  https://doi.org/10.1016/j.preteyeres.2023.101205
  3. Commun Biol. 2023 07 17. 6(1): 740
    P62/SQSTM1 binds with claudin-2 to target for selective autophagy in stressed intestinal epithelium.
    Rizwan Ahmad, Balawant Kumar, Raju Lama Tamang, Geoffrey A Talmon, Punita Dhawan, Amar B Singh.
      Impaired autophagy promotes Inflammatory Bowel Disease (IBD). Claudin-2 is upregulated in IBD however its role in the pathobiology remains uncertain due to its complex regulation, including by autophagy. Irrespective, claudin-2 expression protects mice from DSS colitis. This study was undertaken to examine if an interplay between autophagy and claudin-2 protects from colitis and associated epithelial injury. Crypt culture and intestinal epithelial cells (IECs) are subjected to stress, including starvation or DSS, the chemical that induces colitis in-vivo. Autophagy flux, cell survival, co-immunoprecipitation, proximity ligation assay, and gene mutational studies are performed. These studies reveal that under colitis/stress conditions, claudin-2 undergoes polyubiquitination and P62/SQSTM1-assisted degradation through autophagy. Inhibiting autophagy-mediated claudin-2 degradation promotes cell death and thus suggest that claudin-2 degradation promotes autophagy flux to promote cell survival. Overall, these data inform for the previously undescribed role for claudin-2 in facilitating IECs survival under stress conditions, which can be harnessed for therapeutic advantages.
    DOI:  https://doi.org/10.1038/s42003-023-05116-2
  4. Mol Cancer. 2023 Jul 15. 22(1): 112
    Expression of mTOR in normal and pathological conditions.
    A Marques-Ramos, R Cervantes.
      The mechanistic/mammalian target of rapamycin (mTOR), a protein discovered in 1991, integrates a complex pathway with a key role in maintaining cellular homeostasis. By comprising two functionally distinct complexes, mTOR complex 1 (mTORC1) and mTORC2, it is a central cellular hub that integrates intra- and extracellular signals of energy, nutrient, and hormone availability, modulating the molecular responses to acquire a homeostatic state through the regulation of anabolic and catabolic processes. Accordingly, dysregulation of mTOR pathway has been implicated in a variety of human diseases. While major advances have been made regarding the regulators and effectors of mTOR signaling pathway, insights into the regulation of mTOR gene expression are beginning to emerge. Here, we present the current available data regarding the mTOR expression regulation at the level of transcription, translation and mRNA stability and systematize the current knowledge about the fluctuations of mTOR expression observed in several diseases, both cancerous and non-cancerous. In addition, we discuss whether mTOR expression changes can be used as a biomarker for diagnosis, disease progression, prognosis and/or response to therapeutics. We believe that our study will contribute for the implementation of new disease biomarkers based on mTOR as it gives an exhaustive perspective about the regulation of mTOR gene expression in both normal and pathological conditions.
    Keywords:  mTOR biomarker; mTOR expression; mTOR expression cancer; mTOR expression disease; mTOR mRNA stability; mTOR transcriptional regulation; mTOR translation regulation
    DOI:  https://doi.org/10.1186/s12943-023-01820-z
  5. PLoS Pathog. 2023 Jul 17. 19(7): e1011548
    The mitophagy receptor NIX induces vIRF-1 oligomerization and interaction with GABARAPL1 for the promotion of HHV-8 reactivation-induced mitophagy.
    Mai Tram Vo, Chang-Yong Choi, Young Bong Choi.
      Recently, viruses have been shown to regulate selective autophagy for productive infections. For instance, human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), activates selective autophagy of mitochondria, termed mitophagy, thereby inhibiting antiviral innate immune responses during lytic infection in host cells. We previously demonstrated that HHV-8 viral interferon regulatory factor 1 (vIRF-1) plays a crucial role in lytic replication-activated mitophagy by interacting with cellular mitophagic proteins, including NIX and TUFM. However, the precise molecular mechanisms by which these interactions lead to mitophagy activation remain to be determined. Here, we show that vIRF-1 binds directly to mammalian autophagy-related gene 8 (ATG8) proteins, preferentially GABARAPL1 in infected cells, in an LC3-interacting region (LIR)-independent manner. Accordingly, we identified key residues in vIRF-1 and GABARAPL1 required for mutual interaction and demonstrated that the interaction is essential for mitophagy activation and HHV-8 productive replication. Interestingly, the mitophagy receptor NIX promotes vIRF-1-GABARAPL1 interaction, and NIX/vIRF-1-induced mitophagy is significantly inhibited in GABARAPL1-deficient cells. Moreover, a vIRF-1 variant defective in GABARAPL1 binding substantially loses the ability to induce vIRF-1/NIX-induced mitophagy. These results suggest that NIX supports vIRF-1 activity as a mitophagy mediator. In addition, we found that NIX promotes vIRF-1 aggregation and stabilizes aggregated vIRF-1. Together, these findings indicate that vIRF-1 plays a role as a viral mitophagy mediator that can be activated by a cellular mitophagy receptor.
    DOI:  https://doi.org/10.1371/journal.ppat.1011548
  6. Autophagy. 2023 Jul 18. 1-17
    Aging Differentially Affects Axonal Autophagosome Formation and Maturation.
    Heather Tsong, Erika Lf Holzbaur, Andrea Kh Stavoe.
      Misregulation of neuronal macroautophagy/autophagy has been implicated in age-related neurodegenerative diseases. We compared autophagosome formation and maturation in primary murine neurons during development and through aging to elucidate how aging affects neuronal autophagy. We observed an age-related decrease in the rate of autophagosome formation leading to a significant decrease in the density of autophagosomes along the axon. Next, we identified a surprising increase in the maturation of autophagic vesicles in neurons from aged mice. While we did not detect notable changes in endolysosomal content in the distal axon during early aging, we did observe a significant loss of acidified vesicles in the distal axon during late aging. Interestingly, we found that autophagic vesicles were transported more efficiently in neurons from adult mice than in neurons from young mice. This efficient transport of autophagic vesicles in both the distal and proximal axon is maintained in neurons during early aging, but is lost during late aging. Our data indicate that early aging does not negatively impact autophagic vesicle transport nor the later stages of autophagy. However, alterations in autophagic vesicle transport efficiency during late aging reveal that aging differentially impacts distinct aspects of neuronal autophagy.Abbreviations: ACAP3: ArfGAP with coiled-coil, ankyrin repeat and PH domains 3; ARF6: ADP-ribosylation factor 6; ATG: autophagy related; AVs: autophagic vesicles; DCTN1/p150Glued: dynactin 1; DRG: dorsal root ganglia; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; LAMP2: lysosomal-associated protein 2; LysoT: LysoTracker; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK8IP1/JIP1: mitogen-activated protein kinase 8 interacting protein 1; MAPK8IP3/JIP3: mitogen-activated protein kinase 8 interacting protein 3; mCh: mCherry; PE: phosphatidylethanolamine.
    Keywords:  Autophagy; axonal transport; lysosome; microtubule transport; neuron
    DOI:  https://doi.org/10.1080/15548627.2023.2236485
  7. Autophagy Rep. 2022 ;1(1): 65-69
    Driving arginine catabolism to activate systemic autophagy.
    Yiming Zhang, Brian N Finck, Brian J DeBosch.
      Macroautophagy/autophagy is a conserved cellular self-digestive mechanism to catabolize superfluous or damaged cellular components to maintain cell homeostasis. Impaired autophagy underlies multiple pathophysiological states, including aging, neurodegenerative, inflammatory, and metabolic diseases. Intermittent fasting and caloric restriction are effective means by which to activate autophagy, yet relatively few people can sustain such intensive interventions in real-world settings. Moreover, current pharmacotherapies do not yet fully exploit autophagic flux as a target mechanism. Here, we discuss recent work, which demonstrates that arginine catabolism is a tractable process to activate autophagy with utility to treat obesity and its complications. Hepatocyte-specific transgenic activation of arginine catabolism, or systemic administration of an anti-tumor pharmacotherapy, pegylated arginine deiminase, each promote energy expenditure and insulin sensitivity, and reduce dyslipidemia and hepatic steatosis in obese mice. These effects depend upon hepatocyte Fgf21, and whole-body Becn1 expression. The data suggest that hepatocyte and systemic arginine catabolism drive autophagy, and identify an index pharmacological agent to leverage this process.
    DOI:  https://doi.org/10.1080/27694127.2022.2040763
  8. Autophagy. 2023 Jul 20.
    Noncanonical autophagy is a new strategy to inhibit HSV-1 through STING1 activation.
    Zhihua Zheng, Man Zhao, Hao Shan, Dongmei Fang, Zuyi Jin, Jiuge Tang, Zhiping Liu, Liang Hong, Peiqing Liu, Min Li.
      STING1 (stimulator of interferon response cGAMP interactor 1) plays an essential role in immune responses for virus inhibition via inducing the production of type I interferon, inflammatory factors and macroautophagy/autophagy. In this study, we found that STING1 activation could induce not only canonical autophagy but also non-canonical autophagy (NCA) which is independent of the ULK1 or BECN1 complexes to form MAP1LC3/LC3-positive structures. Whether STING1-induced NCA has similar characters and physiological functions to canonical autophagy is totally unknown. Different from canonical autophagy, NCA could increase single-membrane structures and failed to degrade long-lived proteins, and could be strongly suppressed by interrupting vacuolar-type H+-translocating ATPase (V-ATPase) activity. Importantly, STING1-induced NCA could effectively inhibit DNA virus HSV-1 in cell model. Moreover, STING1[1-340], a STING1 mutant lacking immunity and inflammatory response due to deletion of the tail end of STING1, could degrade virus through NCA alone, suggesting that the antiviral effect of activated STING1 could be separately mediated by inherent immunity, canonical autophagy, and NCA. In addition, the translocation and dimerization of STING1 do not rely on its immunity function and autophagy pathway. Similar to canonical autophagy, LC3-positive structures of NCA induced by STING1 could finally fuse with lysosomes, and the degradation of HSV-1 could be reverted by inhibition of lysosome function, suggesting that the elimination of DNA virus via NCA still requires the lysosome pathway. Collectively, we proved that besides its classical immunity function and canonical autophagy pathway, STING1-induced NCA is also an efficient antiviral pathway for the host cell.
    Keywords:  HSV-1; STING1; autophagy; inherent immunity; lysosome degradation; noncanonical autophagy
    DOI:  https://doi.org/10.1080/15548627.2023.2237794
  9. EMBO Rep. 2023 Jul 19. e57289
    Autophagy orchestrates the crosstalk between cells and organs.
    Klara Piletic, Ghada Alsaleh, Anna Katharina Simon.
      Over the recent years, it has become apparent that a deeper understanding of cell-to-cell and organ-to-organ communication is necessary to fully comprehend both homeostatic and pathological states. Autophagy is indispensable for cellular development, function, and homeostasis. A crucial aspect is that autophagy can also mediate these processes through its secretory role. The autophagy-derived secretome relays its extracellular signals in the form of nutrients, proteins, mitochondria, and extracellular vesicles. These crosstalk mediators functionally shape cell fate decisions, tissue microenvironment and systemic physiology. The diversity of the secreted cargo elicits an equally diverse type of responses, which span over metabolic, inflammatory, and structural adaptations in disease and homeostasis. We review here the emerging role of the autophagy-derived secretome in the communication between different cell types and organs and discuss the mechanisms involved.
    Keywords:  EV secretion; autophagy-derived secretome; intercellular communication; secretory autophagy; unconventional secretion
    DOI:  https://doi.org/10.15252/embr.202357289
  10. Sci Adv. 2023 Jul 21. 9(29): eadh3347
    Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson's disease.
    Wesley Peng, Leonie F Schröder, Pingping Song, Yvette C Wong, Dimitri Krainc.
      Mutations in the E3 ubiquitin ligase parkin are the most common cause of early-onset Parkinson's disease (PD). Although parkin modulates mitochondrial and endolysosomal homeostasis during cellular stress, whether parkin regulates mitochondrial and lysosomal cross-talk under physiologic conditions remains unresolved. Using transcriptomics, metabolomics and super-resolution microscopy, we identify amino acid metabolism as a disrupted pathway in iPSC-derived dopaminergic neurons from patients with parkin PD. Compared to isogenic controls, parkin mutant neurons exhibit decreased mitochondria-lysosome contacts via destabilization of active Rab7. Subcellular metabolomics in parkin mutant neurons reveals amino acid accumulation in lysosomes and their deficiency in mitochondria. Knockdown of the Rab7 GTPase-activating protein TBC1D15 restores mitochondria-lysosome tethering and ameliorates cellular and subcellular amino acid profiles in parkin mutant neurons. Our data thus uncover a function of parkin in promoting mitochondrial and lysosomal amino acid homeostasis through stabilization of mitochondria-lysosome contacts and suggest that modulation of interorganelle contacts may serve as a potential target for ameliorating amino acid dyshomeostasis in disease.
    DOI:  https://doi.org/10.1126/sciadv.adh3347
  11. Sci Rep. 2023 Jul 15. 13(1): 11434
    The human acetylcholinesterase C-terminal T30 peptide activates neuronal growth through alpha 7 nicotinic acetylcholine receptors and the mTOR pathway.
    Alexandru Graur, Patricia Sinclair, Amanda K Schneeweis, Daniel T Pak, Nadine Kabbani.
      Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease.
    DOI:  https://doi.org/10.1038/s41598-023-38637-1
  12. Biochim Biophys Acta Mol Cell Res. 2023 Jul 16. pii: S0167-4889(23)00109-X. [Epub ahead of print]1870(7): 119537
    Therapeutic strategies targeting AMPK-dependent autophagy in cancer cells.
    Wenbin Yuan, Wanyi Fang, Rui Zhang, Hao Lyu, Shuai Xiao, Dong Guo, Declan William Ali, Marek Michalak, Xing-Zhen Chen, Cefan Zhou, Jingfeng Tang.
      Macroautophagy is a health-modifying process of engulfing misfolded or aggregated proteins or damaged organelles, coating these proteins or organelles into vesicles, fusion of vesicles with lysosomes to form autophagic lysosomes, and degradation of the encapsulated contents. It is also a self-rescue strategy in response to harsh environments and plays an essential role in cancer cells. AMP-activated protein kinase (AMPK) is the central pathway that regulates autophagy initiation and autophagosome formation by phosphorylating targets such as mTORC1 and unc-51 like activating kinase 1 (ULK1). AMPK is an evolutionarily conserved serine/threonine protein kinase that acts as an energy sensor in cells and regulates various metabolic processes, including those involved in cancer. The regulatory network of AMPK is complicated and can be regulated by multiple upstream factors, such as LKB1, AKT, PPAR, SIRT1, or noncoding RNAs. Currently, AMPK is being investigated as a novel target for anticancer therapies based on its role in macroautophagy regulation. Herein, we review the effects of AMPK-dependent autophagy on tumor cell survival and treatment strategies targeting AMPK.
    Keywords:  AMPK; Autophagy; Cancer therapy; Metabolism; Tumor
    DOI:  https://doi.org/10.1016/j.bbamcr.2023.119537
  13. Autophagy. 2023 Jul 21. 1-16
    LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles.
    Lei Qiao, Jiayi Hu, Xiaohan Qiu, Chunlin Wang, Jieqiong Peng, Cheng Zhang, Meng Zhang, Huixia Lu, Wenqiang Chen.
      LAMP2 (lysosomal associated membrane protein 2) is one of the major protein components of the lysosomal membrane. There currently exist three LAMP2 isoforms, LAMP2A, LAMP2B and LAMP2C, and they vary in distribution and function. LAMP2A serves as a receptor and channel for transporting cytosolic proteins in a process called chaperone-mediated autophagy (CMA). LAMP2B is required for autophagosome-lysosome fusion in cardiomyocytes and is one of the components of exosome membranes. LAMP2C is primarily implicated in a novel type of autophagy in which nucleic acids are taken up into lysosomes for degradation. In this review, the current evidence for the function of each LAMP2 isoform in various pathophysiological processes and human diseases, as well as their possible mechanisms, are comprehensively summarized. We discuss the evolutionary patterns of the three isoforms in vertebrates and provide technical guidance on investigating these isoforms. We are also concerned with the newly arising questions in this particular research area that remain unanswered. Advances in the functions of the three LAMP2 isoforms will uncover new links between lysosomal dysfunction, autophagy and human diseases.Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; AD: Alzheimer disease; Ag: antigens; APP: amyloid beta precursor protein; ATG14: autophagy related 14; AVSF: autophagic vacuoles with unique sarcolemmal features; BBC3/PUMA: BCL2 binding component 3; CCD: C-terminal coiled coil domain; CMA: chaperone-mediated autophagy; CVDs: cardiovascular diseases; DDIT4/REDD1: DNA damage inducible transcript 4; ECs: endothelial cells; ER: endoplasmic reticulum; ESCs: embryonic stem cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/β-glucocerebrosidase: glucosylceramidase beta; GSCs: glioblastoma stem cells; HCC: hepatocellular carcinoma; HD: Huntington disease; HSCs: hematopoietic stem cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IL3: interleukin 3; IR: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; LDs: lipid droplets; LRRK2: leucine rich repeat kinase 2; MA: macroautophagy; MHC: major histocompatibility complex; MST1: macrophage stimulating 1; NAFLD: nonalcoholic fatty liver disease; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NLRP3: NLR family pyrin domain containing 3; PARK7: Parkinsonism associated deglycase; PD: Parkinson disease; PEA15/PED: proliferation and apoptosis adaptor protein 15; PKM/PKM2: pyruvate kinase M1/2; RA: rheumatoid arthritis; RARA: retinoic acid receptor alpha; RCAN1: regulator of calcineurin 1; RCC: renal cell carcinoma; RDA: RNautophagy and DNautophagy; RNAi: RNA interference; RND3: Rho Family GTPase 3; SG-NOS3/eNOS: deleterious glutathionylated NOS3; SLE: systemic lupus erythematosus; TAMs: tumor-associated macrophages; TME: tumor microenvironment; UCHL1: ubiquitin C-terminal hydrolase L1; VAMP8: vesicle associated membrane protein 8.
    Keywords:  LAMP2A; LAMP2B; LAMP2C; autophagy; chaperone-mediated autophagy; lysosome
    DOI:  https://doi.org/10.1080/15548627.2023.2235196
  14. Cell Death Dis. 2023 Jul 15. 14(7): 436
    LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner.
    Elżbieta Kania, Jaclyn S Long, David G McEwan, Kirsten Welkenhuyzen, Rita La Rovere, Tomas Luyten, John Halpin, Evy Lobbestael, Veerle Baekelandt, Geert Bultynck, Kevin M Ryan, Jan B Parys.
      Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.
    DOI:  https://doi.org/10.1038/s41419-023-05964-0
  15. Nat Cell Biol. 2023 Jul 20.
    Spartin is a receptor for the autophagy of lipid droplets.
      
    DOI:  https://doi.org/10.1038/s41556-023-01201-0
  16. Front Cell Dev Biol. 2023 ;11 1242664
    Editorial: Mitophagy in health and disease, volume II.
    Evandro Fei Fang, Nektarios Tavernarakis, Konstantinos Palikaras.
      
    Keywords:  aging; autophagy; cardiomyopathy; cell death; diabetes; homeostasis; kidney; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2023.1242664
  17. Structure. 2023 Jul 04. pii: S0969-2126(23)00208-3. [Epub ahead of print]
    Structure of the Schizosaccharomyces pombe Gtr-Lam complex reveals evolutionary divergence of mTORC1-dependent amino acid sensing.
    Steven D Tettoni, Shawn B Egri, Dylan D Doxsey, Kristen Veinotte, Christna Ouch, Jeng-Yih Chang, Kangkang Song, Chen Xu, Kuang Shen.
      mTORC1 is a protein kinase complex that controls cellular growth in response to nutrient availability. Amino acid signals are transmitted toward mTORC1 via the Rag/Gtr GTPases and their upstream regulators. An important regulator is LAMTOR, which localizes Rag/Gtr on the lysosomal/vacuole membrane. In human cells, LAMTOR consists of five subunits, but in yeast, only three or four. Currently, it is not known how variation of the subunit stoichiometry may affect its structural organization and biochemical properties. Here, we report a 3.1 Å-resolution structural model of the Gtr-Lam complex in Schizosaccharomyces pombe. We found that SpGtr shares conserved architecture as HsRag, but the intersubunit communication that coordinates nucleotide loading on the two subunits differs. In contrast, SpLam contains distinctive structural features, but its GTP-specific GEF activity toward SpGtr is evolutionarily conserved. Our results revealed unique evolutionary paths of the protein components of the mTORC1 pathway.
    Keywords:  GATOR1; Gtr GTPase; LAMTOR; Rag GTPase; mTOR complex 1 (mTORC1); nutrient sensing
    DOI:  https://doi.org/10.1016/j.str.2023.06.012
  18. Cell Rep. 2023 Jul 13. pii: S2211-1247(23)00818-5. [Epub ahead of print]42(7): 112807
    Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.
    Sarah J Backe, Rebecca A Sager, Jennifer A Heritz, Laura A Wengert, Katherine A Meluni, Xavier Aran-Guiu, Barry Panaretou, Mark R Woodford, Chrisostomos Prodromou, Dimitra Bourboulia, Mehdi Mollapour.
      Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.
    Keywords:  CP: Molecular biology; Hsp90; Ulk1; atg1; autophagy; chaperone; chaperone code; co-chaperone; phosphorylation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112807
  19. iScience. 2023 Jul 21. 26(7): 107218
    Development of small fluorescent probes for the analysis of autophagy kinetics.
    Hajime Tajima Sakurai, Hidefumi Iwashita, Satoko Arakawa, Alifu Yikelamu, Mizuki Kusaba, Satoshi Kofuji, Hiroshi Nishina, Munetaka Ishiyama, Yuichiro Ueno, Shigeomi Shimizu.
      Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.
    Keywords:  Biological sciences tools; Biotechnology; Cell biology; Optical imaging
    DOI:  https://doi.org/10.1016/j.isci.2023.107218
  20. Mol Pain. 2023 Jul 18. 17448069231190815
    Urolithin A alleviates neuropathic pain and activates mitophagy.
    Chenyi Wang, Zizhu Wang, Shiyu Xue, Yutong Zhu, Jiahao Jin, Qiuyu Ren, Xiaodong Shi.
      Neuropathic pain (NP) occurs frequently in the general population and has a negative impact on the quality of life. There is no effective therapy available yet owing to the complex pathophysiology of NP. In our previous study, we found that urolithin A (UA), a naturally occurring microflora-derived metabolite, could relieve NP in mice by inhibiting the activation of microglia and release of inflammation factors. Here in this study, we sought to investigate whether mitophagy would be activated when UA alleviated NP in mice. We showed that the autophagy flow was blocked in the spinal dorsal horn of the chronic constriction injury (CCI) mice when the most obvious pain behavior occurs. Intraperitoneal injection of UA markedly activated the mitophagy mediated by PTEN-induced kinase 1/Parkin, promoted mitobiogenesis in both neurons and microglia, and alleviated NP in the CCI mice. In summary, our data suggest that UA alleviates NP in mice and meanwhile induces mitophagy activation, which highlights a therapeutic potential of UA in the treatment of NP.
    Keywords:  Urolithin A; chronic construction injury model; mitobiogenesis; mitophagy; neuropathic pain
    DOI:  https://doi.org/10.1177/17448069231190815
  21. Trends Microbiol. 2023 Jul 13. pii: S0966-842X(23)00195-6. [Epub ahead of print]
    Plant reoviruses hijack autophagy in insect vectors.
    Wenwen Liu, Taiyun Wei, Xifeng Wang.
      Plant reoviruses, transmitted only by insect vectors, seriously threaten global cereal production. Understanding how insect vectors efficiently transmit the viruses is key to controlling the viral diseases. Autophagy commonly plays important roles in plant host defense against virus infection, but recent studies have shown that plant reoviruses can hijack the autophagy pathway in insect cells to enable their persistence in the insect and continued transmission to plants. Here, we summarize and discuss new insights on viral activation, evasion, regulation, and manipulation of autophagy within the insect vectors and the role of autophagy in virus survival in insect vectors. Deeper knowledge of the functions of autophagy in vectors may lead to novel strategies for blocking transmission of insect-borne plant viruses.
    Keywords:  autophagic pathway; insect transmission; manipulation; mitophagy; plant reoviruses
    DOI:  https://doi.org/10.1016/j.tim.2023.06.008
  22. J Vis Exp. 2023 06 30.
    Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase.
    Denise R B Pilger, Christin Luft, Robin Ketteler.
      Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.
    DOI:  https://doi.org/10.3791/65464
  23. Cell Mol Life Sci. 2023 Jul 18. 80(8): 210
    Autophagy protein 5 controls flow-dependent endothelial functions.
    Pierre Nivoit, Thomas Mathivet, Junxi Wu, Yann Salemkour, Devanarayanan Siva Sankar, Véronique Baudrie, Jennifer Bourreau, Anne-Laure Guihot, Emilie Vessieres, Mathilde Lemitre, Cinzia Bocca, Jérémie Teillon, Morgane Le Gall, Anna Chipont, Estelle Robidel, Neeraj Dhaun, Eric Camerer, Pascal Reynier, Etienne Roux, Thierry Couffinhal, Patrick W F Hadoke, Jean-Sébastien Silvestre, Xavier Guillonneau, Philippe Bonnin, Daniel Henrion, Joern Dengjel, Pierre-Louis Tharaux, Olivia Lenoir.
      Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.
    Keywords:  Autophagy; Endothelium; Flow-mediated dilatation; Mechanosensing; VEGFR2; eNOS
    DOI:  https://doi.org/10.1007/s00018-023-04859-9
  24. Int Immunopharmacol. 2023 Jul 18. pii: S1567-5769(23)00987-6. [Epub ahead of print]122 110662
    Application and value of hydrogen sulfide modulated autophagy in sepsis.
    Yao Sun, Chang Liu.
      Sepsis is is anabnormalhost immune responsecausedbyinfection. Antibiotics, anti-viral drugs, and vasoactive drugs have always been used in the traditional treatment of sepsis, but there are no specific and effective drugs in clinical practice. Autophagy is a highly conservative process in biological evolution, and plays an important role in maintaining intracellular homeostasis and cellular self-renewal. Autophagy can remove and degrade misfolding proteins and damaged organelles in cells, providing materials for cell repair and self-renewal. Hydrogen sulfide (H2S) is a colorless gas that smells likerotteneggs. It is the third endogenous gas signal molecule discovered after nitric oxide and carbon monoxide and has become a research hotspot in recent years. H2S has a variety of biological functions and plays an important role in various physiological and pathological processes. Thereisgrowingevidencethat H2S can regulate autophagy. The intervention of autophagy is a promising therapeutic strategy to improve sepsis organ damage. This article reviews the organ protection of autophagy in sepsis and the role of H2S in regulating autophagy in sepsis, revealing that H2S intervention with autophagy may be a a worthy target in sepsis treatment.
    Keywords:  Autophagy; Hydrogen sulfide (H(2)S); Sepsis
    DOI:  https://doi.org/10.1016/j.intimp.2023.110662
  25. Autophagy. 2023 Jul 17. 1-22
    Analysis of ATG4C function in vivo.
    Isaac Tamargo-Gómez, Gemma G Martínez-García, María F Suárez, Pablo Mayoral, Gabriel Bretones, Aurora Astudillo, Jesús Prieto-Lloret, Christina Sveen, Antonio Fueyo, Nikolai Engedal, Carlos López-Otín, Guillermo Mariño.
      ABBREVIATIONS: ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).
    Keywords:  Animal models; GABARAP; LC3; autophagy; fibrosarcoma; lymphocyte
    DOI:  https://doi.org/10.1080/15548627.2023.2234799
  26. J Inherit Metab Dis. 2023 Jul 21.
    Homocysteine Metabolites Inhibit Autophagy, Elevate Amyloid Beta, and Induce Neuropathy by Impairing Phf8/H4K20me1-dependent Epigenetic Regulation of mTOR in Cystathionine β-Synthase-Deficient Mice.
    Łukasz Witucki, Hieronim Jakubowski.
      The loss of cystathionine β-synthase (CBS), an important homocysteine (Hcy)-metabolizing enzyme or the loss of PHF8, an important histone demethylase participating in epigenetic regulation, causes severe intellectual disability in humans. Similar neuropathies were also observed in Cbs-/- and Phf8-/- mice. How CBS or PHF8 depletion can cause neuropathy was unknown. To answer this question, we examined a possible interaction between PHF8 and CBS using Cbs-/- mouse and neuroblastoma cell models. We quantified gene expression by RT-qPCR and Western blotting, mTOR-bound H4K20me1 by chromatin immunoprecipitation (CHIP) assay, and amyloid β (Aβ) by confocal fluorescence microscopy using anti-Aβ antibody. We found significantly reduced expression of Phf8, increased H4K20me1, increased mTOR expression and phosphorylation, and increased App, both on protein and mRNA levels in brains of Cbs-/- mice vs. Cbs+/- sibling controls. Autophagy-related Becn1, Atg5, and Atg7 were downregulated while p62, Nfl, and Gfap were upregulated on protein and mRNA levels, suggesting reduced autophagy and increased neurodegeneration in Cbs-/- brains. In mouse neuroblastoma N2a or N2a-APPswe cells, treatments with Hcy-thiolactone, N-Hcy-protein or Hcy, or Cbs gene silencing by RNA interference significantly reduced Phf8 expression and increased total H4K20me1 as well as mTOR promoter-bound H4K20me1. This led to transcriptional mTOR upregulation, autophagy downregulation, and significantly increased APP and Aβ levels. The Phf8 gene silencing increased Aβ, but not APP, levels. Taken together, our findings identify Phf8 as a regulator of Aβ synthesis and suggest that neuropathy of Cbs deficiency is mediated by Hcy metabolites, which transcriptionally dysregulate the Phf8- > H4K20me1- > mTOR- > autophagy pathway thereby increasing Aβ accumulation. This article is protected by copyright. All rights reserved.
    Keywords:  APP; Cbs−/− mouse; H4K20me1; N-homocysteinylated protein; Phf8; amyloid beta; autophagy; homocysteine thiolactone; mTOR signaling
    DOI:  https://doi.org/10.1002/jimd.12661
  27. J Tradit Chin Med. 2023 Aug;43(4): 667-675
    Qizhi Jiangtang capsule activates podocyte autophagy in diabetic kidney disease by inhibiting phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways.
    Guo Zhaoan, Sun Lina, Liu Yingying, L I Ruifeng, Liu Chong, Diao Ke, Shi Jing, Sun Jun.
      OBJECTIVE: To investigate the therapeutic action and mechanism of the Qizhi Jiangtang capsule (, QZJT) on diabetic kidney disease (DKD) treatment.METHODS: This experiment used db/db mice and podocytes (MPC5) to develop DKD model. Evaluation of the effect of the QZJT on db/db mice by testing urine and blood biochemical parameters (24-h urinary albumin, serum creatinine, blood urine nitrogen), pathological kidney injury, and podocyte integrity. Moreover, autophagosomes in podocytes of DKD mice and cultured podocytes were detected using electron microscopy. Additionally, Western blotting was applied to detect the expression of podocyte marker protein (podocin), autophagy-associated proteins, and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway changes and .
    RESULTS: QZJT significantly reduced urine protein, blood nitrogen urea, and serum creatinine and showed histological restoration of renal tissues. QZJT also significantly improved the down-regulation of podocin and foot fusion and effacement in db/db mice. QZJT increased autophagic vesicles in mice and cultured podocytes. QZJT also upregulated microtubule-associated protein 1 light chain 3-II (LC3-II) / (LC3-I) and Beclin-1 and downregulated phosphorylated-PI3K (p-PI3K), p-AKT, and p-mTOR in db/db mice and MPC5 cells. However, autophagy inhibitor 3-methyladenine partially alleviated the above effects in MPC5 cells.
    CONCLUSIONS: These results showed that the QZJT can enhance podocyte autophagy and ameliorate podocyte injury in DKD by inhibiting the PI3K/AKT/mTOR signaling pathway.
    Keywords:  AKT; Qizhi Jiangtang capsule; autophagy; diabetic kidney disease; podocytes
    DOI:  https://doi.org/10.19852/j.cnki.jtcm.20230428.001
  28. J Cell Physiol. 2023 Jul 21.
    Autophagy mediates cementoblast mineralization under compression through periostin/β-catenin axis.
    Yuhui Yang, Hao Liu, Ruoxi Wang, Yi Zhao, Yunfei Zheng, Yiping Huang, Weiran Li.
      Repair of orthodontic external root resorption and periodontal tissue dysfunction induced by mechanical force remains a clinical challenge. Cementoblasts are vital in cementum mineralization, a process important for restoring damaged cementum. Despite autophagy plays a role in mineralization under various environmental stimuli, the underlying mechanism of autophagy in mediating cementoblast mineralization remains unclear. Here we verified that murine cementoblasts exhibit compromised mineralization under compressive force. Autophagy was indispensable for cementoblast mineralization, and autophagic activation markedly reversed cementoblast mineralization and prevented cementum damage in mice during tooth movement. Subsequently, messenger RNA sequencing analyses identified periostin (Postn) as a mediator of autophagy and mineralization in cementoblasts. Cementoblast mineralization was significantly inhibited following the knockdown of Postn. Furthermore, Postn silencing suppressed Wnt signaling by modulating the stability of β-catenin. Together our results highlight the role of autophagy in cementoblast mineralization via Postn/β-catenin signaling under compressive force and may provide a new strategy for the remineralization of cementum and regeneration of periodontal tissue.
    Keywords:  autophagy; cementum; mineralization; periostin; signal transduction; ubiquitination
    DOI:  https://doi.org/10.1002/jcp.31075
  29. Protein Sci. 2023 Jul 18. e4731
    Design of negative-regulating proteins of Rheb/mTORC1 with much reduced sizes of the tuberous sclerosis protein complex.
    Wencheng Fu, Geng Wu.
      The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (Short Stabilization domain and GAP domain-Membrane Targeting Motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy. This article is protected by copyright. All rights reserved.
    Keywords:  Rheb; SSG-MTM; SSG-TSC1N; TSC2-GAP; mTORC1; protein engineering; tuberous sclerosis protein complex
    DOI:  https://doi.org/10.1002/pro.4731
  30. Front Mol Neurosci. 2023 ;16 1135015
    Structural and functional understanding of disease-associated mutations in V-ATPase subunit a1 and other isoforms.
    Karen Indrawinata, Peter Argiropoulos, Shuzo Sugita.
      The vacuolar-type ATPase (V-ATPase) is a multisubunit protein composed of the cytosolic adenosine triphosphate (ATP) hydrolysis catalyzing V1 complex, and the integral membrane complex, Vo, responsible for proton translocation. The largest subunit of the Vo complex, subunit a, enables proton translocation upon ATP hydrolysis, mediated by the cytosolic V1 complex. Four known subunit a isoforms (a1-a4) are expressed in different cellular locations. Subunit a1 (also known as Voa1), the neural isoform, is strongly expressed in neurons and is encoded by the ATP6V0A1 gene. Global knockout of this gene in mice causes embryonic lethality, whereas pyramidal neuron-specific knockout resulted in neuronal cell death with impaired spatial and learning memory. Recently reported, de novo and biallelic mutations of the human ATP6V0A1 impair autophagic and lysosomal activities, contributing to neuronal cell death in developmental and epileptic encephalopathies (DEE) and early onset progressive myoclonus epilepsy (PME). The de novo heterozygous R740Q mutation is the most recurrent variant reported in cases of DEE. Homology studies suggest R740 deprotonates protons from specific glutamic acid residues in subunit c, highlighting its importance to the overall V-ATPase function. In this paper, we discuss the structure and mechanism of the V-ATPase, emphasizing how mutations in subunit a1 can lead to lysosomal and autophagic dysfunction in neurodevelopmental disorders, and how mutations to the non-neural isoforms, a2-a4, can also lead to various genetic diseases. Given the growing discovery of disease-causing variants of V-ATPase subunit a and its function as a pump-based regulator of intracellular organelle pH, this multiprotein complex warrants further investigation.
    Keywords:  R740; V-ATPase; developmental and epileptic encephalopahties; neurodevelopmental disorders; progressive myoclonus epilepsy; subunit a1
    DOI:  https://doi.org/10.3389/fnmol.2023.1135015
  31. Cell Mol Life Sci. 2023 Jul 19. 80(8): 216
    Protrudin-mediated ER-endosome contact sites promote phagocytosis.
    Liv Anker Elfmark, Eva Maria Wenzel, Ling Wang, Nina Marie Pedersen, Harald Stenmark, Camilla Raiborg.
      During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis.
    Keywords:  Efferocytosis; Endosomes; FYCO1; Phagocytosis; Protrudin; RAB7; VAMP7
    DOI:  https://doi.org/10.1007/s00018-023-04862-0
  32. Front Aging Neurosci. 2023 ;15 1087072
    APOE ε4 allele, along with G206D-PSEN1 mutation, alters mitochondrial networks and their degradation in Alzheimer's disease.
    Irene Costa-Laparra, Elena Juárez-Escoto, Carlos Vicario, Rosario Moratalla, Patricia García-Sanz.
      Introduction: Alzheimer's disease remains the most common neurodegenerative disorder, depicted mainly by memory loss and the presence in the brain of senile plaques and neurofibrillary tangles. This disease is related to several cellular alterations like the loss of synapses, neuronal death, disruption of lipid homeostasis, mitochondrial fragmentation, or raised oxidative stress. Notably, changes in the autophagic pathway have turned out to be a key factor in the early development of the disease. The aim of this research is to determine the impact of the APOE allele ε4 and G206D-PSEN1 on the underlying mechanisms of Alzheimer's disease.Methods: Fibroblasts from Alzheimer's patients with APOE 3/4 + G206D-PSEN1 mutation and homozygous APOE ε4 were used to study the effects of APOE polymorphism and PSEN1 mutation on the autophagy pathway, mitochondrial network fragmentation, superoxide anion levels, lysosome clustering, and p62/SQSTM1 levels.
    Results: We observed that the APOE allele ε4 in homozygosis induces mitochondrial network fragmentation that correlates with an increased colocalization with p62/SQSTM1, probably due to an inefficient autophagy. Moreover, G206D-PSEN1 mutation causes an impairment of the integrity of mitochondrial networks, triggering high superoxide anion levels and thus making APOE 3/4 + PSEN1 fibroblasts more vulnerable to cell death induced by oxidative stress. Of note, PSEN1 mutation induces accumulation and clustering of lysosomes that, along with an increase of global p62/SQSTM1, could compromise lysosomal function and, ultimately, its degradation.
    Conclusion: The findings suggest that all these modifications could eventually contribute to the neuronal degeneration that underlies the pathogenesis of Alzheimer's disease. Further research in this area may help to develop targeted therapies for the treatment of Alzheimer's disease.
    Keywords:  Alzheimer’s disease; autophagy; lysosomes and mitochondria imaging; mitochondria; neurodegeneration; oxidative stress
    DOI:  https://doi.org/10.3389/fnagi.2023.1087072
  33. Trends Cell Biol. 2023 Jul 18. pii: S0962-8924(23)00128-9. [Epub ahead of print]
    Lysosomal enzyme trafficking: from molecular mechanisms to human diseases.
    Thomas Braulke, Jan E Carette, Wilhelm Palm.
      Lysosomes degrade and recycle macromolecules that are delivered through the biosynthetic, endocytic, and autophagic routes. Hydrolysis of the different classes of macromolecules is catalyzed by about 70 soluble enzymes that are transported from the Golgi apparatus to lysosomes in a mannose 6-phosphate (M6P)-dependent process. The molecular machinery that generates M6P tags for receptor-mediated targeting of lysosomal enzymes was thought to be understood in detail. However, recent studies on the M6P pathway have identified a previously uncharacterized core component, yielded structural insights in known components, and uncovered functions in various human diseases. Here we review molecular mechanisms of lysosomal enzyme trafficking and discuss its relevance for rare lysosomal disorders, cancer, and viral infection.
    Keywords:  LYSET; cancer metabolism; lysosomal enzymes; lysosomal storage disorders; mannose 6-phosphate pathway; viral infections
    DOI:  https://doi.org/10.1016/j.tcb.2023.06.005
  34. Biomed Pharmacother. 2023 Jul 18. pii: S0753-3322(23)00938-1. [Epub ahead of print]165 115147
    Autophagy in sarcopenia: Possible mechanisms and novel therapies.
    Guangyang Xie, Hongfu Jin, Herasimenka Mikhail, Volotovski Pavel, Guang Yang, Bingzhou Ji, Bangbao Lu, Yusheng Li.
      With global population aging, age-related diseases, especially sarcopenia, have attracted much attention in recent years. Characterized by low muscle strength, low muscle quantity or quality and low physical performance, sarcopenia is one of the major factors associated with an increased risk of falls and disability. Much effort has been made to understand the cellular biological and physiological mechanisms underlying sarcopenia. Autophagy is an important cellular self-protection mechanism that relies on lysosomes to degrade misfolded proteins and damaged organelles. Research designed to obtain new insight into human diseases from the autophagic aspect has been carried out and has made new progress, which encourages relevant studies on the relationship between autophagy and sarcopenia. Autophagy plays a protective role in sarcopenia by modulating the regenerative capability of satellite cells, relieving oxidative stress and suppressing the inflammatory response. This review aims to reveal the specific interaction between sarcopenia and autophagy and explore possible therapies in hopes of encouraging more specific research in need and unlocking novel promising therapies to ameliorate sarcopenia.
    Keywords:  Autophagy; Inflammation; Oxidative stress; Sarcopenia; Satellite cell
    DOI:  https://doi.org/10.1016/j.biopha.2023.115147
  35. Cell Mol Life Sci. 2023 Jul 20. 80(8): 219
    Dietary restriction to optimize T cell immunity is an ancient survival strategy conserved in vertebrate evolution.
    Kunming Li, Xiumei Wei, Kang Li, Qian Zhang, Jiansong Zhang, Ding Wang, Jialong Yang.
      Recent advances highlight a key role of transient fasting in optimizing immunity of human and mouse. However, it remains unknown whether this strategy is independently acquired by mammals during evolution or instead represents gradually evolved functions common to vertebrates. Using a tilapia model, we report that T cells are the main executors of the response of the immune system to fasting and that dietary restriction bidirectionally modulates T cell immunity. Long-term fasting impaired T cell immunity by inducing intense autophagy, apoptosis, and aberrant inflammation. However, transient dietary restriction triggered moderate autophagy to optimize T cell response by maintaining homeostasis, alleviating inflammation and tissue damage, as well as enhancing T cell activation, proliferation and function. Furthermore, AMPK is the central hub linking fasting and autophagy-controlled T cell immunity in tilapia. Our findings demonstrate that dietary restriction to optimize immunity is an ancient strategy conserved in vertebrate evolution, providing novel perspectives for understanding the adaptive evolution of T cell response.
    Keywords:  Autophagy; Dietary restriction; Evolution; T cells; Tilapia
    DOI:  https://doi.org/10.1007/s00018-023-04865-x
  36. FEBS Open Bio. 2023 Jul 20.
    Autophagy signaling in hypertrophied muscles of diabetic and control rats.
    M V M Scervino, M A S Fortes, K F Vitzel, D R de Souza, G M Murata, G O Santana, E B da Silva, A C Levada-Pires, W M T Kuwabara, T C A Loureiro, R Curi.
      Autophagy plays a vital role in cell homeostasis by eliminating nonfunctional components and promoting cell survival. Here, we examined the levels of autophagy signaling proteins after seven days of overload hypertrophy in the extensor digitorum longus (EDL) and soleus muscles of control and diabetic rats. We compared control and 3-day streptozotocin-induced diabetic rats, an experimental model for type 1 diabetes mellitus (T1DM). EDL muscles showed increased levels of basal autophagy signaling proteins. The diabetic state did not affect the extent of overload-induced hypertrophy or the levels of autophagy signaling proteins (p-ULK1, Beclin-1, Atg5, Atg12-5, Atg7, Atg3, LC3-I and II, and p62) in either muscle. The p-ULK-1, Beclin-1, and p62 protein expression levels were higher in the EDL muscle than in the soleus before the hypertrophic stimulus. On the other hand, the soleus muscle exhibited increased autophagic signaling after overload-induced hypertrophy, with increases in Beclin-1, Atg5, Atg12-5, Atg7, Atg3, and LC3-I expression in the control and diabetic groups, in addition to p-ULK-1 in the control groups. After hypertrophy, Beclin-1 and Atg5 levels increased in the EDL muscle of both groups, while p-ULK1 and LC3-I increased in the control group. In conclusion, the baseline EDL muscle exhibited higher autophagy than the soleus muscle. Although TDM1 promotes skeletal muscle mass loss and strength reduction, it did not significantly alter the extent of overload-induced hypertrophy and autophagy signaling proteins in EDL and soleus muscles, with the two groups exhibiting different patterns of autophagy activation.
    Keywords:  Autolysosome; Autophagosome; Autophagy-related genes; Hyperglycemia; Protein degradation
    DOI:  https://doi.org/10.1002/2211-5463.13677
  37. Life Sci Alliance. 2023 Oct;pii: e202201634. [Epub ahead of print]6(10):
    The C-terminal tail of CSNAP attenuates the CSN complex.
    Maria G Füzesi-Levi, Gili Ben-Nissan, Dina Listov, Yael Fridmann Sirkis, Zvi Hayouka, Sarel Fleishman, Michal Sharon.
      Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.
    DOI:  https://doi.org/10.26508/lsa.202201634
  38. Front Aging. 2023 ;4 1175601
    Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer's disease patient lymphocytes.
    Hoau-Yan Wang, Zhe Pei, Kuo-Chieh Lee, Boris Nikolov, Tamara Doehner, John Puente, Nadav Friedmann, Lindsay H Burns.
      Introduction: Implicated in both aging and Alzheimer's disease (AD), mammalian target of rapamycin (mTOR) is overactive in AD brain and lymphocytes. Stimulated by growth factors such as insulin, mTOR monitors cell health and nutrient needs. A small molecule oral drug candidate for AD, simufilam targets an altered conformation of the scaffolding protein filamin A (FLNA) found in AD brain and lymphocytes that induces aberrant FLNA interactions leading to AD neuropathology. Simufilam restores FLNA's normal shape to disrupt its AD-associated protein interactions. Methods: We measured mTOR and its response to insulin in lymphocytes of AD patients before and after oral simufilam compared to healthy control lymphocytes. Results: mTOR was overactive and its response to insulin reduced in lymphocytes from AD versus healthy control subjects, illustrating another aspect of insulin resistance in AD. After oral simufilam, lymphocytes showed normalized basal mTOR activity and improved insulin-evoked mTOR activation in mTOR complex 1, complex 2, and upstream and downstream signaling components (Akt, p70S6K and phosphorylated Rictor). Suggesting mechanism, we showed that FLNA interacts with the insulin receptor until dissociation by insulin, but this linkage was elevated and its dissociation impaired in AD lymphocytes. Simufilam improved the insulin-mediated dissociation. Additionally, FLNA's interaction with Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), a negative regulator of mTOR, was reduced in AD lymphocytes and improved by simufilam. Discussion: Reducing mTOR's basal overactivity and its resistance to insulin represents another mechanism of simufilam to counteract aging and AD pathology. Simufilam is currently in Phase 3 clinical trials for AD dementia.
    Keywords:  Alzheimer’s disease; PTEN; aging; filamin A; insulin resistance; mTOR
    DOI:  https://doi.org/10.3389/fragi.2023.1175601
  39. PLoS One. 2023 ;18(7): e0288622
    Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells.
    Amro H Mohammad, Frédéric Couture, Isabelle Gamache, Owen Chen, Wissal El-Assaad, Nelly Abdel-Malak, Anna Kwiatkowska, William Muller, Robert Day, Jose G Teodoro.
      Phosphatase and tensin homolog (PTEN) mutation is common in prostate cancer during progression to metastatic and castration resistant forms. We previously reported that loss of PTEN function in prostate cancer leads to increased expression and secretion of the Prorenin Receptor (PRR) and its soluble processed form, the soluble Prorenin Receptor (sPRR). PRR is an essential factor required for proper assembly and activity of the vacuolar-ATPase (V-ATPase). The V-ATPase is a rotary proton pump required for the acidification of intracellular vesicles including endosomes and lysosomes. Acidic vesicles are involved in a wide range of cancer related pathways such as receptor mediated endocytosis, autophagy, and cell signalling. Full-length PRR is cleaved at a conserved consensus motif (R-X-X-R↓) by a member of the proprotein convertase family to generate sPRR, and a smaller C-terminal fragment, designated M8.9. It is unclear which convertase processes PRR in prostate cancer cells and how processing affects V-ATPase activity. In the current study we show that PRR is predominantly cleaved by PACE4, a proprotein convertase that has been previously implicated in prostate cancer. We further demonstrate that PTEN controls PRR processing in mouse tissue and controls PACE4 expression in prostate cancer cells. Furthermore, we demonstrate that PACE4 cleavage of PRR is needed for efficient V-ATPase activity and prostate cancer cell growth. Overall, our data highlight the importance of PACE4-mediated PRR processing in normal physiology and prostate cancer tumorigenesis.
    DOI:  https://doi.org/10.1371/journal.pone.0288622
  40. Curr Opin Immunol. 2023 Jul 12. pii: S0952-7915(23)00085-7. [Epub ahead of print]84 102366
    Mitochondrial reactive oxygen species: double agents in Mycobacterium tuberculosis infection.
    Lily M Ellzey, Kristin L Patrick, Robert O Watson.
      In addition to housing the major energy-producing pathways in cells, mitochondria are active players in innate immune responses. One critical way mitochondria fulfill this role is by releasing damage-associated molecular patterns (mtDAMPs) that are recognized by innate sensors to activate pathways including, but not limited to, cytokine expression, selective autophagy, and cell death. Mitochondrial reactive oxygen species (mtROS) is a multifunctional mtDAMP linked to pro- and antimicrobial immune outcomes. Formed as a by-product of energy generation, mtROS links mitochondrial metabolism with downstream innate immune responses. As a result, altered cellular metabolism can change mtROS levels and impact downstream antimicrobial responses in a variety of ways. MtROS has emerged as a particularly important mediator of pathogenesis during infection with Mycobacterium tuberculosis (Mtb), an intracellular bacterial pathogen that continues to pose a significant threat to global public health. Here, we will summarize how Mtb modulates mtROS levels in infected macrophages and how mtROS dictates Mtb infection outcomes by controlling inflammation, lipid peroxidation, and cell death. We propose that mtROS may serve as a biomarker to predict tuberculosis patient outcomes and/or a target for host-directed therapeutics.
    DOI:  https://doi.org/10.1016/j.coi.2023.102366
  41. Am J Physiol Cell Physiol. 2023 Jul 17.
    CERS6-derived ceramides aggravate kidney fibrosis by inhibiting PINK1-mediated mitophagy in diabetic kidney disease.
    Xiangyu Wang, Minkai Song, Xiaomin Li, Cailin Su, Yanlin Yang, Kai Wang, Cuiting Liu, Zongji Zheng, Yi-Jie Jia, Shijing Ren, Wenhui Dong, Jiaqi Chen, Ting Wang, Lerong Liu, Meiping Guan, Chao Zhang, Yaoming Xue.
      During diabetic kidney disease (DKD), ectopic ceramide (CER) accumulation in renal tubular epithelial cells (RTECs) is associated with interstitial fibrosis and albuminuria. As RTECs are primarily responsible for renal energy metabolism, their function is intimately linked to mitochondrial quality control. The role of CER synthesis in the progression of diabetic renal fibrosis has not been thoroughly investigated. In this study, we observed a significant upregulation of ceramide synthase 6 (CERS6) expression in the renal cortex of db/db mice, coinciding with increased production of CER (d18:1/14:0) and CER (d18:1/16:0) by Cer6. Concurrently, the number of damaged mitochondria in RTECs rose. Cers6 deficiency reduced the abnormal accumulation of CER (d18:1/14:0) and CER (d18:1/16:0) in the kidney cortex, restoring the PTEN-induced kinase 1 (PINK1)-mediated mitophagy in RTECs, and resulting in a decrease in damaged mitochondria and attenuation of interstitial fibrosis in DKD. Automated docking analysis suggested that both CER (d18:1/14:0) and CER (d18:1/16:0) could bind to the PINK1 protein. Furthermore, inhibiting PINK1 expression in CERS6 knockdown HK-2 cells diminished the therapeutic effect of CERS6 deficiency on DKD. In summary, CERS6-derived CER (d18:1/14:0) and CER (d18:1/16:0) inhibit PINK1-regulated mitophagy by possibly binding to the PINK1 protein, thereby exacerbating the progression of renal interstitial fibrosis in DKD.
    Keywords:  Ceramide synthase 6; Diabetic kidney disease; Interstitial fibrosis; Mitophagy; Renal tubular epithelial cell
    DOI:  https://doi.org/10.1152/ajpcell.00144.2023
  42. Autophagy. 2023 Jul 21. 1-13
    Increased apoptosis of gingival epithelium is associated with impaired autophagic flux in medication-related osteonecrosis of the jaw.
    Xian Dong, Yang He, Jingang An, Linhai He, Yi Zheng, Xinyu Wang, Jie Wang, Shuo Chen, Yi Zhang.
      Macroautophagy/autophagy has both negative and positive aspects in the development of many diseases. Yet, its exact role and specific mechanism in the onset of medication-related osteonecrosis of the jaw (MRONJ) is still not fully understood. Retarded gingiva healing is the primary clinical manifestation in patients with MRONJ. In this study, we aimed to explore the relationship between autophagy and apoptosis in MRONJ gingival epithelium and search for a method to prevent this disease. First, we examined clinical samples from patients diagnosed with MRONJ and healthy controls, finding that autophagy-related markers MAP1LC3/LC3 and SQSTM1/p62 synchronously increased, thus suggesting that autophagic flux was suppressed in MRONJ. Moreover, mRNA sequencing analysis and TUNEL assay showed that the process of apoptosis was upregulated in patients and animals with MRONJ, indicating autophagy and apoptosis participate in the development of MRONJ. Furthermore, the level of autophagy and apoptosis in zoledronic acid (ZA)-treated human keratinocytes cell lines (HaCaT cells) was concentration dependent in vitro. In addition, we also found that RAB7 (RAB7, member RAS oncogene family) activator ML098 could rescue MRONJ gingival lesions in mice by activating the autophagic flux and downregulating apoptosis. To sum up, this study demonstrated that autophagic flux is impaired in the gingival epithelium during MRONJ, and the rescued autophagic flux could prevent the occurrence of MRONJ.Abbreviations: ACTB: actin beta; Baf-A1: bafilomycin A1; CASP3: caspase 3; CASP8: caspase 8; CT: computed tomography; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; HaCaT cells: human keratinocytes cell lines; H&E: hematoxylin and eosin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MRONJ: medication-related osteonecrosis of the jaw; PARP: poly(ADP-ribose) polymerase; RAB7: RAB7, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ZA: zoledronic acid.
    Keywords:  Apoptosis; autophagic flux; gingival epithelium; keratinocytes; medication-related osteonecrosis of the jaw; zoledronic acid
    DOI:  https://doi.org/10.1080/15548627.2023.2234228
  43. Plant J. 2023 Jul 20.
    MdHB7-like positively modulates apple salt tolerance by promoting autophagic activity and Na+ efflux.
    Jie Yang, Lina Qiu, Quanlin Mei, Yunxia Sun, Na Li, Xiaoqing Gong, Fengwang Ma, Ke Mao.
      Salt stress adversely affects the yield and quality of crops and limits their geographical distribution. Studying the functions and regulatory mechanisms of key genes in the salt stress response is important for breeding crops with enhanced stress resistance. Autophagy plays an important role in modulating the tolerance of plants to various types of abiotic stressors. However, the mechanisms underlying salt-induced autophagy are largely unknown. Cation/Ca2+ exchanger proteins enhance apple salt tolerance by inhibiting Na+ accumulation but the mechanism underlying the response to salt stress remains unclear. Here, we show that the autophagy-related gene MdATG18a modulated apple salt tolerance. Under salt stress, the autophagic activity, proline content, and antioxidant enzyme activities were higher and Na+ accumulation was lower in MdATG18a-overexpressing transgenic plants than in control plants. The use of an autophagy inhibitor during the salt treatment demonstrated that the regulatory function of MdATG18a depended on autophagy. The yeast-one-hybrid assay revealed that the homeodomain-leucine zipper (HD-Zip) transcription factor MdHB7-like directly bound to the MdATG18a promoter. Transcriptional regulation and genetic analyses showed that MdHB7-like enhanced salt-induced autophagic activity by promoting MdATG18a expression. The analysis of Na+ efflux rate in transgenic yeast indicated that MdCCX1 expression significantly promoted Na+ efflux. Promoter binding, transcriptional regulation, and genetic analyses showed that MdHB7-like promoted Na+ efflux and apple salt tolerance by directly promoting MdCCX1 expression, which was independent of the autophagy pathway. Overall, our findings provide insight into the mechanism underlying MdHB7-like-mediated salt tolerance in apple through the MdHB7-like-MdATG18a and MdHB7-like-MdCCX1 modules. These results will aid future studies on the mechanisms underlying stress-induced autophagy and the regulation of stress tolerance in plants.
    Keywords:  HD-zip transcription factor; Na+ efflux; apple (Malus domestica); autophagy; cation/Ca2+ exchanger; salt stress
    DOI:  https://doi.org/10.1111/tpj.16395
  44. Cell Rep. 2023 Jul 17. pii: S2211-1247(23)00812-4. [Epub ahead of print]42(7): 112801
    Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis.
    Haijiao Huang, Bo Jing, Feiyan Zhu, Wanxiang Jiang, Ping Tang, Liyang Shi, Huiting Chen, Guoru Ren, Shiyao Xia, Luoling Wang, Yiyuan Cui, Zhiwen Yang, Alexander J Platero, Andrew P Hutchins, Mina Chen, Paul F Worley, Bo Xiao.
      How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction.
    Keywords:  CP: Developmental biology; CP: Neuroscience; DLK1; Raptor; Rheb; hypomyelination; mTORC1; myelination; myelination deficit; neuron-OPC interaction; neuron-glia interaction; oligodendrocyte differentiation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112801
  45. Autophagy. 2023 Jul 20.
    Periplocin suppresses the growth of colorectal cancer cells by triggering LGALS3 (galectin 3)-mediated lysophagy.
    Kui Wang, Shuyue Fu, Lixia Dong, Dingyue Zhang, Mao Wang, Xingyun Wu, Enhao Shen, Li Luo, Changlong Li, Edouard Collins Nice, Canhua Huang, Bingwen Zou.
      Colorectal cancer (CRC) is one of the most common malignancies worldwide and remains a major clinical challenge. Periplocin, a major bioactive component of the traditional Chinese herb Cortex periplocae, has recently been reported to be a potential anticancer drug. However, the mechanism of action is poorly understood. Here, we show that periplocin exhibits promising anticancer activity against CRC both in vitro and in vivo. Mechanistically, periplocin promotes lysosomal damage and induces apoptosis in CRC cells. Notably, periplocin upregulates LGALS3 (galectin 3) by binding and preventing LGALS3 from Lys210 ubiquitination-mediated proteasomal degradation, leading to the induction of excessive lysophagy and resultant exacerbation of lysosomal damage. Inhibition of LGALS3-mediated lysophagy attenuates periplocin-induced lysosomal damage and growth inhibition in CRC cells, suggesting a critical role of lysophagy in the anticancer effects of periplocin. Taken together, our results reveal a novel link between periplocin and the lysophagy machinery, and indicate periplocin as a potential therapeutic option for the treatment of CRC.
    Keywords:  Autophagic flux; LGALS3 (galectin 3); colorectal cancer; lysophagy; lysosomal damage; periplocin
    DOI:  https://doi.org/10.1080/15548627.2023.2239042
  46. Anal Chem. 2023 Jul 18.
    Fluorescence-On Imaging of Reticulophagy Enabled by an Acidity-Reporting Solvatochromic Probe.
    Xiaoxue Zou, Yilong Shi, Shuo Zhang, Jialiang Quan, Jiahuai Han, Shoufa Han.
      Aberrant autophagy of the endoplasmic reticulum (reticulophagy) is engaged in diverse pathological disorders. Herein, we reported sensitive imaging of reticulophagy with ER-Green-proRed, a diad combining a solvatochromic entity of trifluoromethylated naphthalimide for long-term ER tracking by green fluorescence and an entity of rhodamine-lactam fluorogenic to lysosomal acidity. Stringently accumulated in the ER to give green fluorescence, ER-Green-proRed exhibits robust red fluorescence upon codelivery with the ER subdomain into lysosomes. The relevance of turn-on red fluorescence to reticulophagy was validated by reticulophagy modulated by starvation, reticulophagic receptors, and autophagy inhibition. This imaging method was successfully employed to discern reticulophagy induced by various pharmacological agents. These results show the potential of ER-targeted pH probes, as exemplified by ER-Green-proRed, to image reticulophagy and to identify reticulophagy inducers.
    DOI:  https://doi.org/10.1021/acs.analchem.3c02016
  47. Contact (Thousand Oaks). 2023 Jan-Dec;6:6 25152564231183898
    The Organization and Function of the Phagophore-ER Membrane Contact Sites.
    Prado Vargas Duarte, Fulvio Reggiori.
      Macroautophagy is characterized by the de novo formation of double-membrane vesicles termed autophagosomes. The precursor structure of autophagosomes is a membrane cistern called phagophore, which elongates through a massive acquisition of lipids until closure. The phagophore establishes membrane-contact sites (MCSs) with the endoplasmic reticulum (ER), where conserved ATG proteins belonging to the ATG9 lipid scramblase, ATG2 lipid transfer and Atg18/WIPI4 β-propeller families concentrate. Several recent in vivo and in vitro studies have uncovered the relevance of these proteins and MCSs in the lipid supply required for autophagosome formation. Although important conceptual advances have been reached, the functional interrelationship between ATG9, ATG2 and Atg18/WIPI4 proteins at the phagophore-ER MCSs and their role in the phagophore expansion are not completely understood. In this review, we describe the current knowledge about the structure, interactions, localizations, and molecular functions of these proteins, with a particular emphasis on the yeast Saccharomyces cerevisiae and mammalian systems.
    Keywords:  ATG protein; ATG2; ATG9; Atg18/WIPI4; autophagosome; autophagy; lipid transfer; phagophore; scramblase; tethering
    DOI:  https://doi.org/10.1177/25152564231183898
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