bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022‒04‒24
forty-four papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Lysosomes are required for early dorsal signaling in the Xenopus embryo.
  2. Glycoprotein nonmetastatic melanoma protein B regulates lysosomal integrity and lifespan of senescent cells.
  3. Molecular Mechanisms in Lysosomal Storage Diseases: From Pathogenesis to Therapeutic Strategies.
  4. SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-β oligomers.
  5. mTORC1 Regulates High Levels of Protein Synthesis in Retinal Ganglion Cells of Adult Mice.
  6. FKBP5/FKBP51 on weight watch: central FKBP5 links regulatory WIPI protein networks to autophagy and metabolic control.
  7. Evolving therapies in neuronopathic LSDs: opportunities and challenges.
  8. SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.
  9. mTOR deletion ameliorates CD4 + T cell apoptosis during sepsis by improving autophagosome-lysosome fusion.
  10. GBA Variants and Parkinson Disease: Mechanisms and Treatments.
  11. Complex Changes in the Efficiency of the Expression of Many Genes in Monogenic Diseases, Mucopolysaccharidoses, May Arise from Significant Disturbances in the Levels of Factors Involved in the Gene Expression Regulation Processes.
  12. New directions for the clathrin adaptor AP-1 in cell biology and human disease.
  13. Heparan Sulfate, Mucopolysaccharidosis IIIB and Sulfur Metabolism Disorders.
  14. Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis.
  15. Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.
  16. Acetylation in the regulation of autophagy.
  17. TORC1 Signaling Controls the Stability and Function of α-Arrestins Aly1 and Aly2.
  18. Glial Dysfunction and Its Contribution to the Pathogenesis of the Neuronal Ceroid Lipofuscinoses.
  19. Phenotype assessment for neurodegenerative murine models with ataxia and application to Niemann-Pick disease, type C1.
  20. Exploratory Analysis of Selected Components of the mTOR Pathway Reveals Potentially Crucial Associations with Childhood Malnutrition.
  21. Secretory autophagy maintains proteostasis upon lysosome inhibition.
  22. Lysosomal Storage Disease Associated with a CNP Sequence Variant in Dalmatian Dogs.
  23. Cylindromatosis drives synapse pruning and weakening by promoting macroautophagy through Akt-mTOR signaling.
  24. T cell differentiation protein 2 facilitates cell proliferation by enhancing mTOR-mediated ribosome biogenesis in non-small cell lung cancer.
  25. Autophagy in cancer cell remodeling and quality control.
  26. SnapShot: Organelle degradation.
  27. FGF21 alleviates pulmonary hypertension by inhibiting mTORC1/EIF4EBP1 pathway via H19.
  28. DEPDC5-related epilepsy: A comprehensive review.
  29. Pan-mTOR inhibitors sensitize the senolytic activity of Navitoclax via mTORC2 inhibition-mediated apoptotic signaling.
  30. Cholesterol suppresses GOLM1-dependent selective autophagy of RTKs in hepatocellular carcinoma.
  31. Lysosomes Dysfunction Causes Mitophagy Impairment in PBMCs of Sporadic ALS Patients.
  32. Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.
  33. Slc38a9 Deficiency Induces Apoptosis and Metabolic Dysregulation and Leads to Premature Death in Zebrafish.
  34. Efficacy and Safety of Topical Mechanistic Target of Rapamycin Inhibitors for Facial Angiofibromas in Patients with Tuberous Sclerosis Complex: A Systematic Review and Network Meta-Analysis.
  35. mRNA Translation Is Dynamically Regulated to Instruct Stem Cell Fate.
  36. Morphometric Analysis of Lysosomes in the Renal Tubule in Monoclonal Gammopathy Using Transmission Electron Microscopy: "Mottled Appearance" and Beyond.
  37. Calpain-mediated proteolytic production of free amino acids in vascular endothelial cells augments obesity-induced hepatic steatosis.
  38. Snf7 spirals sense and alter membrane curvature.
  39. The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.
  40. Cellular abundance of sodium phosphate co-transporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT.
  41. Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.
  42. Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking.
  43. Codon optimality-mediated mRNA degradation: Linking translational elongation to mRNA stability.
  44. Somatic genomic changes in single Alzheimer's disease neurons.
  1. Proc Natl Acad Sci U S A. 2022 Apr 26. 119(17): e2201008119
    Lysosomes are required for early dorsal signaling in the Xenopus embryo.
    Nydia Tejeda-Muñoz, Edward M De Robertis.
      SignificanceThe dorsal axis of the vertebrate Xenopus embryo is established by an early Wnt signal generated by a rotation of the cortex of the egg toward the opposite side of the sperm entry point. In this study, we report that lysosomal cathepsin D becomes activated on the dorsal marginal zone of the embryo already at the 64-cell stage, and that this asymmetry is enhanced by increasing Wnt signaling levels. We present experiments showing that lysosome acidification, macropinocytosis, and multivesicular body formation are required for the endogenous dorsal signal provided maternally in the egg. The results indicate that the cell biology of lysosomes plays an essential role in vertebrate development.
    Keywords:  Hwa; Wnt signaling; Xenopus laevis; cytoplasmic determinant; lysosomes
    DOI:  https://doi.org/10.1073/pnas.2201008119
  2. Sci Rep. 2022 Apr 20. 12(1): 6522
    Glycoprotein nonmetastatic melanoma protein B regulates lysosomal integrity and lifespan of senescent cells.
    Masayoshi Suda, Ippei Shimizu, Goro Katsuumi, Chieh Lun Hsiao, Yohko Yoshida, Naomi Matsumoto, Yutaka Yoshida, Akihiro Katayama, Jun Wada, Masahide Seki, Yutaka Suzuki, Shujiro Okuda, Kazuyuki Ozaki, Mayumi Nakanishi-Matsui, Tohru Minamino.
      Accumulation of senescent cells in various tissues has been reported to have a pathological role in age-associated diseases. Elimination of senescent cells (senolysis) was recently reported to reversibly improve pathological aging phenotypes without increasing rates of cancer. We previously identified glycoprotein nonmetastatic melanoma protein B (GPNMB) as a seno-antigen specifically expressed by senescent human vascular endothelial cells and demonstrated that vaccination against Gpnmb eliminated Gpnmb-positive senescent cells, leading to an improvement of age-associated pathologies in mice. The aim of this study was to elucidate whether GPNMB plays a role in senescent cells. We examined the potential role of GPNMB in senescent cells by testing the effects of GPNMB depletion and overexpression in vitro and in vivo. Depletion of GPNMB from human vascular endothelial cells shortened their replicative lifespan and increased the expression of negative cell cycle regulators. Conversely, GPNMB overexpression protected these cells against stress-induced premature senescence. Depletion of Gpnmb led to impairment of vascular function and enhanced atherogenesis in mice, whereas overexpression attenuated dietary vascular dysfunction and atherogenesis. GPNMB was upregulated by lysosomal stress associated with cellular senescence and was a crucial protective factor in maintaining lysosomal integrity. GPNMB is a seno-antigen that acts as a survival factor in senescent cells, suggesting that targeting seno-antigens such as GPNMB may be a novel strategy for senolytic treatments.
    DOI:  https://doi.org/10.1038/s41598-022-10522-3
  3. Biomedicines. 2022 Apr 17. pii: 922. [Epub ahead of print]10(4):
    Molecular Mechanisms in Lysosomal Storage Diseases: From Pathogenesis to Therapeutic Strategies.
    Valeria De Pasquale, Melania Scarcella, Luigi Michele Pavone.
      Lysosomal storage diseases (LSDs) are a group of metabolic diseases caused by inborn mutations of lysosomal enzymes, which lead to lysosome substrate accumulation in various cell types [...].
    DOI:  https://doi.org/10.3390/biomedicines10040922
  4. Neurobiol Dis. 2022 Apr 19. pii: S0969-9961(22)00129-2. [Epub ahead of print] 105737
    SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-β oligomers.
    Andrés Norambuena, Xuehan Sun, Horst Wallrabe, Ruofan Cao, Naidi Sun, Evelyn Pardo, Nutan Shivange, Dora Bigler Wang, Lisa A Post, Heather A Ferris, Song Hu, Ammasi Periasamy, George S Bloom.
      Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimer's disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We previously discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-β oligomers (AβOs), a primary factor in AD pathogenesis (Norambuena et al., 2018). Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. AβOs inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from tuberous sclerosis (TS) patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.
    Keywords:  Alzheimer's disease; Amino acids; Insulin; Tau; mTOR
    DOI:  https://doi.org/10.1016/j.nbd.2022.105737
  5. J Biol Chem. 2022 Apr 18. pii: S0021-9258(22)00384-2. [Epub ahead of print] 101944
    mTORC1 Regulates High Levels of Protein Synthesis in Retinal Ganglion Cells of Adult Mice.
    Patrice E Fort, Mandy K Losiewicz, Lynda Elghazi, Dejuan Kong, Corentin Cras-Méneur, Diane C Fingar, Scot R Kimball, Raju V S Rajala, Alexander J Smith, Robin R Ali, Steven F Abcouwer, Thomas W Gardner.
      Mechanistic target of rapamycin (mTOR) and members of mTOR complex 1 (mTORC1), a linchpin of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGC) of rodent and human retinas. However, the role of mTOR complexes in the control of protein synthesis in RGC is unknown. Here we applied the SUnSET method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an AAV2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTOR complexes or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA binding protein with multiple splicing (RBPMS), with little decrease of the total number of cells in the RGC layer, even at 25 weeks after AAV-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.
    Keywords:  eye; gene knockout; mRNA translation; mTOR complex (mTORC); mTORC1; mechanistic target of rapamycin (mTOR); protein synthesis; puromycin; retina; retinal ganglion cell
    DOI:  https://doi.org/10.1016/j.jbc.2022.101944
  6. Autophagy. 2022 Apr 19. 1-3
    FKBP5/FKBP51 on weight watch: central FKBP5 links regulatory WIPI protein networks to autophagy and metabolic control.
    Thomas Bajaj, Alexander S Häusl, Mathias V Schmidt, Nils C Gassen.
      Stress and changes in energy stores are perceived by hormone- and nutrient-sensing nuclei of the hypothalamus, which orchestrate an adaptive physiological body response to maintain homeostasis. Macroautophagy/autophagy is a fundamental lysosomal degradation system contributing to preservation of proteome balance and metabolic homeostasis. Its dysregulation is linked to diverse human pathologies, including neuropsychiatric and metabolic disorders. Autophagy is coordinated by cellular nutrient sensors, including AMPK and MTORC1 that interact with WIPI proteins. Studies suggest that WDR45/WIPI4 interacts with the stress-sensitive co-chaperone FKBP5/FKBP51, which has emerged as a key autophagy scaffold. However, the impact of FKBP5 on autophagy signaling in response to metabolic challenges, such as a high-fat diet, is elusive. Therefore, we manipulated FKBP5 in the mediobasal hypothalamus (MBH) and studied autophagy signaling and protein interactions in their physiological context. We identified FKBP5 as a scaffold of the STK11/LKB1-AMPK complex with WDR45/WIPI4 and TSC2 with WDR45B/WIPI3 in response to metabolic challenges, positioning FKBP5 in major nutrient-sensing and autophagy-regulating networks. Intriguingly, we could demonstrate that FKBP5 deletion in the MBH strongly induces obesity, whereas its overexpression protects against high-fat diet-induced obesity. Our findings suggest a crucial regulatory and adaptive function of FKBP5-regulated autophagy within the MBH in response to metabolic challenges.Abbreviations: AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; BECN1: beclin 1, autophagy related; eWAT: epididymal white adipose tissue; FKBP5/FKBP51: FK506 binding protein 5; KO, knockout; MBH, mediobasal hypothalamus; MTORC1, mechanistic target of rapamycin kinase complex 1; p: phosphorylated; PHLPP: PH domain and leucine rich repeat protein phosphatase; RPS6KB/p70S6K: ribosomal protein S6 kinase; SKP2: S-phase kinase-associated protein 2; SM: soleus muscle; SQSTM1/p62, sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TSC: TSC complex; ULK1: unc-51 like kinase 1; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.
    Keywords:  AMPK; FKBP5/FKBP51; WIPI; autophagy; metabolic stress
    DOI:  https://doi.org/10.1080/15548627.2022.2063006
  7. Metab Brain Dis. 2022 Apr 20.
    Evolving therapies in neuronopathic LSDs: opportunities and challenges.
    Deepa S Rajan, Maria L Escolar.
      Lysosomal storage disorders (LSD) are multisystemic progressive disorders caused by genetic mutations involving lysosomal function. While LSDs are individually considered rare diseases, the overall true prevalence of these disorders is likely higher than our current estimates. More than two third of the LSDs have associated neurodegeneration and the neurological phenotype often defines the course of the disease and treatment outcomes. Addressing the neurological involvement in LSDs has posed a significant challenge in the rapidly evolving field of therapies for these diseases. In this review, we summarize current approaches and clinical trials available for patients with neuronopathic lysosomal storage disorders, exploring the opportunities and challenges that have emerged with each of these.
    Keywords:  Enzyme replacement therapy; Gene therapy; Hematopoietic stem cell therapy; Lysosomal storage disorders (LSDs); Neurodegeneration; Substrate reduction therapy
    DOI:  https://doi.org/10.1007/s11011-022-00939-0
  8. Front Cell Dev Biol. 2022 ;10 854397
    SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.
    Rekha Tripathi, Tanya Aggarwal, Frida A Lindberg, Anna H Klemm, Robert Fredriksson.
      Glutamate acts as a critical regulator of neurotransmitter balance, recycling, synaptic function and homeostasis in the brain and glutamate transporters control glutamate levels in the brain. SLC38A10 is a member of the SLC38 family and regulates protein synthesis and cellular stress responses. Here, we uncover the role of SLC38A10 as a transceptor involved in glutamate-sensing signaling pathways that control both the glutamate homeostasis and mTOR-signaling. The culture of primary cortex cells from SLC38A10 knockout mice had increased intracellular glutamate. In addition, under nutrient starvation, KO cells had an impaired response in amino acid-dependent mTORC1 signaling. Combined studies from transcriptomics, protein arrays and metabolomics established that SLC38A10 is involved in mTOR signaling and that SLC38A10 deficient primary cortex cells have increased protein synthesis. Metabolomic data showed decreased cholesterol levels, changed fatty acid synthesis, and altered levels of fumaric acid, citrate, 2-oxoglutarate and succinate in the TCA cycle. These data suggests that SLC38A10 may act as a modulator of glutamate homeostasis, and mTOR-sensing and loss of this transceptor result in lower cholesterol, which could have implications in neurodegenerative diseases.
    Keywords:  SLC38A10; mTOR; primary cortex cultures; solute carriers transporter; starvation
    DOI:  https://doi.org/10.3389/fcell.2022.854397
  9. Apoptosis. 2022 Apr 18.
    mTOR deletion ameliorates CD4 + T cell apoptosis during sepsis by improving autophagosome-lysosome fusion.
    Hao Wang, Guangxu Bai, Jianwei Chen, Wen Han, Ran Guo, Na Cui.
      Autophagy dysfunction contributes to CD4 + T cell apoptosis during sepsis leading to impairment of adaptive immunity. However, the underlying mechanism is unclear. The mammalian target of rapamycin (mTOR) pathway modulates CD4 + T cell survival during sepsis through mechanisms that are not fully understood. We developed a mouse model of sepsis through cecal ligation and puncture (CLP) to investigate dynamic changes in autophagy in CD4 + T cells. We used T cell specific-mTOR/tuberous sclerosis complex 1 (TSC1)-knockout mice to explore the roles of the mTOR pathway in modulating autophagy during sepsis. We observed reduced fusion of autophagosomes with lysosomes in the CD4 + T cells of CLP mice, which may represent a characteristic feature of autophagy dysfunction. Deletion of mTOR relieved autophagosome-lysosome fusion dysfunction and ameliorated apoptosis of CD4 + T cells in CLP mice, but this rescued phenotype was abolished by treatment with bafilomycin A1, a specific A-L fusion inhibitor. We further explored the underlying molecular mechanism and found that phosphorylation levels of transcription factor EB were significant higher in CLP mice and that expression of A-L fusion protein SNAREs were restricted, both of which were ameliorated by mTOR deletion. Taken together, these results suggest that the mTOR pathway plays a critical role in regulation of CD4 + T-cell apoptosis during sepsis, partly through regulation of A-L fusion-related protein transcription.
    Keywords:  Autophagosome-lysosome fusion; Sepsis; mTOR
    DOI:  https://doi.org/10.1007/s10495-022-01719-y
  10. Cells. 2022 Apr 08. pii: 1261. [Epub ahead of print]11(8):
    GBA Variants and Parkinson Disease: Mechanisms and Treatments.
    Laura Smith, Anthony H V Schapira.
      The GBA gene encodes for the lysosomal enzyme glucocerebrosidase (GCase), which maintains glycosphingolipid homeostasis. Approximately 5-15% of PD patients have mutations in the GBA gene, making it numerically the most important genetic risk factor for Parkinson disease (PD). Clinically, GBA-associated PD is identical to sporadic PD, aside from the earlier age at onset (AAO), more frequent cognitive impairment and more rapid progression. Mutations in GBA can be associated with loss- and gain-of-function mechanisms. A key hallmark of PD is the presence of intraneuronal proteinaceous inclusions named Lewy bodies, which are made up primarily of alpha-synuclein. Mutations in the GBA gene may lead to loss of GCase activity and lysosomal dysfunction, which may impair alpha-synuclein metabolism. Models of GCase deficiency demonstrate dysfunction of the autophagic-lysosomal pathway and subsequent accumulation of alpha-synuclein. This dysfunction can also lead to aberrant lipid metabolism, including the accumulation of glycosphingolipids, glucosylceramide and glucosylsphingosine. Certain mutations cause GCase to be misfolded and retained in the endoplasmic reticulum (ER), activating stress responses including the unfolded protein response (UPR), which may contribute to neurodegeneration. In addition to these mechanisms, a GCase deficiency has also been associated with mitochondrial dysfunction and neuroinflammation, which have been implicated in the pathogenesis of PD. This review discusses the pathways associated with GBA-PD and highlights potential treatments which may act to target GCase and prevent neurodegeneration.
    Keywords:  GBA; Parkinson disease; alpha-synuclein; autophagy; lipids; unfolded protein response
    DOI:  https://doi.org/10.3390/cells11081261
  11. Genes (Basel). 2022 Mar 26. pii: 593. [Epub ahead of print]13(4):
    Complex Changes in the Efficiency of the Expression of Many Genes in Monogenic Diseases, Mucopolysaccharidoses, May Arise from Significant Disturbances in the Levels of Factors Involved in the Gene Expression Regulation Processes.
    Zuzanna Cyske, Lidia Gaffke, Karolina Pierzynowska, Grzegorz Węgrzyn.
      Monogenic diseases are primarily caused by mutations in a single gene; thus, they are commonly recognized as genetic disorders with the simplest mechanisms. However, recent studies have indicated that the molecular mechanisms of monogenic diseases can be unexpectedly complicated, and their understanding requires complex studies at the molecular level. Previously, we have demonstrated that in mucopolysaccharidoses (MPS), a group of monogenic lysosomal storage diseases, several hundreds of genes reveal significant changes in the expression of various genes. Although the secondary effects of the primary biochemical defect and the inefficient degradation of glycosaminoglycans (GAGs) might be considered, the scale of the changes in the expression of a large fraction of genes cannot be explained by a block in one biochemical pathway. Here, we demonstrate that in cellular models of 11 types of MPS, the expression of genes coding for proteins involved in the regulation of the expression of many other genes at various stages (such as signal transduction, transcription, splicing, RNA degradation, translation, and others) is significantly disturbed relative to the control cells. This conclusion was based on transcriptomic studies, supported by biochemical analyses of levels of selected proteins encoded by genes revealing an especially high level of dysregulation in MPS (EXOSC9, SRSF10, RPL23, and NOTCH3 proteins were investigated). Interestingly, the reduction in GAGs levels, through the inhibition of their synthesis normalized the amounts of EXOSC9, RPL23, and NOTCH3 in some (but not all) MPS types, while the levels of SRSF10 could not be corrected in this way. These results indicate that different mechanisms are involved in the dysregulation of the expression of various genes in MPS, pointing to a potential explanation for the inability of some therapies (such as enzyme replacement therapy or substrate reduction therapy) to fully correct the physiology of MPS patients. We suggest that the disturbed expression of some genes, which appears as secondary or tertiary effects of GAG storage, might not be reversible, even after a reduction in the amounts of the storage material.
    Keywords:  gene expression; glycosaminoglycans; mucopolysaccharidosis; transcriptomics
    DOI:  https://doi.org/10.3390/genes13040593
  12. Curr Opin Cell Biol. 2022 Apr 13. pii: S0955-0674(22)00025-4. [Epub ahead of print]76 102079
    New directions for the clathrin adaptor AP-1 in cell biology and human disease.
    Mara C Duncan.
      The clathrin adaptor protein complex-1 (AP-1) is a central player in cell physiology and human health. It is best known for its role in linking clathrin to its cargo at the trans-Golgi network and endosomes. It participates in traffic important for the correct function of a large number of organelles, including the trans-Golgi network, endosomes, lysosomes, lysosome-related organelles, and plasma membrane. Although it was one of the first clathrin adaptors identified, new discoveries about cargo and pathways that depend on AP-1 continue to emerge. This review summarizes new research into AP-1 that further illuminates its roles in the traffic of plasma membrane proteins, in maintaining TGN content, and in human disease.
    DOI:  https://doi.org/10.1016/j.ceb.2022.102079
  13. Antioxidants (Basel). 2022 Mar 30. pii: 678. [Epub ahead of print]11(4):
    Heparan Sulfate, Mucopolysaccharidosis IIIB and Sulfur Metabolism Disorders.
    Marta Kaczor-Kamińska, Kamil Kamiński, Maria Wróbel.
      Mucopolysaccharidosis, type IIIB (MPS IIIB) is a rare disease caused by mutations in the N-alpha-acetylglucosaminidase (NAGLU) gene resulting in decreased or absent enzyme activity. On the cellular level, the disorder is characterized by the massive lysosomal storage of heparan sulfate (HS)-one species of glycosaminoglycans. HS is a sulfur-rich macromolecule, and its accumulation should affect the turnover of total sulfur in cells; according to the studies presented here, it, indeed, does. The lysosomal degradation of HS in cells produces monosaccharides and inorganic sulfate (SO42-). Sulfate is a product of L-cysteine metabolism, and any disruption of its levels affects the entire L-cysteine catabolism pathway, which was first reported in 2019. It is known that L-cysteine level is elevated in cells with the Naglu-/- gene mutation and in selected tissues of individuals with MPS IIIB. The level of glutathione and the Naglu-/- cells' antioxidant potential are significantly reduced, as well as the activity of 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) and the level of sulfane sulfur-containing compounds. The direct reason is not yet known. This paper attempts to identify some of cause-and-effect correlations that may lead to this condition and identifies research directions that should be explored.
    Keywords:  3-mercaptopyruvate sulfurtransferase; Sanfilippo B syndrome; cysteine; glycosaminoglycans; heparin; sulfane sulfur; sulfate; sulfurtransferases
    DOI:  https://doi.org/10.3390/antiox11040678
  14. Mol Ther Methods Clin Dev. 2022 Jun 09. 25 170-189
    Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis.
    Davide Sala, Francesca Ornaghi, Francesco Morena, Chiara Argentati, Manuela Valsecchi, Valeria Alberizzi, Roberta Di Guardo, Alessandra Bolino, Massimo Aureli, Sabata Martino, Angela Gritti.
      Genetic deficiency of β-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.
    Keywords:  CNS; GM2 gangliosidosis; Sandhoff disease; bone marrow transplantation; cell therapy; gene therapy; hexosaminidase; lentiviral vectors; lysosomal storage disorders
    DOI:  https://doi.org/10.1016/j.omtm.2022.03.011
  15. Nat Commun. 2022 Apr 19. 13(1): 2025
    Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.
    Daniel J Ham, Anastasiya Börsch, Kathrin Chojnowska, Shuo Lin, Aurel B Leuchtman, Alexander S Ham, Marco Thürkauf, Julien Delezie, Regula Furrer, Dominik Burri, Michael Sinnreich, Christoph Handschin, Lionel A Tintignac, Mihaela Zavolan, Nitish Mittal, Markus A Rüegg.
      Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.
    DOI:  https://doi.org/10.1038/s41467-022-29714-6
  16. Autophagy. 2022 Apr 18. 1-9
    Acetylation in the regulation of autophagy.
    Yinfeng Xu, Wei Wan.
      Post-translational modifications, such as phosphorylation, ubiquitination and acetylation, play crucial roles in the regulation of autophagy. Acetylation has emerged as an important regulatory mechanism for autophagy. Acetylation regulates autophagy initiation and autophagosome formation by targeting core components of the ULK1 complex, the BECN1-PIK3C3 complex, and the LC3 lipidation system. Recent studies have shown that acetylation occurs on the key proteins participating in autophagic cargo assembly and autophagosome-lysosome fusion, such as SQSTM1/p62 and STX17. In addition, acetylation controls autophagy at the transcriptional level by targeting histones and the transcription factor TFEB. Here, we review the current knowledge on acetylation of autophagy proteins and their regulations and functions in the autophagy pathway with focus on recent findings.Abbreviations : ACAT1: acetyl-CoA acetyltransferase 1; ACSS2: acyl-CoA synthetase short chain family member 2; AMPK: AMP-activated protein kinase; ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCAR2/DBC1: cell cycle and apoptosis regulator 2; BECN1: beclin 1; CMA: chaperone-mediated autophagy; CREBBP/CBP: CREB binding protein; EP300/p300: E1A binding protein p300; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3: glycogen synthase kinase 3; HDAC6: histone deacetylase 6; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PD: Parkinson disease; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKM2: pyruvate kinase M1/2; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: post-translational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RUBCN/Rubicon: rubicon autophagy regulator; RUBCNL/Pacer: rubicon like autophagy enhancer; SIRT1: sirtuin 1; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TFEB: transcription factor EB; TP53/p53: tumor protein p53; TP53INP2/DOR: tumor protein p53 inducible nuclear protein 2; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WIPI2: WD repeat domain, phosphoinositide interacting 2.
    Keywords:  Acetylation; acetyltransferase; autophagy; deacetylase; post-translational modification
    DOI:  https://doi.org/10.1080/15548627.2022.2062112
  17. Biomolecules. 2022 Mar 31. pii: 533. [Epub ahead of print]12(4):
    TORC1 Signaling Controls the Stability and Function of α-Arrestins Aly1 and Aly2.
    Ray W Bowman, Eric M Jordahl, Sydnie Davis, Stefanie Hedayati, Hannah Barsouk, Nejla Ozbaki-Yagan, Annette Chiang, Yang Li, Allyson F O'Donnell.
      Nutrient supply dictates cell signaling changes, which in turn regulate membrane protein trafficking. To better exploit nutrients, cells relocalize membrane transporters via selective protein trafficking. Key in this reshuffling are the α-arrestins, selective protein trafficking adaptors conserved from yeast to man. α-Arrestins bind membrane proteins, controlling the ubiquitination and endocytosis of many transporters. To prevent the spurious removal of membrane proteins, α-arrestin-mediated endocytosis is kept in check through phospho-inhibition. This phospho-regulation is complex, with up to 87 phospho-sites on a single α-arrestin and many kinases/phosphatases targeting α-arrestins. To better define the signaling pathways controlling paralogous α-arrestins, Aly1 and Aly2, we screened the kinase and phosphatase deletion (KinDel) library, which is an array of all non-essential kinase and phosphatase yeast deletion strains, for modifiers of Aly-mediated phenotypes. We identified many Aly regulators, but focused our studies on the TORC1 kinase, a master regulator of nutrient signaling across eukaryotes. We found that TORC1 and its signaling effectors, the Sit4 protein phosphatase and Npr1 kinase, regulate the phosphorylation and stability of Alys. When Sit4 is lost, Alys are hyperphosphorylated and destabilized in an Npr1-dependent manner. These findings add new dimensions to our understanding of TORC1 regulation of α-arrestins and have important ramifications for cellular metabolism.
    Keywords:  Saccharomyces cerevisiae; fluorescence microscopy; kinase; phosphatase; protein degradation; protein trafficking; ubiquitin; vacuole proteases; α-arrestin
    DOI:  https://doi.org/10.3390/biom12040533
  18. Front Neurol. 2022 ;13 886567
    Glial Dysfunction and Its Contribution to the Pathogenesis of the Neuronal Ceroid Lipofuscinoses.
    Keigo Takahashi, Hemanth R Nelvagal, Jenny Lange, Jonathan D Cooper.
      While significant efforts have been made in developing pre-clinical treatments for the neuronal ceroid lipofuscinoses (NCLs), many challenges still remain to bring children with NCLs a cure. Devising effective therapeutic strategies for the NCLs will require a better understanding of pathophysiology, but little is known about the mechanisms by which loss of lysosomal proteins causes such devastating neurodegeneration. Research into glial cells including astrocytes, microglia, and oligodendrocytes have revealed many of their critical functions in brain homeostasis and potential contributions to neurodegenerative diseases. Genetically modified mouse models have served as a useful platform to define the disease progression in the central nervous system across NCL subtypes, revealing a wide range of glial responses to disease. The emerging evidence of glial dysfunction questions the traditional "neuron-centric" view of NCLs, and would suggest that directly targeting glia in addition to neurons could lead to better therapeutic outcomes. This review summarizes the most up-to-date understanding of glial pathologies and their contribution to the pathogenesis of NCLs, and highlights some of the associated challenges that require further research.
    Keywords:  Batten disease; astrocyte; microglia; neuronal ceroid lipofuscinosis; oligodendrocyte
    DOI:  https://doi.org/10.3389/fneur.2022.886567
  19. Biol Open. 2022 Apr 22. pii: bio.059052. [Epub ahead of print]
    Phenotype assessment for neurodegenerative murine models with ataxia and application to Niemann-Pick disease, type C1.
    Julia Yerger, Antony C Cougnoux, Craig B Abbott, Rachel Luke, Tannia S Clark, Niamh X Cawley, Forbes D Porter, Cristin D Davidson.
      Identifying meaningful predictors of therapeutic efficacy from preclinical studies is challenging. However, clinical manifestations occurring in both patients and mammalian models offer significant translational value. Many neurological disorders, including inherited, metabolic Niemann-Pick disease, type C (NPC), exhibit ataxia. Both individuals with NPC and murine models manifest ataxia, and investigational therapies impacting this phenotype in mice have been reported to slow disease progression in patients (e.g. miglustat, intrathecal 2-hydroxypropyl-beta-cyclodextrin, and acetyl-L-leucine). Reproducible phenotypic scoring of animal models can facilitate comparisons between genotypes, sexes, disease course, and therapies. Previously, other groups developed a Composite Phenotypic Scoring System (CPSS) which was subsequently used to distinguish strain dependent phenotypes and, with modifications, to evaluate potential therapies. However, high inter-rater reliability is paramount to widespread use. We have created a comprehensive, easy to follow phenotypic assessment based on the CPSS and have verified its reproducibility using murine models of NPC disease. Application of this scoring system is not limited to NPC disease and may be applicable to other models of neurodegeneration exhibiting motor incoordination, thereby increasing its utility in translational studies.
    Keywords:  Cerebellar ataxia; Lysosomal disease; NPC1; Neurological disease; Niemann-Pick disease type C; Phenotype assessment
    DOI:  https://doi.org/10.1242/bio.059052
  20. Nutrients. 2022 Apr 12. pii: 1612. [Epub ahead of print]14(8):
    Exploratory Analysis of Selected Components of the mTOR Pathway Reveals Potentially Crucial Associations with Childhood Malnutrition.
    Parag Palit, Md Amran Gazi, Subhasish Das, Md Mehedi Hasan, Zannatun Noor, Jafrin Ferdous, Md Ashraful Alam, Sharika Nuzhat, Md Ridwan Islam, Mustafa Mahfuz, Rashidul Haque, Tahmeed Ahmed.
      Dysregulations in the mammalian target of rapamycin (mTOR) pathway are associated with several human anomalies. We aimed to elucidate possible implications for potential aberrations in the mTOR pathway with childhood malnutrition. We analyzed the activity of phospho-mTORC1 and the expressions of several mTOR pathway genes, namely: MTOR, TSC1, LAMTOR2, RPS6K1 and RICTOR from peripheral blood mononuclear cells isolated from venous blood of children suffering from different forms of malnutrition and compared them with those from healthy children. Significant reduction in the phosphorylation of mTORC1 was noted, as well as a decrease in expression of LAMTOR2 gene and increase in TSC1 gene expression were observed between malnourished children in comparison to the healthy children. The deregulation in the activity of the TSC1 and LAMTOR2 gene was significantly associated with all forms of childhood malnutrition. Our findings provide key insights into possible down-modulation in the overall activity of the mTOR pathway in childhood malnutrition. Further studies focusing on the analysis of a multitude of components involved in the mTOR pathway both at the gene and protein expression levels are required for conclusive evidence for the aforementioned proposition.
    Keywords:  childhood malnutrition; gene expression; mTOR pathway
    DOI:  https://doi.org/10.3390/nu14081612
  21. J Cell Biol. 2022 Jun 06. pii: e202110151. [Epub ahead of print]221(6):
    Secretory autophagy maintains proteostasis upon lysosome inhibition.
    Tina A Solvik, Tan A Nguyen, Yu-Hsiu Tony Lin, Timothy Marsh, Eric J Huang, Arun P Wiita, Jayanta Debnath, Andrew M Leidal.
      The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.
    DOI:  https://doi.org/10.1083/jcb.202110151
  22. Gene. 2022 Apr 18. pii: S0378-1119(22)00332-8. [Epub ahead of print] 146513
    Lysosomal Storage Disease Associated with a CNP Sequence Variant in Dalmatian Dogs.
    Garrett Bullock, Gary S Johnson, Tendai Mhlanga-Mutangadura, Scott C Petesch, Samantha Thompson, Sandra Goebbels, Martin L Katz.
      A progressive neurological disorder was identified in purebred Dalmatian dogs. The disease is characterized by anxiety, pacing and circling, hypersensitivity, cognitive decline, sleep disturbance, loss of coordination, loss of control over urination and defecation, and visual impairment. Neurological signs first became apparent when the dogs were approximately 18 months of age and progressed slowly. Two affected littermates were euthanized at approximately 7 years, 5 months and 8 years, 2 months of age due to the severity of neurological impairment. The mother of the affected dogs and four other relatives exhibited milder, later-onset neurological signs. Pronounced accumulations of autofluorescent intracellular inclusions were found in cerebral cortex, cerebellum, optic nerve, and cardiac muscle of the affected dogs. These inclusions co-localized with immunolabeling of the lysosomal marker protein LAMP2, indicating that the dogs suffered from a lysosomal storage disease. Ultrastructural analysis indicated that the storage bodies were surrounded by a single-layer membrane, but the storage granules were distinct from those reported for other lysosomal storage diseases. Whole genome sequences, generated with DNA from the two euthanized Dalmatians, both contained a rare, homozygous single-base deletion and reading-frame shift in CNP which encodes the enzyme CNPase (EC 3.1.4.37). The late-onset disease was exhibited by five of seven related Dalmatians that were heterozygous for the deletion allele and over 8 years of age, whereas none of the 16 age-matched reference-allele homozygotes developed neurologic signs. No CNPase antigen could be detected with immunohistochemical labeling in tissues from the dogs with the earlier-onset disorder. Similar to the latter Dalmatians, autofluorescent storage granules were apparent in brain and cardiac tissue from transgenic mice that were nullizygous for Cnp. Based on the clinical signs, the histopathological, immunohistochemical, ultrastructural, and molecular-genetic findings, and the finding that nullizygous Cnp mice accumulate autofluorescent storage granules, we propose that the earlier-onset Dalmatian disorder is a novel lysosomal storage disease that results from a loss-of-function mutation in CNP and that shares features characteristic of the neuronal ceroid lipofuscinoses. That the later-onset disorder occurred only in dogs heterozygous for the CNP deletion variant suggests that this disorder is a result of the variant allele's presence.
    DOI:  https://doi.org/10.1016/j.gene.2022.146513
  23. Mol Psychiatry. 2022 Apr 21.
    Cylindromatosis drives synapse pruning and weakening by promoting macroautophagy through Akt-mTOR signaling.
    Alexis S Zajicek, Hongyu Ruan, Huihui Dai, Mary C Skolfield, Hannah L Phillips, Wendi J Burnette, Behnam Javidfar, Shao-Cong Sun, Schahram Akbarian, Wei-Dong Yao.
      The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation, and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux, and conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.
    DOI:  https://doi.org/10.1038/s41380-022-01571-1
  24. Discov Oncol. 2022 Apr 18. 13(1): 26
    T cell differentiation protein 2 facilitates cell proliferation by enhancing mTOR-mediated ribosome biogenesis in non-small cell lung cancer.
    Zhenying Lian, Xingyu Yan, Yutao Diao, Dayong Cui, Hongyan Liu.
      Dysregulation of T cell differentiation protein 2 (MAL2) has been observed in multiple cancers, but its exact role in lung cancer is poorly understood. Here we report a role of MAL2 in accelerating cell proliferation in non-small cell lung cancer (NSCLC). MAL2 expression enhances cell proliferation in both cell and nude mouse models. Mechanistically, overexpression of MAL2 results in the hyper-activation of the MAPK/mTOR signaling pathway in NSCLC cells which leads to active ribosome biogenesis. Importantly, pharmacological inhibition of mTOR or MEK lowered the abundance of PCNA, a marker of tumor cell proliferation, and subsequently suppressed ribosome biogenesis, cell growth and xenograft growth in mouse model. MAL2 upregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal that MAL2 is a potential diagnostic biomarker and targeting the MAL2/MAPK/mTOR signaling pathway may improve therapeutic strategy and efficacy for this subset of NSCLC patients.
    Keywords:  Cell proliferation; Phosphoproteomics; Ribosome biogenesis; T cell differentiation protein 2; mTOR targeted-therapy
    DOI:  https://doi.org/10.1007/s12672-022-00488-z
  25. Mol Cell. 2022 Apr 21. pii: S1097-2765(22)00261-1. [Epub ahead of print]82(8): 1514-1527
    Autophagy in cancer cell remodeling and quality control.
    Grace A Hernandez, Rushika M Perera.
      As one of the two highly conserved cellular degradation systems, autophagy plays a critical role in regulation of protein, lipid, and organelle quality control and cellular homeostasis. This evolutionarily conserved pathway singles out intracellular substrates for elimination via encapsulation within a double-membrane vesicle and delivery to the lysosome for degradation. Multiple cancers disrupt normal regulation of autophagy and hijack its degradative ability to remodel their proteome, reprogram their metabolism, and adapt to environmental challenges, making the autophagy-lysosome system a prime target for anti-cancer interventions. Here, we discuss the roles of autophagy in tumor progression, including cancer-specific mechanisms of autophagy regulation and the contribution of tumor and host autophagy in metabolic regulation, immune evasion, and malignancy. We further discuss emerging proteomics-based approaches for systematic profiling of autophagosome-lysosome composition and contents. Together, these approaches are uncovering new features and functions of autophagy, leading to more effective strategies for targeting this pathway in cancer.
    Keywords:  autophagy; cancer; lysosome; quality control; remodeling
    DOI:  https://doi.org/10.1016/j.molcel.2022.03.023
  26. Mol Cell. 2022 Apr 21. pii: S1097-2765(22)00253-2. [Epub ahead of print]82(8): 1604-1604.e1
    SnapShot: Organelle degradation.
    Noboru Mizushima.
      Organelles are continuously turned over as part of cellular homeostasis and adaptation. Most organelles, even including the nucleus, are degraded by lysosomes via different pathways, such as macroautophagy, microautophagy, organelle-derived vesicle degradation, and crinophagy. In some specific cases-for example, in lens fiber cells-organelles are degraded by cytosolic phospholipases. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.molcel.2022.03.015
  27. J Cell Mol Med. 2022 Apr 19.
    FGF21 alleviates pulmonary hypertension by inhibiting mTORC1/EIF4EBP1 pathway via H19.
    Xiuchun Li, Yaxin Zhang, Lihuang Su, Luqiong Cai, Chi Zhang, Jianhao Zhang, Junwei Sun, Mengyu Chai, Mengsi Cai, Qian Wu, Chi Zhang, Xiaoqing Yan, Liangxing Wang, Xiaoying Huang.
      Long non-coding RNAs (lncRNAs) play a significant role in pulmonary hypertension (PH). Our preliminary data showed that hypoxia-induced PH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the regulatory role of long non-coding RNAs in PH treated with FGF21. RNA sequencing analysis and real-time PCR identified a significantly up-regulation of the H19 after FGF21 administration. Moreover, gain- and loss-of-function assays demonstrated that FGF21 suppressed hypoxia-induced proliferation of pulmonary artery smooth muscle cells partially through upregulation of H19. In addition, FGF21 deficiency markedly exacerbated hypoxia-induced increases of pulmonary artery pressure and pulmonary vascular remodelling. In addition, AAV-mediated H19 overexpression reversed the malignant phenotype of FGF21 knockout mice under hypoxia expose. Further investigation uncovered that H19 also acted as an orchestra conductor that inhibited the function of mechanistic target of rapamycin complex 1 (mTORC1) by disrupting the interaction of mTORC1 with eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1). Our work highlights the important role of H19 in PH treated with FGF21 and suggests a mechanism involving mTORC1/EIF4EBP1 inhibition, which may provide a fundamental for clinical application of FGF21 in PH.
    Keywords:  fibroblast growth factor 21; long non-coding RNAs; molecular mechanism; pulmonary hypertension; pulmonary vascular diseases
    DOI:  https://doi.org/10.1111/jcmm.17318
  28. Epilepsy Behav. 2022 Apr 13. pii: S1525-5050(22)00127-5. [Epub ahead of print]130 108678
    DEPDC5-related epilepsy: A comprehensive review.
    Debopam Samanta.
      DEPDC5-related epilepsy, caused by pathogenic germline variants(with or without additional somatic variants in the brain) of DEPDC5 (Dishevelled, Egl-10 and Pleckstrin domain-containing protein 5) gene, is a newly discovered predominantly focal epilepsy linked to enhanced mTORC1 pathway. DEPDC5-related epilepsy includes several familial epilepsy syndromes, including familial focal epilepsy with variable foci (FFEVF) and rare sporadic nonlesional focal epilepsy. DEPDC5 has been identified as one of the more common epilepsy genes linked to infantile spasms and sudden unexpected death (SUDEP). Although intelligence usually is unaffected in DEPDC5-related epilepsy, some people have been diagnosed with intellectual disabilities, autism spectrum disorder, and other psychiatric problems. DEPDC5 variants have also been found in 20% of individuals with various brain abnormalities, challenging the traditional distinction between lesional and nonlesional epilepsies. The most exciting development of DEPDC5 variants is the possibility of precision therapeutics using mTOR inhibitors, as evidenced with phenotypic rescue in many animal models. However, more research is needed to better understand the functional impact of diverse (particularly missense or splice-region) variants, the specific involvement of DEPDC5 in epileptogenesis, and the creation and utilization of precision therapies in humans. Precision treatments for DEPDC5-related epilepsy will benefit not only a small number of people with the condition, but they will also pave the way for new therapeutic approaches in epilepsy (including acquired epilepsies in which mTORC1 activation occurs, for example, post-traumatic epilepsy) and other neurological disorders involving a dysfunctional mTOR pathway.
    Keywords:  Children; Everolimus; Gene; Genomic; Mechanistic target of rapamycin; Seizure
    DOI:  https://doi.org/10.1016/j.yebeh.2022.108678
  29. Biochem Pharmacol. 2022 Apr 16. pii: S0006-2952(22)00139-3. [Epub ahead of print] 115045
    Pan-mTOR inhibitors sensitize the senolytic activity of Navitoclax via mTORC2 inhibition-mediated apoptotic signaling.
    Weitong Xu, Tingting Zhao, Honghan Chen, Ning Huang, Hui Gong, Jian Zhang, Yu Yang, Tiepeng Li, Gongchang Zhang, Chuhui Gong, Ming Yang, Hengyi Xiao.
      Compounds with senolysis activity are discovered in recent years, featuring by their capacity to specifically eliminate senescent cells in vitro or in vivo. These compounds, referring to as Senolytics, provide a new method for aging counteraction and probably for geriatric disease amelioration. However, their clinical application is unpractical still, mainly because of the safety issue. In fact, the effective dose range even of the most potent senolytic cannot guarantee the safety requirements application for human being. Here, we report a study which investigated the combinational application of one potential senolytic molecule navitoclax, a Bcl-2 inhibitor with several mTOR inhibitors, to assess the influence of this combination on the senolytic outcome. Our results reveal that pan-mTOR inhibitors can reduce the dosage or timespan of navitoclax necessary for reaching IC50 and LT50 in senescent cells, also extend the lifespan of premature-aged Drosophila and mitigate the aging-related phenotype. Our results also confirmed that mTOR inhibitor sensitized senolytic cell death is apoptotic and pan-mTOR inhibitors PP242 and AZD8055 works more effectively than mTORC1 inhibitor Rapamycin. Mechanically, we verified the crucial role of mTORC2 inhibition contributes sensitization by increasing the expression of the pro-apoptotic protein Bim. In summary, this study firstly exposes the sensitization effect of pan-mTOR inhibitors on navitoclax-induced senolytic apoptosis, therefore providing novel evidence to show the advantage of drug combination on setting senotherapy. It also provides an intriguing clue to demonstrate the value of mTORC2 inhibition for apoptotic death of senescent cells.
    Keywords:  apoptosis; mTORC2; navitoclax; senolytic
    DOI:  https://doi.org/10.1016/j.bcp.2022.115045
  30. Cell Rep. 2022 Apr 19. pii: S2211-1247(22)00473-9. [Epub ahead of print]39(3): 110712
    Cholesterol suppresses GOLM1-dependent selective autophagy of RTKs in hepatocellular carcinoma.
    Wei-Qing Shao, Wen-Wei Zhu, Meng-Jun Luo, Ming-Hao Fan, Qin Li, Sheng-Hao Wang, Zhi-Fei Lin, Jing Zhao, Yan Zheng, Qiong-Zhu Dong, Lu Lu, Hu-Liang Jia, Ju-Bo Zhang, Ming Lu, Jin-Hong Chen, Lun-Xiu Qin.
      Aberrant activation of receptor tyrosine kinases (RTKs) and the subsequent metabolic reprogramming play critical roles in cancer progression. Our previous study has shown that Golgi membrane protein 1 (GOLM1) promotes hepatocellular carcinoma (HCC) metastasis by enhancing the recycling of RTKs. However, how this RTK recycling process is regulated and coupled with RTK degradation remains poorly defined. Here, we demonstrate that cholesterol suppresses the autophagic degradation of RTKs in a GOLM1-dependent manner. Further mechanistic studies reveal that GOLM1 mediates the selective autophagy of RTKs by interacting with LC3 through an LC3-interacting region (LIR), which is regulated by a cholesterol-mTORC1 axis. Lowering cholesterol by statins improves the efficacy of multiple tyrosine kinase inhibitors (TKIs) in vivo. Our findings indicate that cholesterol serves as a signal to switch GOLM1-RTK degradation to GOLM1-RTK recycling and suggest that lowering cholesterol by statin may be a promising combination strategy to improve the TKI efficiency in HCC.
    Keywords:  CP: Cancer; CP: Cell biology; cholesterol metabolism; liver cancer; lysosomal degradation; statin; tyrosine kinase inhibitor
    DOI:  https://doi.org/10.1016/j.celrep.2022.110712
  31. Cells. 2022 Apr 09. pii: 1272. [Epub ahead of print]11(8):
    Lysosomes Dysfunction Causes Mitophagy Impairment in PBMCs of Sporadic ALS Patients.
    Matteo Bordoni, Orietta Pansarasa, Eveljn Scarian, Riccardo Cristofani, Roberta Leone, Valentina Fantini, Maria Garofalo, Luca Diamanti, Stefano Bernuzzi, Stella Gagliardi, Stephana Carelli, Angelo Poletti, Cristina Cereda.
      Mitochondria alterations are present in tissues derived from patients and animal models, but no data are available for peripheral blood mononuclear cells (PBMCs) of ALS patients. This work aims to investigate mitophagy in PBMCs of sporadic (sALS) patients and how this pathway can be tuned by using small molecules. We found the presence of morphologically atypical mitochondria by TEM and morphological abnormalities by MitoTracker™. We found a decreased number of healthy mitochondria in sALS PBMCs and an impairment of mitophagy with western blot and immunofluorescence. After rapamycin treatment, we found a higher increase in the LC3 marker in sALS PBMCs, while after NH4Cl treatment, we found a lower increase in the LC3 marker. Finally, mTOR-independent autophagy induction with trehalose resulted in a significant decrease in the lysosomes level sALS PBMCs. Our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients rely on the impairment of the mitophagy pathway. We also found that the induction of the mTOR-independent autophagy pathway leads to a decrease in lysosomes level, suggesting a more sensitivity of sALS PBMCs to trehalose. Such evidence suggests that trehalose could represent an effective treatment for ALS patients.
    Keywords:  ALS; PBMCs; autolysosomes; mTOR; mitochondria; trehalose
    DOI:  https://doi.org/10.3390/cells11081272
  32. J Biol Chem. 2022 Apr 13. pii: S0021-9258(22)00356-8. [Epub ahead of print] 101916
    Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.
    Natasha S Clayton, Millie Fox, Jose J Vicenté-Garcia, Courtney M Schroeder, Trevor D Littlewood, Jonathon I Wilde, Kadalmani Krishnan, Murray J B Brown, Claire Crafter, Helen R Mott, Darerca Owen.
      Activated Cdc42-associated kinase (ACK) is an oncogenic non-receptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85β, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85β promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85, and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
    Keywords:  Activated Cdc42 kinase; Cdc42; PI3Kinase; cancer; nuclear signalling; p110-independent p85; p85 dimers; protein degradation; protein phosphorylation; tyrosine kinase
    DOI:  https://doi.org/10.1016/j.jbc.2022.101916
  33. Int J Mol Sci. 2022 Apr 11. pii: 4200. [Epub ahead of print]23(8):
    Slc38a9 Deficiency Induces Apoptosis and Metabolic Dysregulation and Leads to Premature Death in Zebrafish.
    Xiya Wu, Jianyang Chen, Chengdong Liu, Xuan Wang, Huihui Zhou, Kangsen Mai, Gen He.
      Eukaryotic cells control nutritional homeostasis and determine cell metabolic fate through a series of nutrient transporters and metabolic regulation pathways. Lysosomal localized amino acid transporter member 9 of the solute carrier family 38 (SLC38A9) regulates essential amino acids' efflux from lysosomes in an arginine-regulated fashion. To better understand the physiological role of SLC38A9, we first described the spatiotemporal expression pattern of the slc38a9 gene in zebrafish. A quarter of slc38a9-/- mutant embryos developed pericardial edema and died prematurely, while the remaining mutants were viable and grew normally. By profiling the transcriptome of the abnormally developed embryos using RNA-seq, we identified increased apoptosis, dysregulated amino acid metabolism, and glycolysis/gluconeogenesis disorders that occurred in slc38a9-/- mutant fish. slc38a9 deficiency increased whole-body free amino acid and lactate levels but reduced glucose and pyruvate levels. The change of glycolysis-related metabolites in viable slc38a9-/- mutant fish was ameliorated. Moreover, loss of slc38a9 resulted in a significant reduction in hypoxia-inducible gene expression and hypoxia-inducible factor 1-alpha (Hif1α) protein levels. These results improved our understanding of the physiological functions of SLC38A9 and revealed its indispensable role in embryonic development, metabolic regulation, and stress adaption.
    Keywords:  SLC38A9; amino acid homeostasis; apoptosis; glycolysis; hypoxia
    DOI:  https://doi.org/10.3390/ijms23084200
  34. Biomedicines. 2022 Mar 31. pii: 826. [Epub ahead of print]10(4):
    Efficacy and Safety of Topical Mechanistic Target of Rapamycin Inhibitors for Facial Angiofibromas in Patients with Tuberous Sclerosis Complex: A Systematic Review and Network Meta-Analysis.
    Yu-Ting Lin, Chia-Ling Yu, Yu-Kang Tu, Ching-Chi Chi.
      Previous studies have suggested that the topical mechanistic target of rapamycin (mTOR) inhibitors may be effective in treating facial angiofibromas in patients with tuberous sclerosis complex (TSC). Various concentrations of topical sirolimus for TSC have been tested, but their comparative efficacy and safety remained unclear. To assess the effects of topical mTOR inhibitors in treating facial angiofibromas, we conducted a systematic review and network meta-analysis (NMA) and searched MEDLINE, Embase, and Cochrane Library for relevant randomized controlled trials on 14 February 2022. The Cochrane Collaboration tool was used to assess the risk of bias of included trials. Our outcomes were clinical improvement and severe adverse events leading to withdrawal. We included three trials on 261 TSC patients with facial angiofibromas. The NMA found when compared with placebo, facial angiofibromas significantly improved following the application of various concentrations of topical sirolimus (risk ratio being 3.87, 2.70, 4.43, and 3.34 for 0.05%, 0.1%, 0.2%, and 1%, respectively). When compared with placebo, all concentrations of topical sirolimus did not differ in severe adverse events leading to withdrawal. The ranking analysis suggested topical sirolimus 0.2% as the most effective drug. In conclusion, topical sirolimus 0.05-1% are effective and safe in treating facial angiofibromas in patients with TSC, with topical sirolimus 0.2% being the most effective.
    Keywords:  angiofibroma; mechanistic target of rapamycin (mTOR); network meta-analysis; sirolimus; systematic review; tuberous sclerosis complex
    DOI:  https://doi.org/10.3390/biomedicines10040826
  35. Front Mol Biosci. 2022 ;9 863885
    mRNA Translation Is Dynamically Regulated to Instruct Stem Cell Fate.
    Ruoxu Wang, Marc Amoyel.
      Stem cells preserve tissue homeostasis by replacing the cells lost through damage or natural turnover. Thus, stem cells and their daughters can adopt two identities, characterized by different programs of gene expression and metabolic activity. The composition and regulation of these programs have been extensively studied, particularly by identifying transcription factor networks that define cellular identity and the epigenetic changes that underlie the progressive restriction in gene expression potential. However, there is increasing evidence that post-transcriptional mechanisms influence gene expression in stem cells and their progeny, in particular through the control of mRNA translation. Here, we review the described roles of translational regulation in controlling all aspects of stem cell biology, from the decision to enter or exit quiescence to maintaining self-renewal and promoting differentiation. We focus on mechanisms controlling global translation rates in cells, mTOR signaling, eIF2ɑ phosphorylation, and ribosome biogenesis and how they allow stem cells to rapidly change their gene expression in response to tissue needs or environmental changes. These studies emphasize that translation acts as an additional layer of control in regulating gene expression in stem cells and that understanding this regulation is critical to gaining a full understanding of the mechanisms that underlie fate decisions in stem cells.
    Keywords:  differentiation; eIF2 kinase; mTOR; protein synthesis; ribosome biogenesis; self-renewal; stem cell; translation
    DOI:  https://doi.org/10.3389/fmolb.2022.863885
  36. Microsc Microanal. 2022 Apr 19. 1-9
    Morphometric Analysis of Lysosomes in the Renal Tubule in Monoclonal Gammopathy Using Transmission Electron Microscopy: "Mottled Appearance" and Beyond.
    Minsun Jung, Hajeong Lee, Kyung Chul Moon.
      Lysosomal “mottled appearance”, or uneven electron-dense content related to monoclonal gammopathy (MG), has been mainly described in light chain proximal tubulopathy (LCPT). We aimed to determine the ultrastructural characteristics of lysosomal mottled appearance in kidney biopsies and its association with LCPT and MG. Seventy-seven biopsies were grouped into LCPT (n = 5), MG conditions other than LCPT (n = 43), and non-MG conditions (n = 29). The mottled lysosomes in the renal tubules were evaluated using transmission electron microscopy and morphometric analysis. Mottled lysosomes were more prevalent (% of present cases) and frequent (no. of mottled lysosomes/20,000× ultramicroscopic field) in the LCPT group (100% and 8.20 ± 4.15/field) than in the MG (41.9% and 1.13 ± 2.05/field) and non-MG (37.9% and 0.80 ± 1.44/field) groups. In morphometric analysis of all mottled lysosomes (n = 520) detected from the 34 biopsies (5 LCPT, 18 MG, and 11 non-MG), we found that mottled lysosomes were larger, more irregular, and more electron-dense for the LCPT group than for the MG and non-MG groups. Therefore, mottled lysosomes can be present in disorders other than LCPT or even without MG. The morphological characteristics of mottled lysosomes could provide objective guidance for the diagnosis of LCPT.
    Keywords:  light chain proximal tubulopathy; lysosomes; monoclonal gammopathy; morphometry; paraproteinemias; transmission electron microscopy
    DOI:  https://doi.org/10.1017/S1431927622000587
  37. J Biol Chem. 2022 Apr 18. pii: S0021-9258(22)00393-3. [Epub ahead of print] 101953
    Calpain-mediated proteolytic production of free amino acids in vascular endothelial cells augments obesity-induced hepatic steatosis.
    Risako Akasu, Takuro Miyazaki, Mohamed Z Elhussiny, Yuki Sugiura, Yuki Tomitsuka, Shogo Haraguchi, Kinya Otsu, Vishwajit S Chowdhury, Akira Miyazaki.
      Free amino acids that accumulate in the plasma of diabetes and obesity patients influence lipid metabolism and protein synthesis in the liver. The stress-inducible intracellular protease calpain proteolyzes various substrates in vascular endothelial cells (ECs), although its contribution to the supply of free amino acids in the liver microenvironment remains enigmatic. In the present study, we showed that calpains are associated with free amino acid production in cultured ECs. Furthermore, conditioned media derived from calpain-activated ECs facilitated the phosphorylation of ribosomal protein S6 kinase (S6K) and de novo lipogenesis in hepatocytes, which were abolished by the amino acid transporter inhibitor, JPH203, and the mTORC1 inhibitor, rapamycin. Meanwhile, calpain-overexpressing capillary-like ECs were observed in the livers of high-fat diet-fed mice. Conditional knockout of EC/hematopoietic Capns1, which encodes a calpain regulatory subunit, diminished levels of branched chain amino acids in the hepatic microenvironment without altering plasma amino acid levels. Concomitantly, conditional knockout of Capns1 mitigated hepatic steatosis without normalizing body weight and the plasma lipoprotein profile in an amino acid transporter-dependent manner. Mice with targeted Capns1 knockout exhibited reduced phosphorylation of S6K and maturation of lipid homeostasis transcription factor SREBP1 in hepatocytes. Finally, we show that bone marrow transplantation negated the contribution of hematopoietic calpain systems; therefore, calpains are likely responsible for the observed phenotypes of ECs. We conclude that overactivation of calpain systems may be responsible for the production of free amino acid in ECs, which may be sufficient to potentiate S6K/SREBP1-induced lipogenesis in surrounding hepatocytes.
    Keywords:  calpastatin; capillarization; diabetes; inflammation: nonalcoholic fatty liver disease; pathological angiogenesis; protease; sinusoidal liver endothelial cells; steatohepatitis; triglyceride; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2022.101953
  38. Nat Commun. 2022 Apr 21. 13(1): 2174
    Snf7 spirals sense and alter membrane curvature.
    Nebojsa Jukic, Alma P Perrino, Frédéric Humbert, Aurélien Roux, Simon Scheuring.
      Endosomal Sorting Complex Required for Transport III (ESCRT-III) is a conserved protein system involved in many cellular processes resulting in membrane deformation and scission, topologically away from the cytoplasm. However, little is known about the transition of the planar membrane-associated protein assembly into a 3D structure. High-speed atomic force microscopy (HS-AFM) provided insights into assembly, structural dynamics and turnover of Snf7, the major ESCRT-III component, on planar supported lipid bilayers. Here, we develop HS-AFM experiments that remove the constraints of membrane planarity, crowdedness, and support rigidity. On non-planar membranes, Snf7 monomers are curvature insensitive, but Snf7-spirals selectively adapt their conformation to membrane geometry. In a non-crowded system, Snf7-spirals reach a critical radius, and remodel to minimize internal stress. On non-rigid supports, Snf7-spirals compact and buckle, deforming the underlying bilayer. These experiments provide direct evidence that Snf7 is sufficient to mediate topological transitions, in agreement with the loaded spiral spring model.
    DOI:  https://doi.org/10.1038/s41467-022-29850-z
  39. Autophagy Rep. 2022 ;1(1): 88-118
    The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.
    Kiren Baines, Kazuaki Yoshioka, Yoh Takuwa, Jon D Lane.
      Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12-ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12-ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12-ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins.
    Keywords:  ATG12; ATG5; HIP1R; PIK3C2A; autophagy; clathrin; endocytosis; proteomics
    DOI:  https://doi.org/10.1080/27694127.2022.2042054
  40. J Biol Chem. 2022 Apr 18. pii: S0021-9258(22)00385-4. [Epub ahead of print] 101945
    Cellular abundance of sodium phosphate co-transporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT.
    Christoph Zechner, W Mike Henne, Adwait A Sathe, Chao Xing, Genaro Hernandez, Shengyi Sun, Mi Cheong Cheong.
      Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate co-transporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, while mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein down-regulation in phosphate-replete cells. We show that SLC20A1 co-localizes with ESCRT, and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes.
    Keywords:  CRISPR/Cas9; Membrane transport; cell metabolism; cell surface protein; endosomal sorting complexes required for transport (ESCRT); genome-wide forward genetic screen; phosphate transporter; protein degradation
    DOI:  https://doi.org/10.1016/j.jbc.2022.101945
  41. Int J Mol Sci. 2022 Apr 18. pii: 4459. [Epub ahead of print]23(8):
    Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.
    Ai Abe, Masamitsu Maekawa, Toshihiro Sato, Yu Sato, Masaki Kumondai, Hayato Takahashi, Masafumi Kikuchi, Katsumi Higaki, Jiro Ogura, Nariyasu Mano.
      Niemann-Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symptoms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC-MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC-MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology observations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.
    Keywords:  LC–MS/MS; Niemann–Pick disease type C; ammonium solution; metabolic change; mitochondria; steroid hormones
    DOI:  https://doi.org/10.3390/ijms23084459
  42. iScience. 2022 Apr 15. 25(4): 104164
    Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking.
    Hiroyasu Hatakeyama, Ko Kobayashi, Makoto Kanzaki.
      Quantitative features of GLUT4 glucose transporter's behavior deep inside cells remain largely unknown. Our previous analyses with live-cell imaging of intracellular GLUT4 trafficking demonstrated two crucial early events responsible for triggering insulin-responsive translocation processes, namely, heterotypic fusion and liberation. To quantify the regulation, interrelationships, and dynamics of the initial events more accurately and comprehensively, we herein applied three analyses, each based on our distinct dual-color live-cell imaging approaches. With these approaches, heterotypic fusion was found to be the first trigger for insulin-responsive GLUT4 redistributions, preceding liberation, and to be critically regulated by Akt substrate of 160 kDa (AS160) and actin dynamics. In addition, demonstrating the subcellular regional dependence of GLUT4 dynamics revealed that liberated GLUT4 molecules are promptly incorporated into the trafficking itinerary of transferrin receptors. Our approaches highlight the physiological significance of endosomal "GLUT4 molecule trafficking" rather than "GLUT4 vesicle delivery" to the plasma membrane in response to insulin.
    Keywords:  Biological sciences; Biological sciences research methodologies; Biology experimental methods; Cell biology; Optical imaging
    DOI:  https://doi.org/10.1016/j.isci.2022.104164
  43. Mol Cell. 2022 Apr 21. pii: S1097-2765(22)00270-2. [Epub ahead of print]82(8): 1467-1476
    Codon optimality-mediated mRNA degradation: Linking translational elongation to mRNA stability.
    Haneui Bae, Jeff Coller.
      Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.
    Keywords:  codon optimality; deadenylation; decapping; genetic code; mRNA degradation; mRNA stability; protein synthesis; ribosomes; translation
    DOI:  https://doi.org/10.1016/j.molcel.2022.03.032
  44. Nature. 2022 Apr 20.
    Somatic genomic changes in single Alzheimer's disease neurons.
    Michael B Miller, August Yue Huang, Junho Kim, Zinan Zhou, Samantha L Kirkham, Eduardo A Maury, Jennifer S Ziegenfuss, Hannah C Reed, Jennifer E Neil, Lariza Rento, Steven C Ryu, Chanthia C Ma, Lovelace J Luquette, Heather M Ames, Derek H Oakley, Matthew P Frosch, Bradley T Hyman, Michael A Lodato, Eunjung Alice Lee, Christopher A Walsh.
      Dementia in Alzheimer's disease progresses alongside neurodegeneration1-4, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations5 at rates similar to those of dividing cells6,7 which suggests that genetic factors, environmental exposures or disease states might influence this accumulation5. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer's disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer's disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles 'clock-like' mutational signatures that have been previously described in healthy and cancerous cells6-10. In neurons affected by Alzheimer's disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation4,11, which we show is increased in Alzheimer's-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer's disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer's disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer's disease.
    DOI:  https://doi.org/10.1038/s41586-022-04640-1
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