bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒10‒02
forty-four papers selected by
Viktor Korolchuk, Newcastle University
  1. Mitochondria-ER cooperation: NLRX1 detects mitochondrial protein import stress and promotes mitophagy through the ER protein RRBP1.
  2. Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers.
  3. MERS-CoV nsp1 regulates autophagic flux via mTOR signaling and dysfunctional lysosomes.
  4. Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication.
  5. Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.
  6. Retrograde axonal autophagy and endocytic pathways are parallel and separate in neurons.
  7. Development of an autophagy activator from Class III PI3K complexes, Tat-BECN1 peptide: Mechanisms and applications.
  8. α-synuclein build-up is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α.
  9. TIPE2 protects cardiomyocytes from ischemia-reperfusion-induced apoptosis by decreasing cell autophagy via the mTORC1 signaling pathway.
  10. Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase.
  11. SARS-CoV-2 ORF3a inhibits cGAS-STING-mediated autophagy flux and antiviral function.
  12. Ral GTPases are critical regulators of spinal cord myelination and homeostasis.
  13. Editorial: Defective macroautophagy in organelle turnover: From basic mechanisms to human disease.
  14. Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.
  15. Elevated SFXN2 limits mitochondrial autophagy and increases iron-mediated energy production to promote multiple myeloma cell proliferation.
  16. Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.
  17. Spns1 is a lysophospholipid transporter mediating lysosomal phospholipid salvage.
  18. A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.
  19. Mitochondrial-Endoplasmic Reticulum Communication-Mediated Oxidative Stress and Autophagy.
  20. Peli1 contributes to myocardial ischemia/reperfusion injury by impairing autophagy flux via its E3 ligase mediated ubiquitination of P62.
  21. Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication.
  22. Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.
  23. Lotus germ extract rejuvenates aging fibroblasts via restoration of disrupted proteostasis by the induction of autophagy.
  24. Moxidectin induces autophagy arrest in colorectal cancer.
  25. Role of Autophagy in the Pathogenesis of Diabetes and Therapeutic Potential of Autophagy Modulators in the Treatment of Diabetes and Metabolic Syndrome.
  26. AMPK-dependent autophagy activation and alpha-Synuclein clearance: a putative mechanism behind alpha-mangostin's neuroprotection in a rotenone-induced mouse model of Parkinson's disease.
  27. High throughput analysis of vacuolar acidification.
  28. Preclinical evidence of a direct pro-survival role of arginine deprivation in multiple myeloma.
  29. SUMOylation targeting mitophagy in cardiovascular diseases.
  30. TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy.
  31. Autophagy in adipogenesis: Molecular mechanisms and regulation by bioactive compounds.
  32. Inhibition of mTORC1 differentially affects ribosome biogenesis in rat soleus muscle at the early and later stages of hindlimb unloading.
  33. WIPI proteins: Biological functions and related syndromes.
  34. Editorial: Molecular and cellular pathways leading to mitochondrial dysfunction and neurodegeneration: Lessons from in vivo models.
  35. The role of phenothiazine derivatives in autophagy regulation: A systematic review.
  36. Role of autophagy in tumor response to radiation: Implications for improving radiotherapy.
  37. Phosphorylation of Jhd2 by the Ras-cAMP-PKA(Tpk2) pathway regulates histone modifications and autophagy.
  38. Mast cell regranulation requires a metabolic switch involving mTORC1 and a glucose-6-phosphate transporter.
  39. The Cryptococcus neoformans Flc1 Homologue Controls Calcium Homeostasis and Confers Fungal Pathogenicity in the Infected Hosts.
  40. Mitochondrial Localization of SARM1 in Acrylamide Intoxication Induces Mitophagy and Limits Neuropathy.
  41. Transient Systemic Autophagy Inhibition is Selectively and Irreversibly Deleterious to Lung Cancer.
  42. Histocompatibility Minor 13 (HM13), targeted by miR-760, exerts oncogenic role in breast cancer by suppressing autophagy and activating PI3K-AKT-mTOR pathway.
  43. TFEB regulates sulfur amino acid and coenzyme A metabolism to support hepatic metabolic adaptation and redox homeostasis.
  44. Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.
  1. Autophagy. 2022 Sep 28.
    Mitochondria-ER cooperation: NLRX1 detects mitochondrial protein import stress and promotes mitophagy through the ER protein RRBP1.
    Samuel A Killackey, Yuntian Bi, Dana J Philpott, Damien Arnoult, Stephen E Girardin.
      Mitochondria rely on efficient protein import across their membranes for optimal function. We have shown that numerous mitochondrial stressors all converge on a common pathway disrupting this import efficiency. We identified a novel pathway involving NLRX1 and RRBP1 that responds to this import stress, resulting in LC3 lipidation, mitochondrial targeting and ultimate degradation. Furthermore, we demonstrated the relevance of this mitophagy axis in murine skeletal muscle following acute exercise. We propose that mitochondrial protein import stress is an underlying, common trigger for mitophagy, offering a novel avenue for therapeutic exploration and mechanistic insight.
    Keywords:  Autophagy; NLR; exercise; import; mitochondria; mitophagy; proteostasis
    DOI:  https://doi.org/10.1080/15548627.2022.2129763
  2. Life Sci. 2022 Sep 21. pii: S0024-3205(22)00689-0. [Epub ahead of print]308 120989
    Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers.
    Harleen Kaur, Régis Moreau.
      AIMS: As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated.MATERIALS AND METHODS: In this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers.
    KEY FINDINGS: Results showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown.
    SIGNIFICANCE: The study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.
    Keywords:  Autophagy; Claudin; Intestinal epithelium; Rapamycin; Tight junction; mTORC1
    DOI:  https://doi.org/10.1016/j.lfs.2022.120989
  3. Emerg Microbes Infect. 2022 Sep 24. 1-10
    MERS-CoV nsp1 regulates autophagic flux via mTOR signaling and dysfunctional lysosomes.
    Yujie Feng, Zhaoyi Pan, Zhihui Wang, Zhengyang Lei, Songge Yang, Huajun Zhao, Xueyao Wang, Yating Yu, Qiuju Han, Jian Zhang.
      Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signaling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.
    Keywords:  MERS-CoV; autophagic flux; lysosomes; mTOR; nsp1
    DOI:  https://doi.org/10.1080/22221751.2022.2128434
  4. Virulence. 2022 Dec;13(1): 1697-1712
    Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication.
    Jingxiang Wang, Xianjin Kan, Xiaomei Li, Jing Sun, Xiulong Xu.
      Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.
    Keywords:  AMPK; JNK; Porcine epidemic diarrhoea virus; TAK1; autophagy
    DOI:  https://doi.org/10.1080/21505594.2022.2127192
  5. J Biol Chem. 2022 Sep 23. pii: S0021-9258(22)00976-0. [Epub ahead of print] 102533
    Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.
    Amita Pal, Arunkumar Paripati, Pallavi Deolal, Arpan Chatterjee, Pushpa Rani Prasad, Priyanka Adla, Naresh Babu V Sepuri.
      Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of the mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of ROS and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.
    Keywords:  Saccharomyces cerevisiae; autophagy; cell death; mitochondria; mitophagy; protein aggregation; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1016/j.jbc.2022.102533
  6. J Neurosci. 2022 Sep 26. pii: JN-RM-1292-22. [Epub ahead of print]
    Retrograde axonal autophagy and endocytic pathways are parallel and separate in neurons.
    Vineet Vinay Kulkarni, Max Henry Stempel, Anip Anand, David Kader Sidibe, Sandra Maday.
      Autophagy and endocytic trafficking are two key pathways that regulate the composition and integrity of the neuronal proteome. Alterations in these pathways are sufficient to cause neurodevelopmental and neurodegenerative disorders. Thus, defining how autophagy and endocytic pathways are organized in neurons remains a key area of investigation. These pathways share many features and converge on lysosomes for cargo degradation, but what remains unclear is the degree to which the identity of each pathway is preserved in each compartment of the neuron. Here, we elucidate the degree of intersection between autophagic and endocytic pathways in axons of primary mouse cortical neurons of both sexes. Using microfluidic chambers, we labeled newly-generated bulk endosomes and signaling endosomes in the distal axon, and systematically tracked their trajectories, molecular composition, and functional characteristics relative to autophagosomes. We find that newly-formed endosomes and autophagosomes both undergo retrograde transport in the axon, but as distinct organelle populations. Moreover, these pathways differ in their degree of acidification and association with molecular determinants of organelle maturation. These results suggest that the identity of autophagic and newly endocytosed organelles is preserved for the length of the axon. Lastly, we find that expression of a pathogenic form of α-synuclein, a protein enriched in presynaptic terminals, increases merging between autophagic and endocytic pathways. Thus, aberrant merging of these pathways may represent a mechanism contributing to neuronal dysfunction in Parkinson's disease and related α-synucleinopathies.SIGNIFICANCE STATEMENTAutophagy and endocytic trafficking are retrograde pathways in neuronal axons that fulfill critical degradative and signaling functions. These pathways share many features and converge on lysosomes for cargo degradation, but the extent to which the identity of each pathway is preserved in axons is unclear. We find that autophagosomes and endosomes formed in the distal axon undergo retrograde transport to the soma in parallel and separate pathways. These pathways also have distinct maturation profiles along the mid-axon, further highlighting differences in the potential fate of transported cargo. Strikingly, expression of a pathogenic variant of α-synuclein increases merging between autophagic and endocytic pathways, suggesting that mis-sorting of axonal cargo may contribute to neuronal dysfunction in Parkinson's disease and related α-synucleinopathies.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1292-22.2022
  7. Front Cell Dev Biol. 2022 ;10 851166
    Development of an autophagy activator from Class III PI3K complexes, Tat-BECN1 peptide: Mechanisms and applications.
    Yanfei He, Huaqing Lu, Yuting Zhao.
      Impairment or dysregulation of autophagy has been implicated in many human pathologies ranging from neurodegenerative diseases, infectious diseases, cardiovascular diseases, metabolic diseases, to malignancies. Efforts have been made to explore the therapeutic potential of pharmacological autophagy activators, as beneficial health effects from caloric restriction or physical exercise are linked to autophagy activation. However, the lack of specificity remains the major challenge to the development and clinical use of autophagy activators. One candidate of specific autophagy activators is Tat-BECN1 peptide, derived from Beclin 1 subunit of Class III PI3K complexes. Here, we summarize the molecular mechanisms by which Tat-BECN1 peptide activates autophagy, the strategies for optimization and development, and the applications of Tat-BECN1 peptide in cellular and organismal models of physiology and pathology.
    Keywords:  Beclin 1; Class III PI3K complexes; Tat-BECN1 peptide; autophagy; cell-penetrating peptides; drug development
    DOI:  https://doi.org/10.3389/fcell.2022.851166
  8. J Biol Chem. 2022 Sep 23. pii: S0021-9258(22)00974-7. [Epub ahead of print] 102531
    α-synuclein build-up is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α.
    Helena Borland, Izabela Rasmussen, Kaare Bjerregaard-Andersen, Michel Rasmussen, Anders Olsen, Frederik Vilhardt.
      α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build-up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism, but their secretion also propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the NGF-differentiated catecholaminergic PC12 neuronal cell line which was conferred α-synucleinopathic cytotoxicity by inducible expression of αSyn and tubulin-polymerization promoting protein (TPPP) p25α. p25α aggregates αSyn, and by imposing a partial autophagosome-lysosome block, it mimics aspects of lysosomal deficiency common in neurodegenerative disease. Under basal conditions, we determined αSyn was degraded by multiple pathways, but most prominently by macroautophagy and Nedd4/Ndfip1-mediated degradation. We found that expression of p25α induced strong p38MAPK activity. Remarkably, when opposed by inhibitor SB203580 or p38MAPK shRNA knockdown, endolysosomal localization and degradation of αSyn increased several-fold, and αSyn secretion and cytotoxicity decreased. This effect was specifically dependent on Hsc70 and the ESCRT machinery, but different from classical microautophagy, as the αSyn Hsc70 binding motif was unnecessary. Furthermore, in a primary neuron (h)-αSyn seeding model, we showed p38MAPK inhibition decreased pathological accumulation of p-Ser129-αSyn and cytotoxicity. In conclusion, p38MAPK inhibition shifts αSyn degradation from various forms of autophagy to an ESCRT-dependent uptake mechanism, resulting in increased αSyn turn-over and cell viability in p25α-expressing cells. More generally, our results suggest that under conditions of autophagolysosomal malfunction, the uninterrupted endosomal pathway offers a possibility to achieve disease-associated protein degradation.
    Keywords:  Autophagy; Chaperone-mediated autophagy; ESCRT; Endosomes; Lysosomes; Microautophagy; TPPP/p25α; α-Synuclein
    DOI:  https://doi.org/10.1016/j.jbc.2022.102531
  9. Exp Ther Med. 2022 Oct;24(4): 613
    TIPE2 protects cardiomyocytes from ischemia-reperfusion-induced apoptosis by decreasing cell autophagy via the mTORC1 signaling pathway.
    Gong Cheng, Xiaoyan Huang, Penghua You, Panpan Feng, Shuo Jia, Ji Zhang, Hongjun You, Fengjun Chang.
      In cardiac ischemia-reperfusion (I/R), autophagy of hyperactivated cardiomyocytes degrades normal proteins and organelles, destroys cells and causes irreversible cell death. The present study aimed to determine the molecular mechanism through which TNF-α-induced protein 8-like protein 2 (TIPE2) regulates cardiomyocyte apoptosis via autophagy in I/R. The results revealed that the number of apoptotic cells and the protein expression levels of TIPE2 in the heart tissue of I/R model mice were significantly increased. In vitro, the overexpression of TIPE2 decreased oxygen glucose deprivation (OGD)-induced autophagy, apoptosis and activation of the mTOR complex 1 (mTORC1) signaling pathway in H9c2 cells. Treatment with the mTORC1 inhibitor not only inhibited the TIPE2-activated mTORC1 signaling pathway, but also increased OGD-induced autophagy and apoptosis of H9c2 cells. In conclusion, the results of the present study revealed that TIPE2 may protect cardiomyocytes from I/R-induced apoptosis by decreasing cell autophagy via the mTORC1 signaling pathway.
    Keywords:  TNF-α-induced protein 8-like protein 2; autophagy; mTOR complex 1
    DOI:  https://doi.org/10.3892/etm.2022.11550
  10. Stem Cells Transl Med. 2022 Sep 29. pii: szac069. [Epub ahead of print]
    Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase.
    Katherine E Hekman, Kyle M Koss, David Z Ivancic, Congcong He, Jason A Wertheim.
      Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.
    Keywords:  endothelial cell; mitochondria; rapamycin; senescence; tissue engineering; vascular tissue
    DOI:  https://doi.org/10.1093/stcltm/szac069
  11. J Med Virol. 2022 Sep 26.
    SARS-CoV-2 ORF3a inhibits cGAS-STING-mediated autophagy flux and antiviral function.
    Jiaming Su, Si Shen, Ying Hu, Shiqi Chen, Leyi Cheng, Yong Cai, Wei Wei, Yanpu Wang, Yajuan Rui, Xiao-Fang Yu.
      Recognizing aberrant cytoplasmic dsDNA and stimulating cGAS-STING-mediated innate immunity are essential for the host defense against viruses. Recent studies have reported that SARS-CoV-2 infection, responsible for the COVID-19 pandemic, triggers cGAS-STING activation. cGAS-STING activation can trigger IRF3-type I interferon (IFN) and autophagy-mediated antiviral activity. Although viral evasion of STING-triggered IFN-mediated antiviral function has been well studied, studies concerning viral evasion of STING-triggered autophagy-mediated antiviral function are scarce. In the present study, we have discovered that SARS-CoV-2 ORF3a is a unique viral protein that can interact with STING and disrupt the STING-LC3 interaction, thus blocking cGAS-STING-induced autophagy but not IRF3-type I IFN induction. This novel function of ORF3a, distinct from targeting autophagosome-lysosome fusion, is a selective inhibition of STING-triggered autophagy to facilitate viral replication. We have also found that activation of bat STING can induce autophagy and antiviral activity despite its defect in IFN induction. Furthermore, ORF3a from bat coronaviruses can block bat STING-triggered autophagy and antiviral function. Interestingly, the ability to inhibit STING-induced autophagy appears to be an acquired function of SARS-CoV-2 ORF3a, since SARS-CoV ORF3a lacks this function. Taken together, these discoveries identify ORF3a as a potential target for intervention against COVID-19. This article is protected by copyright. All rights reserved.
    Keywords:  Coronavirus; Immune responses; Innate immunity; SARS coronavirus; Virus classification
    DOI:  https://doi.org/10.1002/jmv.28175
  12. Cell Rep. 2022 Sep 27. pii: S2211-1247(22)01254-2. [Epub ahead of print]40(13): 111413
    Ral GTPases are critical regulators of spinal cord myelination and homeostasis.
    Jonathan DeGeer, Anna Lena Datwyler, Chiara Rickenbach, Andrea Ommer, Daniel Gerber, Cristina Fimiani, Joanne Gerber, Jorge A Pereira, Ueli Suter.
      Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.
    Keywords:  CP: Neuroscience; RalA; RalB; mTOR; myelin maintenance; myelination; oligodendrocytes
    DOI:  https://doi.org/10.1016/j.celrep.2022.111413
  13. Front Cell Dev Biol. 2022 ;10 1018778
    Editorial: Defective macroautophagy in organelle turnover: From basic mechanisms to human disease.
    Eloy Bejarano, José Antonio Rodríguez-Navarro, Roberta Filograna, Javier Calvo-Garrido.
      
    Keywords:  SQSTM1; aging; macroautophagy; neurodegeneration; selective autophagy
    DOI:  https://doi.org/10.3389/fcell.2022.1018778
  14. J Cell Physiol. 2022 Sep 27.
    Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.
    Kanchan Phadwal, Eve Koo, Ross A Jones, Rachael O Forsythe, Keyi Tang, Qiyu Tang, Brendan M Corcoran, Andrea Caporali, Vicky E MacRae.
      Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.
    Keywords:  Runx2; VSMCs; autophagy; calcification; metformin
    DOI:  https://doi.org/10.1002/jcp.30887
  15. Cell Death Dis. 2022 Sep 26. 13(9): 822
    Elevated SFXN2 limits mitochondrial autophagy and increases iron-mediated energy production to promote multiple myeloma cell proliferation.
    Ying Chen, Jinjun Qian, Pinggang Ding, Wang Wang, Xinying Li, Xiaozhu Tang, Chao Tang, Ye Yang, Chunyan Gu.
      Human sideroflexin 2 (SFXN2) belongs to the SFXN protein family, which is a mitochondrial outer membrane protein involved in mitochondrial iron metabolism. Mitochondria are indispensable for cellular energy production and iron metabolism. However, it remains elusive how SFXN2 modulates mitochondrial homeostasis and cellular iron metabolism in multiple myeloma (MM). In this study, we first found that SFXN2 was significantly elevated and correlated to poor outcomes in MM patients from clinical datasets. SFXN2 overexpression promoted MM cell proliferation and suppressed starvation-induced autophagy/mitophagy, while SFXN2 knockdown aggravated mitochondria damage and autophagic processes in ARP1 and H929 MM cell lines. Furthermore, inhibition of SFXN2 exerted effectively anti-myeloma activity in vivo by using myeloma xenograft model. Mechanism studies indicated that heme oxygenase 1 (HO1) with anti-oxidant function contributed to the process of autophagy suppression and cellular proliferation mediated by SFXN2. Our study revealed the critical role of SFXN2 in regulating mitochondrial bioenergetics, mitophagy, cellular iron metabolism, and redox homeostasis in interconnected and intricate way. Collectively, these findings not only provide insights into the metabolic reprogramming of tumor cells, but also highlight the therapeutic potential of SFXN2 in combination with iron metabolism as target for prognosis and treatment in MM patients.
    DOI:  https://doi.org/10.1038/s41419-022-05272-z
  16. J Cell Physiol. 2022 Sep 26.
    Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.
    Sayuki Iwahashi, Jiajun Lyu, Kazuya Tokumura, Ryoma Osumi, Manami Hiraiwa, Takuya Kubo, Tetsuhiro Horie, Satoru Demura, Noriaki Kawakami, Taku Saito, Gyujin Park, Kazuya Fukasawa, Takashi Iezaki, Akane Suzuki, Akane Tomizawa, Hiroki Ochi, Hironori Hojo, Shinsuke Ohba, Eiichi Hinoi.
      Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathway but inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.
    Keywords:  L-type amino acid transporter 1; chondrocytes; general amino acid control pathway; idiopathic scoliosis; mechanistic target of rapamycin complex 1
    DOI:  https://doi.org/10.1002/jcp.30883
  17. Proc Natl Acad Sci U S A. 2022 Oct 04. 119(40): e2210353119
    Spns1 is a lysophospholipid transporter mediating lysosomal phospholipid salvage.
    Menglan He, Alvin C Y Kuk, Mei Ding, Cheen Fei Chin, Dwight L A Galam, Jie Min Nah, Bryan C Tan, Hui Li Yeo, Geok Lin Chua, Peter I Benke, Markus R Wenk, Lena Ho, Federico Torta, David L Silver.
      The lysosome is central to the degradation of proteins, carbohydrates, and lipids and their salvage back to the cytosol for reutilization. Lysosomal transporters for amino acids, sugars, and cholesterol have been identified, and the metabolic fates of these molecules in the cytoplasm have been elucidated. Remarkably, it is not known whether lysosomal salvage exists for glycerophospholipids, the major constituents of cellular membranes. By using a transport assay screen against orphan lysosomal transporters, we identified the major facilitator superfamily protein Spns1 that is ubiquitously expressed in all tissues as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, with LPC and LPE being the lysosomal breakdown products of the most abundant eukaryotic phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively. Spns1 deficiency in cells, zebrafish embryos, and mouse liver resulted in lysosomal accumulation of LPC and LPE species with pathological consequences on lysosomal function. Flux analysis using stable isotope-labeled phospholipid apolipoprotein E nanodiscs targeted to lysosomes showed that LPC was transported out of lysosomes in an Spns1-dependent manner and re-esterified back into the cytoplasmic pools of phosphatidylcholine. Our findings identify a phospholipid salvage pathway from lysosomes to the cytosol that is dependent on Spns1 and critical for maintaining normal lysosomal function.
    Keywords:  Mfsd2a; autophagy; lysosome; phospholipid; transporter
    DOI:  https://doi.org/10.1073/pnas.2210353119
  18. Diabetologia. 2022 Oct 01.
    A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.
    Kyota Aoyagi, Shun-Ichi Yamashita, Yoshihiro Akimoto, Chiyono Nishiwaki, Yoko Nakamichi, Haruhide Udagawa, Manabu Abe, Kenji Sakimura, Tomotake Kanki, Mica Ohara-Imaizumi.
      AIMS/HYPOTHESIS: Mitophagy, the selective autophagy of mitochondria, is essential for maintenance of mitochondrial function. Recent studies suggested that defective mitophagy in beta cells caused diabetes. However, because of technical difficulties, the development of a convenient and reliable method to evaluate mitophagy in beta cells in vivo is needed. The aim of this study was to establish beta cell-specific mitophagy reporter mice and elucidate the role of mitophagy in beta cell function under metabolically stressed conditions induced by a high-fat diet (HFD).METHODS: Mitophagy was assessed using newly generated conditional mitochondrial matrix targeting mitophagy reporter (CMMR) mice, in which mitophagy can be visualised specifically in beta cells in vivo using a fluorescent probe sensitive to lysosomal pH and degradation. Metabolic stress was induced in mice by exposure to the HFD for 20 weeks. The accumulation of dysfunctional mitochondria was examined by staining for functional/total mitochondria and reactive oxygen species (ROS) using specific fluorescent dyes and antibodies. To investigate the molecular mechanism underlying mitophagy in beta cells, overexpression and knockdown experiments were performed. HFD-fed mice were examined to determine whether chronic insulin treatment for 6 weeks could ameliorate mitophagy, mitochondrial function and impaired insulin secretion.
    RESULTS: Exposure to the HFD increased the number of enlarged (HFD-G) islets with markedly elevated mitophagy. Mechanistically, HFD feeding induced severe hypoxia in HFD-G islets, which upregulated mitophagy through the hypoxia-inducible factor 1-ɑ (Hif-1ɑ)/BCL2 interacting protein 3 (BNIP3) axis in beta cells. However, HFD-G islets unexpectedly showed the accumulation of dysfunctional mitochondria due to excessive ROS production, suggesting an insufficient capacity of mitophagy for the degradation of dysfunctional mitochondria. Chronic administration of insulin ameliorated hypoxia and reduced ROS production and dysfunctional mitochondria, leading to decreased mitophagy and restored insulin secretion.
    CONCLUSIONS/INTERPRETATION: We demonstrated that CMMR mice enabled the evaluation of mitophagy in beta cells. Our results suggested that metabolic stress induced by the HFD caused the aberrant accumulation of dysfunctional mitochondria, which overwhelmed the mitophagic capacity and was associated with defective maintenance of mitochondrial function and impaired insulin secretion.
    Keywords:  Beta cells; Hypoxia; Insulin secretion; Mitochondria; Mitophagy; ROS
    DOI:  https://doi.org/10.1007/s00125-022-05800-8
  19. Biomed Res Int. 2022 ;2022 6459585
    Mitochondrial-Endoplasmic Reticulum Communication-Mediated Oxidative Stress and Autophagy.
    Xiaoqing Liu, Riaz Hussain, Khalid Mehmood, Zhaoxin Tang, Hui Zhang, Ying Li.
      Oxidative stress is an imbalance between free radicals and the antioxidant system causing overgeneration of free radicals (oxygen-containing molecules) ultimately leading to oxidative damage in terms of lipid peroxidation, protein denaturation, and DNA mutation. Oxidative stress can activate autophagy to alleviate oxidative damage and maintain normal physiological activities of cells by degrading damaged organelles or local cytoplasm. When oxidative stress is not eliminated by autophagy, it activates the apoptosis cascade. This review provides a brief summary of mitochondrial-endoplasmic reticulum communication-mediated oxidative stress and autophagy. Mitochondria and endoplasmic reticulum being important organelles in cells are directly or indirectly connected to each other through mitochondria-associated endoplasmic reticulum membranes and jointly regulate oxidative stress and autophagy. The reactive oxygen species (ROS) produced by the mitochondrial respiratory chain are the main inducers of oxidative stress. Damaged mitochondria can be effectively cleared by the process of mitophagy mediated by PINK1/parkin pathway, Nix/BNIP3 pathways, and FUNDC1 pathway, avoiding excessive ROS production. However, the mechanism of mitochondrial-endoplasmic reticulum communication in the regulation of oxidative stress and autophagy is rarely known. For this reason, this review explores the mutual connection of mitochondria and endoplasmic reticulum in mediating oxidative stress and autophagy through ROS and Ca2+ and aims to provide part of the theoretical basis for alleviating oxidative stress through autophagy mediated by mitochondrial-endoplasmic reticulum communication.
    DOI:  https://doi.org/10.1155/2022/6459585
  20. J Mol Cell Cardiol. 2022 Sep 27. pii: S0022-2828(22)00529-6. [Epub ahead of print]173 30-46
    Peli1 contributes to myocardial ischemia/reperfusion injury by impairing autophagy flux via its E3 ligase mediated ubiquitination of P62.
    Jie Yang, Tingting Tong, Chenghao Zhu, Miao Zhou, Yuqing Jiang, Hao Chen, Linli Que, Li Liu, Guoqing Zhu, Tuanzhu Ha, Qi Chen, Chuanfu Li, Yong Xu, Jiantao Li, Yuehua Li.
      Autophagy flux is impaired during myocardial ischemia/reperfusion (M-I/R) via the accumulation of autophagosome and insufficient clearance, which exacerbates cardiomyocyte death. Peli1 (Pellion1) is a RING finger domain-containing ubiquitin E3 ligase that could catalyze the polyubiquitination of substrate proteins. Peli1 has been demonstrated to play an important role in ischemic cardiac diseases. However, little is known about whether Peli1 is involved in the regulation of autophagy flux during M-I/R. The present study investigated whether M-I/R induced impaired autophagy flux could be mediated through Peli1 dependent mechanisms. We induced M-I/R injury in wild type (WT) and Peli1 knockout mice and observed that M-I/R significantly decreased cardiac function that was associated with increased cardiac Peli1 expression and upregulated autophagy-associated protein LC3II and P62. In contrast, Peli1 knockout mice exhibited significant improvement of M-I/R induced cardiac dysfunction and decreased LC3II and P62 expression. Besides, inhibitors of autophagy also increased the infarct size in Peli1 knockout mice after 24 h of reperfusion. Mechanistic studies demonstrated that in vivo I/R or in vitro hypoxia/reoxygenation (H/R) markedly increased the Peli1 E3 ligase activity which directly promoted the ubiquitination of P62 at lysine(K)7 via K63-linkage to inhibit its dimerization and autophagic degradation. Co-immunoprecipitation and GST-pull down assay indicated that Peli1 interacted with P62 via the Ring domain. In addition, Peli1 deficiency also decreased cardiomyocyte apoptosis. Together, our work demonstrated a critical link between increased expression and activity of Peli1 and autophagy flux blockage in M-I/R injury, providing insight into a promising strategy for treating myocardium M-I/R injury.
    Keywords:  Autophagy flux; E3 ligase; Ischemia/reperfusion; P62; Peli1; Ubiquitination
    DOI:  https://doi.org/10.1016/j.yjmcc.2022.09.004
  21. Front Cell Infect Microbiol. 2022 ;12 979996
    Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication.
    Christoph C Carter, Fred D Mast, Jean Paul Olivier, Natasha M Bourgeois, Alexis Kaushansky, John D Aitchison.
      The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.
    Keywords:  apoptosis; dengue virus (DENV); flavivirus; mechanistic target of rapamycin (mTOR); mechanistic target of rapamycin complex 2 (mTORC2); non-structural protein 5; pathogenesis; viral replication
    DOI:  https://doi.org/10.3389/fcimb.2022.979996
  22. J Cell Biol. 2022 Nov 07. pii: e202207091. [Epub ahead of print]221(11):
    Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.
    Jingyue Jia, Fulong Wang, Zambarlal Bhujabal, Ryan Peters, Michal Mudd, Thabata Duque, Lee Allers, Ruheena Javed, Michelle Salemi, Christian Behrends, Brett Phinney, Terje Johansen, Vojo Deretic.
      We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.
    DOI:  https://doi.org/10.1083/jcb.202207091
  23. Aging (Albany NY). 2022 Sep 26. 14(undefined):
    Lotus germ extract rejuvenates aging fibroblasts via restoration of disrupted proteostasis by the induction of autophagy.
    Kayo Machihara, Sou Kageyama, Shoma Oki, Hiroki Makino, Masamichi Sasaki, Hiroyasu Iwahashi, Takushi Namba.
      Cell aging attenuates cellular functions, resulting in time-dependent disruption of cellular homeostasis, which maintains the functions of proteins and organelles. Mitochondria are important organelles responsible for cellular energy production and various metabolic processes, and their dysfunction is strongly related to the progression of cellular aging. Here we demonstrate that disruption of proteostasis attenuates mitochondrial function before the induction of DNA damage signaling by proliferative and replicative cellular aging. We found that lotus (Nelumbo nucifera Gaertn.) germ extract clears abnormal proteins and agglutinates via autophagy-mediated restoration of mitochondrial function and cellular aging phenotypes. Pharmacological analyses revealed that DAPK1 expression was suppressed in aging cells, and lotus germ extract upregulated DAPK1 expression by stimulating the acetylation of histones and then induced autophagy by activating the DAPK1-Beclin1 signaling pathway. Furthermore, treatment of aging fibroblasts with lotus germ extract stimulated collagen production and increased contractile ability in three-dimensional cell culture. Thus, time-dependent accumulation of abnormal proteins and agglutinates suppressed mitochondrial function in cells in the early stage of aging, and reactivation of mitochondrial function by restoring proteostasis rejuvenated aging cells. Lotus germ extract rejuvenates aging fibroblasts via the DAPK1-Beclin1 pathway-induced autophagy to clear abnormal proteins and agglutinates.
    Keywords:  aging; autophagy; mitochondria; proteostasis
    DOI:  https://doi.org/10.18632/aging.204303
  24. Med Oncol. 2022 Sep 29. 39(12): 211
    Moxidectin induces autophagy arrest in colorectal cancer.
    Yushan Mao, Hanhan Xie, Dan Shu, Lin Cheng, Jingbin Lan, Kejian Pan.
      Colorectal cancer (CRC) is a cancer with a high morbidity and mortality worldwide. Hence, developing new therapeutic drugs for CRC is very important. Moxidectin (MOX) has shown good anti-glioblastoma effect both in vitro and in vivo. This study aimed to elucidate the anti-CRC effect of MOX and its potential mechanism by investigating the influence of MOX on the viability, apoptosis, necrosis and autophagy of colorectal cancer cells (HCT15 and SW620) and its underlying mechanisms. It was found that MOX can induce autophagy arrest, promote autophagy initiation, inhibit autophagic flux and cell proliferation, simultaneously PI3K-Akt-mTOR signaling pathway and microtubule acetylation. Furthermore, MOX suppressed the growth of xenograft tumors, which was consistent with the in vitro results.
    Keywords:  Accumulation; Autophagosome; CFTR; Colorectal cancer; Moxidectin
    DOI:  https://doi.org/10.1007/s12032-022-01799-5
  25. J Korean Med Sci. 2022 Sep 26. 37(37): e276
    Role of Autophagy in the Pathogenesis of Diabetes and Therapeutic Potential of Autophagy Modulators in the Treatment of Diabetes and Metabolic Syndrome.
    Soo-Jin Oh, Myung-Shik Lee.
      Autophagy is critically involved in the maintenance of intracellular nutrient homeostasis and organelle function. Dysregulated autophagy is likely to play a role in the development of metabolic disorders and diabetes because autophagy is critical in the rejuvenation of dysfunctional or stressed endoplasmic reticulum and mitochondria that play a crucial role in the development of diabetes. Indeed, systemic autophagy insufficiency led to the increased tissue lipid content, aggravated metabolic and finally more severe diabetes when metabolic stress was imposed, suggesting that autophagy insufficiency of dysfunction of lysosome, an effector organelle of autophagy, due to aging, genetic predisposition or environmental factors could be an underlying cause of diabetes. Conversely, autophagy enhancer could improve metabolic profile of obese mice by reducing tissue lipid content and ameliorating metabolic inflammation. Furthermore, clearance of human islet amyloid polypeptide (hIAPP) oligomer and amyloid that accumulate in pancreatic islets of > 90% of diabetes patients was also dependent on autophagy. Consistently, autophagy enhancer could improve glucose profile and β-cell function of transgenic mice expressing amyloidogenic hIAPP in pancreatic β-cells, which was accompanied by reduced accumulation of hIAPP oligomer or amyloid, ameliorated β-cell apoptosis and increased β-cell mass. These results suggest that autophagy enhancer could be a novel therapeutic modality against diabetes associated with lipid overload and human diabetes characterized by islet amyloid accumulation.
    Keywords:  Autophagy; ER; IAPP; Islet Amyloid; Metabolic Inflammation; Mitochondria
    DOI:  https://doi.org/10.3346/jkms.2022.37.e276
  26. Metab Brain Dis. 2022 Sep 30.
    AMPK-dependent autophagy activation and alpha-Synuclein clearance: a putative mechanism behind alpha-mangostin's neuroprotection in a rotenone-induced mouse model of Parkinson's disease.
    Pathik Parekh, Nishant Sharma, Monika Sharma, Anagha Gadepalli, Adil Ali Sayyed, Sayan Chatterjee, Abhijeet Kate, Amit Khairnar.
      Alpha-Synuclein (α-Syn) accumulation is central to the pathogenesis of Parkinson's disease (PD), hence the quest for finding potential therapeutics that may promote the α-Syn clearance is the need of the hour. To this, activation of the evolutionarily conserved protein and key regulator of the autophagy, 5'AMP-activated protein kinase (AMPK) is well-known to induce autophagy and subsequently the clearance of α-Syn aggregates. Alpha-mangostin (AM) a polyphenolic xanthone obtained from Garcinia Mangostana L. was previously reported to activate AMPK-dependent autophagy in various pre-clinical cancer models. However, no studies evidenced the effect of AM on AMPK-dependent autophagy activation in the PD. Therefore, the present study aimed to investigate the neuroprotective activity of AM in the chronic rotenone mouse model of PD against rotenone-induced α-Syn accumulation and to dissect molecular mechanisms underlying the observed neuroprotection. The findings showed that AM exerts neuroprotection against rotenone-induced α-Syn accumulation in the striatum and cortex by activating AMPK, upregulating autophagy (LC3II/I, Beclin-1), and lysosomal (TFEB) markers. Of note, an in-vitro study utilizing rat pheochromocytoma cells verified that AM conferred the neuroprotection only through AMPK activation, as the presence of inhibitors of AMPK (dorsomorphin) and autophagy (3-methyl adenine) failed to mitigate rotenone-induced α-Syn accumulation. Moreover, AM also counteracted rotenone-induced behavioral deficits, oxidative stress, and degeneration of nigro-striatal dopaminergic neurons. In conclusion, AM provided neuroprotection by ameliorating the rotenone-induced α-Syn accumulation through AMPK-dependent autophagy activation and it can be considered as a therapeutic agent which might be having a higher translational value in the treatment of PD.
    Keywords:  AMPK; Alpha-mangostin; Autophagy; Chronic rotenone mouse model; Neurodegeneration; Parkinson’s disease
    DOI:  https://doi.org/10.1007/s11011-022-01087-1
  27. Anal Biochem. 2022 Sep 24. pii: S0003-2697(22)00387-6. [Epub ahead of print] 114927
    High throughput analysis of vacuolar acidification.
    Chi Zhang, Adam Balutowski, Yilin Feng, Jorge D Calderin, Rutilio A Fratti.
      Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is lumenal pH. In the endocytic and secretory pathways, lumenal pH is controlled by isoforms and concentration of the vacuolar-type H+-ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes.
    Keywords:  Acridine orange; Gadolinium; Lanthanum; Lysosome; Nickel; V-ATPase; Vph1
    DOI:  https://doi.org/10.1016/j.ab.2022.114927
  28. Front Oncol. 2022 ;12 968208
    Preclinical evidence of a direct pro-survival role of arginine deprivation in multiple myeloma.
    Matteo Trudu, Laura Oliva, Ugo Orfanelli, Alessandra Romano, Francesco Di Raimondo, Francesca Sanvito, Maurilio Ponzoni, Simone Cenci.
      Multiple myeloma grows by establishing multiple interactions with bone marrow cells. These include expansion of myeloid-derived suppressor cells, which drive immunoevasion via mechanisms that include arginase-1-driven depletion of L-arginine, thus indirectly promoting myeloma cell survival and tumor progression. The peculiar biology of malignant plasma cells postulates that arginine depletion may benefit their fitness also directly, e.g., by engaging the integrated stress response, or by stimulating autophagy through mTORC1 inhibition. We thus investigated the direct impact of arginine deprivation on myeloma cells and challenged its pathophysiological relevance in vitro and in vivo. First, we found that partial arginine depletion spared proliferation of human multiple myeloma cells at concentrations that arrest human T cells. Next, we asked if arginine shortage activates putative adaptive pathways in myeloma cells. Low arginine failed to activate the integrated stress response, as indicated by unmodified phosphorylation of the eukaryotic initiation factor 2α, but sizably inhibited mTORC1, as revealed by reduced phosphorylation of ribosomal protein S6. Notably, depressed mTORC1 activity was not sufficient to increase autophagy, as assessed by the lysosomal digestion rate of the autophagosome-associated protein, LC3-II. Rather, it stimulated mTORC2, resulting in increased phosphatidylinositol-3 kinase-dependent AKT phosphorylation and activity, leading to heightened inhibitory phosphorylation of the pro-apoptotic BAD protein. We then tested whether arginine depletion-activated AKT may protect malignant plasma cells from cell death. Indeed, culturing myeloma cells in low arginine medium significantly reduced the apoptotic effect of the first-in-class proteasome inhibitor, bortezomib, an outcome prevented by pharmacological inhibition of AKT phosphorylation. Finally, we challenged the relevance of the identified circuit in vivo. To gauge the pathophysiologic relevance of low arginine to myeloma growth independently of immunoevasion, we xenotransplanted human myeloma cells subcutaneously into T cell-deficient Rag2-/-γc-/- recipient mice and treated palpable tumor-bearing mice with the clinical-grade arginase inhibitor CB1158. Arginase inhibition significantly raised serum arginine concentration, reduced tumor growth by caliper assessment, and decreased intra-tumor AKT phosphorylation in vivo. Altogether, our results reveal a novel direct pro-survival effect of arginine deprivation on myeloma cells, with potential therapeutic implications.
    Keywords:  AKT; arginine; autophagy; mammalian target of rapamycin; multiple myeloma; plasma cell; stress; survival
    DOI:  https://doi.org/10.3389/fonc.2022.968208
  29. J Mol Med (Berl). 2022 Sep 26.
    SUMOylation targeting mitophagy in cardiovascular diseases.
    Hong Xiao, Hong Zhou, Gaofeng Zeng, Zhenjiang Mao, Junfa Zeng, Anbo Gao.
      Small ubiquitin-like modifier (SUMO) plays a key regulatory role in cardiovascular diseases, such as cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. As a multifunctional posttranslational modification molecule in eukaryotic cells, SUMOylation is essentially associated with the regulation of mitochondrial dynamics, especially mitophagy, which is involved in the progression and development of cardiovascular diseases. SUMOylation targeting mitochondrial-associated proteins is admittedly considered to regulate mitophagy activation and mitochondrial functions and dynamics, including mitochondrial fusion and fission. SUMOylation triggers mitochondrial fusion to promote mitochondrial dysfunction by modifying Fis1, OPA1, MFN1/2, and DRP1. The interaction between SUMO and DRP1 induces SUMOylation and inhibits lysosomal degradation of DRP1, which is further involved in the regulation of mitochondrial fission. Both SUMOylation and deSUMOylation contribute to the initiation and activation of mitophagy by regulating the conjugation of MFN1/2 SERCA2a, HIF1α, and PINK1. SUMOylation mediated by the SUMO molecule has attracted much attention due to its dual roles in the development of cardiovascular diseases. In this review, we systemically summarize the current understanding underlying the expression, regulation, and structure of SUMO molecules; explore the biochemical functions of SUMOylation in the initiation and activation of mitophagy; discuss the biological roles and mechanisms of SUMOylation in cardiovascular diseases; and further provide a wider explanation of SUMOylation and deSUMOylation research to provide a possible therapeutic strategy for cardiovascular diseases. Considering the precise functions and exact mechanisms of SUMOylation in mitochondrial dysfunction and mitophagy will provide evidence for future experimental research and may serve as an effective approach in the development of novel therapeutic strategies for cardiovascular diseases. Regulation and effect of SUMOylation in cardiovascular diseases via mitophagy. SUMOylation is involved in multiple cardiovascular diseases, including cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. Since it is expressed in multiple cells associated with cardiovascular disease, SUMOylation can be regulated by numerous ligases, including the SENP family proteins PIAS1, PIASy/4, UBC9, and MAPL. SUMOylation regulates the activation and degradation of PINK1, SERCA2a, PPARγ, ERK5, and DRP1 to mediate mitochondrial dynamics, especially mitophagy activation. Mitophagy activation regulated by SUMOylation further promotes or inhibits ventricular diastolic dysfunction, perfusion injury, ventricular remodelling and ventricular noncompaction, which contribute to the development of cardiovascular diseases.
    Keywords:  Cardiovascular diseases; Mitochondria; Mitophagy; SUMO; SUMOylation
    DOI:  https://doi.org/10.1007/s00109-022-02258-4
  30. Nat Commun. 2022 Sep 26. 13(1): 5658
    TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy.
    Chengyang Li, Patrick Duckney, Tong Zhang, Yanshu Fu, Xin Li, Johan Kroon, Geert De Jaeger, Yunjiang Cheng, Patrick J Hussey, Pengwei Wang.
      ER-mitochondrial contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer membrane proteins, TRB1, TRB2, and the ER protein, VAP27-1. In Arabidopsis, there are three TraB family isoforms and the trb1/trb2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation, and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TRB1 and TRB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trb1/trb2 and VAP27 mutants (vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TRB1-regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB family proteins which are component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy.
    DOI:  https://doi.org/10.1038/s41467-022-33402-w
  31. Biomed Pharmacother. 2022 Sep 21. pii: S0753-3322(22)01104-0. [Epub ahead of print]155 113715
    Autophagy in adipogenesis: Molecular mechanisms and regulation by bioactive compounds.
    Faizullah Khan, Haroon Khan, Ajmal Khan, Masao Yamasaki, Naima Moustaid-Moussa, Ahmed Al-Harrasi, Shaikh Mizanoor Rahman.
      White adipose tissue expands rapidly due to increased adipocyte number (hyperplasia) and size (hypertrophy), which results in obesity. Adipogenesis is a process of the formation of mature adipocytes from precursor cells. Additionally, obesity-related metabolic complications, such as fatty liver and insulin resistance, are linked to adipogenesis. On the contrary, autophagy is a catabolic process; essential to maintain cellular homeostasis via the degradation or recycling of unnecessary or damaged components. Importantly, autophagy dictates obesity and adipogenesis. Hence, a clear understanding of how autophagy regulates adipogenesis is crucial for drug development and the prevention and treatment of obesity and its associated disorders, such as type 2 diabetes, cardiovascular disease, and cancer. In this review, we highlighted recent findings regarding the crosstalk between adipogenesis and autophagy, as well as the molecules involved. Furthermore, the review discussed how bioactive compounds regulate adipogenesis by manipulating autophagy and underlying molecular mechanisms. Based on in vitro and animal studies, we summarized the effects of bioactive compounds on adipogenesis and autophagy. Hence, human studies are necessary to validate the effectiveness and optimal dosage of these bioactive compounds.
    Keywords:  Adipogenesis; Autophagy; Bioactive compounds; Molecular regulation; Transcription factor
    DOI:  https://doi.org/10.1016/j.biopha.2022.113715
  32. Arch Biochem Biophys. 2022 Sep 22. pii: S0003-9861(22)00295-8. [Epub ahead of print]730 109411
    Inhibition of mTORC1 differentially affects ribosome biogenesis in rat soleus muscle at the early and later stages of hindlimb unloading.
    Sergey V Rozhkov, Kristina A Sharlo, Boris S Shenkman, Timur M Mirzoev.
      Prolonged inactivity of skeletal muscles due to limb immobilization, bedrest, and exposure to microgravity results in a significant muscle atrophy. Inactivity-induced muscle atrophy is caused by a downregulation of protein synthesis (PS) and increased proteolysis. Mechanistic target of rapamycin complex 1 (mTORC1) is considered to be one of the main regulators of translational capacity (quantity of ribosomes), a key determinant of PS. Using a specific mTORC1 inhibitor (rapamycin) we aimed to determine if mTORC1 activity would influence ribosome biogenesis in rat soleus muscle at both early and later stages of mechanical unloading. Wistar rats were subjected to 1- and 7-day hindlimb suspension (HS) with and without rapamycin injections (1.5 mg/kg) and compared to weight-bearing control animals. The key markers of ribosome biogenesis were assessed by RT-PCR or agarose gel electrophoresis. The rate of PS was measured by SUnSET method. Both 1-day and 7-day HS resulted in a significant downregulation of ribosome biogenesis markers (c-Myc, 47S pre-rRNA, 18S + 28S rRNAs) and the rate of PS. Rapamycin administration during 1-day HS fully prevented a decrease in 47S pre-rRNA expression and amount of 18S + 28S rRNAs (without affecting c-Myc mRNA expression) and partially attenuated a decline in PS. Rapamycin treatment during 7-day HS significantly decreased p70S6K phosphorylation but failed to rescue a reduction in both the markers of ribosome biogenesis and the rate of PS. All together, our results suggest that mTORC1 inhibition at the initial (1 day), but not later (7 days) stage of HS can be beneficial for the maintenance of translational capacity (ribosome biogenesis) and the rate of PS in rat soleus muscle.
    Keywords:  Hindlimb unloading; Protein synthesis; Rapamycin; Ribosome biogenesis; Soleus muscle; mTORC1
    DOI:  https://doi.org/10.1016/j.abb.2022.109411
  33. Front Mol Neurosci. 2022 ;15 1011918
    WIPI proteins: Biological functions and related syndromes.
    Mohammed Almannai, Dana Marafi, Ayman W El-Hattab.
      WIPI (WD-repeat protein Interacting with PhosphoInositides) are important effectors in autophagy. These proteins bind phosphoinositides and recruit autophagy proteins. In mammals, there are four WIPI proteins: WIPI1, WIPI2, WIPI3 (WDR45B), and WIPI4 (WDR45). These proteins consist of a seven-bladed β-propeller structure. Recently, pathogenic variants in genes encoding these proteins have been recognized to cause human diseases with a predominant neurological phenotype. Defects in WIPI2 cause a disease characterized mainly by intellectual disability and variable other features while pathogenic variants in WDR45B and WDR45 have been recently reported to cause El-Hattab-Alkuraya syndrome and beta-propeller protein-associated neurodegeneration (BPAN), respectively. Whereas, there is no disease linked to WIPI1 yet, one study linked it neural tube defects (NTD). In this review, the role of WIPI proteins in autophagy is discussed first, then syndromes related to these proteins are summarized.
    Keywords:  WD repeat domain; WDR; WIPI; autophagy; neurodevelopment
    DOI:  https://doi.org/10.3389/fnmol.2022.1011918
  34. Front Neurosci. 2022 ;16 1006100
    Editorial: Molecular and cellular pathways leading to mitochondrial dysfunction and neurodegeneration: Lessons from in vivo models.
    Shabab B Hannan, Alvaro Sanchez-Martinez, Gloria Brea-Calvo, Aurora Gomez-Duran, Juan A Navarro.
      
    Keywords:  MAMs; animal models; lysosome; mitochondria; mitochondrial dynamics; mitophagy; neurodegenerative disease
    DOI:  https://doi.org/10.3389/fnins.2022.1006100
  35. J Appl Toxicol. 2022 Sep 27.
    The role of phenothiazine derivatives in autophagy regulation: A systematic review.
    Michał Otręba, Jerzy Stojko, Anna Rzepecka-Stojko.
      In this review, we summarized the current literature on the impact of phenothiazine derivatives on autophagy in vitro. Phenothiazines are antipsychotic drugs used in the treatment of schizophrenia, which is related to altered neurotransmission and dysregulation of neuronal autophagy. Thus, phenothiazine derivatives can impact autophagy. We identified 35 papers, where the use of the phenothiazines in the in vitro autophagy assays on normal and cancer cell lines, C. elegans, and zebrafish were discussed. Chlorpromazine, fluphenazine, mepazine, methotrimeprazine, perphenazine, prochlorperazine, promethazine, thioridazine, trifluoperazine, and novel derivatives can modulate autophagy. Stimulation of autophagy by phenothiazines may be either mTOR-dependent or -independent. The final effect depends on the used concentration as well as the cell line. A further investigation of the mechanisms of autophagy regulation by phenothiazine derivatives is required to understand the biological actions, and to increase the therapeutic potential of this class of drugs.
    Keywords:  autophagy; cancer; cell line; phenothiazine derivatives; tumor
    DOI:  https://doi.org/10.1002/jat.4397
  36. Front Oncol. 2022 ;12 957373
    Role of autophagy in tumor response to radiation: Implications for improving radiotherapy.
    Amrita Roy, Soumen Bera, Luciano Saso, Bilikere S Dwarakanath.
      Autophagy is an evolutionary conserved, lysosome-involved cellular process that facilitates the recycling of damaged macromolecules, cellular structures, and organelles, thereby generating precursors for macromolecular biosynthesis through the salvage pathway. It plays an important role in mediating biological responses toward various stress, including those caused by ionizing radiation at the cellular, tissue, and systemic levels thereby implying an instrumental role in shaping the tumor responses to radiotherapy. While a successful execution of autophagy appears to facilitate cell survival, abortive or interruptions in the completion of autophagy drive cell death in a context-dependent manner. Pre-clinical studies establishing its ubiquitous role in cells and tissues, and the systemic response to focal irradiation of tumors have prompted the initiation of clinical trials using pharmacologic modifiers of autophagy for enhancing the efficacy of radiotherapy. However, the outcome from the Phase I/II trials in many human malignancies has so far been equivocal. Such observations have not only precluded the advancement of these autophagy modifiers in the Phase III trial but have also raised concerns regarding their introduction as an adjuvant to radiotherapy. This warrants a thorough understanding of the biology of the cancer cells, including its spatio-temporal context, as well as its microenvironment all of which might be the crucial factors that determine the success of an autophagy modifier as an anticancer agent. This review captures the current understanding of the interplay between radiation induced autophagy and the biological responses to radiation damage as well as provides insight into the potentials and limitations of targeting autophagy for improving the radiotherapy of tumors.
    Keywords:  DNA damage repair; autophagy; cell death; radiotherapy; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2022.957373
  37. Nat Commun. 2022 Sep 27. 13(1): 5675
    Phosphorylation of Jhd2 by the Ras-cAMP-PKA(Tpk2) pathway regulates histone modifications and autophagy.
    Qi Yu, Xuanyunjing Gong, Yue Tong, Min Wang, Kai Duan, Xinyu Zhang, Feng Ge, Xilan Yu, Shanshan Li.
      Cells need to coordinate gene expression with their metabolic states to maintain cell homeostasis and growth. How cells transduce nutrient availability to appropriate gene expression remains poorly understood. Here we show that glycolysis regulates histone modifications and gene expression by activating protein kinase A (PKA) via the Ras-cyclic AMP pathway. The catalytic subunit of PKA, Tpk2 antagonizes Jhd2-catalyzed H3K4 demethylation by phosphorylating Jhd2 at Ser321 and Ser340 in response to glucose availability. Tpk2-catalyzed Jhd2 phosphorylation impairs its nuclear localization, reduces its binding to chromatin, and promotes its polyubiquitination and degradation by the proteasome. Tpk2-catalyzed Jhd2 phosphorylation also maintains H3K14 acetylation by preventing the binding of histone deacetylase Rpd3 to chromatin. By phosphorylating Jhd2, Tpk2 regulates gene expression, maintains normal chronological life span and promotes autophagy. These results provide a direct connection between metabolism and histone modifications and shed lights on how cells rewire their biological responses to nutrient signals.
    DOI:  https://doi.org/10.1038/s41467-022-33423-5
  38. Cell Rep. 2022 Sep 27. pii: S2211-1247(22)01174-3. [Epub ahead of print]40(13): 111346
    Mast cell regranulation requires a metabolic switch involving mTORC1 and a glucose-6-phosphate transporter.
    Jason A Iskarpatyoti, Jianling Shi, Mathew A Abraham, Abhay P S Rathore, Yuxuan Miao, Soman N Abraham.
      Mast cells (MCs) are granulated cells implicated in inflammatory disorders because of their capacity to degranulate, releasing prestored proinflammatory mediators. As MCs have the unique capacity to reform granules following degranulation in vitro, their potential to regranulate in vivo is linked to their pathogenesis. It is not known what factors regulate regranulation, let alone if regranulation occurs in vivo. We report that mice can undergo multiple bouts of MC regranulation following successive anaphylactic reactions. mTORC1, a nutrient sensor that activates protein and lipid synthesis, is necessary for regranulation. mTORC1 activity is regulated by a glucose-6-phosphate transporter, Slc37a2, which increases intracellular glucose-6-phosphate and ATP during regranulation, two upstream signals of mTOR. Additionally, Slc37a2 concentrates extracellular metabolites within endosomes, which are trafficked into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.
    Keywords:  CP: Metabolism; Slc37a2; mTOR; mast cell; metabolism; regranulation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111346
  39. mBio. 2022 Sep 28. e0225322
    The Cryptococcus neoformans Flc1 Homologue Controls Calcium Homeostasis and Confers Fungal Pathogenicity in the Infected Hosts.
    Piotr R Stempinski, Kristie D Goughenour, Lukas M du Plooy, J Andrew Alspaugh, Michal A Olszewski, Lukasz Kozubowski.
      Cryptococcus neoformans, an opportunistic yeast pathogen, relies on a complex network of stress response pathways that allow for proliferation in the host. In Saccharomyces cerevisiae, stress responses are regulated by integral membrane proteins containing a transient receptor potential (TRP) domain, including the flavin carrier protein 1 (Flc1), which regulates calcium homeostasis and flavin transport. Here, we report that deletion of C. neoformans FLC1 results in cytosolic calcium elevation and increased nuclear content of calcineurin-dependent transcription factor Crz1, which is associated with an aberrant cell wall chitin overaccumulation observed in the flc1Δ mutant. Absence of Flc1 or inhibition of calcineurin with cyclosporine A prevents vacuolar fusion under conditions of combined osmotic and temperature stress, which is reversed in the flc1Δ mutant by the inhibition of TORC1 kinase with rapamycin. Flc1-deficient yeasts exhibit compromised vacuolar fusion under starvation conditions, including conditions that stimulate formation of carbohydrate capsule. Consequently, the flc1Δ mutant fails to proliferate under low nutrient conditions and displays a defect in capsule formation. Consistent with the previously uncharacterized role of Flc1 in vacuolar biogenesis, we find that Flc1 localizes to the vacuole. The flc1Δ mutant presents a survival defect in J774A.1 macrophage cell-line and profound virulence attenuation in both the Galleria mellonella and mouse pulmonary infection models, demonstrating that Flc1 is essential for pathogenicity. Thus, cryptococcal Flc1 functions in calcium homeostasis and links calcineurin and TOR signaling with vacuolar biogenesis to promote survival under conditions associated with vacuolar fusion required for this pathogen's fitness and virulence. IMPORTANCE Cryptococcosis is a highly lethal infection with limited drug choices, most of which are highly toxic or complicated by emerging antifungal resistance. There is a great need for new drug targets that are unique to the fungus. Here, we identify such a potential target, the Flc1 protein, which we show is crucial for C. neoformans stress response and virulence. Importantly, homologues of Flc1 exist in other fungal pathogens, such as Candida albicans and Aspergillus fumigatus, and are poorly conserved in humans, which could translate into wider spectrum therapy associated with minimal toxicity. Thus, Flc1 could be an "Achille's heel" of C. neoformans to be leveraged therapeutically in cryptococcosis and possibly other fungal infections.
    Keywords:  Cryptococcus neoformans; autophagy; calcium signaling; opportunistic fungi; pathogenesis
    DOI:  https://doi.org/10.1128/mbio.02253-22
  40. Mol Neurobiol. 2022 Sep 29.
    Mitochondrial Localization of SARM1 in Acrylamide Intoxication Induces Mitophagy and Limits Neuropathy.
    Shuai Wang, Mingxue Song, Hui Yong, Cuiqin Zhang, Kang Kang, Zhidan Liu, Yiyu Yang, Zhengcheng Huang, Shu'e Wang, Haotong Ge, Xiulan Zhao, Fuyong Song.
      Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the defining molecule and central executioner of programmed axon death, also known as Wallerian degeneration. SARM1 has a mitochondrial targeting sequence, and it can bind to and stabilize PTEN-induced putative kinase 1 (PINK1) for mitophagy induction, but the deletion of the mitochondrial localization sequence is found to disrupt the mitochondrial localization of SARM1 in neurons without altering its ability to promote axon degeneration after axotomy. The biological significance of SARM1 mitochondrial localization remains elusive. In this study, we observed that the pro-degeneration factor, SARM1, was upregulated in acrylamide (ACR) neuropathy, a slow, Wallerian-like, programmed axonal death process. The upregulated SARM1 accumulated on mitochondria, interfered with mitochondrial dynamics, and activated PINK1-mediated mitophagy. Importantly, rapamycin (RAPA) intervention eliminated mitochondrial accumulation of SARM1 and partly attenuated ACR neuropathy. Thus, mitochondrial localization of SARM1 may contribute to its clearance through the SARM1-PINK1 mitophagy pathway, which inhibits axonal degeneration through a negative feedback loop. The mitochondrial localization of SARM1 complements the coordinated activity of the pro-survival factor, nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and SARM1 and is part of the self-limiting molecular mechanisms underpinning programmed axon death in ACR neuropathy. Mitophagy clearance of SARM1 is complementary to the coordinated activity of NMNAT2 and SARM1 in ACR neuropathy.
    Keywords:  Acrylamide; Axon degeneration; Neurotoxin; Peripheral neuropathy; Rapamycin; Wallerian degeneration
    DOI:  https://doi.org/10.1007/s12035-022-03050-8
  41. Cancer Res. 2022 Sep 26. pii: CAN-22-1039. [Epub ahead of print]
    Transient Systemic Autophagy Inhibition is Selectively and Irreversibly Deleterious to Lung Cancer.
    Khoosheh Khayati, Vrushank Bhatt, Taijin Lan, Fawzi Alogaili, Wenping Wang, Enrique Lopez, Zhixian Sherrie Hu, Samantha Gokhale, Liam Cassidy, Masashi Narita, Ping Xie, Eileen White, Jessie Yanxiang Guo.
      Autophagy is a conserved catabolic process that maintains cellular homeostasis. Autophagy supports lung tumorigenesis and is a potential therapeutic target in lung cancer. A better understanding of the importance of tumor cell-autonomous versus systemic autophagy in lung cancer could facilitate clinical translation of autophagy inhibition. Here, we exploited inducible expression of Atg5 shRNA to temporally control Atg5 levels and generate reversible tumor-specific and systemic autophagy loss mouse models of KrasG12D/+;p53-/- (KP) non-small cell lung cancer (NSCLC). Transient suppression of systemic but not tumor Atg5 expression significantly reduced established KP lung tumor growth without damaging normal tissues. In vivo 13C isotope tracing and metabolic flux analyses demonstrated that systemic Atg5 knockdown specifically led to reduced glucose and lactate uptake. As a result, carbon flux from glucose and lactate to major metabolic pathways, including the tricarboxylic acid cycle, glycolysis, and serine biosynthesis, was significantly reduced in KP NSCLC following systemic autophagy loss. Furthermore, systemic Atg5 knockdown increased tumor T cell infiltration, leading to T cell-mediated tumor killing. Importantly, intermittent transient systemic Atg5 knockdown, which resembles what would occur during autophagy inhibition for cancer therapy, significantly prolonged lifespan of KP lung tumor-bearing mice, resulting in recovery of normal tissues but not tumors. Thus, systemic autophagy supports the growth of established lung tumors by promoting immune evasion and sustaining cancer cell metabolism for energy production and biosynthesis, and the inability of tumors to recover from loss of autophagy provides further proof of concept that inhibition of autophagy is a valid approach to cancer therapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-1039
  42. Cell Death Dis. 2022 Sep 25. 13(8): 728
    Histocompatibility Minor 13 (HM13), targeted by miR-760, exerts oncogenic role in breast cancer by suppressing autophagy and activating PI3K-AKT-mTOR pathway.
    Haiyan Yang, Zhi Li, Zhangwei Wang, Xu Zhang, Xinyuan Dai, Guoren Zhou, Qiang Ding.
      Histocompatibility Minor 13 (HM13) is reported to participate in regulating multiple cancers. In the present study, we uncovered that HM13 was highly expressed in breast cancer and correlated with worse prognosis. Downregulation of HM13 could suppress breast cancer cell proliferation and metastasis abilities. Tumorigenicity mediated by HM13 was also observed in the xenograft model. Knockdown of HM13 could activate autophagy by inducing endoplasmic reticulum (ER) stress. Moreover, further experiments demonstrated that downregulated HM13 could inhibit PI3K-AKT-mTOR pathway. We then verified that HM13 was a direct target of miR-760 functioned as a tumor -suppressor in breast cancer. And the tumor suppressive effects of miR-760 could be partially reversed by HM13. Taken together, these findings elucidated that HM13, targeted by miR-760, could play an oncogenic role in breast cancer by inducing autophagic inhibition and facilitating PI3K-AKT-mTOR pathway. Our findings suggested HM13 could act as a novel therapeutic target candidate for breast cancer and supported the idea that autophagy inducers might represent a new approach to treat breast cancer.
    DOI:  https://doi.org/10.1038/s41419-022-05154-4
  43. Nat Commun. 2022 Sep 28. 13(1): 5696
    TFEB regulates sulfur amino acid and coenzyme A metabolism to support hepatic metabolic adaptation and redox homeostasis.
    David Matye, Sumedha Gunewardena, Jianglei Chen, Huaiwen Wang, Yifeng Wang, Mohammad Nazmul Hasan, Lijie Gu, Yung Dai Clayton, Yanhong Du, Cheng Chen, Jacob E Friedman, Shelly C Lu, Wen-Xing Ding, Tiangang Li.
      Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.
    DOI:  https://doi.org/10.1038/s41467-022-33465-9
  44. Cell Rep. 2022 Sep 27. pii: S2211-1247(22)01250-5. [Epub ahead of print]40(13): 111409
    Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.
    Alexander S Dowdell, Ian M Cartwright, David A Kitzenberg, Rachael E Kostelecky, Omemh Mahjoob, Bejan J Saeedi, Nichole Welch, Louise E Glover, Sean P Colgan.
      The intestinal mucosa exists in a state of "physiologic hypoxia," where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed "xenophagy." Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.
    Keywords:  CP: Immunology; CP: Microbiology; autophagy; chromatin immunoprecipitation; colitis; colonoid; hypoxia; inflammation; innate immunity; mucosa; neutrophil; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2022.111409
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