bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021‒09‒19
thirty-nine papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2021 Sep 14. 1-3
      The mammalian ULK kinase complex is the most upstream component in the macroautophagy/autophagy signaling pathway. ULK1 and homolog ULK2, the sole serine/threonine kinases in autophagy, transduce an array of autophagy-inducing stimuli to downstream autophagic machinery, regulating autophagy from autophagosome initiation to fusion of autophagosomes with lysosomes. ULK signaling is also implicated in a diverse array of non-canonical processes from necroptosis to ER-Golgi trafficking to stress granule clearance. However, the exact mechanisms by which ULK regulates these diverse processes remain largely unknown. Most notably, the number of validated ULK substrates is surprisingly low. Our study identifies new ULK substrates from a wide array of protein families and signaling pathways and supports an expanded range of physiological roles for the ULKs. We further characterize several new substrates, including the PIK3C3/VPS34-containing complex subunit PIK3R4/VPS15 and the AMPK component PRKAG2. Finally, by analyzing PIK3R4/VPS15-deficient models we discover novel aspects of ULK signaling with potential relevance in selective autophagy.
    Keywords:  AMPK; PIK3R4; PRKAG2; VPS15; VPS34; p150
  2. Cell Stress. 2021 Sep;5(9): 128-142
      The yeast Atg8 protein and its paralogs in mammals, mammalian Atg8s (mAtg8s), have been primarily appreciated for their participation in autophagy. However, lipidated mAtg8s, including the most frequently used autophagosomal membrane marker LC3B, are found on cellular membranes other than autophagosomes. Here we put forward a hypothesis that the lipidation of mAtg8s, termed 'Atg8ylation', is a general membrane stress and remodeling response analogous to the role that ubiquitylation plays in tagging proteins. Ubiquitin and mAtg8s are related in sequence and structure, and the lipidation of mAtg8s occurs on its C-terminal glycine, akin to the C-terminal glycine of ubiquitin. Conceptually, we propose that mAtg8s and Atg8ylation are to membranes what ubiquitin and ubiquitylation are to proteins, and that, like ubiquitylation, Atg8ylation has a multitude of downstream effector outputs, one of which is autophagy.
    Keywords:  ampk; atg8; autophagy; endosome; exosomes; galectin; lap; lc3; lysosome; microvesicles; mtor; secretion; secretory autophagy; tfeb; ubiquitin; ubiquitylation; unconventional secretion
  3. Int J Biol Sci. 2021 ;17(13): 3634-3645
      Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy in humans, and new therapeutic targets are urgently needed. Yes-associated protein (YAP) plays a significant role in cancer progression. Autophagy is also closely associated with various human cancers. However, the interplay between YAP and autophagy in PDAC remains poorly understood. In this study, we found that YAP was upregulated and activated in PDAC. Further analysis revealed that there is a YAP-autophagy feedback loop in pancreatic cancer. Mechanistically, YAP activates autophagy by promoting Atg5 transcription via TEAD1-mediated binding, while autophagy negatively regulates YAP through autophagic degradation. The hyperactivation of YAP in PDAC unbalances the YAP-autophagy circuit and promotes cancer progression. Inhibition of autophagy enhances the oncogenic activity of YAP in PDAC. The autophagy activator rapamycin promotes the antitumor effect of verteporfin, a YAP inhibitor. Therefore, our study elucidated the interaction between YAP and autophagy in PDAC and our results suggest that targeting the YAP-autophagy circuit may be a new therapeutic strategy for pancreatic cancer.
    Keywords:  Atg5; YAP; autophagy; pancreatic cancer; verteporfin
  4. Autophagy. 2021 Sep 17. 1-23
      Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression.ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4.
    Keywords:  Autophagic cell death; EGFR; ERBB2; cell growth; tumorsphere
  5. Biol Open. 2021 Sep 17. pii: bio.058736. [Epub ahead of print]
      There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.
    Keywords:  Autophagy; Cell biology; GRASP55
  6. Elife. 2021 09 14. pii: e70871. [Epub ahead of print]10
      The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.
    Keywords:  DEPTOR; cancer; cryo-EM; human; mTOR; metabolism; molecular biophysics; signaling; structural biology
  7. Cell Death Dis. 2021 Sep 17. 12(10): 854
      Dopaminergic (DA) cell death in Parkinson's disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles - a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD.
  8. PLoS Comput Biol. 2021 Sep 16. 17(9): e1008513
      The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GβL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.
  9. Elife. 2021 09 14. pii: e68799. [Epub ahead of print]10
      The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.
    Keywords:  DEPTOR; biochemistry; cancer; chemical biology; cryo-EM; human; mTOR; molecular biophysics; partial inhibition; signal transduction; structural biology
  10. Cell Death Differ. 2021 Sep 12.
      Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases.
  11. Neuroscience. 2021 Sep 09. pii: S0306-4522(21)00455-3. [Epub ahead of print]
      Chaperones and cellular degradative mechanisms modulate Tau aggregation. During aging and neurodegenerative disorders, the cellular proteostasis is disturbed due to impaired protective mechanisms. This results in accumulation of aberrant Tau aggregates in the neuron that leads to microtubule destabilization and neuronal degeneration. The intricate mechanisms to prevent Tau aggregation involve chaperones, autophagy, and proteasomal system have gained main focus about concerning to therapeutic intervention. However, the thorough understanding of other key proteins, such as Valosin-containing protein (VCP), is limited. In various neurodegenerative diseases, the chaperone-like activity of VCP is involved in preventing protein aggregation and mediating the degradation of aberrant proteins by proteasome and autophagy. In the case of Tau aggregation associated with Alzheimer's disease, the importance of VCP is poorly understood. VCP is known to co-localize with Tau, and alterations in VCP cause aberrant accumulation of Tau. Nevertheless, the direct mechanism of VCP in altering Tau aggregation is not known. Hence, we speculate that VCP might be one of the key modulators in preventing Tau aggregation and can disintegrate Tau aggregates by directing its clearance by autophagy.
    Keywords:  Tau aggregates; Valosin-containing protein; autophagy; segregase; ubiquitin-proteasome system
  12. Cell Physiol Biochem. 2021 Sep 15. 55(S4): 68-95
      Protein homeostasis strongly depends on the targeted and selective removal of unneeded or flawed proteins, of protein aggregates, and of damaged or excess organelles by the two main intracellular degradative systems, namely the ubiquitin proteasomal system (UPS) and the autophagosomal lysosomal system. Despite representing completely distinct mechanisms of degradation, which underlie differing regulatory mechanisms, growing evidence suggests that the UPS and autophagy strongly interact especially in situations of overwhelming and impairment, and that both are involved in podocyte proteostasis and in the pathogenesis of podocyte injury. The differential impact of autophagy and the UPS on podocyte biology and on podocyte disease development and progression is not understood. Recent advances in understanding the role of the UPS and autophagy in podocyte biology are reviewed here.
    Keywords:  Podocyte; Autophagy; Lysosome; Proteasome; Ubiquitin proteasome system; Crosstalk; Diabetic nephropathy; Membranous nephropathy; Focal segmental glomerulosclerosis; Lupus nephritis; Ageing
  13. Neurochem Int. 2021 Sep 14. pii: S0197-0186(21)00233-3. [Epub ahead of print] 105187
      Hydrogen sulfide (H2S) serves as a neuromodulator and regulator of neuroinflammation. It is reported to be therapeutic for Parkinson's disease (PD) animal and cellular models. However, whether it affects α-synuclein accumulation in dopaminergic cells, the key pathological feature in PD, is poorly understood. In this study we reported that exogenous H2S donors NaHS and GYY4137 (GYY) enhanced the autophagy activity, as indicated by the increases of autophagy marker LC3-II expression and LC3 dots formation even during lysosome inhibition in dopaminergic cell lines and HEK293 cells. The enhancement of H2S donors on autophagic flux was mediated by adenosine 5'-monophosphate-activated protein kinase (AMPK)-dependent mammalian target of rapamycin (mTOR) inhibition, as H2S donors activated AMPK but reduced the mTOR activity and H2S donors-induced LC3-II increase was diminished by mTOR activator. Moreover, point mutation of Cys302 into alanine (C302A) in AMPKα2 subunit abolished the AMPK activation and mTOR inhibition, as well as autophagic flux increase elicited by NaHS. Interestingly, NaHS triggered AMPK S-sulfuration, which was not observed in AMPK C302A-transfected cells. Further, NaHS was able to attenuate α-synuclein accumulation in a cellular model induced by dopamine oxidized metabolite 3, 4-dihydroxyphenylacetaldehyde (DOPAL), and this effect was interfered by autophagy inhibitor wortmannin and also eliminated in AMPK Cys302A-transfected cells. In sum, the findings identified a role of Cys302 S-sulfuration in AMPK activation induced by exogenous H2S and demonstrated that H2S donors could enhance the autophagic flux via AMPK-mTOR signaling and thus reduce α-synuclein accumulation in vitro.
    Keywords:  AMPK; Autophagy; Hydrogen sulfide; S-sulfuration; α-synuclein
  14. Commun Biol. 2021 Sep 17. 4(1): 1093
      TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells.
  15. FEBS J. 2021 Sep 15.
      Parkinson's disease (PD) is a progressive neurodegenerative disorder induced by the loss of dopaminergic neurons in midbrain. The mechanism of neurodegeneration is associated with aggregation of misfolded proteins, oxidative stress and mitochondrial dysfunction. Considering this, the process of removal of unwanted organelles or proteins by autophagy is vitally important in neurons, and activation of these processes could be protective in PD. Short time acidification of the cytosol can activate mitophagy and autophagy. Here, we used sodium pyruvate and sodium lactate to induce changes in intracellular pH in human fibroblasts with PD mutations (Pink1, Pink1/Park2, α-synuclein triplication, A53T). We have found that both lactate and pyruvate in millimolar concentrations can induce a short time acidification of the cytosol in these cells. This induced activation of mitophagy and autophagy in control and PD fibroblasts and protected against cell death. Importantly, application of lactate to acute brain slices of WT and Pink1 KO mice also induced a reduction of pH in neurons and astrocytes that increased the level of mitophagy. Thus, acidification of the cytosol by compounds which play an important role in cell metabolism can also activate mitophagy and autophagy and protect cells in the familial form of PD.
    Keywords:  Parkinson’s disease; autophagy; lactate; mitophagy; pyruvate
  16. J Cell Sci. 2021 Sep 16. pii: jcs.254631. [Epub ahead of print]
      In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMCs homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though, the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMCs proliferation and migration impacting atherosclerosis. In sum, this work shows that: 1) ChA is sufficient to induce lysosomal dysfunction in VSMCs; 2) In ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered; and 3) Recombinant LAL can be a therapeutic approach for lysosomal dysfunction.
    Keywords:  Atherosclerosis; Lysosome adaptation; Lysosome dysfunction; Oxidized lipids; Vascular smooth muscle cells
  17. Autophagy. 2021 Sep 14. 1-21
      Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.
    Keywords:  IGF1R; IGFBP3; LC3; MTORC1; SQSTM1/p62; stroke
  18. Autophagy. 2021 Sep 14. 1-13
      We recently identified FAM134B2, which is an N-terminal truncated reticulophagy receptor highly induced by starvation such as fasting of mice and treatment of mammalian cells with a starvation medium that does not contain amino acids, glucose and growth factors. However, which starvation signal mediates the induction of FAM134B2 is still obscure. In this study, we found that amino acid deficiency (AAD) could mimic the starvation condition to induce FAM134B2 expression. Unexpectedly, EIF2AK4/GCN2-mediated integrated signal response (ISR) and MTOR (mechanistic target of rapamycin kinase) signals, which constitute two major signaling pathways that respond to AAD, did not contribute to AAD-induced FAM134B2 induction. mRNA-seq and siRNA screenings identified two ISR-independent transcription factors, MEF2D (myocyte enhancer factor 2D) and NR4A1 (nuclear receptor subfamily 4 group A member 1), involved in AAD-induced FAM134B2 expression. AAD induces MEF2D, resulting in the induction of NR4A1, which in turn induces FAM134B2-mediated reticulophagy to maintain intracellular amino acid levels. In conclusion, the MEF2D-NR4A1-FAM134B2 cascade is a critical signal in amino acid homeostasis.
    Keywords:  Amino acid deficiency; FAM134B2; MEF2D; NR4A1; reticulophagy
  19. J Biol Chem. 2021 Sep 11. pii: S0021-9258(21)00993-5. [Epub ahead of print] 101191
      Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease (PD) and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high throughput screen (qHTS) of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and preventing PD pathology.
    Keywords:  A-443654; AKT; Parkinson disease; SNCA; STAU1; alpha‐synuclein (α‐synuclein); autophagy; endoplasmic reticulum stress (ER stress); high‐throughput screening (HTS); staufen1
  20. Front Immunol. 2021 ;12 705436
      Autophagy is an evolutionary conserved catabolic pathway that ensures the degradation of intracellular components. The autophagic pathway is regulated by autophagy-related (Atg) proteins that govern formation of double-membraned vesicles called autophagosomes. Autophagy deficiency in regulatory T (Treg) cells leads to increased apoptosis of these cells and to the development of autoimmune disorders, predominantly characterized by intestinal inflammation. Recently, RORγt-expressing Treg cells have been identified as key regulators of gut homeostasis, preventing intestinal immunopathology. To study the role of autophagy in RORγt+ Foxp3+ Treg cells, we generated mice lacking the essential component of the core autophagy machinery Atg5 in Foxp3+ cells. Atg5 deficiency in Treg cells led to a predominant intestinal inflammation. While Atg5-deficient Treg cells were reduced in peripheral lymphoid organs, the intestinal RORγt+ Foxp3+ subpopulation of Treg cells was most severely affected. Our data indicated that autophagy is essential to maintain the intestinal RORγt+ Foxp3+ Treg population, thereby protecting the mice from gut inflammatory disorders.
    Keywords:  Atg5; RORγt+ Foxp3+ Treg cells; autophagy; inflammation; intestinal homeostasis
  21. Invest Ophthalmol Vis Sci. 2021 Sep 02. 62(12): 15
      Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice.Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ.
    Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss.
    Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.
  22. Anal Chem. 2021 Sep 14.
      Autophagy plays a critical role in many vitally important physiological and pathological processes, such as the removal of damaged and aged organelles and redundant proteins. Although autophagy is mainly a protective process for cells, it can also cause cell death. In this study, we employed in situ and ex situ surface-enhanced Raman scattering (SERS) spectroscopies to obtain chemical information of lysosomes of HepG2 cells. Results reveal that the SERS profiles of the isolated lysosomes are different from the in situ spectra, indicating that lysosomes lie in different microenvironments in these two cases. We further investigated the molecular changes of isolated lysosomes according to the autophagy induced by starvation via ex situ SERS. During autophagy, the conformation of proteins and the structures of lipids have been affected, and autophagy-related molecular evidence is given for the first time in the living lysosomes. We expect that this study will provide a reference for understanding the cell autophagy mechanism.
  23. Cancer Drug Resist. 2021 ;4 85-95
      Wild-type p53 is a stress-responsive transcription factor and a potent tumor suppressor. P53 inhibits the growth of incipient cancer cells by blocking their proliferation or inducing their death through apoptosis. Autophagy is a self-eating process that plays a key role in response to stress. During autophagy, organelles and other intracellular components are degraded in autophagolysosomes and the autophagic breakdown products are recycled into metabolic and energy producing pathways needed for survival. P53 can promote or inhibit autophagy depending on its subcellular localization, mutation status, and the level of stress. Blocking autophagy has been reported in several studies to increase p53-mediated apoptosis, revealing that autophagy can influence cell-fate in response to activated p53 and is a potential target to increase p53-dependent tumor suppression.
    Keywords:  Autophagy; histone methylation; metabolism
  24. Commun Biol. 2021 Sep 14. 4(1): 1076
      Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.
  25. J Immunol. 2021 Sep 17. pii: ji2100463. [Epub ahead of print]
      Thymic epithelial cells (TECs) are critical for the development and generation of functionally competent T cells. Until now, the mechanism that regulates the survival of TECs is poorly understood. In the current study, we found that Tsc1 controls the homeostasis of medullary TECs (mTECs) by inhibiting lysosomal-mediated apoptosis pathway in mice. TEC-specific deletion of Tsc1 predominately decreased the cell number of mTECs and, to a lesser content, affected the development cortical TECs. The defect of mTECs caused by Tsc1 deficiency in mice impaired thymocyte development and peripheral T cell homeostasis. Mechanistically, Tsc1 deficiency did not affect the cell proliferation of mTECs but increased the apoptosis of mTECs significantly. RNA-sequencing analysis showed that pathways involved in lysosomal biogenesis, cell metabolism, and apoptosis were remarkably elevated in Tsc1-deficient mTECs compared with their wild-type counterparts. Tsc1-deficient mTECs exhibited overproduction of reactive oxygen species and malfunction of lysosome, with lysosome membrane permeabilization and the release of cathepsin B and cathepsin L to the cytosol, which then lead to Bid cleaved into active truncated Bid and subsequently intrinsic apoptosis. Finally, we showed that the impaired development of mTECs could be partially reversed by decreasing mTORC1 activity via haploinsufficiency of Raptor Thus, Tsc1 is essential for the homeostasis of mTECs by inhibiting lysosomal-mediated apoptosis through mTORC1-dependent pathways.
  26. Surg Oncol Clin N Am. 2021 Oct;pii: S1055-3207(21)00041-7. [Epub ahead of print]30(4): 709-718
      This article provides a brief review of the therapeutic opportunity of inhibiting autophagy in pancreatic cancer. The autophagic process, importance of autophagy in pancreatic cancer, relevant clinical trials, and new agents in preclinical and clinical development are discussed.
    Keywords:  Autophagy; Autophagy clinical trials; Pancreatic cancer; Pancreatic cancer clinical trials; Therapeutics
  27. Aging (Albany NY). 2021 Sep 14. 13(undefined):
      Most EEC cases are associated with activities of the mTOR pathway, which regulates protein synthesis, cell growth and autophagy. While Up-Frameshift 1(UPF1) is a key protein factor in the nonsense-mediated mRNA degradation pathway (NMD), its role in carcinogenesis of EEC remains unclear. In this study, we first evaluated the expression level of UPF1 in EEC tissues and cell lines. Then, we investigated the effect of UPF1 on cellular function and mTOR signaling pathway; these effects were further validated in vivo. Finally, its effect on autophagy was evaluated by western blot and GFP-mRFP-LC3 staining. UPF1 expression in the EEC tissue samples was significantly higher than that of matched normal tissue samples. Overexpression of UPF1 promoted migration and invasion of EEC cells. Conversely, depletion of UPF1 suppressed migration and invasion of EEC cells. In addition, overexpression of UPF1 increased the in vivo growth of our EEC xenograft tumors. Finally, UPF1 increased the activity of the mTOR/P70S6K/4EBP1 signaling pathway and inhibited autophagy in EEC cells. These findings suggest that UPF1 functions as an oncogene to promote EEC carcinogenesis. Our findings propose UPF1 as a new potential therapeutic target for EEC.
    Keywords:  UPF1; autophagy; endometrioid endometrial carcinoma; mTOR
  28. Cell Biol Toxicol. 2021 Sep 15.
      The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. • Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. • Octyl syringate destabilizes the lysosomal function. • Octyl syringate blocks the autophagic flux. • Octyl syringate is a potential candidate compound for cancer therapy.
    Keywords:  Autophagic flux; Lysosomal dysfunction; Lysosome target therapy; Non-apoptotic cell death; Octyl syringate
  29. Aging Cell. 2021 Sep 14. e13471
      During aging, brain performances decline. Cellular senescence is one of the aging drivers and a key feature of a variety of human age-related disorders. The transcriptional repressor RE1-silencing transcription factor (REST) has been associated with aging and higher risk of neurodegenerative disorders. However, how REST contributes to the senescence program and functional impairment remains largely unknown. Here, we report that REST is essential to prevent the senescence phenotype in primary mouse neurons. REST deficiency causes failure of autophagy and loss of proteostasis, increased oxidative stress, and higher rate of cell death. Re-establishment of autophagy reverses the main hallmarks of senescence. Our data indicate that REST has a protective role in physiological aging by regulating the autophagic flux and the senescence program in neurons, with implications for neurological disorders associated with aging.
    Keywords:  REST/NRSF; autophagy; mitochondria; neurons; oxidative stress; rapamycin; senescence; trehalose
  30. Cell Mol Life Sci. 2021 Sep 15.
      Mitochondria-the intracellular powerhouse in which nutrients are converted into energy in the form of ATP or heat-are highly dynamic, double-membraned organelles that harness a plethora of cellular functions that sustain energy metabolism and homeostasis. Exciting new discoveries now indicate that the maintenance of this ever changing and functionally pleiotropic organelle is particularly relevant in terminally differentiated cells that are highly dependent on aerobic metabolism. Given the central role in maintaining metabolic and physiological homeostasis, dysregulation of the mitochondrial network might therefore confer a potentially devastating vulnerability to high-energy requiring cell types, contributing to a broad variety of hereditary and acquired diseases. In this Review, we highlight the biological functions of mitochondria-localized enzymes from the perspective of understanding-and potentially reversing-the pathophysiology of inherited disorders affecting the homeostasis of the mitochondrial network and cellular metabolism. Using methylmalonic acidemia as a paradigm of complex mitochondrial dysfunction, we discuss how mitochondrial directed-signaling circuitries govern the homeostasis and physiology of specialized cell types and how these may be disturbed in disease. This Review also provides a critical analysis of affected tissues, potential molecular mechanisms, and novel cellular and animal models of methylmalonic acidemia which are being used to develop new therapeutic options for this disease. These insights might ultimately lead to new therapeutics, not only for methylmalonic acidemia, but also for other currently intractable mitochondrial diseases, potentially transforming our ability to regulate homeostasis and health.
    Keywords:  Cell damage; Inherited metabolic diseases; Metabolism; Mitochondria; Mitophagy; Oxidative stress
  31. Dev Cell. 2021 Sep 08. pii: S1534-5807(21)00678-X. [Epub ahead of print]
      Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.
    Keywords:  Atg6/Beclin 1; DNA damage response; Gcn2; Rad53; TORC1; amino acids; endosomal trafficking; phosphatydilinositol 3-phosphate; replication stress
  32. Int J Oncol. 2021 Oct;pii: 83. [Epub ahead of print]59(4):
      Mechanistic target of rapamycin (mTOR), which functions via two multiprotein complexes termed mTORC1 and mTORC2, is positioned in the canonical phosphoinositide 3‑kinase‑related kinase (PI3K)/AKT (PI3K/AKT) pathways. These complexes exert their actions by regulating other important kinases, such as 40S ribosomal S6 kinases (S6K), eukaryotic translation initiation factor 4E (elF4E)‑binding protein 1 (4E‑BP1) and AKT, to control cell growth, proliferation, migration and survival in response to nutrients and growth factors. Glioblastoma (GB) is a devastating form of brain cancer, where the mTOR pathway is deregulated due to frequent upregulation of the Receptor Tyrosine Kinase/PI3K pathways and loss of the tumor suppressor phosphatase and tensin homologue (PTEN). Rapamycin and its analogs were less successful in clinical trials for patients with GB due to their incomplete inhibition of mTORC1 and the activation of mitogenic pathways via negative feedback loops. Here, the effects of selective ATP‑competitive dual inhibitors of mTORC1 and mTORC2, Torin1, Torin2 and XL388, are reported. Torin2 exhibited concentration‑dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E‑BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell migration, proliferation and S‑phase entry. Torin1 demonstrated similar effects, but only at higher doses. XL388 suppressed cell proliferation at a higher dose, but failed to inhibit cell migration. Treatment with Torin1 suppressed phosphorylation of proline rich AKT substrate of 40 kDa (PRAS40) at Threonine 246 (PRAS40Thr246) whereas Torin2 completely abolished it. XL388 treatment suppressed the phosphorylation of PRAS40Thr246 only at higher doses. Drug resistance analysis revealed that treatment of GB cells with XL388 rendered partial drug resistance, which was also seen to a lesser extent with rapamycin and Torin1 treatments. However, treatment with Torin2 completely eradicated the tumor cell population. These results strongly suggest that Torin2, compared to Torin1 or XL388, is more effective in suppressing mTORC1 and mTORC2, and therefore in the inhibition of the GB cell proliferation, dissemination and in overcoming resistance to therapy. These findings underscore the significance of Torin2 in the treatment of GB.
    Keywords:  PRAS40; Torin1; Torin2; glioblastoma; mTOR; mTOR inhibitors
  33. Biochem Biophys Res Commun. 2021 Sep 06. pii: S0006-291X(21)01240-7. [Epub ahead of print]578 7-14
      Ubiquitin-conjugating enzyme E2S (UBE2S), an important E2 enzyme in the process of ubiquitination, has exhibited oncogenic activities in various malignant tumors. However, it remains unknown whether UBE2S plays a role in urinary bladder cancer (UBC) development. In the current study, our data confirmed UBE2S upregulation in UBC. In vitro and in vivo experiments demonstrated that UBE2S knockdown resulted in attenuated proliferation and enhanced apoptosis, which was inverse to the phenotypes with UBE2S overexpression. Gain and loss of function assays confirmed that UBE2S exerts oncogenic activities in UBC by mediating the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Furthermore, we discovered that this UBE2S-modulated carcinogenic mechanism was in the consequence of directly targeting tuberous sclerosis 1 (TSC1), which is the upstream inhibitor of mTOR signaling for ubiquitous degradation. Taken together, this study demonstrated that UBE2S is a carcinogen in UBC and promotes UBC progression by ubiquitously degrading TSC1. This consequently mediates the activation of the mTOR pathway, suggesting a potential therapeutic regimen for UBC by targeting the newly identified UBE2S/TSC1/mTOR axis.
    Keywords:  Bladder cancer; TSC1; UBE2S; Ubiquitination
  34. J Immunother Cancer. 2021 Sep;pii: e002954. [Epub ahead of print]9(9):
      BACKGROUND: Metformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated.METHODS: MitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8+ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer.
    RESULTS: Met administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs.
    CONCLUSIONS: We found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs' proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.
    Keywords:  adaptive immunity; cd8-positive T-lymphocytes; immunomodulation; lymphocytes; tumor microenvironment; tumor-infiltrating
  35. FEBS J. 2021 Sep 12.
      Mitochondrial dysfunction is increasingly appreciated as a central contributor to human disease. Oxidative metabolism at the mitochondrial respiratory chain produces ATP and is intricately tied to redox homeostasis and biosynthetic pathways. Metabolic stress arising from genetic mutations in mitochondrial genes and environmental factors such as malnutrition or overnutrition is perceived by the cell and leads to adaptive and maladaptive responses that can underlie pathology. Here, we will outline cellular sensors that react to alterations in energy production, organellar redox, and metabolites stemming from mitochondrial disease (MD) mutations. MD is a heterogenous group of disorders primarily defined by defects in mitochondrial oxidative phosphorylation from nuclear or mitochondrial-encoded gene mutations. Pre-clinical therapies that improve fitness of MD mouse models have been recently identified. Targeting metabolic/energetic deficiencies, maladaptive signaling processes, and hyper-oxygenation of tissues are all strategies aside from direct genetic approaches that hold therapeutic promise. A further mechanistic understanding of these curative processes as well as the identification of novel targets will significantly impact mitochondrial biology and disease research.
    Keywords:  Mitochondrial dysfunction; hypoxia; mTORC1; metabolism; mitochondrial disease; mitochondrial signaling; oxidative stress; reactive oxygen species; redox homeostasis
  36. Brain. 2021 Sep 17. pii: awab300. [Epub ahead of print]
      Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation into non-canonical dipeptide repeats, which can then accumulate as aggregates and contribute to these pathologies. There are five different dipeptide repeat proteins (polyGA, polyGR, polyPR, polyPA and polyGP), some of which are known to be neurotoxic. In the present study, we used BioID2 proximity labelling to identify the interactomes of all five dipeptide repeat proteins consisting of 125 repeats each. We identified 113 interacting partners for polyGR, 90 for polyGA, 106 for polyPR, 25 for polyPA, and 27 for polyGP. Gene Ontology enrichment analysis of the proteomic data revealed that these target interaction partners are involved in a variety of functions, including protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes, and RNA-binding. Using autopsy brain tissue from patients with C9orf72 expansion complemented with cell culture analysis, we evaluated the interactions between polyGA and valosin-containing protein (VCP). Functional analysis of this interaction revealed sequestration of VCP with polyGA aggregates, altering levels of soluble VCP protein. VCP also functions in autophagy processes, and consistent with this, we observed altered autophagy in cells expressing polyGA. We also observed altered co-localisation of polyGA aggregates and p62 in cells depleted of the VCP protein. Altogether these data suggest that sequestration of VCP with polyGA aggregates contributes to the loss of VCP function, and consequently to alterations in autophagy processes in C9orf72 expansion disorders.
    Keywords:  ALS; C9orf72; VCP; autophagy
  37. Autophagy. 2021 Sep 14. 1-16
      Mutations in the macroautophagy/autophagy gene EPG5 are responsible for Vici syndrome, a human genetic disease characterized by combined immunodeficiency. Previously, we found that epg5-/- mice exhibit hyperinflammation in the lungs mediated by IL1B/IL-1β and TNF/TNFα, resulting in resistance to influenza. Here, we find that disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. Gene expression analysis reveals IFNL/IFN-λ responsive genes as a key alteration. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free mouse studies indicate Epg5-associated inflammation of both the intestine and lung is microbiota-independent. Genetic studies support IFNL signaling as the primary mediator of resistance to enteric viruses, but not of microbial dysbiosis, in epg5-/- mice. This study unveils an important role, unexpectedly independent of the microbiota, for autophagy gene Epg5 in host organism protection by modulating intestinal IFNL responses.Abbreviations: CTNNB1: catenin (cadherin associated protein), beta 1; DAPI: 4',6-diamidino-2-phenylindole; EPG5: ectopic P-granules autophagy protein 5 homolog (C. elegans); FT: fecal transplant; IFI44: interferon-induced protein 44; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; IFNG/IFN-γ: interferon gamma; IFNL/IFN-λ: interferon lambda; IFNLR1: interferon lambda receptor 1; IL1B/IL-1β: interleukin 1 beta; ISG: interferon stimulated gene; GF: germ-free; LEfSe: linear discriminant analysis effect size; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MNoV: murine norovirus; MX2: MX dynamin-like GTPase 2; OAS1A: 2'-5' oligoadenylate synthetase 1A; RV: rotavirus; SPF: specific-pathogen free; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TNF/TNFα: tumor necrosis factor.
    Keywords:  Antiviral; Epg5; IFN-λ; microbiota; norovirus; rotavirus
  38. Autophagy. 2021 Sep 15. 1-19
      Epidemiological and clinical studies have shown that exposure to particulate matter (PM) is associated with an increased incidence of lung cancer and metastasis. However, the underlying mechanism remains unclear. Here, we demonstrated the central role of PM-induced neutrophil recruitment in promoting lung cancer metastasis. We found that reactive oxygen species (ROS)-mediated alveolar epithelial macroautophagy/autophagy was essential for initiating neutrophil chemotaxis and pre-metastatic niche formation in the lungs in response to PM exposure. During PM-induced autophagy, the E3 ubiquitin ligase TRIM37 was degraded and protected TRAF6 from proteasomal degradation in lung epithelial cells, which promoted the NFKB-dependent production of chemokines to recruit neutrophils. Importantly, ROS blockade, autophagy inhibition or TRAF6 knockdown abolished PM-induced neutrophil recruitment and lung metastasis enhancement. Our study indicates that host lung epithelial cells and neutrophils coordinate to promote cancer metastasis to the lungs in response to PM exposure and provides ideal therapeutic targets for metastatic progression.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ATII: alveolar type II; Cho-Traf6 siRNA: 5'-cholesterol-Traf6 siRNA; EMT: epithelial-mesenchymal transition; HBE: human bronchial epithelial; HCQ: hydroxychloroquine; MAPK: mitogen-activated protein kinase; NAC: N-acetyl-L-cysteine; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NS: normal saline; PM: particulate matter; ROS: reactive oxygen species; TRAF6: TNF receptor-associated factor 6; TRIM37: tripartite motif-containing 37.
    Keywords:  Autophagy; NFKB; ROS; TRAF6; TRIM37; lung metastasis; neutrophils; particulate matter
  39. Biochem J. 2021 Sep 13. pii: BCJ20210375. [Epub ahead of print]
      Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.
    Keywords:  Kinase; Kinase inhibitor; Parkinsons disease; Rab GTPase; leucine rich repeat kinase