bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021‒02‒07
thirty-one papers selected by
Viktor Korolchuk
Newcastle University

  1. Mol Cell. 2021 Jan 28. pii: S1097-2765(21)00009-5. [Epub ahead of print]
      Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis-challenging conditions in human cell lines. We identified various autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow dissection of the proteostasis contribution of autophagy to numerous individual proteins.
    Keywords:  APEX2; SQSTM1/p62; TOLLIP; aggrephagy; autophagosomes; autophagy; endosomal microautophagy; proteostasis imbalance; proximity labeling; selective autophagy receptors
  2. J Biol Chem. 2020 Jan 03. pii: S0021-9258(17)49565-2. [Epub ahead of print]295(1): 263-274
      Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.
    Keywords:  amino acid; cancer biology; cancer therapy; growth factor; lysosome; mTOR complex (mTORC); phosphatidic acid; phospholipase D; phospholipid vesicle
  3. Cells. 2021 Feb 02. pii: 305. [Epub ahead of print]10(2):
      Cells have developed response systems to constantly monitor environmental changes and accordingly adjust growth, differentiation, and cellular stress programs. The evolutionarily conserved, nutrient-responsive, mechanistic target of rapamycin signaling (mTOR) pathway coordinates basic anabolic and catabolic cellular processes such as gene transcription, protein translation, autophagy, and metabolism, and is directly implicated in cellular and organismal aging as well as age-related diseases. mTOR mediates these processes in response to a broad range of inputs such as oxygen, amino acids, hormones, and energy levels, as well as stresses, including DNA damage. Here, we briefly summarize data relating to the interplays of the mTOR pathway with DNA damage response pathways in fission yeast, a favorite model in cell biology, and how these interactions shape cell decisions, growth, and cell-cycle progression. We, especially, comment on the roles of caffeine-mediated DNA-damage override. Understanding the biology of nutrient response, DNA damage and related pharmacological treatments can lead to the design of interventions towards improved cellular and organismal fitness, health, and survival.
    Keywords:  Rad3; TORC1; TORC2; caffeine; nutrients; rapamycin
  4. Elife. 2021 Feb 03. pii: e60969. [Epub ahead of print]10
      Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.
    Keywords:  Autophagy; GATOR1; GATOR2; Gcn2; Rag GTPase; S. pombe; TORC1; cell biology
  5. J Cell Sci. 2021 Feb 03. pii: jcs.256255. [Epub ahead of print]
      Mitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses-embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.
    Keywords:  Autophagy; Fasting; Hypoxia; Lysosome; Mitochondria
  6. J Biol Chem. 2020 May 22. pii: S0021-9258(17)50273-2. [Epub ahead of print]295(21): 7418-7430
      Autophagy and lysosomal activities play a key role in the cell by initiating and carrying out the degradation of misfolded proteins. Transcription factor EB (TFEB) functions as a master controller of lysosomal biogenesis and function during lysosomal stress, controlling most but, importantly, not all lysosomal genes. Here, we sought to better understand the regulation of lysosomal genes whose expression does not appear to be controlled by TFEB. Sixteen of these genes were screened for transactivation in response to diverse cellular insults. mRNA levels for lysosomal-associated membrane protein 3 (LAMP3), a gene that is highly up-regulated in many forms of cancer, including breast and cervical cancers, were significantly increased during the integrated stress response, which occurs in eukaryotic cells in response to accumulation of unfolded and misfolded proteins. Of note, results from siRNA-mediated knockdown of activating transcription factor 4 (ATF4) and overexpression of exogenous ATF4 cDNA indicated that ATF4 up-regulates LAMP3 mRNA levels. Finally, ChIP assays verified an ATF4-binding site in the LAMP3 gene promoter, and a dual-luciferase assay confirmed that this ATF4-binding site is indeed required for transcriptional up-regulation of LAMP3. These results reveal that ATF4 directly regulates LAMP3, representing the first identification of a gene for a lysosomal component whose expression is directly controlled by ATF4. This finding may provide a key link between stresses such as accumulation of unfolded proteins and modulation of autophagy, which removes them.
    Keywords:  activating transcription factor 4 (ATF4); autophagy; cell stress; eukaryotic initiation factor 2 (eIF2); lysosomal-associated membrane protein 3 (LAMP3); lysosome; mammalian target of rapamycin complex 1 (mTORC1); protein misfolding; transcription factor EB (TFEB); unfolded protein response (UPR)
  7. J Lipid Res. 2020 Dec;pii: S0022-2275(20)60023-2. [Epub ahead of print]61(12): 1617-1628
      The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.
    Keywords:  Mycobacterium tuberculosis; adenosine 5′-monophosphate-activated protein kinase-mechanistic target of rapamycin complex 1-transcription factor EB axis; immunology; lipids; macrophages/monocytes; mechanistic target of rapamycin complex 1 regulation; statins
  8. Front Cell Dev Biol. 2020 ;8 611938
      Maintenance of neuronal homeostasis is a challenging task, due to unique cellular organization and bioenergetic demands of post-mitotic neurons. It is increasingly appreciated that impairment of mitochondrial homeostasis represents an early sign of neuronal dysfunction that is common in both age-related neurodegenerative as well as in neurodevelopmental disorders. Mitochondrial selective autophagy, known as mitophagy, regulates mitochondrial number ensuring cellular adaptation in response to several intracellular and environmental stimuli. Mounting evidence underlines that deregulation of mitophagy levels has an instructive role in the process of neurodegeneration. Although mitophagy induction mediates the elimination of damaged mitochondria and confers neuroprotection, uncontrolled runaway mitophagy could reduce mitochondrial content overstressing the remaining organelles and eventually triggering neuronal cell death. Unveiling the molecular mechanisms of neuronal mitophagy and its intricate role in neuronal survival and cell death, will assist in the development of novel mitophagy modulators to promote cellular and organismal homeostasis in health and disease.
    Keywords:  aging; cell death; energy metabolism; homeostasis; mitochondria; mitophagy; neurodegeneration; neuroprotection
  9. Oncogene. 2021 Feb 02.
      Previous work has suggested androgen receptor (AR) signaling mediates prostate cancer progression in part through the modulation of autophagy. However, clinical trials testing autophagy inhibition using chloroquine derivatives in men with castration-resistant prostate cancer (CRPC) have yet to yield promising results, potentially due to the side effects of this class of compounds. We hypothesized that identification of the upstream activators of autophagy in prostate cancer could highlight alternative, context-dependent targets for blocking this important cellular process during disease progression. Here, we used molecular, genetic, and pharmacological approaches to elucidate an AR-mediated autophagy cascade involving Ca2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2; a kinase with a restricted expression profile), 5'-AMP-activated protein kinase (AMPK), and Unc-51 like autophagy activating kinase 1 (ULK1), but independent of canonical mechanistic target of rapamycin (mTOR) activity. Increased CAMKK2-AMPK-ULK1 signaling correlated with disease progression in genetic mouse models and patient tumor samples. Importantly, CAMKK2 disruption impaired tumor growth and prolonged survival in multiple CRPC preclinical mouse models. Similarly, an inhibitor of AMPK-ULK1 blocked autophagy, cell growth, and colony formation in prostate cancer cells. Collectively, our findings converge to demonstrate that AR can co-opt the CAMKK2-AMPK-ULK1 signaling cascade to promote prostate cancer by increasing autophagy. Thus, this pathway may represent an alternative autophagic target in CRPC.
  10. FASEB J. 2021 Feb;35(2): e21361
      Bcl-2-associated athanogen-6 (BAG6) is a nucleocytoplasmic shuttling protein involved in protein quality control. We previously demonstrated that BAG6 is essential for autophagy by regulating the intracellular localization of the acetyltransferase EP300, and thus, modifying accessibility to its substrates (TP53 in the nucleus and autophagy-related proteins in the cytoplasm). Here, we investigated BAG6 localization and function in the cytoplasm. First, we demonstrated that BAG6 is localized in the mitochondria. Specifically, BAG6 is expressed in the mitochondrial matrix under basal conditions, and translocates to the outer mitochondrial membrane after mitochondrial depolarization with carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial uncoupler that induces mitophagy. In SW480 cells, the deletion of BAG6 expression abrogates its ability to induce mitophagy and PINK1 accumulation. On the reverse, its ectopic expression in LoVo colon cancer cells, which do not express endogenous BAG6, reduces the size of the mitochondria, induces mitophagy, leads to the activation of the PINK1/PARKIN pathway and to the phospho-ubiquitination of mitochondrial proteins. Finally, BAG6 contains two LIR (LC3-interacting Region) domains specifically found in receptors for selective autophagy and responsible for the interaction with LC3 and for autophagosome selectivity. Site-directed mutagenesis showed that BAG6 requires wild-type LIRs domains for its ability to stimulate mitophagy. In conclusion, we propose that BAG6 is a novel mitophagy receptor or adaptor that induces PINK1/PARKIN signaling and mitophagy in a LIR-dependent manner.
    Keywords:  BAG6; mitophagy; receptor; signaling
  11. Cell Mol Neurobiol. 2021 Feb 02.
      Parkinson's disease (PD) is a severe neurodegenerative disorder caused by the progressive loss of dopaminergic neurons in the substantia nigra and affects millions of people. Currently, mitochondrial dysfunction is considered as a central role in the pathogenesis of both sporadic and familial forms of PD. Mitophagy, a process that selectively targets damaged or redundant mitochondria to the lysosome for elimination via the autophagy devices, is crucial in preserving mitochondrial health. So far, aberrant mitophagy has been observed in the postmortem of PD patients and genetic or toxin-induced models of PD. Except for mitochondrial dysfunction, mitophagy is involved in regulating several other PD-related pathological mechanisms as well, e.g., oxidative stress and calcium imbalance. So far, the mitophagy mechanisms induced by PD-related proteins, PINK1 and Parkin, have been studied widely, and several other PD-associated genes, e.g., DJ-1, LRRK2, and alpha-synuclein, have been discovered to participate in the regulation of mitophagy as well, which further strengthens the link between mitophagy and PD. Thus, in this view, we reviewed mitophagy pathways in belief and discussed the interactions between mitophagy and several PD's pathological mechanisms and how PD-related genes modulate the mitophagy process.
    Keywords:  Mitochondrial dynamics; Mitochondrial function; Mitochondrial quality control; Mitophagy; Parkinson’s disease
  12. Autophagy. 2021 Feb 02.
      Macroautophagy is a catabolic process critical for the degradation of intracellular material, but its physiological functions in vertebrates are not fully understood. Here, we discuss our recent finding that macroautophagy plays a role in lamellar body maturation. The lamellar body is a lysosome-related organelle and stores phospholipid-containing surfactant complexes that reduce the surface tension of the air-water interface in order to inflate the airspace in lungs and swim bladders. In the epithelial cells of these organs, autophagosomes fuse with immature lamellar bodies to increase their size and lipid contents. This function is essential for respiration after birth in mice and for maintaining buoyancy in zebrafish. These findings unveil a novel function of macroautophagy in the maturation of surfactant-containing lamellar bodies.
    Keywords:  autophagy; lamellar body; lung; lysosome-related organelle; swim bladder; zebrafish
  13. Autophagy. 2021 Feb 02.
      Mitochondrial autophagy (mitophagy) selectively degrades mitochondria and plays an important role in mitochondrial homeostasis. In the yeast Saccharomyces cerevisiae, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy, whereas this phosphorylation is counteracted by the protein phosphatase Ppg1. Although Ppg1 functions cooperatively with the Far complex (Far3, Far7, Far8, Vps64/Far9, Far10 and Far11), their relationship and the underlying phosphoregulatory mechanism of Atg32 remain unclear. Our recent study revealed: (i) the Far complex plays its localization-dependent roles, regulation of mitophagy and target of rapamycin complex 2 (TORC2) signaling, via the mitochondria- and endoplasmic reticulum (ER)-localized Far complexes, respectively; (ii) Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required to assemble the sub- and core-Far complexes; (iii) association and dissociation between the Far complex and Atg32 are crucial determinants for mitophagy regulation. Here, we summarize our findings and discuss unsolved issues.
    Keywords:  Atg32; Far complex; Ppg1; mitochondria; mitophagy; yeast
  14. Cell Rep. 2021 Feb 02. pii: S2211-1247(21)00002-4. [Epub ahead of print]34(5): 108689
      The epidermis regenerates continually to maintain a protective barrier at the body's surface composed of differentiating keratinocytes. Maturation of this stratified tissue requires that keratinocytes undergo wholesale organelle degradation upon reaching the outermost tissue layers to form compacted, anucleate cells. Through live imaging of organotypic cultures of human epidermis, we find that regulated breakdown of mitochondria is critical for epidermal development. Keratinocytes in the upper layers initiate mitochondrial fragmentation, depolarization, and acidification upon upregulating the mitochondrion-tethered autophagy receptor NIX. Depleting NIX compromises epidermal maturation and impairs mitochondrial elimination, whereas ectopic NIX expression accelerates keratinocyte differentiation and induces premature mitochondrial fragmentation via the guanosine triphosphatase (GTPase) DRP1. We further demonstrate that inhibiting DRP1 blocks NIX-mediated mitochondrial breakdown and disrupts epidermal development. Our findings establish mitochondrial degradation as a key step in terminal keratinocyte differentiation and define a pathway operating via the mitophagy receptor NIX in concert with DRP1 to drive epidermal morphogenesis.
    Keywords:  autophagy; cornification; differentiation; epidermis; epithelial morphogenesis; fission; keratinocyte; live microscopy; mitochondria; mitophagy
  15. EMBO J. 2021 Feb 02. e105268
      Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.
    Keywords:  GAPDH; NAD+; Nmnat3; lysosome; mitochondria
  16. Mol Metab. 2021 Jan 28. pii: S2212-8778(21)00013-2. [Epub ahead of print] 101173
      OBJECTIVE: Brown adipose tissue (BAT) thermogenesis offers the potential to improve metabolic health in mice and men. However, humans predominantly live under thermoneutral conditions, leading to BAT whitening - a reduction in BAT mitochondrial content and metabolic activity. Recent studies have established mitophagy as a major driver of mitochondrial degradation in the whitening of thermogenic brite/beige adipocytes, yet the pathways mediating mitochondrial breakdown in whitening of classical BAT remain largely elusive. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy belonging to the MIT family of transcription factors, is the only member of this family that is upregulated during whitening, pointing towards a role of TFEB in whitening-associated mitochondrial breakdown.METHODS: We generated brown adipocyte-specific TFEB knockout mice, and induced BAT whitening by thermoneutral housing. We characterized gene and protein expression patterns, BAT metabolic activity, systemic metabolism as well as mitochondrial localization using in vivo and in vitro approaches.
    RESULTS: Under conditions of low thermogenic activation, deletion of TFEB preserved mitochondrial mass independently of mitochondriogenesis in BAT and primary brown adipocytes. This did however not translate into elevated thermogenic capacity or protection from diet-induced obesity. Autophagosomal/lysosomal marker levels were altered in TFEB-deficient BAT and primary adipocytes, and lysosomal markers co-localized and co-purified with mitochondria in TFEB-deficient BAT, indicating trapping of mitochondria in late stages of mitophagy.
    CONCLUSION: We here identify TFEB as a driver of BAT whitening, mediating mitochondrial degradation via the autophagosomal and lysosomal machinery. This study provides proof of concept that interfering with the mitochondrial degradation machinery can increase mitochondrial mass in classical BAT under human-relevant conditions. It must however be considered that interfering with autophagy may result in accumulation of non-functional mitochondria. Future studies targeting earlier steps of mitophagy or target recognition are therefore warranted.
    Keywords:  TFEB; UCP1; brown adipose tissue; mitophagy; thermogenesis; whitening
  17. Front Cell Neurosci. 2020 ;14 620020
      Autophagy dysfunction has been directly linked with the onset and progression of Parkinson's disease (PD), but the underlying mechanisms are not well understood. High-mobility group A1 (HMGA1), well-known chromatin remodeling proteins, play pivotal roles in diverse biological processes and diseases. Their function in neural cell death in PD, however, have not yet been fully elucidated. Here, we report that HMGA1 is highly induced during dopaminergic cell death in vitro and mice models of PD in vivo. Functional studies using genetic knockdown of endogenous HMGA1 show that HMGA1 signaling inhibition accelerates neural cell death, at least partially through aggravating MPP+-induced autophagic flux reduction resulting from partial block in autophagic flux at the terminal stages, indicating a novel potential neuroprotective role for HMGA1 in dopaminergic neurons death. MicroRNA-103/107 (miR-103/107) family, which is highly expressed in neuron, coordinately ensures proper end-stage autophagy. We further illustrate that MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced HMGA1 elevation counterparts the effect of miR-103/107 downregulation by directly binding to their promoters, respectively, sustaining their expression in MPP+-damaged MN9D cells and modulates autophagy through CDK5R1/CDK5 signaling pathway. We also find that HMGA1 is a direct target of miR-103/107 family. Thus, our results suggest that HMGA1 forms a negative feedback loop with miR-103/107-CDK5R1/CDK5 signaling to regulate the MPP+/MPTP-induced autophagy impairment and neural cell death. Collectively, we identify a paradigm for compensatory neuroprotective HMGA1 signaling in dopaminergic neurons that could have important therapeutic implications for PD.
    Keywords:  HMGA1; Parkinson’s disease; autophagy; dopaminergic neuron; miR-103/107
  18. Plant Cell Physiol. 2021 Feb 02. pii: pcab017. [Epub ahead of print]
      Zinc (Zn) is a nutritionally essential metal element, but excess Zn in the environment is toxic to plants. Autophagy is a major pathway responsible for intracellular degradation. Here, we demonstrate the important role of autophagy in adaptation to excess Zn stress. We found that autophagy-defective Arabidopsis thaliana (atg2 and atg5) exhibited marked excess Zn-induced chlorosis and growth defects relative to wild-type. Imaging and biochemical analyses revealed that autophagic activity was elevated under excess Zn. Interestingly, the excess Zn symptoms of atg5 were alleviated by supplementation of high levels of iron (Fe) to the media. Under excess Zn, in atg5, Fe starvation was especially severe in juvenile true leaves. Consistent with this, accumulation levels of Fe3+ near the shoot apical meristem was remarkably reduced in atg5. Furthermore, excision of cotyledons induced severe excess Zn symptoms in wild-type, similar to those observed in atg5. Our data suggest that Fe3+ supplied from source leaves (cotyledons) via autophagy is distributed to sink leaves (true leaves) to promote healthy growth under excess Zn, revealing a new dimension, the importance of heavy-metal stress responses by the intracellular recycling.
    Keywords:   Arabidopsis thaliana ; atg mutant; autophagy; chlorosis; excess zinc; iron starvation
  19. Biochim Biophys Acta Gen Subj. 2021 Feb 02. pii: S0304-4165(21)00017-9. [Epub ahead of print] 129858
      Mitochondria are dynamic organelles functioning in diverse reactions and processes such as energy metabolism, apoptosis, innate immunity, and aging, whose quality and quantity control is critical for cell homeostasis. Mitochondria-specific autophagy, termed mitophagy, is an evolutionarily conserved process that selectively degrades mitochondria via autophagy, thereby contributing to mitochondrial quality and quantity control. In the budding yeast Saccharomyces cerevisiae, the single-pass membrane protein Atg32 accumulates on the surface of mitochondria and recruit the autophagy machinery to initiate mitophagy. This catabolic process is elaborately regulated through transcriptional induction and post-translational modifications of Atg32. Notably, other factors acting in manifold pathways including protein N-terminal acetylation, phospholipid methylation, stress signaling, and endoplasmic reticulum-localized protein dephosphorylation and membrane protein insertion are also linked to mitophagy. Here we review recent discoveries of molecules regulating mitophagy in yeast.
    Keywords:  Atg32; Autophagy; Mitochondria; Mitophagy; Yeast
  20. Proc Natl Acad Sci U S A. 2021 Feb 09. pii: e2021475118. [Epub ahead of print]118(6):
      Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.
    Keywords:  SLC7A11; autophagy; lysosome; pancreatic cancer; pancreatic ductal adenocarcinoma
  21. Pharmacol Res. 2021 Feb 01. pii: S1043-6618(21)00011-6. [Epub ahead of print] 105428
      Autophagy is a ubiquitous mechanism for maintaining cellular homeostasis through the degradation of long-lived proteins, insoluble protein aggregates, and superfluous or damaged organelles. Dysfunctional autophagy is observed in a variety of human diseases. With advanced research into the role that autophagy plays in physiological and pathological conditions, targeting autophagy is becoming a novel tactic for disease management. Saponins are naturally occurring glycosides containing triterpenoids or steroidal sapogenins as aglycones, and some saponins are reported to modulate autophagy. Research suggests that saponins may have therapeutic and preventive efficacy against many autophagy-related diseases. Therefore, this review comprehensively summarizes and discusses the reported saponins that exhibit autophagy regulating activities. In addition, the relevant signaling pathways that the mechanisms involved in regulating autophagy and the targeted diseases were also discussed. By regulating autophagy and related pathways, saponins exhibit bioactivities against cancer, neurodegenerative diseases, atherosclerosis and other cardiac diseases, kidney diseases, liver diseases, acute pancreatitis, and osteoporosis. This review provides an overview of the autophagy-regulating activity of saponins, the underlying mechanisms and potential applications for managing various diseases.
    Keywords:  Atherosclerosis; Autophagy; Cancer; Neurodegenerative diseases; Saponins
  22. J Cell Sci. 2021 Feb 03. pii: jcs.253682. [Epub ahead of print]
      Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded by autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 hr of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the PAS (pre-autophagosomal structure/phagophore assembly site). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.
    Keywords:  Atg1; Autophagy; Yeast
  23. Int J Mol Sci. 2021 Jan 28. pii: 1318. [Epub ahead of print]22(3):
      Autophagy is a process essential for cellular energy consumption, survival, and defense mechanisms. The role of autophagy in several types of human cancers has been explicitly explained; however, the underlying molecular mechanism of autophagy in glioblastoma remains ambiguous. Autophagy is thought to be a "double-edged sword", and its effect on tumorigenesis varies with cell type. On the other hand, autophagy may play a significant role in the resistance mechanisms against various therapies. Therefore, it is of the utmost importance to gain insight into the molecular mechanisms deriving the autophagy-mediated therapeutic resistance and designing improved treatment strategies for glioblastoma. In this review, we discuss autophagy mechanisms, specifically its pro-survival and growth-suppressing mechanisms in glioblastomas. In addition, we try to shed some light on the autophagy-mediated activation of the cellular mechanisms supporting radioresistance and chemoresistance in glioblastoma. This review also highlights autophagy's involvement in glioma stem cell behavior, underlining its role as a potential molecular target for therapeutic interventions.
    Keywords:  autophagy; chemoresistance; glioblastoma; glioma stem cells; molecular targets; radioresistance
  24. Cell Mol Life Sci. 2021 Feb 05.
      Mitochondrial quality control depends upon selective elimination of damaged mitochondria, replacement by mitochondrial biogenesis, redistribution of mitochondrial components across the network by fusion, and segregation of damaged mitochondria by fission prior to mitophagy. In this review, we focus on mitochondrial dynamics (fusion/fission), mitophagy, and other mechanisms supporting mitochondrial quality control including maintenance of mtDNA and the mitochondrial unfolded protein response, particularly in the context of the heart.
    Keywords:  Cardiac; Fission; Fusion; Mitochondria; Mitophagy
  25. Biochem Biophys Res Commun. 2021 Feb 02. pii: S0006-291X(21)00142-X. [Epub ahead of print]545 69-74
      Peroxisomes play an essential role in cellular homeostasis by regulating lipid metabolism and the conversion of reactive oxygen species (ROS). Several peroxisomal proteins, known as peroxins (PEXs), control peroxisome biogenesis and degradation. Various mutations in the PEX genes are genetic causes for the development of inheritable peroxisomal-biogenesis disorders, such as Zellweger syndrome. Among the peroxins, PEX1 defects are the most common mutations in Zellweger syndrome. PEX1 is an AAA-ATPase that regulates the recycling of PEX5, which is essential for importing peroxisome matrix proteins. However, the post-transcriptional regulation of PEX1 is largely unknown. Here, we showed that heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) controls PEX1 expression. In addition, we found that depletion of HNRNPA1 induces autophagic degradation of peroxisome, which is blocked in ATG5-knockout cells. In addition, depletion of HNRNPA1 increased peroxisomal ROS levels. Inhibition of the generation of peroxisomal ROS by treatment with NAC significantly suppressed pexophagy in HNRNPA1-deficient cells. Taken together, our results suggest that depletion of HNRNPA1 increases peroxisomal ROS and pexophagy by downregulating PEX1 expression.
    Keywords:  HNRNPA1; PEX1; Pexophagy; ROS
  26. Int J Mol Med. 2021 Mar;pii: 27. [Epub ahead of print]47(3):
      Diabetic nephropathy (DN) is the primary cause of end‑stage renal disease, which is closely associated with dysfunction of the podocytes, the main component of the glomerular filtration membrane; however, the exact underlying mechanism is unknown. Polyamines, including spermine, spermidine and putrescine, have antioxidant and anti‑aging properties that are involved in the progression of numerous diseases, but their role in DN has not yet been reported. The present study aimed to explore the role of polyamines in DN, particularly in podocyte injury, and to reveal the molecular mechanism underlying the protective effect of exogenous spermine. Streptozotocin intraperitoneal injection‑induced type 1 diabetic (T1D) rat models and high glucose (HG)‑stimulated podocyte injury models were established. It was found that in T1D rat kidneys and HG‑induced podocytes, ornithine decarboxylase (a key enzyme for polyamine synthesis) was downregulated, while spermidine/spermine N1‑acetyltransferase (a key enzyme for polyamines degradation) was upregulated, which suggested that reduction of the polyamine metabolic pool particularly decreased spermine content, is a major factor in DN progression. In addition, hyperglycemia can induce an increased rat kidney weight ratio, serum creatinine, urea, urinary albumin excretion and glomerular cell matrix levels, and promote mesangial thickening and loss or fusion of podocytes. The expression levels of podocyte marker proteins (nephrin, CD2‑associated protein and podocin) and autophagy‑related proteins [autophagy protein 5, microtube‑associated proteins 1A/1B light chain 3 (LC3)II/LC3I, Beclin 1 and phosphorylated (p)‑AMPK] were downregulated, while cleaved caspase‑3, P62 and p‑mTOR were increased. These changes could be improved by pretreatment with exogenous spermine or rapamycin (autophagic agonist). In conclusion, spermine may have the potential to prevent diabetic kidney injury in rats by promoting autophagy via regulating the AMPK/mTOR signaling pathway.
  27. Sci Rep. 2021 Feb 03. 11(1): 2879
      Alzheimer's disease (AD) is a common neurodegenerative disease with poor prognosis. New options for drug discovery targets are needed. We developed an imaging based arrayed CRISPR method to interrogate the human genome for modulation of in vitro correlates of AD features, and used this to assess 1525 human genes related to tau aggregation, autophagy and mitochondria. This work revealed (I) a network of tau aggregation modulators including the NF-κB pathway and inflammatory signaling, (II) a correlation between mitochondrial morphology, respiratory function and transcriptomics, (III) machine learning predicted novel roles of genes and pathways in autophagic processes and (IV) individual gene function inferences and interactions among biological processes via multi-feature clustering. These studies provide a platform to interrogate underexplored aspects of AD biology and offer several specific hypotheses for future drug discovery efforts.
  28. Life Sci. 2021 Feb 01. pii: S0024-3205(21)00124-7. [Epub ahead of print] 119139
      AIMS: Complicated mechanisms in cancer cells have been restricting the medicinal value of resveratrol (Res). The mechanisms by which Res exerts its anti-tumor activity in lung cancer cells have diverged among reports in recent years, whether cells choose to undergo autophagic cell death or apoptosis remains controversial. Yet, whether Res-induced autophagic cell death transforms into apoptosis is still unknown, and by which autophagy regulates programmed cell death is still undefined.MAIN METHODS: Here, A549 cells were treated with Res to investigate the mechanisms of autophagy and apoptosis using western blot, immunofluorescence staining for LC3B.
    KEY FINDINGS: Non-canonical autophagy was induced by Res-treatment in a Beclin-1- and ATG5-independent manner, with apoptosis being activated simultaneously. Autophagy induced by Res was activated by rapamycin with decreased apoptosis, suggesting that autophagy may serve as a protective pathway in cells. Mitophagy was found to be induced by Res using fluorescence co-localization of mitochondria with lysosomes. Subsequently, it was identified that mitophagy was mediated by LC3B/p62 interaction and could be inhibited by LC3B knockout and p62 knockdown following increased apoptosis.
    SIGNIFICANCE: In conclusion, the current results demonstrate that Res-induced non-canonical autophagy in A549 lung cancer cells with apoptosis activation simultaneously, while LC3B/p62-mediated mitophagy protects tumor cells against apoptosis, providing novel mechanisms about the critical role of mitophagy in regulating cell fate.
    Keywords:  A549; Apoptosis; Autophagy; LC3B; Mitophagy; Resveratrol; p62
  29. Neurobiol Aging. 2020 Dec 24. pii: S0197-4580(20)30424-3. [Epub ahead of print]100 91-105
      Development of efficacious treatments for Parkinson's disease (PD) demands an improved understanding of mechanisms underlying neurodegeneration. Two cellular death pathways postulated to play key roles in PD are autophagy and apoptosis. Molecular overlap between these pathways was investigated through identifying studies that used therapeutic compounds to alter expression of specific molecular components of the pathways. Bcl-2 was identified as an important protein with the ability to suppress autophagy and apoptosis through inhibiting Beclin-1 and Bax, respectively. Involvement of c-Jun N-terminal kinases (JNK) and p38, was evident in the activation of apoptosis through increasing the Bax/Bcl-2 ratio. JNK-mediated phosphorylation also suppresses the inhibiting functions of Bcl-2, indicating an ability to induce not only apoptosis but also autophagy. Additionally, a p38-mediated increase in heme oxygenase-1 expression inhibits apoptosis. Moreover, besides inhibiting mammalian target of rapamycin, Akt is associated with decreased Bax expression, thereby acting as both an autophagy inducer and apoptosis inhibitor. Ultimately, manipulation of molecular components involved in autophagy and apoptosis regulation could be targeted as possible therapies for PD.
    Keywords:  Apoptosis; Autophagy; Bcl-2; Parkinson's disease; Therapy
  30. Autophagy. 2021 Feb 01.
      As a self-degradative mechanism, macroautophagy/autophagy has a role in the maintenance of energy homeostasis during critical periods in the development of cells. It also controls cellular damage through the eradication of damaged proteins and organelles. This process is accomplished by tens of ATG (autophagy-related) proteins. Recent studies have shown the involvement of non-coding RNAs in the regulation of autophagy. These transcripts mostly modulate the expression of ATG genes. Both long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to modulate the autophagy mechanism. Levels of several lncRNAs and miRNAs are altered in this process. In the present review, we discuss the role of lncRNAs and miRNAs in the regulation of autophagy in diverse contexts such as cancer, deep vein thrombosis, spinal cord injury, diabetes and its complications, acute myocardial infarction, osteoarthritis, pre-eclampsia and epilepsy.
    Keywords:  Autophagy; biomarker; cancer; lncRNAs; microRNAs
  31. Nature. 2021 Jan 27.
      Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson's disease1-5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoKGF. LysoKGF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson's disease and reduces channel currents. Reduction in lysoKGF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217-another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson's disease-produces a gain-of-function in lysoKGF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson's disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson's disease.