So Many Roads: the Multifaceted Regulation of Autophagy Induction
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Abstract:
Autophagy is an evolutionary conserved, degradative process from single-cell eukaryotes, such asKeywords:
BECN1
ULK1
Autophagy-related protein 13
BAG3
TOR signaling
ATG16L1
ATG8
VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.
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Organelle biogenesis
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Autophagy is an evolutionary conserved, degradative process from single-cell eukaryotes, such as
BECN1
ULK1
Autophagy-related protein 13
BAG3
TOR signaling
ATG16L1
ATG8
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Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.
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TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.
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The PIK3C3/VPS34-containing phosphatidylinositol 3-kinase (PtdIns3K) initiation complex (complex I) is necessary for macroautophagy/autophagy initiation and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-ATG14, while the endosomal trafficking complex (complex II) is necessary for vesicle trafficking and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-UVRAG. This composition difference was exploited to identify novel and specific autophagy inhibitors that disrupted the BECN1-ATG14 protein-protein interaction, without affecting vesicle trafficking. A cellular NanoBRET assay was implemented to identify these inhibitors, and one compound was able to successfully disrupt the BECN1-ATG14 interaction and inhibit autophagy, with limited impact on vesicle trafficking. These results reveal the first protein-protein interaction inhibitor targeting the autophagy initiation machinery and demonstrate the viability of targeting protein-protein interactions for the discovery of autophagy-specific modulators.
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Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.
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ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.
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Trypanosomatid protozoa parasites are responsible for tropical diseases, and undergo complex life cycles involving developmental forms adapted to insect vectors and vertebrate hosts. During their life cycle these parasites proceed through different forms in response to dramatic environmental changes and/or developmentally regulated programs. Successful progression of the parasite through its life cycle is highly dependent on the capacity of adaptation to distinct stresses involving processes such as autophagy. In eukaryotes, Target Of Rapamycin (TOR) protein kinases act as a sensor, which integrates the nutritional and energetic status, adjusting cell metabolism and growth. Compromising cell viability in yeast and mammals leads to a reduction of TOR function, triggering processes aimed to overcome unfavourable conditions. This is partly achieved by TOR-mediated regulation of protein synthesis and recycling of cellular components by autophagy. In the last few years, autophagy has been described during developmental differentiation processes in Trypanosomatids. However, no link between TOR signalling, autophagy and differentiation has been described so far. This addendum is a commentary to the work published by our group,1 where we discuss the possible role of TOR kinases, as a controller of cell growth and autophagy, in the regulation of differentiation processes during Trypanosomatids life cycles.
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ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle's balanced salt solution; DAPI: 4ʹ-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.
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ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.
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