The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra.

Hydra vulgaris (Hv) has a high regenerative potential and negligible senescence, as its stem cell populations divide continuously. In contrast, the cold-sensitive H. oligactis (Ho_CS) rapidly develop an aging phenotype under stress, with epithelial stem cells deficient for autophagy, unable to maintain their self-renewal. Here we tested in aging, non-aging and regenerating Hydra the activity and regulation of the ULK1 kinase involved in autophagosome formation. In vitro kinase assays show that human ULK1 activity is activated by Hv extracts but repressed by Ho_CS extracts, reflecting the ability or inability of their respective epithelial cells to initiate autophagosome formation. The factors that keep ULK1 inactive in Ho_CS remain uncharacterized. Hv_Basel1 animals exposed to the ULK1 inhibitor SBI-0206965 no longer regenerate their head, indicating that the sustained autophagy flux recorded in regenerating Hv_AEP2 transgenic animals expressing the DsRed-GFP-LC3A autophagy tandem sensor is necessary. The SBI-0206965 treatment also alters the contractility of intact Hv_Basel1 animals, and leads to a progressive reduction of animal size in Hv_AEP2, similarly to what is observed in ULK1(RNAi) animals. We conclude that the evolutionarily-conserved role of ULK1 in autophagy initiation is crucial to maintain a dynamic homeostasis in Hydra, which supports regeneration efficiency and prevents aging.

Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma.

Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

Toxoplasma gondii induces prolonged host epidermal growth factor receptor signalling to prevent parasite elimination by autophagy: Perspectives for in vivo control of the parasite.

Toxoplasma gondii causes retinitis and encephalitis. Avoiding targeting by autophagosomes is key for its survival because T. gondii cannot withstand lysosomal degradation. During invasion of host cells, T. gondii triggers epidermal growth factor receptor (EGFR) signalling enabling the parasite to avoid initial autophagic targeting. However, autophagy is a constitutive process indicating that the parasite may also use a strategy operative beyond invasion to maintain blockade of autophagic targeting. Finding that such a strategy exists would be important because it could lead to inhibition of host cell signalling as a novel approach to kill the parasite in previously infected cells and treat toxoplasmosis. We report that T. gondii induced prolonged EGFR autophosphorylation. This effect was mediated by PKCα/PKCß âž” Src because T. gondii caused prolonged activation of these molecules and their knockdown or incubation with inhibitors of PKCα/PKCß or Src after host cell invasion impaired sustained EGFR autophosphorylation. Addition of EGFR tyrosine kinase inhibitor (TKI) to previously infected cells led to parasite entrapment by LC3 and LAMP-1 and pathogen killing dependent on the autophagy proteins ULK1 and Beclin 1 as well as lysosomal enzymes. Administration of gefitinib (EGFR TKI) to mice with ocular and cerebral toxoplasmosis resulted in disease control that was dependent on Beclin 1. Thus, T. gondii promotes its survival through sustained EGFR signalling driven by PKCα/ß âž” Src, and inhibition of EGFR controls pre-established toxoplasmosis.

Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-ß (Aß) production. Treatment with Aß increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism.

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration.

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration. Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

Autophagy plays a pro-survival role against methamphetamine-induced apoptosis in H9C2 cells.

Methamphetamine (METH) is a commonly abused psychostimulant that can induce severe neurotoxicity. Cardiovascular injury caused by METH has recently gained increasing attention; however, the underlying mechanisms remain unclear. As autophagy has been shown to be associated with cell injury, the association between autophagy and METH-mediated cell apoptosis was investigated in the present study. METH treatment significantly increased the expression of two key autophagy proteins, i.e., Beclin-1 and LC3-II, in the cardiomyocyte cell line H9C2. Furthermore, according to western blot and flow cytometry analyses, METH contributed to cell injury and markedly enhanced cleaved-caspase 3 and PARP expression. In addition, the corresponding AKT-mTOR survival pathway axis was appeared deactivated. The autophagic activity was closely associated with METH-mediated cell injury because rapamycin, which is an autophagy inducer, markedly attenuated METH-induced cell injury, while 3-Methyladenine (3-MA), which is an autophagy inhibitor, and bafilomycinA1 (Baf-A1), which is a blocker of autophagosome-lysosome fusion, markedly exacerbated METH-induced cell injury. Notably, defective autophagosome-lysosome fusion might be partially involved in the METH-induced enhancement of LC3-II expression and cell injury. However, the underlying mechanisms require further investigation.

mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition.

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In the present study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein leucine rich repeat kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity.

AMPK Inhibits ULK1-Dependent Autophagosome Formation and Lysosomal Acidification via Distinct Mechanisms.

Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

Gastrodin protects myocardial cells against hypoxia/reoxygenation injury in neonatal rats by inhibiting cell autophagy through the activation of mTOR signals in PI3K-Akt pathway.

OBJECTIVES: This study aimed to investigate the protective effect of gastrodin (GAS) on myocardial cells with hypoxia/reoxygenation (H/R) injury in neonatal rats and explore the underlying mechanism. METHODS: Myocardial cells were extracted from neonatal rats and divided into six groups: control, H/R, H/R + Low-Concentration GAS, H/R + Middle-Concentration GAS, H/R + High-Concentration GAS and H/R + High-Concentration GAS + AKT Inhibitor groups. After 48-h treatment, cell viability, autophagosome quantity and the expression levels of LC3-II, p62, Akt, pAkt, mammalian target of rapamycin (mTOR) and uncoordinated 51-like kinase 1 (ULK1) in myocardial cells were made comparisons among each group. KEY FINDINGS: Gastrodin improved the proliferation activity of myocardial cells under H/R injury in a dose-dependent manner and inhibited the level of cell autophagy. However, when AKT inhibitor was added, the effect of GAS was partly inhibited (P < 0.05). Gene and protein expressions showed that GAS made no significant effect on the expression quantity of Akt and mTOR genes (P > 0.05) but could significantly promote the phosphorylation of Akt and mTOR (P < 0.05). GAS had significant inhibiting effect on the expression of ULK1 (P < 0.05). CONCLUSIONS: Gastrodin could protect against H/R injury of myocardial cells in neonatal rats by reducing the level of autophagy through the activation of mTOR signals in PI3K-Akt pathway.

Impaired Protein Quality Control During Left Ventricular Remodeling in Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor.

BACKGROUND: Sustained inflammation in the heart is sufficient to provoke left ventricular dysfunction and left ventricular remodeling. Although inflammation has been linked to many of the biological changes responsible for adverse left ventricular remodeling, the relationship between inflammation and protein quality control in the heart is not well understood. METHODS AND RESULTS: To study the relationship between chronic inflammation and protein quality control, we used a mouse model of dilated cardiomyopathy driven by cardiac restricted overexpression of TNF (tumor necrosis factor; Myh6-sTNF). Myh6-sTNF mice develop protein aggregates containing ubiquitin-tagged proteins within cardiac myocytes related to proteasome dysfunction and impaired autophagy. The 26S proteasome was dysfunctional despite normal function of the core 20S subunit. We found an accumulation of autophagy substrates in Myh6-sTNF mice, which were also seen in tissue from patients with end-stage heart failure. Moreover, there was evidence of impaired autophagosome clearance after chloroquine administration in these mice indicative of impaired autophagic flux. Finally, there was increased mammalian target of rapamycin complex 1 (mTORC1) activation, which has been linked to inhibition of both the proteasome and autophagy. CONCLUSIONS: Myh6-sTNF mice with sustained inflammatory signaling develop proteasome dysfunction and impaired autophagic flux that is associated with enhanced mTORC1 activation.

The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation.

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

Rab2 promotes autophagic and endocytic lysosomal degradation.

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.

Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17.

Class III PI3-kinase (PI3KC3) is essential for autophagy initiation, but whether PI3KC3 participates in other steps of autophagy remains unknown. The HOPS complex mediates the fusion of intracellular vesicles to lysosome, but how HOPS specifically tethers autophagosome to lysosome remains elusive. Here, we report Pacer (protein associated with UVRAG as autophagy enhancer) as a regulator of autophagy. Pacer localizes to autophagic structures and positively regulates autophagosome maturation. Mechanistically, Pacer antagonizes Rubicon to stimulate Vps34 kinase activity. Next, Pacer recruits PI3KC3 and HOPS complexes to the autophagosome for their site-specific activation by anchoring to the autophagosomal SNARE Stx17. Furthermore, Pacer is crucial for the degradation of hepatic lipid droplets, the suppression of Salmonella infection, and the clearance of protein aggregates. These results not only identify Pacer as a crucial multifunctional enhancer in autophagy but also uncover both the involvement of PI3KC3 and the mediators of HOPS's specific tethering activity in autophagosome maturation.

Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation.

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration.

Phosphatidylinositol-3-phosphate in the regulation of autophagy membrane dynamics.

Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence.

Kallikrein-related peptidase 6 induces chemotherapeutic resistance by attenuating auranofin-induced cell death through activation of autophagy in gastric cancer.

Kallikrein-related peptidase 6 (KLK6) is a biomarker of gastric cancer associated with poor prognosis. Mechanisms by which KLK6 could be exploited for chemotherapeutic use are unclear. We evaluated auranofin (AF), a compound with cytotoxic effects, in KLK6-deficient cells, and we investigated whether KLK6 expression induces autophagy and acquisition of drug resistance in gastric cancer. Using cultured human cells and a mouse xenograft model, we investigated how KLK6 affects antitumor-reagent-induced cell death and autophagy. Expression levels of KLK6, p53, and autophagy marker LC3B were determined in gastric cancer tissues. We analyzed the effects of knockdown/overexpression of KLK6, LC3B, and p53 on AF-induced cell death in cancer cells. Increased KLK6 expression in human gastric cancer tissues and cells inhibited AF-induced cell motility due to increased autophagy and p53 levels. p53 dependent induction of KLK6 expression increased autophagy and drug resistance, whereas KLK6 silencing decreased the autophagy level and increased drug sensitivity. During AF-induced cell death, KLK6 and LC3B colocalized to autophagosomes, associated with p53, and were then trafficked to the cytosol. In the xenograft model of gastric cancer, KLK6 expression decreased AF-induced cell death and KLK6-induced autophagy increased AF resistance. Taken together, the data suggest that the induction of autophagic processes through KLK6 expression may increase acquisition of resistance to AF. Our findings may contribute to a new paradigm for tumor therapeutics.