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Autophagy is an essential degradation pathway that assists eukaryote survival under multiple stress conditions. Autophagosomes engulfing cargoes accomplish degradation only when they have matured through fusing with lysosomes or vacuoles. However, the molecular machinery mediating autophagosome maturation in plants remains unknown. Using the combined approaches of mass spectrometry, biochemistry, reverse genetics and microscopy, we uncover that UVRAG, a subunit of the class III phosphatidylinositol 3-kinase complexes in Nicotiana benthamiana, plays an essential role in autophagsome maturation via ATG14-assisted recruitment to autophagosomes and by facilitating RAB7 activation. An interaction between N. benthamiana UVRAG and ATG14 was observed in vitro and in vivo, which strikingly differed from their mutually exclusive appearance in different PI3KC3 complexes in yeast and mammals. This interaction increased the localisation of UVRAG on autophagosomes and enabled the convergence of autophagic and late endosomal structures, where they contributed to fusions between these two types of organelles by recruiting the essential membrane fusion factors RAB7 GTPase and the homotypic fusion and protein sorting (HOPS) complex. In addition, we uncovered a joint contribution of ATG14 and UVRAG to geminiviral infection, beyond autophagy. Our study provides insights into the mechanisms of autophagosome maturation in plants and expands the understanding of organisations and roles of the PI3KC3 complexes.

High levels of reactive oxygen species (ROS) result in oxidative stress, which damages cells and leads to the development of many diseases. Macroautophagy/autophagy plays an important role in protecting cells from diverse stress stimuli including oxidative stress. However, the molecular mechanisms of autophagy activation in response to oxidative stress remain largely unclear. In this study, we showed that TRAF6 mediates oxidative stress-induced ATG9A ubiquitination at two C-terminal lysine residues (K581 and K838). ATG9A ubiquitination promotes its association with BECN1, BECN1-PIK3C3/VPS34-UVRAG complex assembly and PIK3C3/VPS34 activation, thereby activating autophagy and endocytic trafficking. We also identified TNFAIP3/A20 as a negative regulator of oxidative-induced autophagy by counteracting TRAF6-mediated ATG9A ubiquitination. Moreover, ATG9A depletion attenuates LPS-induced autophagy and causes aberrant TLR4 signaling and inflammatory responses. Our findings revealed a critical role of ATG9A ubiquitination in oxidative stress-induced autophagy, endocytic trafficking and innate immunity.

Increasing evidence shows that autophagy impairment is involved in the pathogenesis and progression of neurodegenerative diseases including Parkinson's disease (PD). We previously identified a natural alkaloid named corynoxine B (Cory B) as a neuronal autophagy inducer. However, its brain permeability is relatively low, which hinders its potential use in treating PD. Thus we synthesized various derivatives of Cory B to find more potent autophagy inducers with improved brain bioavailability. In this study, we evaluated the autophagy-enhancing effect of CB6 derivative and its neuroprotective action against PD in vitro and in vivo. We showed that CB6 (5-40 μM) dose-dependently accelerated autophagy flux in cultured N2a neural cells through activating the PIK3C3 complex and promoting PI3P production. In MPP

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.

Purpose The role of vacuolar protein sorting 34 (Vps34), an indispensable protein required for cell vesicular trafficking, in the biological behavior of hepatocellular carcinoma (HCC) has yet to be studied.Materials and Methods In the present study, the expression of Vps34 in HCC and the effect of Vps34 on HCC cell invasion was detected both in vivo and in vitro. Furthermore, by modulating the RILP and Rab11, which regulate juxtanuclear lysosome aggregation and recycling endosome respectively, the underlying mechanism was investigated.Results Vps34 was significantly decreased in HCC and negatively correlated with the HCC invasiveness both in vivo and in vitro. Moreover, Vps34 could promote lysosomal juxtanuclear accumulation, reduce the invasive ability of HCC cells via the Rab7-RILP pathway. In addition, the deficiency of Vps34 in HCC cells affected the endosome-lysosome system, resulting in enhanced Rab11 mediated endocytic recycling of cell surface receptor and increased invasion of HCC cells.Conclusion Our study reveals that Vps34 acts as an invasion suppressor in HCC cells, and more importantly, the endosome-lysosome trafficking regulated by Vps34 has the potential to become a target pathway in HCC treatment.

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Specific changes in phospholipid content are a hallmark of the membranes of maturing endosomes and phagosomes, but is it unclear how this is controlled. Naufer et al. now show that acidification of the lumen of endosomes and phagosomes triggers dissociation of the Vps34 lipid kinase from these organelles, which terminates PtdIns(3)P synthesis and signaling., Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired “browning” of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

VPS34 has attracted attention in oncology as a target to modulate autophagy. However, the generation of selective VPS34 inhibitors with suitable PK properties has been a difficult task. The discovery of compound

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

Breast cancer is a heterogeneous disease that represents the most common cancer around the world; it comprises 12% of new cases according to the World Health Organization. Despite new approaches in early diagnosis and current treatment, breast cancer is still the leading cause of death for cancer mortality. New targeted therapies against key signalling transduction molecules are required. Phosphoinositide 3-kinase (PI3K) regulates multiple biological functions such as proliferation, survival, migration, and growth. It is well established that PI3K isoform-selective inhibitors show fewer toxic side effects compared to broad spectrum inhibition of PI3K (pan-PI3K inhibitors). Therefore, we tested the PI3K p110δ-selective inhibitor, IC87114, and Vps34-selective inhibitor, Vps34-IN1, on the breast cancer cell lines MCF-7 and MDA-MB-231, representing hormone-responsive and triple-negative breast cancer cells, respectively. Our data show that both inhibitors decreased migration of MCF-7 and MDA-MB-231 cells, and Vps34 also significantly impacted MCF-7 cell proliferation. Three-dimensional (3D) in vitro culture models show that IC87114 and Vps34-IN1 treatment reduced the growth of MCF-7 and MDA-MB-231 cells in 3D tumour spheroid cultures. This study identifies IC87114 and Vps34-IN1 as potential therapeutic approaches in breast cancer.

Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible., This research was funded by H2020 MarieSkłodowska-Curie Actions (765912 - DRIVE).

Endosome fission is essential for cargo sorting and targeting in the endosomal system. However, whether organelles other than the endoplasmic reticulum (ER) participate in endosome fission through membrane contacts is unknown. Here, we characterize a Golgi-derived vesicle, the SEC14L2 compartment, that plays a unique role in facilitating endosome fission through ternary contacts with endosomes and the ER. Localized to the ER-mediated endosome fission site, the phosphatidylinositol transfer protein SEC14L2 promotes phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 3-phosphate (PtdIns3P) conversion before endosome fission. In the absence of SEC14L2, endosome fission is attenuated and more enlarged endosomes arise due to endosomal accumulation of PtdIns4P and reduction in PtdIns3P. Collectively, our data suggest roles of the Golgi network in ER-associated endosome fission and a mechanism involving ER–endosome contacts in the regulation of endosomal phosphoinositide conversion. Gong et al. uncover a role for the Golgi network in ER-associated endosome fission, showing that a Golgi-derived SEC14L2 compartment mediates ER-associated endosome fission by promoting PtdIns4P to PtdIns3P conversion.

Autophagy is an evolutionarily conserved intracellular degradation system and is regulated by various signaling pathways including the Beclin 1/Vacuolar protein sorting 34 (Vps34) complex. Protein phosphatase 6 (PP6) is an essential serine/threonine phosphatase that regulates various biological processes. Recently, we found that PP6 protein is degraded by p62-dependent selective autophagy. In this study, we show that PP6 conversely inhibits autophagy. PP6 associate with the C-terminal region of Beclin 1, which is close to the binding region of Vps34. The protein levels of PP6 affect Beclin 1/Vps34 complex formation and phosphatase activity of PP6 is not involved in this. We also show that chemically induced PP6/Beclin 1 association leads to Vps34 dissociation from Beclin 1. Overall, our data reveal a novel regulatory mechanism for autophagy by PP6.

Background Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. Methods Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA–protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. Results We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-β/Smad2 pathway. Conclusions Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.

The blood flow restoration of ischemic tissues causes myocardial injury. Dexmedetomidine (Dex) protects multi-organs against ischemia/reperfusion (I/R) injury. This study investigated the protective mechanism of Dex post-treatment in myocardial I/R injury. The rat model of myocardial I/R was established. The effects of Dex post-treatment on cardiac function and autophagy flow were observed. Dex attenuated myocardial I/R injury and reduced I/R-induced autophagy in rats. Dex weakened the interactions between Beclin1 and Vps34 and Beclin1 and Atg14L, thus downregulating Vps34 kinase activity. In vitro, the cardiomyocytes subjected to oxygen glucose deprivation/reoxygenation were treated with Dex and PI3K inhibitor LY294002. LY294002 attenuated the myocardial protective effect of DEX, indicating that Dex protected against cardiac I/R by activating the PI3K/Akt pathway. In conclusion, Dex upregulated the phosphorylation of Beclin1 at S295 site by activating the PI3K/Akt pathway and reduced the interactions of Atg14L-Beclin1-Vps34 complex, thus inhibiting autophagy and protecting against myocardial I/R injury.

Macroautophagy/autophagy is special because the double-layer lipid-formed autophagosome is formed by de novo generation. Phosphatidylinositol-3-phosphate (PtdIns3P) produced by class III phosphatidylinositol 3-kinase complex I (PtdIns3K-CI) is an essential source lipid for the formation of autophagosomes. However, how autophagy is initiated is unknown. In other words, the mechanism by which PtdIns3K-CI is recruited to the phagophore assembly site (PAS) to initiate autophagosome formation is unclear. We recently uncovered the pivotal role of yeast Vac8 in autophagy initiation through the recruitment of PtdIns3K-CI to the PAS. N-terminal palmitoylation of Vac8 anchors it to the vacuole membrane, and the middle ARM domains bind PtdIns3K-CI, leading to the generation of PtdIns3P at the PAS and subsequent autophagosome formation. We found that mouse ARMC3 is the homolog of yeast Vac8 and that its autophagic roles are conserved. Interestingly, spermatids from mice with Armc3 deletion showed blocked ribophagy, low energy levels of mitochondria and motionless flagella, which caused male infertility. These findings revealed a germ tissue-specific autophagic function of ARMC3 in complex eukaryotic species.

The mammalian ULK kinase complex is the most upstream component in the macroautophagy/autophagy signaling pathway. ULK1 and homolog ULK2, the sole serine/threonine kinases in autophagy, transduce an array of autophagy-inducing stimuli to downstream autophagic machinery, regulating autophagy from autophagosome initiation to fusion of autophagosomes with lysosomes. ULK signaling is also implicated in a diverse array of non-canonical processes from necroptosis to ER-Golgi trafficking to stress granule clearance. However, the exact mechanisms by which ULK regulates these diverse processes remain largely unknown. Most notably, the number of validated ULK substrates is surprisingly low. Our study identifies new ULK substrates from a wide array of protein families and signaling pathways and supports an expanded range of physiological roles for the ULKs. We further characterize several new substrates, including the PIK3C3/VPS34-containing complex subunit PIK3R4/VPS15 and the AMPK component PRKAG2. Finally, by analyzing PIK3R4/VPS15-deficient models we discover novel aspects of ULK signaling with potential relevance in selective autophagy.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex plays a role in both canonical and noncanonical autophagy, key processes that control immune-cell responsiveness to a variety of stimuli. Our previous studies found that PIK3C3 is a critical regulator that controls the development, homeostasis, and function of dendritic and T cells. In this study, we investigated the role of PIK3C3 in myeloid cell biology using myeloid cell-specific Pik3c3-deficient mice. We found that Pik3c3-deficient macrophages express increased surface levels of major histocompatibility complex (MHC) class I and class II molecules. In addition, myeloid cell-specific Pik3c3 ablation in mice caused a partial impairment in the homeostatic maintenance of macrophages expressing the apoptotic cell uptake receptor TIM-4. Pik3c3 deficiency caused phenotypic changes in myeloid cells that were dependent on the early machinery (initiation/nucleation) of the classical autophagy pathway. Consequently, myeloid cell-specific Pik3c3-deficient animals showed significantly reduced severity of experimental autoimmune encephalomyelitis (EAE), a primarily CD4(+) T-cell-mediated mouse model of multiple sclerosis (MS). This disease protection was associated with reduced accumulation of myelin-specific CD4(+) T cells in the central nervous system and decreased myeloid cell IL-1β production. Further, administration of SAR405, a selective PIK3C3 inhibitor, delayed disease progression. Collectively, our studies establish PIK3C3 as an important regulator of macrophage functions and myeloid cell-mediated regulation of EAE. Our findings also have important implications for the development of small-molecule inhibitors of PIK3C3 as therapeutic modulators of MS and other autoimmune diseases.

B‑cell lymphomas (BCLs) are malignant lymphoid tumours originating from the malignant proliferation and transformation of mature lymphocytes at various stages of differentiation and clonal expansion of the lymphatic and circulatory systems. Efforts to control or even eradicate BCLs are frequently hampered by the development of drug resistance. Autophagy is an evolutionarily conserved biological process of the energy metabolism. By degrading intracellular organelles and proteins, autophagy provides cells with biochemical reaction substrates for the maintenance of homeostasis under nutrient deprivation or other stressful conditions. Accumulating evidence indicates that autophagy plays an important role in chemotherapy resistance. S100A8 is an important member of the calcium‑binding protein family that plays an important role in regulating tumour resistance to chemotherapy, while the specific molecular regulatory mechanisms remain unclear. In the present study, by employing three BCL cell lines (Daudi, SUDHL‑4 and JeKo‑1), it was demonstrated that BCL cells with a strong drug resistance also exhibited active autophagy. In addition, S100A8 was found to be crucial for regulating drug resistance and promoting autophagy in BCL cells. Interference of S100A8 significantly downregulated Bcl‑2/adenovirus E1B 19‑kDa protein‑interacting protein 3 located in the mitochondria and endoplasmic reticulum to further inhibit autophagy. In addition, S100A8 interference markedly inhibited the formation of the BECN1‑PI3KC3 complex and promoted B‑cell lymphoma 2 expression, which collectively inhibited autophagy.

Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

New findings What is the central question of the study? Is Vps34 a nutrient-sensitive activator of mTORC1 in human skeletal muscle? What is the main finding and its importance? We show that altering nutrient availability, via protein-carbohydrate feeding, does not increase Vps34 kinase activity in human skeletal muscle. Instead, feeding increased Vps34-mTORC1 co-localization in parallel to increased mTORC1 activity. These findings may have important implications in the understanding nutrient-induced mTORC1 activation in skeletal muscle via interaction with Vps34. Abstract The Class III PI3Kinase, Vps34, has recently been proposed as a nutrient sensor, essential for activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (21 ± 0.5 yrs, 77.7 ± 9.9 kg, 25.9 ± 2.7 kg/m2 , mean ± SD) ingested a PRO-CHO beverage containing 20/44/1 g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1 h and 3 h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean ± SD) - 0.273 ± 0.040, 1 h - 0.348 ± 0.061, Pearson's Coefficient (r)) where it co-localized with mTOR (PRE - 0.312 ± 0.040, 1 h - 0.348 ± 0.069, Pearson's Coefficient (r)). These alterations occurred in parallel to an increase in S6K1 kinase activity (941 ± 466% of PRE at 1 h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle.

PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. In vivo data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis. Abbreviations: AKT1: AKT serine/threonine kinase 1; ATG14: autophagy related 14; Co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; hpi: hours post-infection; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; pAb: polyclonal antibody; PDPK1: 3-phosphoinositide dependent protein kinase 1; PI3K: phosphoinositide 3-kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic, subunit type 3; RPS6KB1: ribosomal protein S6 kinase B1; SGK: serum/glucocorticoid regulated kinase; SQSTM1: sequestosome 1; SUMO: small ubiquitin like modifier; UBE2I/UBC9: ubiquitin conjugating enzyme E2 I; UVRAG: UV radiation resistance associated

Differentiation of trophoblast stem (TS) cells into various cell lineages of the placenta during mammalian development is accompanied by dynamic changes in its proteome for exerting the highly specialized functions of various cell subtypes. In the present study, we demonstrate that the autophagic machinery, which includes proteins for initiation, vesicle nucleation, and autophagosome maturation are robustly upregulated during differentiation of TS cells. Interestingly, basal levels of autophagy were detectable in the developing mouse placenta as well as TS cells. However, autophagic flux was actively triggered by induction of differentiation evident from LC3 maturation. Formation of Beclin1, Vps34, and PIK3R4 ternary complex at the phagophore assembly site that is typically known to induce autophagy was also enhanced during differentiation. Degradation of the p62/SQSTM1 cargo protein and its colocalization with LC3, a mature autophagosome marker, was most prevalent in the trophoblast giant cells (TGCs) and negligible in other trophoblast cells at day 6 of differentiation. Furthermore, disruption of autophagy by impairing lysosomal fusion in TS cells before induction of differentiation led to a decrease in the giant cell and spongiotrophoblast cell markers Prl3d1, Prl2c2, Prl4a1, and Tpbpα upon differentiation. In addition, inhibition of autophagy was associated with a decrease in nuclear size of TGCs. Taken together, these data highlight that autophagy is a necessary prelude in commitment of trophoblast differentiation from the multipotent TS cells probably by regulating protein turnover at the onset of differentiation.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

1-phenyl 2-thiourea (PTU) is a Tyr (tyrosinase) inhibitor that is extensively used to block pigmentation and improve optical transparency in zebrafish (Danio rerio) embryo. Here, we reported a previously undescribed effect of PTU on macroautophagy/autophagy in zebrafish embryos. Upon 0.003% PTU treatment, aberrant autophagosome and autolysosome formation, accumulation of lysosomes, and elevated autophagic flux were observed in various tissues and organs of zebrafish embryos, such as skin, brain, and muscle. Similar to PTU treatment, autophagic activation and lysosomal accumulation were also observed in the somatic tyr mutant zebrafish embryos, which suggest that Tyr inhibition may contribute to PTU-induced autophagic activation. Furthermore, we demonstrated that autophagy contributes to pigmentation inhibition, but is not essential to the PTU-induced pigmentation inhibition. With the involvement of autophagy in a wide range of physiological and pathological processes and the routine use of PTU in zebrafish research of autophagy-related processes, these observations raise a novel concern in autophagy-related studies using PTU-treated zebrafish embryos. Abbreviations: 3-MA: 3-methyladenine; Atg: autophagy-related; BSA: bovine serum albumin; CHT: caudal hematopoietic tissue; CQ: chloroquine; GFP: green fluorescent protein; hpf: hour-post-fertilization; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NGS: normal goat serum; PtdIns3K: class III phosphatidylinositol 3-kinase; PTU: 1-phenyl 2-thiourea; RFP: red fluorescent protein; Sqstm1: sequestosome 1; tyr: tyrosinase.

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic ( Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic ( Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type ( Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.