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Autophagy is an important catabolic process, which sustains intracellular homeostasis and lengthens cell survival under stress. Here we identify the ankyrin-repeat-containing, SH3-domain-containing, and proline-rich region-containing protein 2 (ASPP2), a haploinsufficient tumor suppressor, as a molecular regulator of starvation-induced autophagy in hepatocellular carcinoma (HCC). ASPP2 expression is associated with an autophagic response upon nutrient deprivation and downregulation of ASPP2 facilitates autophagic flux, whereas overexpression of ASPP2 blocks this starvation-induced autophagy in HCC cells. Mechanistically, ASPP2 inhibits autophagy through regulating BECN1 transcription and formation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) complex. Firstly, ASPP2 inhibits p65/RelA-induced transcription of BECN1, directly by an ASPP2-p65/RelA-IκBα complex which inhibits phosphorylation of IκBα and the translocation of p65/RelA into the nucleus. Secondly, ASPP2 binds to BECN1, leading to decreased binding of PIK3C3 and UV radiation resistance-associated gene (UVRAG), and increased binding of Rubicon in PIK3C3 complex. Downregulation of ASPP2 enhances the pro-survival and chemoresistant property via autophagy in HCC cells in vitro and in vivo. Decreased ASPP2 expression was associated with increased BECN1 and poor survival in HCC patients. Therefore, ASPP2 is a key regulator of BECN1-dependent autophagy, and decreased ASPP2 may contribute to tumor progression and chemoresistance via promoting autophagy. Show less

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34 Show less

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies. Show less

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. Show less

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation. Show less

Autophagy is a lysosome-dependent degradation pathway that maintains cellular homeostasis in response to a variety of cellular stresses. Accumulating reports based on animal models have indicated the importance of this catabolic program in many human pathophysiological conditions, including diabetes, neurodegenerative diseases, aging, and cancers. Therefore, autophagy has been highlighted as a novel therapeutic target with a wide range of beneficial effects on human diseases. Here, we review the recent advances of our knowledge toward autophagy, as well as the efforts for developing autophagy modulators. Areas covered: The relevant patents (published at 2012-2015) and the research literature claiming the pharmacological modulation of autophagy are reviewed. Also, their molecular mechanisms and potential therapeutic utilities are discussed. Expert opinion: Considering the molecular machinery involved in autophagy induction, the targeting of autophagy-specific protein is very important to design the therapeutic interventions for specifically treating a variety of autophagy-associated disorders. Many patents and the research literature described in this review have shown promising applications of the relevant autophagy modulators for cancer or neurodegeneration treatments, a few of which are already being considered for clinical evaluation. However, most patents have claimed the modulators of autophagy with little information regarding their mechanisms of action. To design highly potent therapeutics, further work, such as developing compounds that specifically target the autophagy-specific machinery, are required. Show less

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD). Show less

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets. Show less

The formation of the autophagosome is controlled by an orderly action of ATG proteins. However, how these proteins are recruited to autophagic membranes remain poorly clarified. In this study, we have provided a line of evidence confirming that EVA1A (eva-1 homolog A)/TMEM166 (transmembrane protein 166) is associated with autophagosomal membrane development. This notion is based on dotted EVA1A structures that colocalize with ZFYVE1, ATG9, LC3B, ATG16L1, ATG5, STX17, RAB7 and LAMP1, which represent different stages of the autophagic process. It is required for autophagosome formation as this phenotype was significantly decreased in EVA1A-silenced cells and Eva1a KO MEFs. EVA1A-induced autophagy is independent of the BECN1-PIK3C3 (phosphatidylinositol 3-kinase, catalytic subunit type 3) complex but requires ATG7 activity and the ATG12-ATG5/ATG16L1 complex. Here, we present a molecular mechanism by which EVA1A interacts with the WD repeats of ATG16L1 through its C-terminal and promotes ATG12-ATG5/ATG16L1 complex recruitment to the autophagic membrane and enhances the formation of the autophagosome. We also found that both autophagic and apoptotic mechanisms contributed to EVA1A-induced cell death while inhibition of autophagy and apoptosis attenuated EVA1A-induced cell death. Overall, these findings provide a comprehensive view to our understanding of the pathways involved in the role of EVA1A in autophagy and programmed cell death. Show less

Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin. Show less

PI3Kδ has been found to be over-expressed in B-Cell-related malignancies. Despite the clinical success of the first selective PI3Kδ inhibitor, CAL-101, inhibition of PI3Kδ itself did not show too much cytotoxic efficacy against cancer cells. One possible reason is that PI3Kδ inhibition induced autophagy that protects the cells from death. Since class III PI3K isoform PIK3C3/Vps34 participates in autophagy initiation and progression, we predicted that a PI3Kδ and Vps34 dual inhibitor might improve the anti-proliferative activity observed for PI3Kδ-targeted inhibitors. We discovered a highly potent ATP-competitive PI3Kδ/Vps34 dual inhibitor, PI3KD/V-IN-01, which displayed 10-1500 fold selectivity over other PI3K isoforms and did not inhibit any other kinases in the kinome. In cells, PI3KD/V-IN-01 showed 30-300 fold selectivity between PI3Kδ and other class I PI3K isoforms. PI3KD/V-IN-01 exhibited better anti-proliferative activity against AML, CLL and Burkitt lymphoma cell lines than known selective PI3Kδ and Vps34 inhibitors. Interestingly, we observed FLT3-ITD AML cells are more sensitive to PI3KD/V-IN-01 than the FLT3 wt expressing cells. In AML cell inoculated xenograft mouse model, PI3KD/V-IN-01 exhibited dose-dependent anti-tumor growth efficacies. These results suggest that dual inhibition of PI3Kδ and Vps34 might be a useful approach to improve the PI3Kδ inhibitor's anti-tumor efficacy. Show less

Macroautophagy/autophagy is a conserved catabolic process that recycles cytoplasmic material during low energy conditions. BECN1/Beclin1 (Beclin 1, autophagy related) is an essential protein for function of the class 3 phosphatidylinositol 3-kinase (PtdIns3K) complexes that play a key role in autophagy nucleation and elongation. Here, we show that AMP-activated protein kinase (AMPK) regulates autophagy by phosphorylating BECN1 at Thr388. Phosphorylation of BECN1 is required for autophagy upon glucose withdrawal. BECN1(T388A), a phosphorylation defective mutant, suppresses autophagy through decreasing the interaction between PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and ATG14 (autophagy-related 14). The BECN1(T388A) mutant has a higher affinity for BCL2 than its wild-type counterpart; the mutant is more prone to dimer formation. Conversely, a BECN1 phosphorylation mimic mutant, T388D, has stronger binding to PIK3C3 and ATG14, and promotes higher autophagy activity than the wild-type control. These findings uncover a novel mechanism of autophagy regulation. Show less

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition. Show less

Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons. Show less

As a central node of the macroautophagy/autophagy process, the BECN1/Beclin1-PIK3C3/VPS34 complex participates in different steps of autophagy by interacting with multiple molecules. The ATG14-associated PIK3C3 complex is involved in autophagy initiation, whereas the UVRAG-associated complex mainly modulates autophagosome maturation and endosome fusion. However, the molecular mechanism that coordinates the sequential execution of the autophagy program remains unknown. We have recently discovered that a Golgi-resident protein, PAQR3, regulates autophagy initiation as it preferentially facilitates the formation of the ATG14-linked PIK3C3 complex instead of the UVRAG-associated complex. Upon glucose starvation, AMPK directly phosphorylates T32 of PAQR3, which is crucial for the activation of the ATG14-associated class III PtdIns3K. Furthermore, Paqr3-deleted mice have a deficiency in exercise-induced autophagy as well as behavioral disorders. Thus, this work not only uncovers the regulatory mechanism of PAQR3 on autophagy initiation, but also provides a potential candidate therapeutic target for neurodegenerative diseases. Show less

The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this study (see the related punctum in Autophagy 12-5), Liu et al. report a function of the CUL3-KLHL20 ubiquitin ligase in feedback regulation, leading to the downregulation of autophagy through the degradation of the ULK1 and PIK3C3/VPS34 complexes. Show less

Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors. Show less

Women are under-represented in research on suicidality to date. Although women have a lower rate of suicide completion than men, due in part to the less-violent methods used, they have a higher rate of suicide attempts. Our group has previously identified genomic (blood gene expression biomarkers) and clinical information (apps) predictors for suicidality in men. We now describe pilot studies in women. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation (no SI) and high suicidal ideation (high SI) states (n=12 participants out of a cohort of 51 women psychiatric participants followed longitudinally, with diagnoses of bipolar disorder, depression, schizoaffective disorder and schizophrenia). We then used a Convergent Functional Genomics (CFG) approach to prioritize the candidate biomarkers identified in the discovery step by using all the prior evidence in the field. Next, we validated for suicidal behavior the top-ranked biomarkers for SI, in a demographically matched cohort of women suicide completers from the coroner's office (n=6), by assessing which markers were stepwise changed from no SI to high SI to suicide completers. We then tested the 50 biomarkers that survived Bonferroni correction in the validation step, as well as top increased and decreased biomarkers from the discovery and prioritization steps, in a completely independent test cohort of women psychiatric disorder participants for prediction of SI (n=33) and in a future follow-up cohort of psychiatric disorder participants for prediction of psychiatric hospitalizations due to suicidality (n=24). Additionally, we examined how two clinical instruments in the form of apps, Convergent Functional Information for Suicidality (CFI-S) and Simplified Affective State Scale (SASS), previously tested in men, perform in women. The top CFI-S item distinguishing high SI from no SI states was the chronic stress of social isolation. We then showed how the clinical information apps combined with the 50 validated biomarkers into a broad predictor (UP-Suicide), our apriori primary end point, predicts suicidality in women. UP-Suicide had a receiver-operating characteristic (ROC) area under the curve (AUC) of 82% for predicting SI and an AUC of 78% for predicting future hospitalizations for suicidality. Some of the individual components of the UP-Suicide showed even better results. SASS had an AUC of 81% for predicting SI, CFI-S had an AUC of 84% and the combination of the two apps had an AUC of 87%. The top biomarker from our sequential discovery, prioritization and validation steps, BCL2, predicted future hospitalizations due to suicidality with an AUC of 89%, and the panel of 50 validated biomarkers (BioM-50) predicted future hospitalizations due to suicidality with an AUC of 94%. The best overall single blood biomarker for predictions was PIK3C3 with an AUC of 65% for SI and an AUC of 90% for future hospitalizations. Finally, we sought to understand the biology of the biomarkers. BCL2 and GSK3B, the top CFG scoring validated biomarkers, as well as PIK3C3, have anti-apoptotic and neurotrophic effects, are decreased in expression in suicidality and are known targets of the anti-suicidal mood stabilizer drug lithium, which increases their expression and/or activity. Circadian clock genes were overrepresented among the top markers. Notably, PER1, increased in expression in suicidality, had an AUC of 84% for predicting future hospitalizations, and CSNK1A1, decreased in expression, had an AUC of 96% for predicting future hospitalizations. Circadian clock abnormalities are related to mood disorder, and sleep abnormalities have been implicated in suicide. Docosahexaenoic acid signaling was one of the top biological pathways overrepresented in validated biomarkers, which is of interest given the potential therapeutic and prophylactic benefits of omega-3 fatty acids. Some of the top biomarkers from the current work in women showed co-directionality of change in expression with our previous work in men, whereas others had changes in opposite directions, underlying the issue of biological context and differences in suicidality between the two genders. With this study, we begin to shed much needed light in the area of female suicidality, identify useful objective predictors and help understand gender commonalities and differences. During the conduct of the study, one participant committed suicide. In retrospect, when the analyses were completed, her UP-Suicide risk prediction score was at the 100 percentile of all participants tested. Show less

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. Show less

Autophagy is a dynamic and self-limiting process. The amplitude and duration of this process need to be properly controlled to maintain cell homeostasis, and excessive or insufficient autophagy activity could each lead to disease states. Compared to our understanding of the molecular mechanisms of autophagy induction, little is known about how the autophagy process is turned off after its activation. We recently identified KLHL20 as a key regulator of autophagy termination. By functioning as a substrate-binding subunit of CUL3 ubiquitin ligase, KLHL20 targets the activated ULK1 and phagophore-residing PIK3C3/VPS34 and BECN1 for ubiquitination and proteasomal degradation, which in turn triggers a destabilization of their complex components ATG13 and ATG14. These hierarchical degradation events cause the exhaustion of the autophagic pool of ULK1 and PIK3C3/VPS34 complexes, thereby preventing persistent and excessive autophagy activity. Impairment of KLHL20-dependent feedback regulation of autophagy enhances cell death under prolonged starvation and aggravates muscle atrophy in diabetic mice, which highlights the pathophysiological significance of this autophagy termination mechanism in cell survival and tissue homeostasis. Modulation of this autophagy termination pathway may be effective for treating diseases associated with deregulation of autophagy activity. Show less

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions. Show less

The oncogenic herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is known to encode four viral interferon regulatory factors (vIRF1 to -4) to subvert the host antiviral immune response, but their detailed DNA-binding profiles as transcription factors in the host remain uncharacterized. Here, we first performed genome-wide vIRF2-binding site mapping in the human genome using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). vIRF2 was capable of binding to the promoter regions of 100 putative target genes. Importantly, we confirmed that vIRF2 can specifically interact with the promoters of the genes encoding PIK3C3, HMGCR, and HMGCL, which are associated with autophagosome formation or tumor progression and metastasis, and regulate their transcription in vivo. The crystal structure of the vIRF2 DNA-binding domain (DBD) (referred to here as vIRF2DBD) showed variable loop conformations and positive-charge distributions different from those of vIRF1 and cellular IRFs that are associated with DNA-binding specificities. Structure-based mutagenesis revealed that Arg82 and Arg85 are required for the in vitro DNA-binding activity of vIRF2DBD and can abolish the transcription regulation function of vIRF2 on the promoter reporter activity of PIK3C3, HMGCR, and HMGCL. Collectively, our study provided unique insights into the DNA-binding potency of vIRF2 and suggested that vIRF2 could act as a transcription factor of its target genes in the host antiviral immune response. The oncogenic herpesvirus KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV has developed a unique mechanism to subvert the host antiviral immune responses by encoding four homologues of cellular interferon regulatory factors (vIRF1 to -4). However, none of their DNA-binding profiles in the human genome have been characterized until now, and the structural basis for their diverse DNA-binding properties remain poorly understood. In this study, we performed the first genome-wide vIRF2-binding site mapping in the human genome and found vIRF2 can bind to the promoter regions of 100 target cellular genes. X-ray structure analysis and functional studies provided unique insights into its DNA-binding potency and regulation of target gene expression. Our study suggested that vIRF2 could act as a transcription factor of its target genes and contribute to KSHV infection and pathogenesis through versatile functions. Show less

The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis. Show less

Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives. Show less

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility. Show less

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation. Show less

Cytolethal distending toxin B (cdtB) is a conserved virulence factor in Salmonella enterica serovar Typhi. Here we report the presence and functionality of cdtB in some nontyphoidal Salmonella (NTS) serovars, including Salmonella Javiana (cdtB+wt S. Javiana), isolated from imported food. To understand the role of cdtB in NTS serovars, a deletion mutant (cdtB(-)ΔS. Javiana) was constructed. Macrophages were infected with cdtB+wt S. Javiana (wild type), cdtB(-)Δ S. Javiana (mutant), and cdtB-negative NTS serovar (S. Typhimurium). Cytotoxic activity and transcription level of genes involved in cell death (apoptosis, autophagy, and necrosis) were assessed in infected macrophages. The cdtB+wt S. Javiana caused cellular distension as well as high degree of vacuolization and presence of the autophagosome marker LC3 in infected macrophages as compared with cdtB(-)ΔS. Javiana. The mRNA expression of genes involved in the induction of autophagy in response to toxin (Esr1 and Pik3C3) and coregulators of autophagy and apoptosis (Bax and Cyld) were significantly upregulated in cdtB(+)wt S. Javiana-infected macrophages. As autophagy destroys internalized pathogens in addition to the infected cell, it may reduce the spread of infection. Show less

Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive. Show less

Recently, we reported that saturated and unsaturated fatty acids trigger autophagy through distinct signal transduction pathways. Saturated fatty acids like palmitate (PA) induce autophagic responses that rely on phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, best known as VPS34) and beclin 1 (BECN1). Conversely, unsaturated fatty acids like oleate (OL) promote non-canonical, PIK3C3- and BECN1-independent autophagy. Here, we explored the metabolic effects of autophagy-inducing doses of PA and OL in mice. Mass spectrometry coupled to principal component analysis revealed that PA and OL induce well distinguishable changes in circulating metabolites as well as in the metabolic profile of the liver, heart, and skeletal muscle. Importantly, PA (but not OL) causes the depletion of multiple autophagy-inhibitory amino acids in the liver. Conversely, OL (but not PA) increased the hepatic levels of nicotinamide adenine dinucleotide (NAD), an obligate co-factor for autophagy-stimulatory enzymes of the sirtuin family. Moreover, PA (but not OL) raised the concentrations of acyl-carnitines in the heart, a phenomenon that perhaps is linked to its cardiotoxicity. PA also depleted the liver from spermine and spermidine, 2 polyamines have been ascribed with lifespan-extending activity. The metabolic changes imposed by unsaturated and saturated fatty acids may contribute to their health-promoting and health-deteriorating effects, respectively. Show less

Autophagy is a catabolic process that maintains cellular homeostasis by degradation of protein aggregates and selective removal of damaged organelles, e.g. mitochondria (mitophagy). Insulin resistance in skeletal muscle has been linked to mitochondrial dysfunction and altered protein metabolism. Here, we investigated whether abnormalities in autophagy are present in human muscle in obesity and type 2 diabetes. Using a case-control design, skeletal muscle biopsies obtained in the basal and insulin-stimulated states from patients with type 2 diabetes during both euglycaemia and hyperglycaemia, and from glucose-tolerant lean and obese individuals during euglycaemia, were used for analysis of mRNA levels, protein abundance and phosphorylation of autophagy-related proteins. Muscle transcript levels of autophagy-related genes (ULK1, BECN1, PIK3C3, ATG5, ATG7, ATG12, GABARAPL1, MAP1LC3B, SQSTM1, TP53INP2 and FOXO3A [also known as FOXO3]), including some specific for mitophagy (BNIP3, BNIP3L and MUL1), and protein abundance of autophagy-related gene (ATG)7 and Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), as well as content and phosphorylation of forkhead box O3A (FOXO3A) were similar among the groups. Insulin reduced lipidation of microtubule-associated protein light chain 3 (LC3)B-I to LC3B-II, a marker of autophagosome formation, with no effect on p62/sequestosome 1 (SQSTM1) content in muscle of lean and obese individuals. In diabetic patients, insulin action on LC3B was absent and p62/SQSTM1 content increased when studied under euglycaemia, whereas the responses of LC3B and p62/SQSTM1 to insulin were normalised during hyperglycaemia. Our results demonstrate that the levels of autophagy-related genes and proteins in muscle are normal in obesity and type 2 diabetes. This suggests that muscle autophagy in type 2 diabetes has adapted to hyperglycaemia, which may contribute to preserve muscle mass. Show less