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The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by ∼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy., Competing Interests: Declaration of competing interest None. The authors have no competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) belongs to the family of Picornaviridae ; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development., Competing Interests: The authors declare no conflict of interest.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology. NEW & NOTEWORTHY This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.

Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Autophagy is a highly conserved catabolic mechanism by which unnecessary or dysfunctional cellular components are removed. The dysregulation of autophagy has been implicated in various neurodegenerative diseases, including Alzheimer's disease (AD). Understanding the molecular mechanism(s)/molecules that influence autophagy may provide important insights into developing therapeutic strategies against AD and other neurodegenerative disorders. Engulfment adaptor phosphotyrosine-binding domain-containing protein 1 (GULP1) is an adaptor that interacts with amyloid precursor protein (APP) to promote amyloid-β peptide production via an unidentified mechanism. Emerging evidence suggests that GULP1 has a role in autophagy. Here, we show that GULP1 is involved in autophagy through an interaction with autophagy-related 14 (ATG14), which is a regulator of autophagosome formation. GULP1 potentiated the stimulatory effect of ATG14 on autophagy by modulating class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1) activity. The effect of GULP1 is attenuated by a GULP1 mutation (GULP1m) that disrupts the GULP1-ATG14 interaction. Conversely, PI3KC3-C1 activity is enhanced in cells expressing APP but not in those expressing an APP mutant that does not bind GULP1, which suggests a role of GULP1-APP in regulating PI3KC3-C1 activity. Notably, GULP1 facilitates the targeting of ATG14 to the endoplasmic reticulum (ER). Moreover, the levels of both ATG14 and APP are elevated in the autophagic vacuoles (AVs) of cells expressing GULP1, but not in those expressing GULP1m. APP processing is markedly enhanced in cells co-expressing GULP1 and ATG14. Hence, GULP1 alters APP processing by promoting the entry of APP into AVs. In summary, we unveil a novel role of GULP1 in enhancing the targeting of ATG14 to the ER to stimulate autophagy and, consequently, APP processing., (© 2024. The Author(s).)

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Cell death-inducing p53-target protein 1 (CDIP1) is a proapoptotic protein that is normally expressed at low levels and is upregulated by genotoxic and endoplasmic reticulum stresses. CDIP1 has been reported to be localized to endosomes and to interact with several proteins, including B-cell receptor-associated protein 31 (BAP31) and apoptosis-linked gene 2 (ALG-2). However, the cellular and molecular mechanisms underlying CDIP1 expression-induced apoptosis remain unclear. In this study, we first demonstrated that CDIP1 was upregulated after treatment with the anticancer drug adriamycin in human breast cancer MCF-7 cells but was degraded rapidly in the lysosomal pathway. We also demonstrated that treatment with the cyclin-dependent kinase 5 (CDK5) inhibitor roscovitine led to an increase in the electrophoretic mobility of CDIP1. In addition, a phosphomimetic mutation at Ser-32 in CDIP1 resulted in an increase in CDIP1 expression-induced apoptosis. We also found that CDIP1 expression led to the induction of autophagy prior to apoptosis. Treatment of cells expressing CDIP1 with SAR405, an inhibitor of the class III phosphatidylinositol 3-kinase VPS34, caused a reduction in autophagy and promoted apoptosis. Therefore, autophagy is thought to be a defense mechanism against CDIP1 expression-induced apoptosis.

Vps34 is the unique member of the class III phosphoinositide 3-kinase family that performs both vesicular transport and autophagy. Its role in natural killer (NK) cells remains uncertain. In this study, a model without Vps34 (Vps34 fl/fl /CD122 Cre/+ ) is generated, deleting Vps34 during and after NK-cell commitment. These mice exhibit a nearly 90% decrease in NK cell count and impaired differentiation. A mechanistic study reveals that the absence of Vps34 disrupts the transport of IL-15 receptor subunit alpha CD122 to the cell membrane, resulting in reduced responsiveness of NK cells to IL-15. In mice lacking Vps34 at the terminal stage of NK-cell development (Vps34 fl/fl /Ncr1 Cre/+ ), NK cells gradually diminish during aging. This phenotype is associated with autophagy deficiency and the stress induced by reactive oxygen species (ROS). Therefore, terminally differentiated NK cells lacking Vps34 display an accelerated senescence phenotype, while the application of antioxidants effectively reverses the senescence caused by Vps34 deletion by neutralizing ROS. In summary, this study unveils the dual and unique activity of Vps34 in NK cells. Vps34-mediated vesicular transport is crucial for CD122 membrane trafficking during NK cell commitment, whereas Vps34-mediated autophagy can delay NK cell senescence., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: The role of autophagy in coronaviruses infection and replication has a lot of debate. Autophagy involves the catalytic breakdown of intracellular components to be subsequently recycled by the lysosome. The aim of the study was to evaluate autophagy genes; PIK3C3 and RAB7A expressions in COVID-19 patients, and identify if PIK3C3 and RAB7A can be used as markers for monitoring COVID-19 patients., Methods: A case-control study was carried out on 50 patients and 50 healthy controls. Genes expression was performed using quantitative real-time polymerase chain reaction., Results: Compared to controls, PIK3C3 and RAB7A gene expression levels were significantly lower in patients (p < 0.001) with approximately with 9.4 and 2.3 decreased fold in PIK3C3 and RAB7A respectively. The ROC curve of PIK3C3 and RAB7A expressions showed sensitivity of 84 % and 74 % and specificity of 98 % and 78 % respectively. There was a positive correlation between PIK3C3 expression and WBCs, absolute neutrophil count, interleukin-6, D-dimer, and ALT among patients and between RAB7A expression and WBCs, CRP, IL-6, D-dimer and ALT in patients., Conclusions: The study showed reduction of PIK3C3 and RAB7A expressions in COVID-19 patients. However, further studies are recommended to clarify their roles in the disease pathogenies as autophagy genes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Macroautophagy/autophagy research often involves overexpressing proteins to investigate their localization, function and activity. However, this approach can disturb the inherent balance of cellular components, potentially affecting the integrity of the autophagy process. With the advent of genome-editing techniques like CRISPR-Cas9, it is now possible to tag endogenous proteins with fluorescent markers, enabling the study of their behaviors under more physiologically relevant conditions. Nevertheless, conventional microscopy methods have limitations in characterizing the behaviors of proteins expressed at endogenous levels. This challenge can be overcome by single-molecule localization microscopy (SMLM) methods, which provide single-molecule sensitivity and super-resolution imaging capabilities. In our recent study, we used SMLM in combination with genome editing to explore the behavior of endogenous ULK1 during autophagy initiation, yielding unprecedented insights into the autophagy initiation process. Abbreviation: ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; ER: endoplasmic reticulum; GABARAPL1: GABA type A receptor associated protein like 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; PALM: photo-activated localization microscopy; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P: phosphatidylinositol-3-phosphate; SMLM: single-molecule localization microscopy; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Autophagy is a ubiquitous pathological/physiological antioxidant cellular reaction in eukaryotic cells. Vacuolar protein sorting 34 (Vps34 or PIK3C3), which plays a crucial role in autophagy, has received much attention. As the only Class III phosphatidylinositol-3 kinase in mammals, Vps34 participates in vesicular transport, nutrient signaling and autophagy. Dysfunctionality of Vps34 induces carcinogenesis, and abnormal autophagy mediated by dysfunction of Vps34 is closely related to the pathological progression of various human diseases, which makes Vps34 a novel target for tumor immunotherapy. In this review, we summarize the molecular mechanisms underlying macroautophagy, and further discuss the structure-activity relationship of Vps34 inhibitors that have been reported in the past decade as well as their potential roles in anticancer immunotherapy to better understand the antitumor mechanism underlying the effects of these inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Lactate is a glycolysis product that is produced from pyruvate by LDH (lactate dehydrogenase) and plays an important role in physiological and pathological processes. However, whether lactate regulates autophagy is still unknown. We recently reported that LDHA is phosphorylated at serine 196 by ULK1 (unc-51 like kinase 1) under nutrient-deprivation conditions, promoting lactate production. Then, lactate mediates PIK3C3/VPS34 lactylation at lysine 356 and lysine 781 via acyltransferase KAT5/TIP60. PIK3C3/VPS34 lactylation enhances the association of PIK3C3/VPS34 with BECN1 (beclin 1, autophagy related), ATG14 and UVRAG, increases PIK3C3/VPS34 lipid kinase activity, promotes macroautophagy/autophagy and facilitates the endolysosomal degradation pathway. PIK3C3/VPS34 hyperlactylation induces autophagy and plays an essential role in skeletal muscle homeostasis and cancer progression. Overall, this study describes an autophagy regulation mechanism and the integration of two highly conserved life processes: glycolysis and autophagy.

Sertoli cells are highly polarized testicular cells that provide a nurturing environment for germ cell development and maturation during spermatogenesis. The class III phosphatidylinositol 3-kinase (PtdIns3K) plays core roles in macroautophagy in various cell types; however, its role in Sertoli cells remains unclear. Here, we generated a mouse line in which the gene encoding the catalytic subunit, Pik3c3 , was specifically deleted in Sertoli cells (cKO) and found that after one round of normal spermatogenesis, the cKO mice quickly became infertile and showed disruption of Sertoli cell polarity and impaired spermiogenesis. Subsequent proteomics and phosphoproteomics analyses enriched the F-actin cytoskeleton network involved in the disorganized Sertoli-cell structure in cKO testis which we identified a significant increase of the F-actin negative regulator SCIN (scinderin) and the reduced phosphorylation of HDAC6, an α-tubulin deacetylase. Our results further demonstrated that the accumulation of SCIN in cKO Sertoli cells caused the disorder and disassembly of the F-actin cytoskeleton, which was related to the failure of SCIN degradation through the autophagy-lysosome pathway. Additionally, we found that the phosphorylation of HDAC6 at site S59 by PIK3C3 was essential for its degradation through the ubiquitin-proteasome pathway. As a result, the HDAC6 that accumulated in cKO Sertoli cells deacetylated SCIN at site K189 and led to a disorganized F-actin cytoskeleton. Taken together, our findings elucidate a new mechanism for PIK3C3 in maintaining the polarity of Sertoli cells, in which both its autophagy regulation or protein kinase activities are required for the stabilization of the actin cytoskeleton. Abbreviations: ACTB: actin, beta; AR: androgen receptor; ATG14: autophagy related 14; BafA1: bafilomycin A 1 ; BECN1: beclin 1, autophagy related; BTB: blood-testis barrier; CASP3: caspase 3; CDC42: cell division cycle 42; CDH2: cadherin 2; CHX: cycloheximide; CTNNA1: catenin (cadherin associated protein), alpha 1; CYP11A1: cytochrome P450, family 11, subfamily A, polypeptide 1; EBSS: Earle's balanced salt solution; ES: ectoplasmic specialization; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCNA: germ cell nuclear acidic protein; GJA1: gap junction protein, alpha 1; H2AX: H2A.X variant histone; HDAC6: histone deacetylase 6; KIT: KIT proto-oncogene, receptor tyrosine kinase; LAMP1: lysosomal associated membrane protein 1; MAP3K5: mitogen-activated protein kinase kinase kinase 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OCLN: occludin; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PNA: arachis hypogaea lectin; RAC1: Rac family small GTPase 1; SCIN: scinderin; SQSTM1/p62: sequestosome 1; SSC: spermatogonia stem cell; STK11: serine/threonine kinase 11; TJP1: tight junction protein 1; TubA: tubastatin A; TUBB3: tubulin beta 3 class III; TUNEL: TdT-mediated dUTP nick-end labeling; UB: ubiquitin; UVRAG: UV radiation resistance associated gene; VIM: vimentin; WT1: WT1 transcription factor; ZBTB16: zinc finger and BTB domain containing 16.

Nearly fifty million older people suffer from neurodegenerative diseases, including Alzheimer (AD) and Parkinson (PD) disease, a global burden expected to triple by 2050. Such an imminent "neurological pandemic" urges the identification of environmental risk factors that are hopefully avoided to fight the disease. In 2022, strong evidence in mouse models incriminated defective lysosomal acidification and impairment of the autophagy pathway as modifiable risk factors for dementia. To date, the most prescribed lysosomotropic drugs are proton pump inhibitors (PPIs), chloroquine (CQ), and the related hydroxychloroquine (HCQ), which belong to the group of disease-modifying antirheumatic drugs (DMARDs). This commentary aims to open the discussion on the possible mechanisms connecting the long-term prescribing of these drugs to the elderly and the incidence of neurodegenerative diseases. Abbreviations : AD: Alzheimer disease; APP-βCTF: amyloid beta precursor protein-C-terminal fragment; BACE1: beta-secretase 1; BBB: brain blood barrier; CHX: Ca 2+ /H + exchanger; CMI: cognitive mild impairment; CQ: chloroquine; DMARD: disease-modifying antirheumatic drugs; GBA1: glucosylceramidase beta 1; HCQ: hydroxychloroquine; HPLC: high-performance liquid chromatography; LAMP: lysosomal associated membrane protein; MAPK/JNK: mitogen-activated protein kinase; MAPT: microtubule associated protein tau; MCOLN1/TRPML1: mucolipin TRP cation channel 1; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NRBF2: nuclear receptor binding factor 2; PANTHOS: poisonous flower; PD: Parkinson disease; PIK3C3: phosphatIdylinositol 3-kinase catalytic subunit type 3; PPI: proton pump inhibitor; PSEN1: presenilin 1, RUBCN: rubicon autophagy regulator; RUBCNL: rubicon like autophagy enhancer; SQSTM1: sequestosome 1; TMEM175: transmembrane protein 175; TPCN2: two pore segment channel 2; VATPase: vacuolar-type H + -translocating ATPase; VPS13C: vacuolar protein sorting ortholog 13 homolog C; VPS35: VPS35 retromer complex component; WDFY3: WD repeat and FYVE domain containing 3; ZFYVE1: zinc finger FYVE-type containing 1.

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections., Competing Interests: The authors declare no conflict of interest.

VPS34 is well-known to be the unique member of the class III phosphoinositide 3-kinase (PI3K) family, forming VPS34 complex 1 and complex 2, which are involved in several key physiological processes. Of note, VPS34 complex 1 is an important node of autophagosome generation, which controls T cell metabolism and maintains cellular homeostasis through the autophagic pathway. And, VPS34 complex 2 is involved in endocytosis as well as vesicular transport, and is closely related to neurotransmission, antigen presentation and brain development. Due to the two important biological functions of VPS34, its dysregulation can lead to the development of cardiovascular disease, cancer, neurological disorders, and many types of human diseases by altering normal human physiology. Thus, in this review, we not only summarize the molecular structure and function of VPS34, but demonstrate the relationships between VPS34 and human diseases. Moreover, we further discuss the current small molecule inhibitors targeting VPS34 based upon the structure and function of VPS34, which may provide an insight into the future targeted drug development., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Adipose tissue, or body fat, plays a critical role in the maintenance of health and the development of metabolic diseases. The pathological expansion of adipose tissue during obesity and the pathological reduction of adipose tissue during lipodystrophy can lead to a similar array of metabolic diseases that include diabetes, but mechanisms remain to be fully defined. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to adipose tissue health and metabolic disease. We found that adipocyte-specific PIK3C3/VPS34 deficiency causes defects in the differentiation, survival and functional properties of adipocytes, resulting in reduced adipose tissue mass, altered blood lipid levels, fatty liver disease, diabetes, and defective body temperature control. These abnormalities mirror those observed in patients with lipodystrophy. These findings identify adipocyte PIK3C3/VPS34 as a potential target for therapeutic intervention in metabolic diseases.

Autophagy and glycolysis are highly conserved biological processes involved in both physiological and pathological cellular programs, but the interplay between these processes is poorly understood. Here, we show that the glycolytic enzyme lactate dehydrogenase A (LDHA) is activated upon UNC-51-like kinase 1 (ULK1) activation under nutrient deprivation. Specifically, ULK1 directly interacts with LDHA, phosphorylates serine-196 when nutrients are scarce and promotes lactate production. Lactate connects autophagy and glycolysis through Vps34 lactylation (at lysine-356 and lysine-781), which is mediated by the acyltransferase KAT5/TIP60. Vps34 lactylation enhances the association of Vps34 with Beclin1, Atg14L, and UVRAG, and then increases Vps34 lipid kinase activity. Vps34 lactylation promotes autophagic flux and endolysosomal trafficking. Vps34 lactylation in skeletal muscle during intense exercise maintains muscle cell homeostasis and correlates with cancer progress by inducing cell autophagy. Together, our findings describe autophagy regulation mechanism and then integrate cell autophagy and glycolysis.

The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1 WSN and H3N8 JL89 . We found that the promotion of self-replication could be mainly attributed to NS1 pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1 pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines. Abbreviations: 293T: human embryonic kidney 293 cells; 293T&#95;LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1 JL89 : non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1 pdm09 : non-structural protein 1 of A/(H1N1) pdm09; NS1 SC09 : non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1 WSN : non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1 JL89 : WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1 SC09 : WSN recombinant strain in which NS1 was replaced with that of SC09.

The COVID-19 pandemic has caused substantial losses worldwide in people's lives, health, and property. Currently, COVID-19 is still prominent worldwide without any specific drug treatment. The SARS-CoV-2 pathogen is the cause of various systemic diseases, mainly acute pneumonia. Within the pathological process, neutrophils are recruited to infected sites, especially in the lungs, for the first stage of removing invading SARS-CoV-2 through a range of mechanisms. Macroautophagy/autophagy, a conserved autodegradation process in neutrophils, plays a crucial role in the neutrophil phagocytosis of pathogens. NETosis refers to neutrophil cell death, while auto-inflammatory factors and antigens release NETs. This review summarizes the latest research progress and provides an in-depth explanation of the underlying mechanisms of autophagy and NETosis in COVID-19. Furthermore, after exploring the relationship between autophagy and NETosis, we discuss potential targets and treatment options. This review keeps up with the latest research on COVID-19 from neutrophil autophagy and NETosis with a new perspective, which can guide the urgent development of antiviral drugs and provide guidance for the clinical treatment of COVID-19. Abbreviations: AKT1: AKT serine/threonine kinase 1; AMPK: AMP-activated protein kinase; AP: autophagosome; ARDS: acute respiratory distress syndrome; ATG: autophagy related; BECN1: beclin 1; cfDNA: cell-free DNA; COVID-19: coronavirus disease 2019; CQ: chloroquine; DMVs: double-membrane vesicles; ELANE/NE: elastase, neutrophil expressed; F3: coagulation factor III, tissue factor; HCQ: hydroxychloroquine; MAP1LC3/LC3: microtubule associated protein 1 light chain of 3; MPO: myeloperoxidase; MTORC1: mechanistic target of rapamycin kinase complex 1; NETs: neutrophil traps; NSP: nonstructural protein; PI3K: class I phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; ROS: reactive oxygen species; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SKP2: S-phase kinase associated protein 2; TCC: terminal complement complex; ULK1: unc-51 like.

Post-translational modifications, such as phosphorylation, ubiquitination and acetylation, play crucial roles in the regulation of autophagy. Acetylation has emerged as an important regulatory mechanism for autophagy. Acetylation regulates autophagy initiation and autophagosome formation by targeting core components of the ULK1 complex, the BECN1-PIK3C3 complex, and the LC3 lipidation system. Recent studies have shown that acetylation occurs on the key proteins participating in autophagic cargo assembly and autophagosome-lysosome fusion, such as SQSTM1/p62 and STX17. In addition, acetylation controls autophagy at the transcriptional level by targeting histones and the transcription factor TFEB. Here, we review the current knowledge on acetylation of autophagy proteins and their regulations and functions in the autophagy pathway with focus on recent findings. Abbreviations : ACAT1: acetyl-CoA acetyltransferase 1; ACSS2: acyl-CoA synthetase short chain family member 2; AMPK: AMP-activated protein kinase; ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCAR2/DBC1: cell cycle and apoptosis regulator 2; BECN1: beclin 1; CMA: chaperone-mediated autophagy; CREBBP/CBP: CREB binding protein; EP300/p300: E1A binding protein p300; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3: glycogen synthase kinase 3; HDAC6: histone deacetylase 6; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PD: Parkinson disease; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKM2: pyruvate kinase M1/2; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: post-translational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RUBCN/Rubicon: rubicon autophagy regulator; RUBCNL/Pacer: rubicon like autophagy enhancer; SIRT1: sirtuin 1; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TFEB: transcription factor EB; TP53/p53: tumor protein p53; TP53INP2/DOR: tumor protein p53 inducible nuclear protein 2; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WIPI2: WD repeat domain, phosphoinositide interacting 2.

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.

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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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., (© 2022 Postoak et al.)

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)P 2 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)P 2 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 3 H-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., Competing Interests: Conflict of interests The authors declare no competing or financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Acquired chemotherapy resistance is one of the main culprits in the relapse of breast cancer. But the underlying mechanism of chemotherapy resistance remains elusive. Here, we demonstrate that a small adaptor protein, SH3BGRL, is not only elevated in the majority of breast cancer patients but also has relevance with the relapse and poor prognosis of breast cancer patients. Functionally, SH3BGRL upregulation enhances the chemoresistance of breast cancer cells to the first-line doxorubicin treatment through macroautophagic/autophagic protection. Mechanistically, SH3BGRL can unexpectedly bind to ribosomal subunits to enhance PIK3C3 translation efficiency and sustain ATG12 stability. Therefore, inhibition of autophagy or silence of PIK3C3 or ATG12 can effectively block the driving effect of SH3BGRL on doxorubicin resistance of breast cancer cells in vitro and in vivo. We also validate that SH3BGRL expression is positively correlated with that of PIK3C3 or ATG12, as well as the constitutive occurrence of autophagy in clinical breast cancer tissues. Taken together, our data reveal that SH3BGRL upregulation would be a key driver to the acquired chemotherapy resistance through autophagy enhancement in breast cancer while targeting SH3BGRL could be a potential therapeutic strategy against breast cancer. Abbreviations: ABCs: ATP-binding cassette transporters; Act D: actinomycin D; ACTB/β-actin: actin beta; ATG: autophagy-related; Baf A 1 : bafilomycin A 1 ; CASP3: caspase 3; CHX: cycloheximide; CQ: chloroquine; Dox: doxorubicin; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GEO: gene expression omnibus; GFP: green fluorescent protein; G6PD: glucose-6-phosphate dehydrogenase; GSEA: gene set enrichment analysis; IHC: immunochemistry; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3-MA: 3-methyladenine; mRNA: messenger RNA; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; SH3BGRL: SH3 domain binding glutamate-rich protein-like; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

Abbreviations: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Hypoxia is a component of both physiological and pathological conditions, including inflammation, solid tumors, and lymphoid tissues, where O 2 demand is not balanced by O 2 supply. During their lifespan, dendritic cells (DCs) are exposed to different pO 2 and activate different adaptive responses, including autophagy, to preserve their viability and functions. Autophagy plays multiple roles in DC physiology. Very recently, we demonstrated that hypoxia shapes autophagy in DCs upon their differentiation state. Here, we proposed a role for PI3Ks, and especially class III PI3K/Vps34, that could be relevant in hypoxia-induced autophagy, in either immature or mature DCs. Hypoxia inhibited mTOR phosphorylation and activated a pro-autophagic program. By using different pharmacological inhibitors, we demonstrated that hypoxia-induced autophagy was mediated by PI3Ks, especially by Vps34. Furthermore, Vps34 expression was enhanced by LPS, a TLR4 ligand, along with the promotion of autophagy under hypoxia. The Vps34 inhibitor, SAR405, abolished hypoxia-induced autophagy, inhibited pro-survival signaling and viability, and increased the expression of proinflammatory cytokines. Our results underlined the impact of autophagy in the maintenance of DC homeostasis at both cell survival and inflammatory response levels, therefore, contributing to a better understanding of the significance of autophagy in DC physiology and pathology.

Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable because of old age or the advanced stage of the disease; thus radio- and chemotherapy are believed as the standard treatments for these patients. However, due to the radio-resistance of tumor cells that may develop during radiotherapy, results remain unsatisfactory. In this article, the possible relationship between the expression of lysine demethylase 5B (KDM5B) and ESCC radio-resistance is clarified, and the underlying mechanism is evaluated. Using the GSE75241 microarray, we identified KDM5B as a potential oncogene in ESCC. KDM5B was overexpressed in ESCC patients and cells. Inhibition of KDM5B enhanced the H3K4me3 methylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) promoter and induced the expression of PIK3C3. Knockdown of KDM5B or overexpression of PIK3C3 in KYSE-150 and TE-10 cells promoted apoptosis, cell cycle arrest, autophagy, and increased sensitivity to radiotherapy. Silencing of PIK3C3 attenuated the promoting effect of sh-KDM5B on the sensitivity of ESCC cells to radiotherapy. The inhibition of sh-KDM5B in radio-resistance of ESCC cells was also reproduced in vivo. Taken together, our findings provide evidence that reduced expression of KDM5B has a critical role in promoting ESCC radio-sensitivity by upregulating PIK3C3, suggesting KDM5B may function as an oncogene in ESCC., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

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., (© 2022. The Author(s).)

Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.

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. Abbreviations : ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

Triple‑negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX‑induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA‑MB‑231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX‑induced autophagy through a transcriptional activation pattern in MDA‑MB‑231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3‑kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3β. Knocking down FOXO1 attenuated the survival of MDA‑MB‑231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC.

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., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

S-adenosyl-l-homocysteine (SAH), an amino acid derivative, is a key intermediate metabolite in methionine metabolism, which is normally considered as a harmful by-product and hydrolyzed quickly once formed. AHCY (adenosylhomocysteinase) converts SAH into homocysteine and adenosine. There are two other members in the AHCY family, AHCYL1 (adenosylhomocysteinase like 1) and AHCYL2 (adenosylhomocysteinase like 2). Here we define AHCYL1 function as a SAH sensor to inhibit macroautophagy/autophagy through PIK3C3. The C terminus of AHCYL1 interacts with SAH specifically and the interaction with SAH promotes the binding of the N terminus to the catalytic domain of PIK3C3, resulting in inhibition of PIK3C3. More importantly, this observation was further validated in vivo , indicating that SAH functions as a signaling molecule. Our study uncovers a new axis of SAH-AHCYL1-PIK3C3, which senses the intracellular level of SAH to inhibit autophagy in an MTORC1-independent manner. Abbreviations: ADOX: adenosine dialdehyde; AHCY: adenosylhomocysteinase; AHCYL1: adenosylhomocysteinase like 1; cLEU: cycloleucine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; SAH: S-adenosyl-l-homocysteine; SAM: S-adenosyl-l-methionine.

Cell surface receptors control how cells respond to their environment. Many cell surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH, and CCC complexes. Here, using mammalian cells, we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces phosphatidylinositol 3-phosphate (PI3P), which is the substrate for PIKfyve to generate PI3,5P 2 . We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH, and CCC complexes on endosomes. Importantly, PIKfyve inhibition results in displacement of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration., Competing Interests: SG, GL, PR, NS, HT, AS, EB, DB, MS, LW No competing interests declared, (© 2022, Giridharan et al.)

PIK3C3/VPS34 is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), which play critical roles in dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3 -deficient DCs exhibit attenuated capacity to reactivate encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3 -deficient mice. Additionally, animals with a DC-specific deficiency in Rb1cc1/Fip200 but not Rubcn were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3 -deficient mice is due to defective canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and the pathogenicity of these cells during EAE, with important implications for the development of immunotherapies for autoimmune diseases such as MS. Abbreviations: ATG: autophagy-related; CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; ROS: reactive oxygen species.

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.

Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases. Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens ; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus ; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Macroautophagy/autophagy is triggered by various starvation and stress conditions. The phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) is essential for the formation of the autophagosome both in yeast and mammals. The class III phosphatidylinositol 3-kinase, PIK3C3C in humans or Vps34 in yeast, produces PtdIns3P by phosphorylating the 3'-OH position of phosphatidylinositol (PtdIns). In order to synthesize PtdIns3P for the initiation of autophagy, PIK3C3/Vps34 has a heterotetrameric core, the PIK3C3 complex I (hereafter complex I) composed of PIK3C3/Vps34, PIK3R4/Vps15, BECN1/Vps30, and ATG14/Atg14. A fifth component of complex I, NRBF2 in mammals and Atg38 in yeast, was found and has been characterized in the past decade. The field has been expanding from cell and structural biology to mouse model and cohort studies. Here I will summarize the structures and models of complex I binding NRBF2/Atg38, its intracellular roles, and its involvement in health and disease. Along with this expansion of the field, different conclusions have been drawn in several topics. I will clarify what has and has not been agreed, and what is to be clarified in the future.

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.

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 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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

Phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), the mammalian ortholog of yeast vesicular protein sorting 34 (Vps34), belongs to the phosphoinositide 3-kinase (PI3K) family. PIK3C3 can phosphorylate phosphatidylinositol (PtdIns) to generate phosphatidylinositol 3-phosphate (PI3P), a phospholipid central to autophagy. Inhibition of PIK3C3 successfully inhibits autophagy. Autophagy maintains cell survival when modifications occur in the cellular environment and helps tumor cells resist metabolic stress and cancer treatment. In addition, PIK3C3 could induce oncogenic transformation and enhance tumor cell proliferation, growth, and invasion through mechanisms independent of autophagy. This review addresses the structural and functional features, tissue distribution, and expression pattern of PIK3C3 in a variety of human tumors and highlights the underlying mechanisms involved in carcinogenesis. The implications in cancer biology, patient prognosis prediction, and cancer therapy are discussed. Altogether, the discovery of pharmacological inhibitors of PIK3C3 could reveal novel strategies for improving treatment outcomes for PIK3C3-mediated human diseases.

A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267-284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy. Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A 1 ; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks' balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.