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Neuropathic pain (NP) is a chronic pain state with a complex etiology that currently lacks effective treatment in clinical practice. Studies have found that exercise training can alleviate NP hyperalgesia, but the specific mechanism remains unclear. Here, we sought to identify proteins and signaling pathways critical for mediating the effects of treadmill training on NP in a mouse model of spared nerve injury (SNI). We used Tandem Mass Tag (TMT) technology for proteins and signaling pathways identification. Functional enrichment analyses were conducted using DAVID and Metascape software. Ingenuity pathway analysis was used to conduct functional annotation and analyze alterations in canonical pathways and molecular networks. Reverse transcription quantitative PCR (RT-qPCR) was used to confirm the results of proteomics analysis. A total of 270 differentially expressed proteins were screened in the detrained and trained groups ( Our results suggest that treadmill training may alleviate nociceptive hyperalgesia in NP mice by modulating the autophagic pathway, providing unique mechanistic insights into the analgesic effects of exercise. Show less

p38 mitogen-activated protein kinases (MAPKs) regulate early endocytic trafficking, but their effects on late endocytic trafficking remain unclear. Herein, we report that the pyridinyl imidazole p38 MAPK inhibitors, SB203580 and SB202190, induce a rapid but reversible Rab7-dependent accumulation of large cytoplasmic vacuoles. While SB203580 did not induce canonical autophagy, phosphatidylinositol 3-phosphate [PI(3)P] accumulated on vacuole membranes, and inhibition of the class III PI3-kinase (PIK3C3/VPS34) suppressed vacuolation. Ultimately, vacuolation resulted from the fusion of ER/Golgi-derived membrane vesicles with late endosomes and lysosomes (LELs), combined with an osmotic imbalance in LELs that led to severe swelling and a decrease in LEL fission. Since PIKfyve inhibitors induce a similar phenotype by preventing the conversion of PI(3)P to PI(3,5)P2, we performed Show less

The development of a broad repertoire of T cells in the immune system requires interaction of T cell receptors expressed by immature T cells with peptide/major histocompatibility complexes (MHCs) displayed by specialized epithelial cells in the thymus, in a process called T cell positive selection. Thymic epithelial cells (TECs) display unique antigen processing machinery which shapes the collection of self-peptides that drive positive selection. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to the generation of positively selecting peptides in TECs. We found that TEC-specific PIK3C3/VPS34 facilitates the positive selection of CD4 but not CD8 T lineage cells, in a mechanism independent of its role in canonical macroautophagy/autophagy. Instead, we propose that PIK3C3/VPS34 alters vesicle trafficking in TECs that modulates lysosomal protease activity which, in turn, controls the generation of MHC class II-presented peptides optimized for positive selection of CD4 T cells. Show less

VPS35 and VPS13 have been associated with Parkinson's disease (PD), and their shared phenotype in yeast when reduced in function is abnormal vacuolar transport. We aim to test if additional potentially deleterious variants in other genes that share this phenotype can modify the risk for PD. 77 VPS and VPS-related genes were analyzed using whole-genome-sequencing data from 202 PD patients of Ashkenazi Jewish (AJ) ancestry. Filtering was done based on quality and functionality scores. Ten variants in nine genes were further genotyped in 1200 consecutively recruited unrelated AJ-PD patients, and allele frequencies and odds ratio calculated compared to gnomAD-AJ-non-neuro database, in un-stratified (n = 1200) and stratified manner (LRRK2-G2019S-PD patients (n = 145), GBA-PD patients (n = 235), and non-carriers of these mutations (NC, n = 787)). Five variants in PIK3C3, VPS11, AP1G2, HGS and VPS13D were significantly associated with PD-risk. PIK3C3-R768W showed a significant association in an un-stratified (all PDs) analysis, as well as in stratified (LRRK2, GBA, and NC) analyses (Odds ratios = 2.71, 5.32, 3.26. and 2.19 with p = 0.0015, 0.002, 0.0287, and 0.0447, respectively). AP1G2-R563W was significantly associated in LRRK2-carriers (OR = 3.69, p = 0.006) while VPS13D-D2932N was significantly associated in GBA-carriers (OR = 5.45, p = 0.0027). VPS11-C846G and HGS-S243Y were significantly associated in NC (OR = 2.48 and 2.06, with p = 0.022 and 0.0163, respectively). Variants in genes involved in vesicle-mediated protein transport and recycling pathways, including autophagy and mitophagy, may differentially modify PD-risk in LRRK2-carriers, GBA carriers, or NC. Specifically, PIK3C3-R768W is a PD-risk allele, with the highest effect size in LRRK2-G2019S carriers. These results suggest oligogenic effect that may depends on the genetic background of the patient. An unbiased burden of mutations approach in these genes should be evaluated in additional PD and control groups. The mechanisms by which these novel variants interact and increase PD-risk should be researched in depth for better tailoring therapeutic intervention for PD prevention or slowing disease progression. Show less

Skeletal muscle turnover helps support the physiological needs of dairy cows during the transition into lactation. We evaluated effects of feeding ethyl-cellulose rumen-protected methionine (RPM) during the periparturient period on abundance of proteins associated with transport AA and glucose, protein turnover, metabolism, and antioxidant pathways in skeletal muscle. Sixty multiparous Holstein cows were used in a block design and assigned to a control or RPM diet from -28 to 60 d in milk. The RPM was fed at a rate of 0.09% or 0.10% of dry matter intake (DMI) during the prepartal and postpartal periods to achieve a target Lys:Met ratio in the metabolizable protein of ∼2.8:1. Muscle biopsies from the hind leg of 10 clinically healthy cows per diet collected at -21, 1, and 21 d relative to calving were used for western blotting of 38 target proteins. Statistical analysis was performed using the PROC MIXED statement of SAS version 9.4 (SAS Institute Inc.) with cow as random effect, whereas diet, time, and diet × time were the fixed effects. Diet × time tended to affect prepartum DMI, with RPM cows consuming 15.2 kg/d and controls 14.6 kg/d. However, diet had no effect on postpartum DMI (17.2 and 17.1 ± 0.4 kg/d for control and RPM, respectively). Milk yield during the first 30 d in milk was also not affected by diet (38.1 and 37.5 ± 1.9 kg/d for control and RPM, respectively). Diet or time did not affect the abundance of several AA transporters or the insulin-induced glucose transporter (SLC2A4). Among evaluated proteins, feeding RPM led to lower overall abundance of proteins associated with protein synthesis (phosphorylated EEF2, phosphorylated RPS6KB1), mTOR activation (RRAGA), proteasome degradation (UBA1), cellular stress responses (HSP70, phosphorylated MAPK3, phosphorylated EIF2A, ERK1/2), antioxidant response (GPX3), and de novo synthesis of phospholipids (PEMT). Regardless of diet, there was an increase in the abundance of the active form of the master regulator of protein synthesis phosphorylated MTOR and the growth-factor-induced serine/threonine kinase phosphorylated AKT1 and PIK3C3, whereas the abundance of a negative regulator of translation (phosphorylated EEF2K) decreased over time. Compared with d 1 after calving and regardless of diet, the abundance of proteins associated with endoplasmic reticulum stress (XBP1 spliced), cell growth and survival (phosphorylated MAPK3), inflammation (transcription factor p65), antioxidant responses (KEAP1), and circadian regulation (CLOCK, PER2) of oxidative metabolism was upregulated at d 21 relative to parturition. These responses coupled with the upregulation of transporters for Lys, Arg, and His (SLC7A1) and glutamate/aspartate (SLC1A3) over time were suggestive of dynamic adaptations in cellular functions. Overall, management approaches that could take advantage of this physiological plasticity may help cows make a smoother transition into lactation. Show less

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, Show less

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) regulates autophagic flux by blocking the fusion of autophagosomes with lysosomes, causing the accumulation of membranous vesicles for replication. Multiple SARS-CoV-2 proteins regulate autophagy with significant roles attributed to ORF3a. Mechanistically, open reading frame 3a (ORF3a) forms a complex with UV radiation resistance associated, regulating the functions of the PIK3C3-1 and PIK3C3-2 lipid kinase complexes, thereby modulating autophagosome biogenesis. ORF3a sequesters VPS39 onto the late endosome/lysosome, inhibiting assembly of the soluble NSF attachement protein REceptor (SNARE) complex and preventing autolysosome formation. ORF3a promotes the interaction between BECN1 and HMGB1, inducing the assembly of PIK3CA kinases into the ER (endoplasmic reticulum) and activating reticulophagy, proinflammatory responses, and ER stress. ORF3a recruits BORCS6 and ARL8B to lysosomes, initiating the anterograde transport of the virus to the plasma membrane. ORF3a also activates the SNARE complex (STX4-SNAP23-VAMP7), inducing fusion of lysosomes with the plasma membrane for viral egress. These mechanistic details can provide multiple targets for inhibiting SARS-CoV-2 by developing host- or host-pathogen interface-based therapeutics. Show less

Osimertinib is a promising approved third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for treating patients with lung adenocarcinoma (LUAD) harboring EGFR-activating mutations, however, almost all patients develop resistance to Osimertinib eventually limiting the long-term efficacy. Autophagy is a vital cellular recycling process promoting Osimertinib resistance. Identifying accurate and efficient autophagy-regulatory factors is of great significance in reducing Osimertinib resistance. This study identified Cezanne, a member of the ovarian tumor protease (OTU)-deubiquitinating family, as an autophagy regulator. Cezanne was highly expressed in Osimertinib-resistant cells, and Cezanne overexpression promoted Osimertinib resistance, while chloroquine (CQ), an autophagy inhibitor, reverted this process. In the Cezanne-overexpressing cells, autophagy was activated even in the absence of autophagy inducers rapamycin and Earle's Balanced Salt Solution (EBSS). Further study showed that Cezanne stabilized PIK3C3 by deubiquitinating K48-linked ubiquitination at Lysine 322. Surprisingly, as a compensatory mechanism of PI3P generation, PIK3C2A was shown to be upregulated by Cezanne by promoting its transcription in a POLR2A-dependent way. Based on these results, Cezanne also accelerates EGFR recycling which may explain the mechanism mediating Cezanne expression and Osimertinib resistance. In conclusion, this study establishes a new model connecting Cezanne, autophagy, and Osimertinib resistance, opening new avenues to explore the effect of Cezanne and autophagy in LUAD. Show less

Dysfunctional autophagy and impairment of adult hippocampal neurogenesis (AHN) each contribute to the pathogenesis of major depressive disorder (MDD). However, whether dysfunctional autophagy is linked to aberrant AHN underlying MDD remains unclear. Here we demonstrate that the expression of nuclear receptor binding factor 2 (NRBF2), a component of autophagy-associated PIK3C3/VPS34-containing phosphatidylinositol 3-kinase complex, is attenuated in the dentate gyrus (DG) under chronic stress. NRBF2 deficiency inhibits the activity of the VPS34 complex and impairs autophagic flux in adult neural stem cells (aNSCs). Moreover, loss of NRBF2 disrupts the neurogenesis-related protein network and causes exhaustion of aNSC pool, leading to the depression-like phenotype. Strikingly, overexpressing NRBF2 in aNSCs of the DG is sufficient to rescue impaired AHN and depression-like phenotype of mice. Our findings reveal a significant role of NRBF2-dependent autophagy in preventing chronic stress-induced AHN impairment and suggest the therapeutic potential of targeting NRBF2 in MDD treatment. Show less

The decline of endothelial autophagy is closely related to vascular senescence and disease, although the molecular mechanisms connecting these outcomes in vascular endothelial cells (VECs) remain unclear. Here, we identify a crucial role for CD44, a multifunctional adhesion molecule, in controlling autophagy and ageing in VECs. The CD44 intercellular domain (CD44ICD) negatively regulates autophagy by reducing PIK3R4 and PIK3C3 levels and disrupting STAT3-dependent PtdIns3K complexes. CD44 and its homologue clec-31 are increased in ageing vascular endothelium and Caenorhabditis elegans, respectively, suggesting that an age-dependent increase in CD44 induces autophagy decline and ageing phenotypes. Accordingly, CD44 knockdown ameliorates age-associated phenotypes in VECs. The endothelium-specific CD44ICD knock-in mouse is shorter-lived, with VECs exhibiting obvious premature ageing characteristics associated with decreased basal autophagy. Autophagy activation suppresses the premature ageing of human and mouse VECs overexpressing CD44ICD, function conserved in the CD44 homologue clec-31 in C. elegans. Our work describes a mechanism coordinated by CD44 function bridging autophagy decline and ageing. Show less

Cyclic adenosine monophosphate (cAMP) levels are directly activated by adenylate cyclase (AC) and play an anti-inflammatory role in chronic obstructive pulmonary disease (COPD). Previously, we have shown that isoforskolin (ISOF) can effectively activate AC1 and AC2 in vitro, improve pulmonary ventilation and reduce the inflammatory response in COPD model rats, supporting that ISOF may be a potential drug for the prevention and treatment of COPD, but the mechanism has not been explored in detail. The potential pharmacological mechanisms of ISOF against COPD were analyzed by network pharmacology and multi-omics based on pharmacodynamic study. To use specific agonists, inhibitors and/or SiRNA for gene regulation function studies, combined qPCR, WB were applied to detect changes in mRNA and protein expression of important targets PIK3C3, AKT, mTOR, SPP1 and AQP4 which related to ISOF effect on COPD. And the key inflammatory factors detected by ELISA. Bioinformatics suggested that the anti-COPD pharmacological mechanism of ISOF was related to PI3K-AKT signaling pathway, and suggested target protein like PIK3C3, AQP4, SPP1, AKT, mTOR. Using the AQP4 inhibitor,or inhibiting SPP1 expression by siRNA-SPP1 could block the PIK3C3-AKT-mTOR pathway and ameliorate chronic inflammation. ISOF showed cAMP-promoting effect then suppressed AQP4 expression, together with decreased level of IL-1β, IL-6, and IL-8. These findings demonstrate ISOF controlled the cAMP-regulated PIK3C3-AKT-mTOR pathway, thereby alleviating inflammatory development in COPD. The cAMP/AQP4/PIK3C3 axis also modulate Th17/Treg differentiation, revealed potential therapeutic targets for this disease. Show less

Copy number variation (CNV) is a genetic structural polymorphism important for phenotypic diversity and important economic traits of livestock breeds, and it plays an important role in the desired genetic variation. This study used whole genome sequencing to detect the CNV variation in the genome of 6 local Tibetan sheep groups. We detected 69,166 CNV events and 7230 copy number variable regions (CNVRs) after merging the overlapping CNVs, accounting for 2.72% of the reference genome. The CNVR length detected ranged from 1.1 to 1693.5 Kb, with a total length of 118.69 Mb and an average length of 16.42 Kb per CNVR. Functional GO cluster analysis showed that the CNVR genes were mainly involved in sensory perception systems, response to stimulus, and signal transduction. Through CNVR-based Vst analysis, we found that the CACNA2D3 and CTBP1 genes related to hypoxia adaptation, the HTR1A gene related to coat color, and the TRNAS-GGA and PIK3C3 genes related to body weight were all strongly selected. The findings of our study will contribute novel insights into the genetic structural variation underlying hypoxia adaptation and economically important traits in Tibetan sheep. Show less

Antileishmanial chemotherapy is currently limited due to severe toxic side effects and drug resistance. Hence, new antileishmanial compounds based on alternative approaches, mainly to avoid the emergence of drug resistance, are needed. The present work aims to decipher the mechanism of action of an antileishmanial drug candidate, named VP343, inhibiting intracellular Show less

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro. Show less

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. Show less

AMBRA1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN1 beclin 1; BECN2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4',6-diamidino-2-phenylindole; dCCD delete CCD; DRD2/D2R dopamine receptor D2 GPRASP1/GASP1 G protein-coupled receptor associated sorting protein 1 GPCR G-protein coupled receptor; ITC isothermal titration calorimetry; IP immunoprecipitation; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NRBF2 nuclear receptor binding factor 2; OPRD1/DOR opioid receptor delta 1 PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RUBCN rubicon autophagy regulator; SQSTM1/p62 sequestosome 1; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WT wild type. Show less

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 Show less

Atg11 is an adaptor protein required for the induction of selective autophagy via receptor binding. However, our understanding of the molecular mechanisms by which it regulates selective autophagy remains incomplete. Here, we show that Atg11 is phosphorylated by Atg1. Rapamycin treatment or starvation conditions induced slower electrophoretic mobility of Atg11 in an Atg1 kinase activity-dependent manner. Through Show less

Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy. Show less

Copper is an essential trace element in biological systems, maintaining the activity of enzymes and the function of transcription factors. However, at high concentrations, copper ions show increased toxicity by inducing regulated cell death, such as apoptosis, paraptosis, pyroptosis, ferroptosis, and cuproptosis. Furthermore, copper ions can trigger macroautophagy/autophagy, a lysosome-dependent degradation pathway that plays a dual role in regulating the survival or death fate of cells under various stress conditions. Pathologically, impaired copper metabolism due to environmental or genetic causes is implicated in a variety of human diseases, such as rare Wilson disease and common cancers. Therapeutically, copper-based compounds are potential chemotherapeutic agents that can be used alone or in combination with other drugs or approaches to treat cancer. Here, we review the progress made in understanding copper metabolic processes and their impact on the regulation of cell death and autophagy. This knowledge may help in the design of future clinical tools to improve cancer diagnosis and treatment. Show less

Single cell-based analysis of macroautophagy/autophagy is largely achieved through the use of fluorescence microscopy to detect autophagy-related proteins that associate with autophagic membranes and therefore can be quantified as fluorescent puncta. In this context, an automated analysis of the number and size of recognized puncta is preferable to a manual count, because more reliable results can be generated in a short time. Here we present a method for open source CellProfiler software-based analysis for quantitative autophagy assessments using GFP-tagged WIPI1 (WD repeat domain, phosphoinositide interacting 1) images acquired with Airyscan or confocal laser-scanning microscopy. The CellProfiler protocol is provided as a ready-to-use software pipeline, and the creation of this pipeline is detailed in both text and video formats. In addition, we provide CellProfiler pipelines for endogenous SQSTM1/p62 (sequestosome 1) or intracellular lipid droplet (LD) analysis, suitable to assess forms of selective autophagy. All protocols and software pipelines can be quickly and easily adapted for the use of alternative autophagy markers or cell types, and can also be used for high-throughput purposes. Show less

Macroautophagy/autophagy occurs basally under nutrient-rich conditions in most mammalian cells, contributing to protein and organelle quality control, and protection against aging and neurodegeneration. During autophagy, lysosomes are heavily utilized via their fusion with autophagosomes and must be repopulated to maintain autophagic degradative capacity. During starvation-induced autophagy, lysosomes are generated via Show less

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. Show less

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. Show less

Common genetic factors likely contribute to multiple psychiatric diseases including mood and substance use disorders. Certain stable, heritable traits reflecting temperament, termed externalizing or internalizing, play a large role in modulating vulnerability to these disorders. To model these heritable tendencies, we selectively bred rats for high and low exploration in a novel environment [bred High Responders (bHR) vs. Low Responders (bLR)]. To identify genes underlying the response to selection, we phenotyped and genotyped 538 rats from an F Show less

Histone deacetylases (HDACs) inhibit the acetylation of crucial autophagy genes, thereby deregulating autophagy and autophagic cell death (ACD) and facilitating cancer cell survival. Vorinostat, a broad-spectrum pan-HDAC inhibitor, inhibits the deacetylation of key autophagic markers and thus interferes with ACD. Vorinostat-regulated ACD can have an autophagy-mediated, -associated or -dependent mechanism depending on the involvement of apoptosis. Molecular insights revealed that hyperactivation of the PIK3C3/VPS34-BECN1 complex increases lysosomal disparity and enhances mitophagy. These changes are followed by reduced mitochondrial biogenesis and by secondary signals that enable superactivated, nonselective or bulk autophagy, leading to ACD. Although the evidence is limited, this review focuses on molecular insights into vorinostat-regulated ACD and describes critical concepts for clinical translation. Show less

mTOR pathway is known to promote cancer malignancy and influence cancer immunity but is unknown for its role in immune checkpoint inhibitors (ICI) therapy. Using Memorial Sloan-Kettering Cancer Center dataset (MSKCC), we extracted mTOR pathway gene mutations for stepwise Cox regression in 1661 cancer patients received ICI. We associated the mutation of the gene signature resulted from the stepwise Cox regression with the 1661 patients' survival. Other 553 ICI-treated patients were collected from 6 cohorts for validation. We also performed this survival association in patients without ICI treatment from MSKCC as discovery (n = 2244) and The Cancer Genome Atlas (TCGA) as validation (n = 763). Pathway enrichment analysis were performed using transcriptome profiles from TCGA and IMvigor210 trial to investigate the potential mechanism. We identified 8 genes involved in mTOR pathway, including FGFR2, PIK3C3, FGFR4, FGFR1, FGF3, AKT1, mTOR, and RPTOR, resulted from stepwise Cox regression in discovery (n = 1661). In both discovery (n = 1661) and validation (n = 553), the mutation of the 8-gene signature was associated with better survival of the patients treated with ICI, which was independent of tumor mutation burden (TMB) and mainly attributed to the missense mutations. This survival association was not observed in patients without ICI therapy. Intriguingly, the mutation of the 8-gene signature was associated with increased TMB and PD1/PD-L1 expression. Immunologically, pathways involved in anti-tumor immune response were enriched in presence of this mutational signature in mTOR pathway, leading to increased infiltration of immune effector cells (e.g., CD8 + T cells, NK cells, and M1 macrophages), but decreased infiltration of immune inhibitory M2 macrophages. These results suggested that mTOR pathway gene mutations were predictive of better survival upon ICI treatment in multiple cancers, likely by its association with enhanced anti-tumor immunity. Larger studies are warranted to validate our findings. Show less

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. Show less

Macroautophagy/autophagy is an adaptable pathway involved in the degradation of very different targets that include proteins, organelles, or even invading intracellular microorganisms. The regulation of this complex pathway depends on a great number of proteins, some common for the majority of the processes and others specific for a particular autophagic event. Nevertheless, the kind of interaction between the players contributes to determining the specificity of the regulation. In a recent study, we found a new regulatory protein of starvation-activated autophagy called FKBP8. The absence of this protein impairs autophagy activation produced by serum starvation and its overexpression can activate the pathway in cells incubated in full media. Besides, we found that the FKBP8 function is mediated by the interaction with the PIK3C3/VPS34-containing complex. Previously, FKBP8 has been shown to participate in mitophagy. In the latter process, FKBP8 works inducing mitochondrial fission, and also it functions as a receptor protein through its LIR domain to direct autophagy. In contrast to mitophagy, in starvation-activated autophagy, not the LIR but the transmembrane domain of FKBP8 is necessary for the regulatory function and interaction with the PIK3C3 complex. Show less