ULK1 Complex Research Articles

The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization

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.

Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane’s lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

TBC1D4 antagonizes RAB2A-mediated autophagic and endocytic pathways

ABSTRACT Macroautophagic/autophagic and endocytic pathways play essential roles in maintaining homeostasis at different levels. It remains poorly understood how both pathways are coordinated and fine-tuned for proper lysosomal degradation of diverse cargoes. We and others recently identified a Golgi-resident RAB GTPase, RAB2A, as a positive regulator that controls both autophagic and endocytic pathways. In the current study, we report that TBC1D4 (TBC1 domain family member 4), a TBC domain-containing protein that plays essential roles in glucose homeostasis, suppresses RAB2A-mediated autophagic and endocytic pathways. TBC1D4 bound to RAB2A through its N-terminal PTB2 domain, which impaired RAB2A-mediated autophagy at the early stage by preventing ULK1 complex activation. During the late stage of autophagy, TBC1D4 impeded the association of RUBCNL/PACER and RAB2A with STX17 on autophagosomes by direct interaction with RUBCNL via its N-terminal PTB1 domain. Disruption of the autophagosomal trimeric complex containing RAB2A, RUBCNL and STX17 resulted in defective HOPS recruitment and eventually abortive autophagosome-lysosome fusion. Furthermore, TBC1D4 inhibited RAB2A-mediated endocytic degradation independent of RUBCNL. Therefore, TBC1D4 and RAB2A form a dual molecular switch to modulate autophagic and endocytic pathways. Importantly, hepatocyte- or adipocyte-specific tbc1d4 knockout in mice led to elevated autophagic flux and endocytic degradation and tissue damage. Together, this work establishes TBC1D4 as a critical molecular brake in autophagic and endocytic pathways, providing further mechanistic insights into how these pathways are intertwined both in vitro and in vivo. Abbreviations: ACTB: actin beta; ATG9: autophagy related 9; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; CLEM: correlative light electron microscopy; Ctrl: control; DMSO: dimethyl sulfoxide; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FL: full length; GAP: GTPase-activating protein; GFP: green fluorescent protein; HOPS: homotypic fusion and protein sorting; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OE: overexpression; PG: phagophore; PtdIns3K: class III phosphatidylinositol 3-kinase; SLC2A4/GLUT4: solute carrier family 2 member 4; SQSTM1/p62: sequestosome 1; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TAP: tandem affinity purification; TBA: total bile acid; TBC1D4: TBC1 domain family member 4; TUBA1B: tubulin alpha 1b; ULK1: unc-51 like autophagy activating kinase 1; VPS39: VPS39 subunit of HOPS complex; WB: western blot; WT: wild type.

Abstract 5379: Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC

Abstract BACKGROUND: Inactivating mutations and deletions in STK11 are present in 9-20% of NSCLC and result in immunologically cold tumors that respond poorly to therapy, despite having a high tumor mutational burden and neoantigen load. STK11 mutations in NSCLC are associated with higher levels of ULK1/2-mediated macroautophagy, resulting in localization of MHC-I to autophagosomes and lysosomes leading to its degradation and decreased antigen presentation. Knockdown of ULK1/2 can restore surface MHC-I in highly autophagic cell lines. Here, we show that inhibition of ULK1/2 decreases macroautophagy, resulting in increased antigen processing and presentation (APP) and MHC-I levels in cancer cells, supporting the hypothesis that inhibition of ULK1/2 will restore antigen presentation in NSCLC STK11mut tumors. METHODS: TCGA datasets were analyzed to determine transcriptional differences in autophagy and APP between STK11 WT and mutant NSCLC tumors. CRISPR was used to stably knockout STK11 in multiple human NSCLC cell lines to generate isogenic pairs. mRNA and protein levels of APP machinery in NSCLC cell lines were assessed using qPCR, western blots, and flow cytometry. CRISPR was additionally used to knockout ULK1 and ULK2 in human STK11 mutant A549 cells. ULK1/2 tool compounds were synthesized based on existing patent literature. Effects of the combination of ULK1/2 inhibition with low levels of IFNγ on surface MHC-I levels were evaluated in several NSCLC cell lines by flow cytometry. RESULTS: NSCLC tumors with loss of function mutations or deletion of STK11 display increased transcription of genes in the ULK complex and reduced transcription of genes necessary for APP. STK11mut and KO NSCLC cell lines also display lower levels of APP at the mRNA and protein levels. Double knockout of ULK1 and ULK2 in A549 cells is not cytotoxic and results in decreased autophagy as well as increased immunoproteasome components and cell surface MHC-I. ULK inhibition reduces autophagy and restores APP at the protein level, but not at the transcriptional level, in all NSCLC cell lines evaluated. Combination of very low concentrations of IFNγ with ULK inhibition demonstrates additive increases in MHC-I greater than either single-agent treatment alone. CONCLUSIONS: STK11mut/del is associated with poor prognosis in NSCLC and leads to increased ULK-induced autophagy and decreased APP. ULK inhibition increases APP and surface MHC-I levels in STK11mut cell lines. In the presence of low concentrations of IFNγ, ULK1/2 inhibition restores APP in NSCLC STK11mut cell lines to the levels seen in NSCLC STK11 WT cell lines. The data presented here provides a rationale for targeting ULK1/2 to amplify immune recognition in immunologically cold tumors with high autophagy to increase responses to immunotherapies. Citation Format: Samantha C. Schwager, Eugene Park, Anne Van Abbema, Yihong Guan, Connor Rosen, Sean Cho, Michael Black, Inna Kurilyak, Jenna Jeffrey, Juan Jose Fung, Susan L. Paprcka, Ester Fernandez-Salas. Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5379.

Local membrane source gathering by p62 body drives autophagosome formation.

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

Site-specific Atg13 methylation-mediated autophagy regulates epithelial inflammation in PM2.5-induced pulmonary fibrosis

Fine particulate matters (PM2.5) increased the risk of pulmonary fibrosis. However, the regulatory mechanisms of lung epithelium in pulmonary fibrosis remained elusive. Here we developed PM2.5-exposure lung epithelial cells and mice models to investigate the role of autophagy in lung epithelia mediating inflammation and pulmonary fibrosis. PM2.5 exposure induced autophagy in lung epithelial cells and then drove pulmonary fibrosis by activation of NF-κB/NLRP3 signaling pathway. PM2.5-downregulated ALKBH5 protein expression promotes m6A modification of Atg13 mRNA at site 767 in lung epithelial cells. Atg13-mediated ULK complex positively regulated autophagy and inflammation in epithelial cells with PM2.5 treatment. Knockout of ALKBH5 in mice further accelerated ULK complex-regulated autophagy, inflammation and pulmonary fibrosis. Thus, our results highlighted that site-specific m6A methylation on Atg13 mRNA regulated epithelial inflammation-driven pulmonary fibrosis in an autophagy-dependent manner upon PM2.5 exposure, and it provided target intervention strategies towards PM2.5-induced pulmonary fibrosis.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

Mitochondrial Lon-induced mitophagy benefits hypoxic resistance via Ca2+-dependent FUNDC1 phosphorylation at the ER-mitochondria interface

During hypoxia, FUNDC1 acts as a mitophagy receptor and accumulates at the ER (endoplasmic reticulum)-mitochondria contact sites (EMC), also called mitochondria-associated membranes (MAM). In mitophagy, the ULK1 complex phosphorylates FUNDC1(S17) at the EMC site. However, how mitochondria sense the stress and send the signal from the inside to the outside of mitochondria to trigger mitophagy is still unclear. Mitochondrial Lon was reported to be localized at the EMC under stress although the function remained unknown. In this study, we explored the mechanism of how mitochondrial sensors of hypoxia trigger and stabilize the FUNDC1-ULK1 complex by Lon in the EMC for cell survival and cancer progression. We demonstrated that Lon is accumulated in the EMC and associated with FUNDC1-ULK1 complex to induce mitophagy via chaperone activity under hypoxia. Intriguingly, we found that Lon-induced mitophagy is through binding with mitochondrial Na+/Ca2+ exchanger (NCLX) to promote FUNDC1-ULK1-mediated mitophagy at the EMC site in vitro and in vivo. Accordingly, our findings highlight a novel mechanism responsible for mitophagy initiation under hypoxia by chaperone Lon in mitochondria through the interaction with FUNDC1-ULK1 complex at the EMC site. These findings provide a direct correlation between Lon and mitophagy on cell survival and cancer progression.

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation.

The assembly of the autophagy initiation machinery nucleates autophagosome biogenesis, including in the PINK1- and Parkin-dependent mitophagy pathway implicated in Parkinson's disease. The structural interaction between the sole transmembrane autophagy protein, autophagy-related protein 9A (ATG9A), and components of the Unc-51-like autophagy activating kinase (ULK1) complex is one of the major missing links needed to complete a structural map of autophagy initiation. We determined the 2.4-Å x-ray crystallographic structure of the ternary structure of ATG9A carboxyl-terminal tail bound to the ATG13:ATG101 Hop1/Rev7/Mad2 (HORMA) dimer, which is part of the ULK1 complex. We term the interacting portion of the extreme carboxyl-terminal part of the ATG9A tail the "HORMA dimer-interacting region" (HDIR). This structure shows that the HDIR binds to the HORMA domain of ATG101 by β sheet complementation such that the ATG9A tail resides in a deep cleft at the ATG13:ATG101 interface. Disruption of this complex in cells impairs damage-induced PINK1/Parkin mitophagy mediated by the cargo receptor NDP52.

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

Network analysis of the autophagy biochemical network in relation to various autophagy-targeted proteins found among SARS-CoV-2 variants of concern

Autophagy is an important cellular process that triggers a coordinated action involving multiple individual proteins and protein complexes while SARS-CoV-2 (SARS2) was found to both hinder autophagy to evade host defense and utilize autophagy for viral replication. Interestingly, the possible significant stages of the autophagy biochemical network in relation to the corresponding autophagy-targeted SARS2 proteins from the different variants of concern (VOC) were never established. In this study, we performed the following: autophagy biochemical network design and centrality analyses; generated autophagy-targeted SARS2 protein models; and superimposed protein models for structural comparison. We identified 2 significant biochemical pathways (one starts from the ULK complex and the other starts from the PI3P complex) within the autophagy biochemical network. Similarly, we determined that the autophagy-targeted SARS2 proteins (Nsp15, M, ORF7a, ORF3a, and E) are structurally conserved throughout the different SARS2 VOC suggesting that the function of each protein is preserved during SARS2 evolution. Interestingly, among the autophagy-targeted SARS2 proteins, the M protein coincides with the 2 significant biochemical pathways we identified within the autophagy biochemical network. In this regard, we propose that the SARS2 M protein is the main determinant that would influence autophagy outcome in regard to SARS2 infection.

The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation.

Mitophagy, a type of selective autophagy, specifically targets damaged mitochondria. The ULK complex regulates Parkin-mediated mitophagy, but the mechanism through which the ULK complex initiates mitophagosome formation remains unknown. The Rab7 GTPase (herein referring to Rab7a) is a key initiator of mitophagosome formation, and Ser-72 phosphorylation of Rab7 is important for this process. We have previously identified LRRK1 as a protein kinase responsible for Rab7 Ser-72 phosphorylation. In this study, we investigated the role of LRRK1 in mitophagy. We showed that LRRK1 functions downstream of ULK1 and ULK2 in Parkin-mediated mitophagy. Furthermore, we demonstrated that ectopic targeting of active LRRK1 to mitochondria is sufficient to induce the Ser-72 phosphorylation of Rab7, circumventing the requirement for ATG13, a component of the ULK complex. Thus, the ULK complex recruits LRRK1 to mitochondria by interacting with ATG13 to initiate mitophagosome formation. This study highlights the crucial role of the ULK complex-LRRK1 axis in the regulation of Parkin-mediated mitophagy.

ULK1 Blockade Preferentially Targets Germinal Centre B-Cell - Diffuse Large B-Cell Lymphoma Subtype By Attenuating Autophagy, c-MYC and Inflammation

Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous lymphoid malignancy. Gene Expression profiling (GEP) has characterized two distinct subsets: Germinal Centre B-cell (GCB) and Activated B-cell (ABC) DLBCL. Oncogenic PI3K-AKT signaling has been identified in primary DLBCL subtypes[1]. PI3K-AKT is activated in response to the loss of PTEN in GCB-DLBCL. In ABC-DLBCL the activation is associated with CD79B mutations and concomitant MYD88 alterations. The Bruton Tyrosine Kinase (BTK) functions as an intermediary kinase. Ibrutinib (BTK inhibitor) has demonstrated promising capability in ABC-DLBCL unlike GCB tumors, warranting further investigations[2]. PI3K-AKT-mTOR signaling is also a known regulator of macro-autophagy (herein referred to as autophagy). ULK1 complex is critical for the initiation of autophagy. Aberrant signaling in this pathway promotes nutritional adaptation to malignant cells and contributes to drug resistance. We hypothesize autophagy facilitates cytoprotection and contributes to drug resistance including R-CHOP and Ibrutinib in GCB-DLBCL. Dual targeting of the BTK with Ibrutinib and autophagy initiating complex(es) would be a strategic method to impair these oncogenic functions. To determine the presence of increased autophagy related genes (ATGs) and its association to R-CHOP treatment outcome, we analyzed the GEP of patients from the REMODL-B trial (R-CHOP: GCB n=240 and ABC n=121)[3]. Gene set enrichment analysis (GSEA) showed no difference between the DLBCL patients indicating mutual autophagy dependency. Genes associated to worst overall survival (OS) in autophagy initiating complex I/ ULK1 complex (ULK1, RB1CC1/FIP200 and ATG13) and complex II (AMBRA1, PIK3C3/VPS34, PIK3R4/VPS15 and USO1/p115) were identified using Kaplan-Meier curves and overlapped according to their respective complexes. Statistical analysis confirmed ULK1 complex was significantly dominate in response to R-CHOP treatment leading to poor OS (p=0.0403). These findings provide a rational to inhibit autophagy via the ULK1 complex in GCB patients. Alike, multiple GCB (n=7) and ABC (n=5) cell lines showed substantial autophagy dependency using LC3B autophagy marker expression. Ibrutinib treatment enhanced autophagic flux in GCB cell lines. Addition of ULK1 complex inhibitor (MRT68921) augmented the anti-tumor activity of Ibrutinib in HT, Oci-Ly1, Oci-Ly18 and SUDHL-6 GCB cell lines and activated caspase dependent apoptosis. Interestingly, ULK1 complex inhibitors (MRT68921/ ULK1-101) induced acute NF-ҡB activation in multiple GCB and ABC lymphoma lines irrespective of their NF-ҡB mutational status. RNA sequencing of GCB and ABC cell lines treated with MRT68921, or vehicle reported diverse ULK1 inhibitory signaling pathways. Oci-Ly1 (GCB, c-MYC amplification) treated cells significantly downregulated genes associated to PI3K-mTOR activity/ signaling, autophagy, metabolism, and IL-6. GSEA reported a substantial decrease in c-MYC transcriptional activation/ pathway and TNF signaling. Immunoblotting data confirmed ULK1 inhibition dephosphorylated c-myc(ser62) in a time dependent manner in Oci-Ly1 and Oci-Ly18. In contrast, Oci-Ly3 (ABC, c-MYC amplification) treated cells upregulated genes involved in JAK-STAT pathway, IL-6, and apoptosis. C-MYC remained unaffected. In summary, GCB-DLBCL patients treated with R-CHOP harbor increased autophagy signatures that potentially dictate treatment outcome. Our in vitro data demonstrates ULK1 inhibition sensitizes GCB cell lines to Ibrutinib due to diverse role in regulating NF-ҡB, c-MYC and inflammation. Unlike, ABC, where ULK1 blockade promotes immunogenic-dependent cell death.

Mapping the proximity interactome of ATG9A reveals unexpected dynamics of ULK1 complex proteins

ABSTRACT ATG9A is essential for macroautophagy/autophagy and considered to be one of the earliest ATG (autophagy related) proteins recruited to sites of autophagosome biogenesis. Recent data suggest ATG9A vesicles may even form the lipid seed of the autophagosome. However, ATG9A regulation is still poorly understood, which is likely at least partly due to challenges inherent to studying an intracellular transmembrane protein with no apparent enzymatic activity. To help overcome these challenges, we used BioID and quantitative LC-MS/MS to map the proximity interactome of ATG9A, which included entire protein complexes involved in protein trafficking, and proteins implicated in autophagy but previously lacking any physical link to core autophagy machinery. We also unexpectedly found an ATG9A interaction with an ULK1-independent ATG13-ATG101 dimer that promotes autophagy in fed cells.

Lipopolysaccharide Inhibits Autophagy and Promotes Inflammatory Responses via p38 MAPK-Induced Proteasomal Degradation of Atg13 in Hepatic Stellate Cells.

Background Inflammation plays a critical role in the progression of acute-on-chronic liver failure (ACLF). Atg13 is a vital regulatory component of the ULK1 complex, which plays an essential role in the initiation of autophagy. Previously, hepatic stellate cells (HSCs) were considered to be noninflammatory cells that contribute only to hepatic fibrosis. Recently, it has been found that HSCs can secrete inflammatory cytokines and participate in hepatic inflammation. Autophagy and proteasome-mediated degradation constitute two major means of protein turnover in cells. Autophagy has been shown to regulate inflammation, but it is unclear whether ubiquitin (Ub)-proteasome system (UPS) is involved in inflammatory responses in HSCs during ACLF. Methods Clinical data were collected from ACLF patients, and surgically resected paraffin-embedded human ACLF liver tissue specimens were collected. The expression of Atg13 was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Secretion of IL-1β was assessed by ELISA. Atg13 was knocked down by siRNA in LX2 cells. Coimmunoprecipitation assay was used to detect protein binding and polyubiquitination of Atg13. In vitro tests with LX2 cells were performed to explore the effects and regulation of p38 MAPK, Atg13, UPS, autophagy, and inflammation. Results Serum lipopolysaccharide (LPS) was positively associated with disease severity in ACLF patients, and p38 MAPK was overexpressed in ACLF liver tissue. We evaluated the role of Atg13 in HSC inflammation and explored the possible underlying mechanisms. Inflammatory factors were upregulated via activation of p38 MAPK and inhibition of autophagy in LX-2 cells. Expression of Atg13 was decreased in LPS-incubated LX2 cells. Atg13 knockdown markedly inhibited autophagy and promoted LPS-induced inflammation in LX2 cells. Our in vitro experiments also showed that LPS induced depletion of Atg13 via UPS, and this process was dependent on p38 MAPK. Conclusions LPS induces proteasomal degradation of Atg13 via p38 MAPK, thereby participating in the aggravation of LPS-induced autophagy inhibition and inflammatory responses in LX2 cells. Atg13 serves as a mediator between autophagy and proteasome. Modulation of Atg13 or proteasome activity might be a novel strategy for treating HSC inflammation.

Salmonella Promotes Its Own Survival in B Cells by Inhibiting Autophagy.

Salmonella is a Gram-negative bacterium known to be the major cause of gastrointestinal diseases and systemic infections. During infection of murine B cells, Salmonella activates the PI3K/Akt pathway through its effector, SopB. This signaling pathway induces the downregulation of NLRC4 transcription, resulting in reduced secretion of IL-1β. Thus, Salmonella-infected B cells do not progress to pyroptosis; consequently, the bacteria can survive inside these cells. However, the mechanism by which Salmonella evades the control of B cells has not yet been elucidated. In this study, we found that SopB activates mTORC1, which is necessary for bacterial survival, since B cells cultured with the mTORC1 inhibitor rapamycin and B cells lacking raptor can control Salmonella infection. A similar result was observed in B cells when they were infected with the Salmonella SopB mutant (Δsopb). Salmonella also promoted the phosphorylation of the ULK1 complex at serine 757 (Ser757) by mTORC1, resulting in decreased levels of LC3-II in infected B cells. In this study, we did not observe these results when B cells were infected with Δsopb Salmonella. Our results demonstrated that Salmonella survival within B cells depends on the inhibition of autophagy by mTORC1 activation.