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Sepsis, a life-threatening condition resulting from a dysregulated response to pathogen infection, poses a significant challenge in clinical management. Here, we report a novel role for the autophagy receptor NCOA4 in the pathogenesis of sepsis. Activated macrophages and monocytes secrete NCOA4, which acts as a mediator of septic death in mice. Mechanistically, lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, induces NCOA4 secretion through autophagy-dependent lysosomal exocytosis mediated by ATG5 and MCOLN1. Moreover, bacterial infection with E. coli or S. enterica leads to passive release of NCOA4 during GSDMD-mediated pyroptosis. Upon release, extracellular NCOA4 triggers the activation of the proinflammatory transcription factor NFKB/NF-κB by promoting the degradation of NFKBIA/IκB molecules. This process is dependent on the pattern recognition receptor AGER, rather than TLR4. In vivo studies employing endotoxemia and polymicrobial sepsis mouse models reveal that a monoclonal neutralizing antibody targeting NCOA4 or AGER delays animal death, protects against organ damage, and attenuates systemic inflammation. Furthermore, elevated plasma NCOA4 levels in septic patients, particularly in non-survivors, correlate positively with the sequential organ failure assessment score and concentrations of lactate and proinflammatory mediators, such as TNF, IL1B, IL6, and HMGB1. These findings demonstrate a previously unrecognized role of extracellular NCOA4 in inflammation, suggesting it as a potential therapeutic target for severe infectious diseases. Abbreviation: BMDMs: bone marrow-derived macrophages; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; ELISA: enzyme-linked immunosorbent assay; LPS: lipopolysaccharide; NO: nitric oxide; SOFA: sequential organ failure assessment. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is an abdominal inflammatory disease initiated by damaged pancreatic acinar cells and developed by systemic inflammation. SQSTM1 (sequestosome 1) has an intracellular function in mediating substrate degradation during macroautophagy/autophagy, and it can be released by macrophages and monocytes to trigger lethal inflammation during bacterial infection. Here, we report that extracellular SQSTM1 acts as a mediator of AP by enhancing the sensitivity to autophagy-dependent ferroptotic cell death. Serum SQSTM1 is elevated in AP patients as well as in mice that have cerulein-induced AP. The administration of SQSTM1-neutralizing antibodies protects against experimental AP in mice. Mechanistically, recombinant SQSTM1 protein (rSQSTM1) increases AGER (advanced glycosylation end-product specific receptor)-dependent ACSL4 (acyl-CoA synthetase long chain family member 4) expression, leading to polyunsaturated fatty acid production for autophagosome formation and subsequent ferroptosis. The rSQSTM1-elicited pathological responses during AP are attenuated in mice with the conditional deletion of Ager in the pancreas. These findings may provide not only new insights into the mechanism of autophagy-dependent cell death, but also suggest that targeting the extracellular SQSTM1 pathway is a potential strategy for the treatment of AP. 5-HETE, 5-hydroxyeicosatetraenoic acid; ACSL4, acyl-CoA synthetase long chain family member 4; AP, acute pancreatitis; ATG, autophagy related; AGER, advanced glycosylation end-product specific receptor; DAMPs, danger/damage-associated molecular patterns; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; IL, interleukin; INSR, insulin receptor; MAP1LC3B, microtubule associated protein 1 light chain 3 beta; MDA, malondialdehyde; MPO, myeloperoxidase; PRRs, pattern recognition receptors; PUFA, polyunsaturated fatty acid; RNAi, RNA interference; SQSTM1, sequestosome 1; TNF, tumor necrosis factor; TLR, toll like receptor [ABSTRACT FROM AUTHOR]

Selective macroautophagy/autophagy maintains cellular homeostasis through the lysosomal degradation of specific cellular proteins or organelles. The pro-survival effect of selective autophagy has been well-characterized, but the mechanism by which it drives cell death is still poorly understood. Here, we use a quantitative proteomic approach to identify HPCAL1 (hippocalcin like 1) as a novel autophagy receptor for the selective degradation of CDH2 (cadherin 2) during ferroptosis. HPCAL1-dependent CDH2 depletion increases susceptibility to ferroptotic death by reducing membrane tension and favoring lipid peroxidation. Site-directed mutagenesis aided by bioinformatic analyses revealed that the autophagic degradation of CDH2 requires PRKCQ (protein kinase C theta)-mediated HPCAL1 phosphorylation on Thr149, as well as a non-classical LC3-interacting region motif located between amino acids 46–51. An unbiased drug screening campaign involving 4208 small molecule compounds led to the identification of a ferroptosis inhibitor that suppressed HPCAL1 expression. The genetic or pharmacological inhibition of HPCAL1 prevented ferroptosis-induced tumor suppression and pancreatitis in suitable mouse models. These findings provide a framework for understanding how selective autophagy promotes ferroptotic cell death.Abbreviations: ANXA7: annexin A7; ARNTL: aryl hydrocarbon receptor nuclear translocator like; CCK8: cell counting kit-8; CDH2: cadherin 2; CETSAs: cellular thermal shift assays; CPT2: carnitine palmitoyltransferase 2; DAMP, danger/damage-associated molecular pattern; DPPH: 2,2-diphenyl-1-picrylhydrazyl; DFO: deferoxamine; EBNA1BP2: EBNA1 binding protein 2; EIF4G1: eukaryotic translation initiation factor 4 gamma 1; FBL: fibrillarin; FKBP1A: FKBP prolyl isomerase 1A; FTH1: ferritin heavy chain 1; GPX4: glutathione peroxidase 4; GSDMs: gasdermins; HBSS: Hanks' buffered salt solution; HMGB1: high mobility group box 1; HNRNPUL1: heterogeneous nuclear ribonucleoprotein U like 1; HPCAL1: hippocalcin like 1; H1-3/HIST1H1D: H1.3 linker histone, cluster member; IKE: imidazole ketone erastin; KD: knockdown; LDH: lactate dehydrogenase; LIR: LC3-interacting region; MAGOH: mago homolog, exon junction complex subunit; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehyde; MLKL: mixed lineage kinase domain like pseudokinase; MPO: myeloperoxidase; MTOR: mechanistic target of rapamycin kinase; OE: overexpressing; OSTM1: osteoclastogenesis associated transmembrane protein 1; PRKC/PKC: protein kinase C; PRKAR1A: protein kinase cAMP-dependent type I regulatory subunit alpha; PRDX3: peroxiredoxin 3; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; SLC40A1: solute carrier family 40 member 1; SPTAN1: spectrin alpha, non-erythrocytic 1; STS: staurosporine; UBE2M: ubiquitin conjugating enzyme E2 M; ZYX: zyxin [ABSTRACT FROM AUTHOR]

Pancreatic cancer tends to be highly resistant to current therapy and remains one of the great challenges in biomedicine with very low 5-year survival rates. Here, we report that zalcitabine, an antiviral drug for human immunodeficiency virus infection, can suppress the growth of primary and immortalized human pancreatic cancer cells through the induction of ferroptosis, an iron-dependent form of regulated cell death. Mechanically, this effect relies on zalcitabine-induced mitochondrial DNA stress, which activates the STING1/TMEM173-mediated DNA sensing pathway, leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation, but not a type I interferon response. Consequently, the genetic and pharmacological inactivation of the autophagy-dependent ferroptosis pathway diminishes the anticancer effects of zalcitabine in cell culture and animal models. Together, these findings not only provide a new approach for pancreatic cancer therapy but also increase our understanding of the interplay between autophagy and DNA damage response in shaping cell death. Abbreviations: ALOX: arachidonate lipoxygenase; ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATM: ATM serine/threonine kinase; ATG: autophagy-related; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; FANCD2: FA complementation group D2; GPX4: glutathione peroxidase 4; IFNA1/IFNα: interferon alpha 1; IFNB1/IFNβ: interferon beta 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MDA: malondialdehyde; mtDNA: mitochondrial DNA; NCOA4: nuclear receptor coactivator 4; PDAC: pancreatic ductal adenocarcinoma; POLG: DNA polymerase gamma, catalytic subunit; qRT-PCR: quantitative polymerase chain reaction; RCD: regulated cell death; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; TFAM: transcription factor A, mitochondrial [ABSTRACT FROM AUTHOR]