Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury

Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury. Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic–pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

巨自噬（Macroautophagy）/自噬（autophagy）在脓毒症诱导的肝损伤中发挥保护作用。作为B类清道夫受体家族成员，CD36在动脉粥样硬化、脂肪性肝病等多种疾病中发挥重要作用。本研究发现，脓毒症患者及脓毒症小鼠模型的肝细胞中CD36表达上调，同时伴随自噬流受损。进一步研究显示，肝细胞特异性CD36敲除（cd36-HKO）可显著改善脂多糖（LPS）诱导的脓毒症小鼠的肝损伤及自噬体-溶酶体融合障碍。肝细胞中UBQLN1（ubiquilin 1）过表达（OE）可阻断cd36-HKO对LPS诱导的小鼠肝损伤的保护作用。机制研究表明，经LPS刺激后，细胞膜上的CD36发生去棕榈酰化并转运至溶酶体，在此处CD36作为桥接分子将UBQLN1连接至可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE），进而促进SNARE蛋白的蛋白酶体降解，最终导致融合障碍。综上，本研究数据证实，CD36可通过UBQLN1依赖的方式调控自噬相关SNARE蛋白的蛋白酶体降解，这一过程对其功能至关重要。靶向肝细胞中的CD36可有效改善脓毒症状态下的自噬流，因此有望成为脓毒症肝损伤临床治疗的潜在策略。

缩写说明：AAV8：腺相关病毒8型（adeno-associated virus 8）；AOSC：急性梗阻性化脓性胆管炎（acute obstructive suppurative cholangitis）；ATP1A1：ATP酶Na+/K+转运亚基α1多肽（ATPase, Na+/K+ transporting, alpha 1 polypeptide）；CASP3：半胱天冬酶3（caspase 3）；CASP8：半胱天冬酶8（caspase 8）；CCL2：趋化因子（C-C基序）配体2（chemokine (C-C motif) ligand 2）；cd36-HKO：肝细胞特异性CD36敲除（hepatocyte-specific cd36 knockout）；Co-IP：免疫共沉淀（co-immunoprecipitation）；CQ：氯喹（chloroquine）；Cys：半胱氨酸（cysteine）；GOT1：可溶性谷草转氨酶1（glutamic-oxaloacetic transaminase 1, soluble）；GPT：可溶性谷氨酸丙酮酸转氨酶（glutamic–pyruvic transaminase, soluble）；IL1B：白细胞介素1β（interleukin 1 beta）；IL6：白细胞介素6（interleukin 6）；KO：敲除（knockout）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；LDH：乳酸脱氢酶（lactate dehydrogenase）；LPS：脂多糖（lipopolysaccharide）；LYPLA1：溶酶体磷脂酶1（lysophospholipase 1）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；OE：过表达（overexpression）；qPCR：定量聚合酶链反应（quantitative polymerase chain reaction）；SNAP29：突触体相关蛋白29（synaptosome associated protein 29）；SNARE：可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（soluble N-ethylmaleimide-sensitive factor attachment protein receptor）；SQSTM1/p62：自噬底物衔接蛋白p62（sequestosome 1）；STX17：突触融合蛋白17（syntaxin 17）；TNF：肿瘤坏死因子（tumor necrosis factor）；TRIM：三结构域蛋白（tripartite motif-containing）；UBA：泛素相关结构域（ubiquitin-associated）；UBL：泛素样结构域（ubiquitin-like）；UBQLN：泛素蛋白（ubiquilin）；VAMP8：囊泡相关膜蛋白8（vesicle associated membrane protein 8）；WT：野生型（wild-type）