ABSTRACT

SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to treat dyslipidemia and fatty liver disease. However, the current known SCAP inhibitors, such as oxysterols, induce endoplasmic reticulum (ER) stress and NR1H3/LXRα (nuclear receptor subfamily 1 group H member 3)-SREBF1/SREBP-1 c-mediated hepatic steatosis, which severely limited the clinical application of this inhibitor. In this study, we identified a small molecule, lycorine, which binds to SCAP, which suppressed the SREBF pathway without inducing ER stress or activating NR1H3. Mechanistically, lycorine promotes SCAP lysosomal degradation in a macroautophagy/autophagy-independent pathway, a mechanism completely distinct from current SCAP inhibitors. Furthermore, we determined that SQSTM1 captured SCAP after its exit from the ER. The interaction of SCAP and SQSTM1 requires the WD40 domain of SCAP and the TB domain of SQSTM1. Interestingly, lycorine triggers the lysosome translocation of SCAP independent of autophagy. We termed this novel protein degradation pathway as the SQSTM1-mediated autophagy-independent lysosomal degradation (SMAILD) pathway. In vivo, lycorine ameliorates high-fat diet-induced hyperlipidemia, hepatic steatosis, and insulin resistance in mice. Our study demonstrated that the inhibition of SCAP through the SMAILD pathway could be employed as a useful therapeutic strategy for treating metabolic diseases.

Abbreviation: 25-OHD: 25-hydroxyvitamin D; 3-MA: 3-methyladenine; ABCG5: ATP binding cassette subfamily G member 5; ABCG8: ATP binding cassette subfamily G member 8; ACACA: acetyl-CoA carboxylase alpha; AEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AHI: anhydroicaritin; AKT/protein kinase B: AKT serine/threonine kinase; APOE: apolipoprotein E; ATF6: activating transcription factor 6; ATG: autophagy-related; BAT: brown adipose tissue; CD274/PD-L1: CD274 molecule; CETSA: cellular thermal shift assay; CMA: chaperone-mediated autophagy; COPII: cytoplasmic coat protein complex-II; CQ: chloroquine; DDIT3/CHOP: DNA damage inducible transcript 3; DNL: de novo lipogenesis; EE: energy expenditure; EGFR: epithelial growth factor receptor; eMI: endosomal microautophagy; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FADS2: fatty acid desaturase 2; FASN: fatty acid synthase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvate transaminase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSP90B1/GRP94: heat shock protein 90 beta family member 1; HSPA5/GRP78: heat hock protein family A (Hsp70) member 5; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INSIG1: insulin induced gene 1; LAMP2A: lysosomal associated membrane protein 2A; LDLR: low density lipoprotein receptor; LyTACs: lysosome targeting chimeras; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MBTPS1: membrane bound transcription factor peptidase, site 1; MEF: mouse embryonic fibroblast; MST: microscale thermophoresis; MTOR: mechanistic target of rapamycin kinase; MVK: mevalonate kinase; PROTAC: proteolysis targeting chimera; RQ: respiratory quotient; SCAP: SREBF chaperone; SCD1: stearoyl-coenzemy A desaturase 1; SMAILD: sequestosome 1 mediated autophagy-independent lysosomal degradation; SQSTM1: sequestosome 1; SREBF: sterol regulatory element binding transcription factor; TNFRSF10B/DR5: TNF receptor superfamily member 10b; TRAF6: TNF receptor associated factor 6; UPR: unfolded protein response; WAT: white adipose tissue; XBP1: X-box binding protein 1

摘要

SCAP（SREBF 伴侣）调节 SREBF（甾醇调节元件结合转录因子）的加工和稳定性，因此成为治疗血脂异常和脂肪肝疾病的新兴药物靶点。然而，目前已知的 SCAP 抑制剂，如氧甾醇，可诱导内质网 (ER) 应激和 NR1H3/LXRα（核受体亚家族 1 组 H 成员 3）-SREBF1/SREBP-1 c 介导的肝脂肪变性，这严重限制了临床应用。这种抑制剂的应用。在这项研究中，我们发现了一种小分子石蒜碱，它与 SCAP 结合，抑制 SREBF 通路，而不诱导 ER 应激或激活 NR1H3。从机制上讲，lycorine 在巨自噬/自噬非依赖性途径中促进 SCAP 溶酶体降解，这种机制与目前的 SCAP 抑制剂完全不同。此外，我们确定 SQSTM1 在退出 ER 后捕获了 SCAP。SCAP和SQSTM1的交互需要SCAP的WD40域和SQSTM1的TB域。有趣的是，lycorine 独立于自噬触发 SCAP 的溶酶体易位。我们将这种新的蛋白质降解途径称为 SQSTM1 介导的自噬非依赖性溶酶体降解 (SMAILD) 途径。在体内，lycorine 可改善高脂饮食引起的小鼠高脂血症、肝脂肪变性和胰岛素抵抗。我们的研究表明，通过 SMAILD 途径抑制 SCAP 可用作治疗代谢疾病的有用治疗策略。

缩写： 25-OHD：25-羟基维生素D；3-MA：3-甲基腺嘌呤；ABCG5：ATP结合盒亚科G成员5；ABCG8：ATP结合盒亚科G成员8；ACACA：乙酰辅酶A羧化酶α；AEBSF：4-(2-氨基乙基)苯磺酰氟盐酸盐；AHI：脱水淫羊藿；AKT/蛋白激酶B：AKT丝氨酸/苏氨酸激酶；APOE：载脂蛋白E；ATF6：激活转录因子 6；ATG：自噬相关；BAT：棕色脂肪组织；CD274/PD-L1：CD274分子；CETSA：细胞热位移测定；CMA：伴侣介导的自噬；COPII：细胞质外壳蛋白复合物-II；CQ：氯喹；DDIT3/CHOP：DNA 损伤诱导转录本 3；DNL：从头脂肪生成; EE：能量消耗；EGFR：上皮生长因子受体；eMI：内体微自噬；ERN1/IRE1α：内质网到核信号 1；FADS2：脂肪酸去饱和酶2；FASN：脂肪酸合酶；GOT1/AST：谷氨酸-草酰乙酸转氨酶1；GPT/ALT：谷氨酸-丙酮酸转氨酶；HMGCR：3-羟基-3-甲基戊二酰-CoA还原酶；HMGCS1：3-羟基-3-甲基戊二酰-CoA合酶1；HSP90B1/GRP94：热休克蛋白 90 β 家族成员 1；HSPA5/GRP78：热休克蛋白家族 A (Hsp70) 成员 5；HSPA8/HSC70：热休克蛋白家族 A (Hsp70) 成员 8；INSIG1：胰岛素诱导基因1；LAMP2A：溶酶体相关膜蛋白 2A；LDLR：低密度脂蛋白受体；LyTACs：溶酶体靶向嵌合体；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MBTPS1：膜结合转录因子肽酶，位点 1；MEF：小鼠胚胎成纤维细胞；MST：微尺度热泳；MTOR：雷帕霉素激酶的机制靶点；MVK：甲羟戊酸激酶；PROTAC：蛋白水解靶向嵌合体；RQ：呼吸商；SCAP：SREBF 伴侣；SCD1：硬脂酰辅酶 A 去饱和酶 1；SMAILD：sequestosome 1 介导的不依赖自噬的溶酶体降解；SQSTM1：隔离体 1；SREBF：甾醇调节元件结合转录因子；TNFRSF10B/DR5：TNF受体超家族成员10b；TRAF6：TNF受体相关因子6；UPR：未折叠蛋白反应；WAT：白色脂肪组织；XBP1：X-box 结合蛋白 1 RQ：呼吸商；SCAP：SREBF 伴侣；SCD1：硬脂酰辅酶 A 去饱和酶 1；SMAILD：sequestosome 1 介导的不依赖自噬的溶酶体降解；SQSTM1：隔离体 1；SREBF：甾醇调节元件结合转录因子；TNFRSF10B/DR5：TNF受体超家族成员10b；TRAF6：TNF受体相关因子6；UPR：未折叠蛋白反应；WAT：白色脂肪组织；XBP1：X-box 结合蛋白 1 RQ：呼吸商；SCAP：SREBF 伴侣；SCD1：硬脂酰辅酶 A 去饱和酶 1；SMAILD：sequestosome 1 介导的不依赖自噬的溶酶体降解；SQSTM1：隔离体 1；SREBF：甾醇调节元件结合转录因子；TNFRSF10B/DR5：TNF受体超家族成员10b；TRAF6：TNF受体相关因子6；UPR：未折叠蛋白反应；WAT：白色脂肪组织；XBP1：X-box 结合蛋白 1