心磷脂介导的线粒体自噬中的 LC3 亚家族：LC3A、LC3B 和 LC3C 同系物的比较,Autophagy

摘要

已提出将磷脂心磷脂 (CL) 外化到线粒体外膜作为线粒体自噬触发因素。CL 将作为结合 LC3 巨自噬/自噬蛋白的信号。到目前为止，LC3 亚家族成员的行为还没有直接进行详细的比较。在目前的贡献中，描述了 LC3A、LC3B 和 LC3C 与含 CL 模型膜的相互作用的分析，以及它们转移到线粒体的能力。LC3A与CL的结合强于LC3B；在 SH-SY5Y 细胞中诱导 CL 外化后，两种蛋白质都显示出与线粒体共定位的相似能力。此外，LC3A 和 LC3B 蛋白的双重沉默被认为可以减少 CCCP 诱导的线粒体自噬。位于 LC3A N 末端区域的残基 14 和 18 被证明对于其在鱼藤酮或 CCCP 诱导的线粒体自噬过程中识别受损线粒体非常重要。此外，体外结果表明，LC3A（而非 LC3B）可能在氧化 CL 识别中发挥作用，作为过度细胞凋亡激活的平衡物。在 LC3C 的情况下，即使该蛋白质显示出比 LC3B 或 LC3A 更强的 CL 结合，相互作用的特异性较低，并且 LC3C 与线粒体的共定位不依赖于鱼藤酮。这些结果表明，与 LC3A 不同，LC3C 在 CL 介导的线粒体自噬过程中不参与货物识别。这些数据支持这样一种观点，即各种 LC3 亚家族成员可能在自噬启动过程中发挥不同的作用，将 LC3A 确定为 CL 介导的线粒体自噬中的新型利益相关者。缩略语: ACTB/β-肌动蛋白：肌动蛋白β；Atg8：自噬相关8；CL：心磷脂；CCCP：羰基氰间氯苯腙；DMSO：二甲基亚砜；DOPE：1,2-二油酰-sn-甘油-3-磷酸乙醇胺；DTT：DL-二硫苏糖醇；FKBP8：FKBP脯氨酰异构酶8；GABARAP：GABA A 型受体相关蛋白；GABARAPL1：GABA A 型受体相关蛋白 like 1；GABARAPL2：GABA A 型受体相关蛋白 2；GFP：绿色荧光蛋白；IMM：线粒体内膜；LUV/LUVs：大单层囊泡；MAP1LC3A/LC3A：微管相关蛋白 1 轻链 3 α；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3 β；MAP1LC3C/LC3C：微管相关蛋白 1 轻链 3 γ；NME4/NDPK-D/Nm23-H4：NME/NM23 核苷二磷酸激酶 4；O/A：寡霉素 A + 抗霉素 A；欧姆：线粒体外膜；PA：磷脂酸；PC：磷脂酰胆碱；PG：磷脂酰甘油；PINK1：PTEN 诱导的推定激酶 1；PtdIns4P：磷脂酰肌醇-4-磷酸；Rho-PE：丽丝胺罗丹明磷脂酰乙醇胺；SUV/SUV：小单层囊泡

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ABSTRACT

Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s