ABSTRACT

Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that does not involve CASP9 apoptotic activity. We report here that a non-apoptotic activity of endosomal membrane CASP9 facilitates the retrograde transport of IGF2R/CI-MPR from the endosomes to the trans-Golgi network, indicating the involvement of CASP9 in endosomal sorting and lysosomal biogenesis. CASP9-deficient cells demonstrate the missorting of CTSD (cathepsin D) and other acid hydrolases, accumulation of late endosomes, and reduced degradation of bafilomycin A₁-sensitive proteins. In the absence of CASP9, IGF2R undergoes significant degradation, and its rescue is achieved by the re-expression of a non-catalytic CASP9 mutant. This endosomal activity of CASP9 is potentially mediated by herein newly identified interactions of CASP9 with the components of the endosomal membrane transport complexes. These endosomal complexes include the retromer VPS35 and the SNX dimers, SNX1-SNX5 and SNX2-SNX6, which are involved in the IGF2R retrieval mechanism. Additionally, CASP9 interacts with HGS/HRS/ESCRT-0 and the CLTC (clathrin heavy chain) that participate in the initiation of the endosomal ESCRT degradation pathway. We propose that endosomal CASP9 inhibits the endosomal membrane degradative subdomain(s) from initiating the ESCRT-mediated degradation of IGF2R, allowing its retrieval to transport-designated endosomal membrane subdomain(s). These findings are the first to identify a cell survival, non-apoptotic function for CASP9 at the endosomal membrane, a site distinctly removed from the cytoplasmic apoptosome. Via its non-apoptotic endosomal function, CASP9 impacts the retrograde transport of IGF2R and, consequently, lysosomal biogenesis.

Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A₁; CASP: caspase; CLTC/CHC: clathrin, heavy chain; CTSD: cathepsin D; ESCRT: endosomal sorting complexes required for transport; HEXB: hexosaminidase subunit beta; HGS/HRS/ESCRT-0: hepatocyte growth factor-regulated tyrosine kinase substrate; IGF2R/CI-MPR: insulin like growth factor 2 receptor; ILV: intraluminal vesicles; KD: knockdown; KO: knockout; M6PR/CD-MPR: mannose-6-phosphate receptor, cation dependent; MEF: murine embryonic fibroblasts; MWU: Mann-Whitney U test; PepA: pepstatin A; RAB7A: RAB7, member RAS oncogene family; SNX-BAR: sorting nexin dimers with a Bin/Amphiphysin/Rvs (BAR) domain each; TGN: trans-Golgi network; TUBB: tubulin beta; VPS26: VPS26 retromer complex component; VPS29: VPS29 retromer complex component; VPS35: VPS35 retromer complex component.

摘要

最近，我们报道了内体膜上 CASP9 前域的表达增加，以响应 HSP90 抑制，介导不涉及 CASP9 凋亡活性的细胞保护作用。我们在此报告，内体膜 CASP9 的非凋亡活性促进 IGF2R/CI-MPR 从内体到反式高尔基体网络的逆行运输，表明 CASP9 参与内体分选和溶酶体生物发生。CASP9 缺陷细胞表现出 CTSD（组织蛋白酶 D）和其他酸性水解酶的错配、晚期内体的积累以及巴弗洛霉素 A ₁敏感蛋白的降解减少。在没有 CASP9 的情况下，IGF2R 经历了显着的降解，它的拯救是通过重新表达非催化CASP9 来实现的突变体。CASP9 的这种内体活性可能是由本文新发现的 CASP9 与内体膜转运复合物组分的相互作用介导的。这些内体复合物包括逆向体 VPS35 和 SNX 二聚体、SNX1-SNX5 和 SNX2-SNX6，它们参与 IGF2R 修复机制。此外，CASP9 与参与内体 ESCRT 降解途径启动的 HGS/HRS/ESCRT-0 和 CLTC（网格蛋白重链）相互作用。我们建议内体 CASP9 抑制内体膜降解亚域启动 ESCRT 介导的 IGF2R 降解，允许其恢复到转运指定的内体膜亚域。这些发现是第一个确定内体膜上 CASP9 的细胞存活、非凋亡功能的发现，从细胞质凋亡体中明显去除的位点。通过其非凋亡的内体功能，CASP9 影响 IGF2R 的逆行转运，从而影响溶酶体的生物发生。

缩写：ACTB：肌动蛋白β；ATG7：自噬相关7；BafA1：巴弗洛霉素 A ₁; CASP：半胱天冬酶；CLTC/CHC：网格蛋白，重链；CTSD：组织蛋白酶 D；ESCRT：运输所需的内体分选复合物；HEXB：氨基己糖苷酶亚基β；HGS/HRS/ESCRT-0：肝细胞生长因子调节的酪氨酸激酶底物；IGF2R/CI-MPR：胰岛素样生长因子 2 受体；ILV：腔内囊泡；KD：击倒；KO：淘汰赛；M6PR/CD-MPR：6-磷酸甘露糖受体，阳离子依赖性；MEF：鼠胚胎成纤维细胞；MWU：Mann-Whitney U 检验；PepA：胃蛋白酶抑制剂 A；RAB7A：RAB7，RAS癌基因家族成员；SNX-BAR：用 Bin/Amphiphysin/Rvs (BAR) 域对连接蛋白二聚体进行排序；TGN：跨高尔基网络；TUBB：微管蛋白β；VPS26：VPS26逆向复合组件；VPS29：VPS29逆向复合组件；VPS35：VPS35 逆转录器复杂组件。