探討Cullin蛋白的連接器在細胞自噬之功能

Abstract

細胞自噬（autophagy）是一個將受損的細胞物質以及一些長壽的蛋白經由溶酶體降解的一個過程。在細胞自噬進行的過程當中，細胞內的物質會受到雙層膜的構造包覆，此構造稱為細胞自噬體（autophagosome），最終與溶酶體結合並藉由水解酶降解包覆的物質並回收。果蠅的CG12084是哺乳動物ZYG11家族的同源物，其最主要的功能在於它會作為Cullin 2泛素連接酶複合物的受體辨認次單位，並且ZYG11在線蟲當中被發現會去調控細胞分裂以及胚胎發育。近期，我們的研究發現在果蠅幼蟲的脂肪組織藉由RNA干擾的方式敲落CG12084基因，會導致作為細胞自噬指標的Atg8a的量會有堆積的現象，然而，CG12084在細胞自噬當中所扮演的角色以及其調控的相關機制尚未釐清。 根據GST pull-down以及質譜的分析，我們發現許多與CG12084有相互作用的蛋白，其中一個為chaperonin containing TCP-1 (CCT, 也稱作TRiC)，其在調節所有真核生物中的細胞骨架蛋白折疊中發揮功能。此外，CCT複合物也參與在細胞自噬的調節過程當中。深入研究發現，CCT複合物的許多次單位都會與CG12084有直接的相互作用，並且能夠在CG12084敲除的條件下，利用CCT2的過度表達去挽救Atg8a積累的現象。綜合以上，我們認為CG12084及CCT之間會互相影響，並且調控細胞自噬。我們將在進一步探討CG12084及CCT調控細胞自噬的分子機制。

Autophagy is a lysosomal degradation pathway by which damaged cellular materials and long-lived proteins are engulfed into double-membraned structures named autophagosomes, which finally fuse with lysosomes where their contents are degraded. Our recent study found that knockdown of CG12084 in Drosophila larval fat body caused the accumulation of Atg8a puncta, a marker of autophagosomes. CG12084 is a Drosophila homolog of mammalian ZYG11 family protein. It has been shown that ZYG11 family proteins function as conserved substrate recognition subunit of Cullin-2 ubiquitin-ligase complexes. ZYG11 regulates cell division and embryonic development in C. elegans. To explore the functions of CG12084 in autophagy, we identified several potential CG12084 interactive protein by GST pull-down assay and mass spectrometric analyses. One potential substrate is chaperonin containing TCP-1 (CCT, also known as TRiC), which functions in regulating cytoskeletal protein folding in all eukaryotes. In addition, CCT complex was also reported to regulate autophagy process. Our data showed that most of the components of the CCT complex interact with CG12084, and CCT2 overexpression rescues the Atg8a puncta accumulation in CG12084 knockdown conditions. Taken together, our study indicates that there is a correlation between CG12084 and CCT and may regulate autophagy machinery together. We will further investigate the molecular mechanisms underlie the regulation of autophagy between CG12084 and CCT.