DIP-2泛素化修飾抑癌蛋白PML之降解機制探討

Abstract

Promyelocytic leukemia protein (簡稱PML)為一種抑癌蛋白。其會於細胞核中形成特殊的斑點狀構造，稱為PML-nuclear body (PML-NB)。PML-NB參與許多不同細胞生理功能的調控，例如誘發細胞凋亡、抑制細胞生長與促使細胞老化。當細胞受到壓力時，PML蛋白表現量會上升；反之，在人類腫瘤細胞中常出現PML蛋白消失的現象。然而，目前調控PML蛋白穩定性的因子之研究尚未透徹。在這篇論文中我們發現，BTB-Kelch蛋白DIP-2扮演著負調節者的角色，它會促使PML蛋白穩定性下降。DIP-2蛋白由一個BTB區塊、一個BACK區塊以及六個Kelch區塊所組成。根據本實驗室先前的研究中發現，DIP-2蛋白會與Cul3蛋白形成一個完整的泛素連接酶複合體，在此我們更進一步發現DIP-2會以其受質辨識區Kelch區塊去辨識PML蛋白，這樣一來，PML蛋白會被牽引到完整的Cul3-DIP-2泛素連接酶複合體中而被進行泛素化修飾，進而導致蛋白質降解。除此之外，有別於PML，DIP-2並不會進行小類泛素化修飾，而其促使PML蛋白泛素化的能力亦不受PML被小類泛素化修飾與否所影響；同時PML K490的小類泛素化與否，也不影響DIP-2造成PML蛋白泛素化修飾的能力。而後我們發現脯胺酸異構酶Pin1的活性會調控DIP-2所導致的PML蛋白泛素化修飾，並進一步證實PML蛋白上可與Pin1結合的pS/T-P區間中，絲胺酸518與527這兩個位置的磷酸化對於DIP-2促使PML蛋白泛素化的能力是必需的。總而言之，我們除了發現DIP-2-Cul3泛素連接酶複合體可將PML蛋白泛素化進而降解，更找到了一項新的PML蛋白降解之機制。

The promyelocytic leukemia (PML) is a tumor suppressor protein and is critical for the formation PML-nuclear body (PML-NB), a subnuclear macromolecular structure implicated in the regulation of various cellular functions, such as induction of apoptosis, growth suppression and cellular senescence. PML is activated in response to various stress signals and is frequently downregulated in human tumors. However, the factors regulating PML stability have not been completely understood. In this thesis, we identify the BTB-Kelch protein DIP-2 as a negative regulator of PML stability. DIP-2 contains an N-terminal BTB/POZ domain, an internal BACK domain and C-terminal six Kelch repeats. Previous study in our laboratory indicates that DIP-2 associates with Cul3 to form an ubiquitin ligase complex. Here, we show that DIP-2 interacts with PML through its substrate recognition domain. This interaction targets PML to the Cul3-DIP-2 complex for ubiquitination and, consequently, degradation. Unlike PML, DIP-2 is not sumoylated and DIP-2 mediates PML ubiquitination through a PML sumoylation-independent manner. Furthermore, PML sumoylation at K490 does not interfere with its ubiquitination by DIP-2-Cul3. However, DIP-2-mediated PML ubiquitination requires the activity of peptidyl prolyl cis/trans isomerase Pin1, which is known to trigger PML degradation. DIP-2-meditated PML ubiquitination also requires the Ser518/527 residues of PML, both of which are within the Pin1 targeting motif, pS/T-P. Together, this study not only identifies the DIP-2-Cul3 complex as an ubiquitin ligase for PML, but also reveals a novel PML degradation pathway.