Substrate targeting mechanism of a DesCEND pathway

项目摘要 泛素介导的蛋白质降解是维持蛋白质稳定所必需的，从而控制不同的 细胞过程。蛋白质泛素化的特异性在很大程度上取决于对短肽的识别 E3泛素连接酶在蛋白质底物中称为退化基序。F-box蛋白作为底物- Skp1-Cul1-F-box-Protein(SCF)E3超家族的识别成分。F-box蛋白FBXO31 在DNA损伤后，介导细胞周期蛋白D1的降解以诱导G1期停滞。细胞周期蛋白D1的C末端区域 含有富含Pro、Glu/Asp、Ser和Thr的序列。这个所谓的害虫主题丰富了高度动态的 真核蛋白质的无序区域，是许多不稳定蛋白质的降解信号。我们有 最近测定了结合于C端的含有PEST的细胞周期蛋白D1多肽的X射线晶体结构 Skp1-FBXO31。该结构揭示了特定的识别是通过复杂的分子间实现的 涉及中占据开放表面空腔的细胞周期蛋白D1羧基尾巴的相互作用网络- FBXO31的桶形图案。我们发现，这种害虫降解分子介导了细胞周期蛋白D1与FBXO31之间的相互作用 体外泛素化和细胞内蛋白分解。我们假设FBXO31负责识别C- 含有末端害虫的底物通过一种新的下降方式退化(通过C端退化而破坏) 路径。通过在系统水平和分子水平上鉴定和表征FBXO31底物 水平上，我们的目的是阐明FBXO31-degron对的互补结构决定因素 底物专一性决定蛋白质的识别和命运。为了实现我们的目标，我们开发了 具体目的如下：1)鉴定和鉴定FBXO31在人体内靶向的C端底物退化 蛋白质；2)表征候选FBXO31降解子的结构和生化性质；以及3) 鉴定内切蛋白裂解产生的FBXO31降解产物。

Project Summary Ubiquitin-mediated protein degradation is essential to maintaining proteostasis, thereby controlling diverse cellular processes. The specificity of protein ubiquitination is largely determined by recognition of short peptide motifs known as degrons in protein substrates by E3 ubiquitin ligases. F-box proteins serve as substrate- recognition components of the Skp1–Cul1–F-box-protein (SCF) E3 superfamily. The F-box protein FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage. The C-terminal region of cyclin D1 contains sequences rich in Pro, Glu/Asp, Ser and Thr. This so-called PEST motif is enriched in highly dynamic disordered regions of eukaryotic proteins and serves as degradation signal for many unstable proteins. We have recently determined the X-ray crystal structure of a C-terminal PEST-containing cyclin D1 peptide bound to Skp1–FBXO31. The structure reveals that specific recognition is achieved by an intricate inter-molecular interaction network involving as well the cyclin D1 carboxyl tail that occupies an open surface cavity in the - barrel motif of FBXO31. We show that this PEST degron mediates the interaction of cyclin D1 with FBXO31 for ubiquitination in vitro and proteolysis in cells. We hypothesize that FBXO31 is responsible for recognition of C- terminal PEST-containing substrate degrons through a novel DesCEND (destruction via C-end degrons) pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we aim to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, we have developed the following specific aims to: 1) identify and characterize C-end substrate degrons targeted by FBXO31 in human proteins; 2) characterize the structural and biochemical properties of candidate FBXO31 degrons; and 3) characterize endoproteolytic cleavage-generated FBXO31 degrons.