Substrate targeting mechanism of a DesCEND pathway

DesCEND 通路的底物靶向机制

• 批准号: 10238152
• 负责人: Bing Hao
• 金额: $ 44.27万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238152
• 关键词: Address; Affinity; Apoptotic; Binding; Biochemical; Bioinformatics; Biological Assay; C-terminal; Calorimetry; Calpain; Cell Culture System; Cell physiology; Cells; Co-Immunoprecipitations; Complement; Complex; Crystallization; Cyclin D1; DNA Damage; Disease; Drug Targeting; Elements; Endopeptidases; F Box Domain; F-Box Proteins; Fluorescence Polarization; G1 Arrest; Genotoxic Stress; Goals; Human; Imaging Techniques; In Vitro; Knowledge; LGLA; Libraries; MAP2K6 gene; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Molecular; Mutagenesis; Mutation; N-terminal; Pathogenesis; Pathway interactions; Peptides; Play; Property; Proteins; Proteolysis; Research; Roentgen Rays; Role; SKP Cullin F-Box Protein Ligases; Signal Transduction; Site; Specificity; Stimulus; Structure; Substrate Specificity; Surface; System; Tail; Testing; Therapeutic; Titrations; Ubiquitin; Ubiquitination; Work; base; biophysical analysis; biophysical properties; c new; design; experimental study; improved; in vitro Assay; insight; intermolecular interaction; live cell imaging; mutant; novel; prolylglutamic acid; protein aminoacid sequence; protein degradation; proteostasis; reconstitution; recruit; response; treatment strategy; ubiquitin-protein ligase

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.

项目摘要 泛素介导的蛋白质降解对于维持蛋白质稳态是必不可少的，从而控制多种多样的蛋白质代谢。 细胞过程蛋白质泛素化的特异性很大程度上取决于对短肽的识别 通过E3泛素连接酶在蛋白质底物中被称为降解决定子的基序。F-box蛋白作为底物- Skp 1-Cul 1-F-box-蛋白（SCF）E3超家族的识别组分。F-box蛋白FBXO 31 介导细胞周期蛋白D1降解，诱导DNA损伤后的G1期阻滞。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。这种所谓的PEST基序富含高度动态的 真核生物蛋白质的无序区域，并作为许多不稳定蛋白质的降解信号。我们有 最近确定了C-末端含有PEST的细胞周期蛋白D1肽的X-射线晶体结构， Skp1-FBXO 31.其结构表明，特异性识别是通过一个复杂的分子间 相互作用网络，以及涉及细胞周期蛋白D1羧基尾巴，占据了一个开放的表面空腔， FBXO 31的桶图案。我们发现，这种PEST降解决定子介导细胞周期蛋白D1与FBXO 31的相互作用， 体外泛素化和细胞内蛋白水解。我们假设FBXO 31负责识别C- 通过新的DesCEND（通过C-末端降解决定子的破坏）的末端含PEST的底物降解决定子 通路通过在系统水平和分子水平上识别和表征FBXO 31的底物， 水平，我们的目标是阐明FBXO 31-降解决定子对的互补结构决定因素，赋予 底物特异性和决定蛋白质识别和命运。为了实现我们的目标，我们制定了 以下具体目的是：1）鉴定和表征人体中FBXO 31靶向的C端底物降解决定子 蛋白质; 2）表征候选FBXO 31降解决定子的结构和生物化学性质;以及3） 表征内切蛋白水解裂解产生的FBXO 31降解决定子。