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Structure and Function of the Essential Cell Cycle Regulator Cdc34

重要细胞周期调节因子 Cdc34 的结构和功能

基本信息

批准号：
10291075
负责人：
Shaun Olsen
金额：
$ 19.68万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-15 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10291075
关键词：
Active Sites Address Affinity Binding Biochemical Biological Biological Assay Biological Process Biology Biophysics C-terminal Cell Cycle Cell Cycle Progression Cell Cycle Proteins Cell Cycle Regulation Cell division Cell physiology Cells Cellular Structures Cellular biology Chemicals Complex Crystallization Cullin Proteins Cyclin-Dependent Kinase Inhibitor Data Development Disease Ensure Enzymes Eukaryotic Cell Event F Box Domain FDA approved Foundations Generations Genetic Materials Goals Hand Human In Vitro Link Lysine Malignant Neoplasms Methionine Mitosis Molecular Multiple Myeloma N-terminal Normal Cell Pathway interactions Patients Pharmaceutical Preparations Play Polymers Polyubiquitin Positioning Attribute Post-Translational Protein Processing Protein Engineering Proteins Regulation Role Secure Signal Transduction Specificity Structure Techniques Testing Therapeutic Therapeutic Intervention Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitination Work X-Ray Crystallography Yeasts base cancer cell design experimental study functional outcomes insight intermolecular interaction lens molecular recognition multicatalytic endopeptidase complex novel protein crosslink reconstitution recruit single molecule targeted treatment ubiquitin-protein ligase

项目摘要

Project Summary The ability of the cell to control progression through the cell cycle in a regulated manner is of fundamental importance to eukaryotic biology. Breakdown of the checkpoints controlling the cell cycle results in catastrophic effects on normal cell function and is a defining feature of cancer cells. Cell division cycle 34 (Cdc34) is a ubiquitin (Ub) conjugating enzyme (E2) that plays an essential role in controlling progression of the cell cycle at both the G1/S and G2/M checkpoints by ubiquitinating key cell cycle regulatory proteins and signaling for their proteasomal destruction at defined timepoints. E2 enzymes such as Cdc34 are the central players in the Ub conjugation cascade in which the sequential interactions and activities of three enzymes (E1, E2, and E3) are required for ubiquitination of target proteins. This cascade is initiated by E1, which activates and transfers Ub to tens of different E2s. The resulting E2~Ub intermediates next interact with a repertoire of hundreds of Ub E3 ligases that catalyze Ub conjugation to target proteins by distinct mechanisms. While in most cases the cognate E2/E3 pair responsible for regulation of a particular biological process is unknown, the Skp, Cullin, F-box (SCF) subfamily of RING E3 ligases is well- established to function with Cdc34 to control cell cycle progression by catalyzing the extension of Lysine 48- linked polyubiquitin (polyUb) chains on target proteins that serve as a signal for proteasomal destruction. Despite its fundamental importance, many key questions remain about how Cdc34 performs its functions in the cell including: 1) how Cdc34 is recruited to E1 during formation of the Cdc34~Ub intermediate, 2) how Cdc34~Ub is subsequently recruited to SCF, and 3) how the Cdc34/SCF complex specifically catalyzes Lys48-linked polyUb chains that signal for proteasomal destruction of target proteins as opposed to other types of polyUb chains. Cdc34 harbors a number of unique sequence and structural features compared to other E2s that are crucial for its function, though the structural basis for these observations are unknown. These features include an acidic loop insertion proximal to the active site and a large C-terminal extension, both of which are required for Lys48-linked polyUb chain specificity and maximal enzymatic activity. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to establish the rules governing molecular recognition in E1/Cdc34 and Cdc34/SCF interactions (Aim 1) and to determine the structural basis by which Cdc34 and SCF function together to specifically assemble Lys48- linked polyUb chains (Aim 2). Ub signaling is a validated target for therapeutic intervention in cancer with FDA-approved medications targeting the proteasome currently extending the lives of multiple myeloma patients. A molecular understanding of how E1, Cdc34, and SCF work together to control the cell cycle resulting from the proposed studies could provide a platform for the development of novel cancer therapeutics.

项目总结