Structural and biochemical characterization of VCPIP1 and VCP complex

VCPIP1 和 VCP 复合物的结构和生化表征

基本信息

批准号：
10675974
负责人：
Binita Shah
金额：
$ 4.19万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10675974
关键词：
3-Dimensional ATP phosphohydrolase Affinity Binding Binding Sites Biochemical Biological Assay Biology Body of uterus Bortezomib Breast Cancer Biology Carcinoma Caspase Cells Chemicals Classification Client Complex Computing Methodologies Coupled Cryoelectron Microscopy Cysteine Data Data Collection Deubiquitinating Enzyme Deubiquitination Development Disease Docking Drug Design Drug Targeting Endometrial Carcinoma Endoplasmic Reticulum Engineering Enzymatic Biochemistry Enzyme Inhibitor Drugs Enzyme Kinetics Enzymes Experimental Designs FDA approved Family Fluorescence Glues Golgi Apparatus Health High Prevalence Homeostasis Human Immunoprecipitation In Vitro Knowledge Lead Ligase Location Malignant Neoplasms Mammalian Cell Maps Mass Spectrum Analysis Mediating Methods Mitosis Modeling Molecular Monitor Mutagenesis Mutate Oncoproteins Pharmaceutical Preparations Play Precision therapeutics Process Proteasome Inhibitor Proteins Reaction Regulation Resolution Role Sampling Signal Transduction Site Specificity Structure Substrate Interaction System Testing Therapeutic Titan Ubiquitin Ubiquitin-Activating Enzymes Ubiquitin-Conjugating Enzymes Ubiquitination X-Ray Crystallography anti-cancer clinical development computerized data processing crosslink density drug discovery enzyme activity improved inhibitor insight lenalidomide member multicatalytic endopeptidase complex mutant ovarian neoplasm prevent protein complex protein structure function proteostasis reconstitution reconstruction scaffold stoichiometry structural biology targeted agent therapeutic target tumor ubiquitin isopeptidase ubiquitin-protein ligase valosin-containing protein

项目摘要

Project Abstract/Summary The ubiquitin proteasome system (UPS) is an integral part of determining protein fate. Proteins are marked with ubiquitin for degradation by the proteasome through the activity of an enzymatic cascade (ubiquitination). Deubiquitinating enzymes (DUBs) remove ubiquitin from proteins rescuing them from degradation1. Together ubiquitination and deubiquitination control protein stability and homeostasis and are essential for metazoan development and aberrant regulation implicated in disease such as cancer. Inhibitors of the UPS, such as Bortezomib, are effective cancer therapeutics. With knowledge of the structure and function of DUBs, inhibitors for DUBs can be developed as precision therapies. An example of an application of DUB inhibitors in cancer biology is stabilization of oncoproteins, such as c-Myc1, that are stabilized by a DUB. Valosin containing protein p97/p47 complex interacting protein 1 (VCPIP1), a member of the OTU family of DUBs2, is a cysteine protease that, like many of the DUBs, has been identified as a therapeutic vulnerability in human cancers3. Its known close interactor, valosin containing protein (VCP)4, is a key player in the UPS system as it unfolds a variety of poly-ubiquitinated substrates prior to their degradation by the proteosome, and is itself a promising therapeutic target5. The protein unfolding by VCP is remarkable and unique as it is able to unfold ubiquitin, a notoriously stable protein that initiates the unfolding of poly-ubiquitinated substrates6 prior to DUB activity at the bottom of the channel. There is no knowledge as to how and why VCPIP1 binds to VCP. Preliminary data from a co-purification from mammalian cells and immuno-precipitation mass spectrometry (IP-MS) confirm that VCPIP1 interacts with VCP. Using cryo-electron microscopy (cryo-EM), initial data collection using a Talos Arctica and data processing with cryoSPARC7 resulted in high quality 2D classes with secondary structure features and, ultimately, a 3.3 Å 3D reconstruction. Titan Krios data collection of chemically crosslinked sample resulted in a 3.0 Å reconstruction of the complex without substrate. This provided initial insights into the interaction site of VCPIP1 and VCP and the general stoichiometry of the complex. Using structural and biochemical methods, this proposal aims to elucidate the structure of VCPIP1 and its mechanism of interaction with VCP, why VCPIP1 forms a complex with VCP and how it acts on clients. This effort together with initial hit compounds will enable the development of inhibitors for VCPIP1. Specifically, Specific Aim 1 aims to solve a high-resolution structure of VCPIP1 bound to VCP using cryo-EM and to biochemically characterize the interaction between VCPIP1 and VCP by structure-function studies. Specific Aim 2 aims to determine a high-resolution structure of the VCPIP1/VCP complex bound to a substrate and adaptors, fluorescence/enzymology assays will be used to demonstrate deubiquitylation by VCPIP1 after substrate unfolding by VCP. Specific Aim 3 aims to use structure-based drug design (SBDD), both computationally and experimentally, to discover and improve VCPIP1 inhibitors.

项目摘要/总结泛素蛋白酶体系统 (UPS) 是决定蛋白质命运的重要组成部分。带有泛素标记，可通过酶级联活性被蛋白酶体降解（去泛素化酶（DUB）从蛋白质中去除泛素，从而将它们从蛋白质中拯救出来。降解1。泛素化和去泛素化共同控制蛋白质的稳定性和稳态。对于后生动物的发育和与癌症等疾病有关的异常调节至关重要。 UPS，如硼替佐米，是有效的癌症治疗药物，了解其结构和功能。 DUB、DUB 抑制剂可作为精准疗法开发。DUB 的应用示例。癌症生物学中的抑制剂是通过 Valosin 稳定癌蛋白，例如 c-Myc1。含有蛋白质 p97/p47 复合物相互作用蛋白 1 (VCPIP1)，是 DUBs2 OTU 家族的成员，是一种半胱氨酸蛋白酶，与许多 DUB 一样，已被确定为人类的治疗脆弱性癌症3。其已知的紧密相互作用蛋白，含缬洛辛蛋白 (VCP)4，是 UPS 系统中的关键角色，因为它在被蛋白酶体降解之前展开多种多聚泛素化底物，并且其本身是治疗靶点 5. 有希望通过 VCP 解折叠的蛋白质是显着且独特的，因为它能够解折叠。泛素，一种众所周知的稳定蛋白质，在 DUB 之前启动多泛素化底物6的展开通道底部的活动尚不清楚 VCPIP1 如何以及为何与 VCP 结合。哺乳动物细胞和免疫沉淀团块共纯化的初步数据使用冷冻电子显微镜 (cryo-EM) 初步确认 VCPIP1 与 VCP 相互作用。使用 Talos Arctica 收集数据并使用 CryoSPARC7 进行数据处理，生成高质量的 2D 类具有二级结构特征，以及最终的 3.3 Å 3D 重建的 Titan Krios 数据集。化学交联的样品产生了 3.0 Å 的复合物重建，无需底物。对 VCPIP1 和 VCP 相互作用位点以及复合物的一般化学计量的初步了解。该提案旨在利用结构和生化方法阐明 VCPIP1 的结构和它与VCP的交互机制，为什么VCPIP1与VCP形成复合体以及它如何作用于客户端。与最初的热门化合物一起努力将有助于开发 VCPIP1 抑制剂。具体目标 1 旨在使用冷冻电镜解析 VCPIP1 与 VCP 结合的高分辨率结构，并通过结构功能研究对 VCPIP1 和 VCP 之间的相互作用进行生化表征。图2旨在确定与底物和接头结合的VCPIP1/VCP复合物的高分辨率结构，荧光/酶学检测将用于证明底物后 VCPIP1 的去泛素化 VCP 正在展开的具体目标 3 旨在使用基于结构的药物设计 (SBDD)，包括计算和计算。通过实验发现和改进 VCPIP1 抑制剂。