Structural and biochemical characterization of VCPIP1 and VCP complex

VCPIP1 和 VCP 复合物的结构和生化表征

• 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
• 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

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/摘要 泛素蛋白质组系统（UPS）是确定蛋白质命运的组成部分。蛋白质是 用泛素标记蛋白酶体通过酶促级联反应降解 （泛素化）。去泛素化酶（DUB）从蛋白质中清除泛素，从蛋白质中拯救它们 降解1。泛素化和去泛素化控制蛋白稳定性和稳态，并且是 对于在癌症等疾病中实施的后生动物发展和异常调节所必需的。抑制剂 UPS，例如硼替佐米，是有效的癌症治疗。了解的结构和功能 配音，配音的抑制剂可以作为精度疗法发展。配音应用的一个示例 癌症生物学的抑制剂是通过DUB稳定的癌蛋白（例如C-MYC1）的稳定。 valosin 含有蛋白质p97/p47复合物相互作用蛋白1（VCPIP1），dubs2家族的成员是一个 半胱氨酸蛋白酶与许多配音一样，已被确定为人类的治疗脆弱性 CANCERS3。它已知的近距离交互剂含有蛋白质（VCP）4，是UPS系统中的关键参与者 在蛋白质体降解之前，将各种多泛素化底物展开，并且本身就是一个 有希望的治疗靶标5。 VCP展开的蛋白质是显着和独特的，因为它能够展开 泛素是一种臭名昭著的稳定蛋白 在通道底部的活动。对于如何以及为什么VCPIP1与VCP结合，尚无知识。 来自哺乳动物细胞和免疫沉淀量的共纯化的初步数据 光谱法（IP-MS）确认VCPIP1与VCP相互作用。使用冷冻电子显微镜（Cryo-EM），初始 使用TALOS ARCTICA和CRYOSPARC7的数据处理收集数据，导致高质量的2D类 具有二级结构特征，最终具有3.3Å3D重建。 Titan Krios数据收集 化学交联样品导致复合物的3.0Å重建，没有底物。提供了 对VCPIP1和VCP的相互作用的初步见解以及复合物的一般化学计量。 使用结构和生化方法，该建议旨在阐明VCPIP1和 它与VCP互动的机制，为什么VCPIP1与VCP形成复杂的及其对客户的作用。这 努力与初始命中化合物一起将使VCPIP1的抑制剂开发。具体来说， 特定目标1旨在解决使用Cryo-EM与VCP结合的VCPIP1的高分辨率结构，并 通过结构功能研究，生化表征VCPIP1与VCP之间的相互作用。具体目标 2旨在确定与基板和适配器结合的VCPIP1/VCP复合物的高分辨率结构， 荧光/酶学测定法将用于证明底物的VCPIP1去泛素化 VCP展开。特定目标3的目的是在计算和 在实验上，发现和改善VCPIP1抑制剂。