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 泛素蛋白酶体系统（UPS）是决定蛋白质命运的组成部分。蛋白 标记有泛素，通过酶级联反应的活性被蛋白酶体降解 （ubiquitination）。去泛素化酶（DUBs）从蛋白质中去除泛素， 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， 对于后生动物发育和与疾病如癌症有关的异常调节是必需的。的抑制剂 UPS如硼替佐米是有效癌症治疗剂。随着知识的结构和功能， DUBs，DUBs的抑制剂可以作为精确疗法开发。DUB的应用示例 癌症生物学中的抑制剂是癌蛋白的稳定化，例如c-Myc 1，其由DUB稳定。瓦洛辛 含有蛋白质p97/p47复合物相互作用蛋白1（VCPIP 1），DUBs 2的OTU家族的成员，是一种 半胱氨酸蛋白酶，像许多DUB一样，已被鉴定为人类中的治疗脆弱性， 癌症3.其已知的密切相互作用物，包含蛋白质的valosin（VCP）4，是UPS系统中的关键参与者，因为它 在被蛋白体降解之前展开各种多聚泛素化底物，并且本身是一种 有希望的治疗靶点5. VCP的蛋白质解折叠是显著的和独特的，因为它能够解折叠 泛素，一种众所周知的稳定蛋白，在DUB之前启动多聚泛素化底物的解折叠6 海峡底部的活动。目前还不知道VCPIP 1如何以及为什么与VCP结合。 来自哺乳动物细胞和免疫沉淀质量的共纯化的初步数据 质谱（IP-MS）证实VCPIP 1与VCP相互作用。使用冷冻电子显微镜（cryo-EM），初始 使用Talos Arctica进行数据收集，并使用cryoSPARC 7进行数据处理，从而获得高质量的2D类 二级结构特征，并最终进行3.3 μ m三维重建。Titan Krios数据收集 化学交联的样品导致在没有底物的情况下复合物的3.0 μ m重构。这提供 初步了解VCPIP 1和VCP的相互作用位点和复合物的一般化学计量。 使用结构和生物化学方法，该提案旨在阐明VCPIP 1的结构， 它与VCP的相互作用机制，为什么VCPIP 1与VCP形成复合体以及它如何作用于客户端。这 与最初的命中化合物一起努力将能够开发VCPIP 1的抑制剂。具体地说， 具体目标1旨在使用cryo-EM解决与VCP结合的VCPIP 1的高分辨率结构， 通过结构-功能研究来生化表征VCPIP 1和VCP之间的相互作用。具体目标 2旨在确定与底物和衔接子结合的VCPIP 1/VCP复合物的高分辨率结构， 将使用荧光/酶学测定来证明VCPIP 1在底物 通过VCP展开。具体目标3旨在使用基于结构的药物设计（SBDD），无论是计算还是 实验上，发现和改善VCPIP 1抑制剂。