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Defining the nuclear envelope-resident protein turnover machinery implicated in diseases affecting cholesterol metabolism

定义与影响胆固醇代谢的疾病有关的核膜驻留蛋白周转机制

基本信息

批准号：
10063878
负责人：
Christian Dirk Schlieker
金额：
$ 30.94万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063878
关键词：
ATP phosphohydrolase Affect Affinity Aging Area Biological Assay CRISPR/Cas technology Candidate Disease Gene Cell Line Cells Cholesterol Cholesterol Homeostasis Co-Immunoprecipitations Complement Congenital Disorders Deubiquitination Development Disease Dislocations Dysplasia Endoplasmic Reticulum Degradation Pathway Etiology Eukaryota Fluorescence Genes Genetic Genetic Screening Goals Human Knock-out Lead Mammalian Cell Mass Spectrum Analysis Membrane Membrane Proteins Metabolic Methodology Modeling Molecular Chaperones Mutation Nuclear Nuclear Envelope Nuclear Extract Nuclear Inner Membrane Nuclear Lamina Nucleoplasm Oxidoreductase Pathway interactions Pelger-Huet Anomaly Pharmacology Physiologic pulse Positioning Attribute Proteins Proteomics Quality Control RNA Interference Role Saccharomycetales Site Sterols Time Ubiquitin Ubiquitination Variant Work base disease-causing mutation experimental study gene discovery gene product genome-wide improved knock-down lamin B receptor multicatalytic endopeptidase complex mutant next generation sequencing novel protein degradation protein protein interaction proteostasis small hairpin RNA success ubiquitin-protein ligase

项目摘要

Project Summary/Abstract The Lamin B receptor (LBR) is a polytopic membrane protein residing at the nuclear envelope in close association with the nuclear lamina. We discovered that human LBR has an essential role in cholesterol synthesis, which is strongly perturbed by LBR mutations responsible for the congenital disorders Greenberg Dysplasia and Pelger-Huët anomaly. Several of these disease-associated mutations lead to the rapid degradation of LBR at the inner nuclear membrane (INM), a site that is poorly understood from the perspective of protein quality control. Progress in this area has been hampered by the absence of suitable methodologies, with the lack of model substrates being the major limitation. This deficiency in our understanding of cellular quality control presents a major obstacle towards understanding and treating congenital diseases caused by mutations in nuclear envelope-resident proteins, commonly referred to as nuclear envelopathies. A detailed characterization of the protein turnover mechanisms responsible for the elimination of LBR disease mutants has allowed us to directly observe inner nuclear membrane protein turnover (INMPT) in mammalian cells for the first time. We have since established LBR-based model substrates and readouts, allowing us to initiate an unprecedented interrogation of protein turnover mechanisms in the nuclear envelope. As a logical extension of these endeavors, we now propose to exploit our newly established experimental platform to identify the machinery responsible for protein quality control at the INM. Two independent yet synergistic approaches will be pursued to achieve this goal: (1) Based on our established strategies to intersect the degradation of LBR derivatives at distinct stages of membrane extraction and proteasomal degradation, we will isolate stalled degradation intermediates to identify associated factors involved in INMPT via mass spectrometry; (2) We will conduct an unbiased, genome-wide CRISPR/Cas9-based screen to discover genes implicated in INMPT. To this end, we will utilize a cell line expressing a degradation-prone, split-GFP derivative of LBR. Mutant stabilization upon functional inactivation of a gene required for INMPT will lead to an increase in fluorescence, allowing us to identify the targeted gene via FACS analysis and next- generation sequencing. For candidates arising from both approaches (1) and (2), we will (3) functionally characterize these factors using our firmly established experimental platform including (i) readouts for localization to the nuclear envelope; (ii) assays for membrane dislocation/extraction and (iii) ubiquitination and proteasomal degradation, thus allowing us to assign a function to each validated candidate gene or interacting protein. The results of these studies will allow us to define the protein homeostasis network that safeguards protein integrity at the nuclear envelope. Apart from closing a major gap in our understanding of cellular protein quality control–an area of significant biomedical relevance–our findings are expected to have important ramifications for our understanding and the treatment of nuclear envelopathies.

项目总结/摘要核纤层蛋白B受体（Lamin B receptor，LBR）是一种位于细胞核被膜上的多位膜蛋白，与核纤层蛋白受体密切相关。与核纤层的联系。我们发现，人类LBR在胆固醇中起着重要作用，合成，这是强烈干扰LBR突变负责先天性疾病格林伯格发育不良和Pelger-Huët异常。这些疾病相关的突变中有几种导致了快速的 LBR在内核膜（INM）的降解，这是一个从生物学角度了解甚少的位点。蛋白质质量控制。由于缺乏适当的方法，这一领域的进展受到阻碍，缺少模型基质是主要的限制。我们对细胞的理解不足质量控制是了解和治疗由以下原因引起的先天性疾病的主要障碍核内蛋白质的突变，通常被称为核内蛋白质病。负责消除LBR的蛋白质周转机制的详细表征疾病突变体使我们能够直接观察细胞内核膜蛋白周转（INMPT），哺乳动物细胞首次。从那时起，我们建立了基于LBR的模型基板和读数，使我们能够对核膜中的蛋白质周转机制进行前所未有的研究。作为这些努力的逻辑延伸，我们现在建议利用我们新建立的实验性平台，以确定负责蛋白质质量控制的机构在INM。两个独立的为实现这一目标，将采取协同增效的方法：（1）根据我们既定的战略，在膜提取和蛋白酶体的不同阶段交叉LBR衍生物的降解降解，我们将分离停滞的降解中间体，以确定参与INMPT的相关因素（2）我们将进行一个无偏见的，基于全基因组CRISPR/Cas9的筛选，发现与INMPT有关的基因。为此，我们将利用表达易降解的， LBR的分裂-GFP衍生物。INMPT所需基因功能失活后的突变体稳定化将导致荧光增加，使我们能够通过FACS分析识别靶基因，然后- 世代排序对于从方法（1）和（2）中产生的候选人，我们将（3）功能上使用我们牢固建立的实验平台表征这些因素，包括（i）定位于核膜;（ii）膜移位/提取的测定和（iii）泛素化，蛋白酶体降解，从而使我们能够为每个验证的候选基因分配功能或相互作用蛋白这些研究的结果将使我们能够定义保障蛋白质稳态的网络核膜上的蛋白质完整性除了填补了我们对细胞蛋白质理解的一个主要空白之外质量控制-一个重要的生物医学相关领域-我们的发现预计将具有重要意义对我们理解和处理核灾难的影响。