The integral membrane protease ZMPSTE24, lamin A processing, and the premature aging disease progeria

整合膜蛋白酶ZMPSTE24、核纤层蛋白A加工与早衰症早衰症

• 批准号: 10207666
• 负责人: Susan D. Michaelis
• 金额: $ 45.69万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207666
• 关键词: Age; Aging; Alleles; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biology; Catalysis; Catalytic Domain; Cell Line; Cells; Child; Disease; Enzymes; Genes; Health; Human; Laboratories; Lamin Type A; Light; Longevity; Membrane; Metalloproteases; Molecular; Mutation; Nuclear Matrix; Partner in relationship; Peptide Hydrolases; Pheromone; Physiological; Play; Population; Premature aging syndrome; Prevention; Process; Progeria; Proteins; Proteolysis; Proteolytic Processing; Publishing; Quality Control; Research Project Grants; Research Subjects; Role; Series; Side; Structure; Symptoms; Syndrome; System; Water; Work; Yeasts; Zinc; healthy aging; in vivo; insight; misfolded protein; normal aging; novel; prelamin A; tool

Project Summary This project focuses on ZMPSTE24, an intriguing integral membrane zinc metalloprotease important for human health and longevity. ZMPSTE24 plays a critical role in the proteolytic processing of the farnesylated CAAX protein prelamin A, precursor of the nuclear scaffold component lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that block cleavage cause the severe premature aging disorder Hutchinson- Gilford Progeria syndrome (HGPS) and a set of related progeroid diseases in which an aberrant permanently farnesylated form of lamin A is the “molecular culprit” that promotes aging-related symptoms. Importantly, diminished prelamin A processing by ZMPSTE24 may also be a critical factor in normal physiological aging. My laboratory pioneered the study of ZMPSTE24 in the early years of this project. We discovered this protease in yeast, where it is called Ste24, and showed that it has two distinct proteolytic activities in the biogenesis of the mating pheromone a-factor (cleavage of the CAAX motif and a second upstream cleavage). Importantly, we demonstrated that mammalian ZMPSTE24 performs these same cleavages in prelamin A maturation. Through this sustained body of work, together with a powerful new humanized yeast system for prelamin A cleavage recently developed in my laboratory, and our recent work showing a quality control role for ZMPSTE24 in removing misfolded proteins from “clogged” translocons, we have at our fingertips a full arsenal of tools, including a variety of biochemical and in vivo assays, cell lines, and disease alleles that will facilitate the proposed studies. The recently published structure of human ZMPSTE24 and the nearly superimposable yeast Ste24 revealed surprising features, defining ZMPSTE24/Ste24p as truly novel class of intramembrane proteases. The seven transmembrane spans of ZMPSTE24/Ste24p form a voluminous water- filled intramembrane chamber with the zinc metalloprotease catalytic site facing the chamber interior, so that substrate access is restricted and must occur through one of several side portals. One major challenge in the field is to establish how this novel intramembrane protease works. We propose to mechanistically dissect human ZMPSTE24 and its substrate prelamin A to define precisely how proteolysis of prelamin A occurs inside of an intramembrane chamber. We will identify features of ZMPSTE24 and prelamin A important for substrate recognition, entry, binding, catalysis, and product release, and will determine the step at which ZMPSTE24 and lamin A disease alleles malfunction. We will also deploy powerful designer screens possible in yeast to identify new Ste24 substrates that may shed light on its mechanism. We will begin an exciting new series of studies aimed at exploring a second major challenge, namely whether and how diminished ZMPSTE24 activity and prelamin A accumulation may drive physiological aging. Together, these studies will reveal fundamental principles relevant to intramembrane protease biology, premature aging, and normal physiological aging. 1

项目摘要 本项目以ZMPSTE24为研究对象，ZMPSTE24是一种对人类具有重要意义的完整膜锌金属蛋白酶 健康长寿。ZMPSTE24在法尼化CAAX蛋白降解过程中起关键作用 蛋白质前层蛋白A，核支架成分层蛋白A的前体。编码基因突变 阻止卵裂的Prelamin A或ZMPSTE24会导致严重的早衰障碍Hutchinson- Gilford Progeria综合征(HGPS)和一组相关的早衰性疾病，在这些疾病中，永久的异常 法尼化形式的层蛋白A是促进衰老相关症状的“分子罪魁祸首”。重要的是 ZMPSTE24对Prelamin A的处理减少也可能是正常生理衰老的一个关键因素。 在这个项目的早期，我的实验室开创了ZMPSTE24研究的先河。我们发现了这个 酵母菌中的蛋白水解酶，它被称为Ste24，并表明它在 交配信息素a因子的生物发生(CAAX基序的切割和第二次上游切割)。 重要的是，我们证明了哺乳动物ZMPSTE24在前层蛋白A中执行同样的切割 成熟。通过这一持续的工作实体，以及强大的新的人性化酵母系统 Prelamin A是我的实验室最近开发的一种裂解，我们最近的工作表明它具有质量控制作用 对于ZMPSTE24来说，在从“堵塞的”转座子中去除错误折叠的蛋白质方面，我们指尖上有一个完整的 武器库，包括各种生化和体内分析，细胞系和疾病等位基因，将 促进拟议的研究。最新发表的人类ZMPSTE24的结构和近 可叠加酵母Ste24揭示了令人惊讶的特征，将ZMPSTE24/Ste24p定义为真正新颖的一类 膜内蛋白酶。ZMPSTE24/Ste24P的七个跨膜形成了一个巨大的水- 在膜内室中填充面向室内部的锌金属蛋白酶催化部位，从而 衬底接触受到限制，必须通过几个侧门之一进行。面临的一个主要挑战是 FIELD是为了确定这种新型的膜内蛋白酶是如何工作的。我们建议机械地 解剖人ZMPSTE24及其底物Prelamin A以精确确定Prelamin A的蛋白分解 发生在膜内小室内。我们将确定ZMPSTE24和Prelamin A的重要功能 用于底物识别、进入、结合、催化和产物释放，并将决定 ZMPSTE24和lamin A病等位基因功能不全。我们还将尽可能部署功能强大的设计器屏幕 在酵母中寻找新的Ste24底物，可能有助于揭示其机制。我们将开始一个激动人心的新时代 一系列研究旨在探讨第二个重大挑战，即是否减少以及如何减少 ZMPSTE24活性和Prelamin A的积累可能导致生理性衰老。总而言之，这些研究 将揭示与膜内蛋白酶生物学、早衰和正常相关的基本原理 生理性衰老。 1