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: 10654442
• 负责人: Susan D. Michaelis
• 金额: $ 7.45万
• 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/10654442
• 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

项目概要 该项目重点关注 ZMPSTE24，这是一种对人类很重要的有趣的整合膜锌金属蛋白酶 健康长寿。 ZMPSTE24 在法尼基化 CAAX 的蛋白水解过程中发挥着关键作用 蛋白质前核纤层蛋白 A，核支架成分核纤层蛋白 A 的前体。编码基因的突变 阻止裂解的 prelamin A 或 ZMPSTE24 会导致严重的早衰症 Hutchinson- 吉尔福德早衰综合症 (HGPS) 和一系列相关的早衰疾病，其中永久性异常 核纤层蛋白 A 的法尼基化形式是促进衰老相关症状的“分子罪魁祸首”。重要的是， ZMPSTE24 减少的前核纤层蛋白 A 加工也可能是正常生理衰老的一个关键因素。 我的实验室在该项目的早期率先开展了 ZMPSTE24 的研究。我们发现了这个 酵母中的蛋白酶，称为 Ste24，并表明它在酵母中具有两种不同的蛋白水解活性 交配信息素 a 因子的生物发生（CAAX 基序的裂解和第二个上游裂解）。 重要的是，我们证明哺乳动物 ZMPSTE24 在前核纤层蛋白 A 中进行相同的切割 成熟。通过这种持续的工作，加上强大的新型人源化酵母系统， 我的实验室最近开发了前核纤层蛋白 A 裂解，我们最近的工作显示了质量控制作用 对于 ZMPSTE24 从“堵塞的”易位子中去除错误折叠的蛋白质，我们触手可及的完整信息 工具库，包括各种生化和体内测定、细胞系和疾病等位基因， 促进拟议的研究。最近发表的人类ZMPSTE24结构和近 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 的七个跨膜跨度形成大量的水- 充满膜内室，锌金属蛋白酶催化位点面向室内部，以便 基材的进入受到限制，必须通过几个侧门之一进行。其中一项重大挑战是 该领域的目标是确定这种新型膜内蛋白酶的工作原理。我们建议机械地 剖析人 ZMPSTE24 及其底物 prelamin A，以精确定义 prelamin A 的蛋白水解作用 发生在膜内室内。我们将确定 ZMPSTE24 和 prelamin A 的重要特征 用于底物识别、进入、结合、催化和产物释放，并将确定其中的步骤 ZMPSTE24 和核纤层蛋白 A 疾病等位基因发生故障。我们还将尽可能部署强大的设计师屏幕 在酵母中鉴定新的 Ste24 底物，这可能有助于阐明其机制。我们将开始一段激动人心的新旅程 旨在探索第二个主要挑战的一系列研究，即是否以及如何减少 ZMPSTE24 活性和 prelamin A 积累可能会导致生理衰老。这些研究共同 将揭示与膜内蛋白酶生物学、过早衰老和正常现象相关的基本原理 生理老化。 1