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

Parent R35 Grant "The integral membrane protease ZMPSTE24，lamin A processing，and the premature 衰老性疾病"。 项目摘要 这个项目的重点是ZMPSTE24，一个有趣的整体膜锌金属蛋白酶的重要性， 人类健康和长寿。ZMPSTE24在法尼基化蛋白的蛋白水解加工中起关键作用。 CAAX蛋白前核纤层蛋白A，核支架组分核纤层蛋白A的前体。的基因中的突变 编码前层蛋白A或ZMPSTE24，其阻断导致严重过早衰老病症的裂解 Hutchinson-Gilford早老综合征（HGPS）和一组相关的早老性疾病，其中异常的 核纤层蛋白A的永久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减少的前层蛋白A加工也可能是正常细胞增殖的关键因素。 生理老化 我的实验室在这个项目的早期率先研究了ZMPSTE24。我们发现这 蛋白酶，其中它被称为Ste24，并表明它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基序的切割和第二上游切割）。 重要的是，我们证明了哺乳动物ZMPSTE24在前层蛋白A中进行这些相同的切割。 成熟通过这一持续的工作，加上一个强大的新的人性化酵母系统， 前核层蛋白A卵裂最近在我的实验室开发，我们最近的工作显示了质量控制的作用 对于ZMPSTE24从"堵塞"的translocons中去除错误折叠的蛋白质，我们有一个完整的 工具库，包括各种生化和体内测定、细胞系和疾病等位基因， 为拟议的研究提供便利。最近发表的人ZMPSTE24的结构和近 可重叠酵母Ste24揭示了令人惊讶的特征，将ZMPSTE24/Ste24p定义为真正的新型酵母菌。 膜内蛋白酶ZMPSTE24/Ste24p的七个跨膜跨度形成了大量的水- 填充的膜内室，其中锌金属蛋白酶催化位点面向内室内部，使得 衬底的接近受到限制，并且必须通过几个侧入口中的一个进行。一个主要的挑战， 该领域的主要任务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及其底物前层蛋白A，以精确定义前层蛋白A的蛋白水解如何在 一个膜内室。我们将鉴定ZMPSTE24和前层蛋白A对底物的重要性， 识别，进入，结合，催化和产物释放，并将决定ZMPSTE24和 核纤层蛋白A疾病等位基因功能障碍。我们还将部署强大的设计师屏幕可能在酵母，以确定 新的Ste24底物，可能揭示其机制。我们将开始一系列激动人心的新研究 旨在探索第二个主要挑战，即是否以及如何减少ZMPSTE24活性， 核纤层蛋白A前体的积累可驱动生理老化。总之，这些研究将揭示基本的 与膜内蛋白酶生物学、过早衰老和正常生理衰老相关的原理。

Parent R35 Grant “The integral membrane protease ZMPSTE24, lamin A processing, and the premature aging disease progeria”. 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.