Lamin A biogenesis, processing and progeria

描述（由申请人提供）：本项目主要研究核膜的关键结构成分Lamin A的生物发生。令人惊讶的是，最近的研究表明，编码Lamin A的基因突变会导致一系列被称为“laminopathies”的疾病（包括心肌病、肌肉营养不良、脂肪营养不良和类早衰症），导致Lamin A生物学兴趣的重新出现。Lamin A前体，prelamin A，经历一系列的翻译后加工事件，包括：1) C端CaaX修饰（前置化、蛋白水解和羧基甲基化），随后2)由锌金属蛋白酶ZmpSte24介导的蛋白内水解裂解事件，去除CaaX修饰的C端，生成成熟的Lamin a。在本项目的前几年，我们实验室发现ZmpSte24是酵母a因子生物发生的关键酶。从这些发现开始，这个项目已经发展到研究哺乳动物细胞中纤层蛋白A的生物发生。最严重的椎板病是早老性疾病哈钦森-吉尔福德早衰综合征（HGPS）。引人注目的是，基于以下发现，ZmpSte24介导的前纤层蛋白a内蛋白水解加工缺陷与类早老性疾病之间似乎存在直接联系：1)HGPS是由纤层蛋白a内ZmpSte24切割位点缺失的突变引起的；2)类早老性疾病下颌骨端发育不良（MAD）和限制性皮病（RD）与ZmpSte24相关。因此，似乎存在于HGPS或zmpste24-/-细胞中的持续戊酰化形式的Lamin A导致加速衰老病理。在本提案中，我们将定义Lamin A加工的关键细胞生物学方面，并解决加工缺乏在疾病机制中发挥的作用。我们将使用分子、细胞生物学、遗传学和生化方法来解决以下目标：目标1)确定层粘胶蛋白A加工的细胞位置（核质面vs.内质面）；目的2)确定剪断的C端尾的命运；目的3)确定对ZmpSte24裂解前纤层蛋白A重要的识别序列，并确定ZmpSte24如何使用纯化酶裂解前纤层蛋白A；目的4)研究未切割前纤层蛋白A尾部导致细胞和疾病表型的分子机制。特别是，我们将测试甲基化是否可能对HGPS中Lamin A的毒性起作用。这一有趣的发现，即类早衰疾病是由prelamin A加工不完全引起的，这强调了全面了解整个加工途径的重要性，我们在本提案中提出了这一点。我们的研究将为类早衰疾病的治疗选择提供见解。最近的研究发现，抑制zmpste24介导的prelamin A加工可能有助于HIV治疗诱导的副作用，并可能与正常衰老的机制有关，这一研究的意义得到了加强。

DESCRIPTION (provided by applicant): This project focuses on the biogenesis of Lamin A, a critical structural component of the nuclear envelope. Surprisingly, recent studies have revealed that mutations in the gene encoding Lamin A result in a wide range of diseases called the "laminopathies" (encompassing cardiomyopathy, muscular dystrophy, lipodystrophy, and progeroid disorders), leading to a resurgence of interest in the biology of Lamin A. The Lamin A precursor, prelamin A, undergoes a series of post-translational processing events, including: 1) C- terminal CaaX modification (prenylation, proteolysis, and carboxyl methylation), followed by 2) an endoproteolytic cleavage event, mediated by the zinc metalloprotease ZmpSte24, that removes the CaaX- modified C-terminus to yield mature Lamin A. In previous years of this project, our laboratory discovered ZmpSte24 as a key enzyme in the biogenesis of yeast a-factor. From these findings this project has evolved to study the biogenesis of Lamin A in mammalian cells. The most severe laminopathy is the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). Strikingly, there appears to be a direct link between defective Zmpste24-mediated endoproteolytic processing of prelamin A and progeroid diseases based on the findings that: 1) HGPS results from a mutation in which the ZmpSte24 cleavage site within Lamin A is deleted, and 2) the progeroid disorders mandibuloacral dysplasia (MAD) and restrictive dermopathy (RD) map to ZmpSte24. Thus, it appears that the persistently prenylated form of Lamin A that is present in HGPS or zmpste24-/- cells leads to accelerated aging pathologies. In this proposal we will define key cell biological aspects of Lamin A processing and address the role that a lack of processing plays in disease mechanisms. We will use molecular, cell biological, genetic, and biochemical approaches to address the following aims: Aim 1) determine the cellular location of Lamin A processing (nucleoplasmic vs. the cytosolic face of the ER); Aim 2) determine the fate of the cleaved C- terminal tail; Aim 3) determine the recognition sequences within prelamin A important for ZmpSte24 cleavage, and define how Zmpste24 cleaves prelamin A using purified enzyme; and Aim 4) investigate the molecular mechanisms by which failure to cleave the prelamin A tail leads to cellular and disease phenotypes. In particular we will test whether methylation may contribute to the toxicity of Lamin A in HGPS. The intriguing finding that progeroid diseases are caused by incomplete processing of prelamin A has underscored the importance of a comprehensive understanding of the entire processing pathway, which we address in this proposal. Our studies will provide insight into therapeutic options for progeroid disorders. The significance of this research is heightened by recent findings that inhibition of ZmpSte24-mediated processing of prelamin A may contribute to HIV therapy-induced side effects, and possibly to the mechanisms of normal aging. PUBLIC HEALTH RELEVANCE: Mutations in the nuclear structural protein Lamin A cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and a spectrum of diseases known as "laminopathies". This project addresses fundamental unanswered questions about Lamin A biology, including how it is processed within the cell and how abnormal processing can cause disease, as in HGPS. Our studies will provide insight into potential therapeutic options for premature aging disorders, and may also shed light on the mechanisms underlying the normal aging process.