Lamin A biogenesis, processing and progeria

描述(由申请者提供)：本项目重点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，Lamin A编码基因的突变会导致一系列称为“板层病”的疾病(包括心肌病、肌营养不良症、脂肪营养不良症和节段性疾病)，从而重新引起人们对Lamin A生物学的兴趣。Lamin A的前体，前Lam in A，经历了一系列翻译后加工事件，包括：1)C端的CAAX修饰(预烯化、蛋白分解和羧甲基化)，然后是2)由锌金属蛋白酶ZmpSte24介导的内切蛋白切割事件，该事件使CAAX修饰的C末端产生成熟的Lamin A。本实验室发现ZmpSte24是酵母a因子生物发生过程中的关键酶。根据这些发现，这个项目已经演变成研究哺乳动物细胞中Lamin A的生物发生。最严重的椎板病变是早衰障碍Hutchinson-Gilford Progeria综合征(HGPS)。值得注意的是，有缺陷的Zmpste24介导的前层蛋白A的内切蛋白处理与孕激素类疾病之间似乎有直接的联系，基于以下发现：1)HGPS是由Lamin A内的ZmpSte24裂解位点缺失的突变所致，以及2)孕激素紊乱症下颌颈发育不良(MAD)和限制性皮肤病(RD)映射到ZmpSte24。因此，在HGPS或zmpste24-/-细胞中存在的持续的前烯基化形式的Lamin A似乎会导致加速衰老的病理。在这项提案中，我们将定义Lamin A加工的关键细胞生物学方面，并解决缺乏加工在疾病机制中所起的作用。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：目的1)确定Lamin A加工的细胞位置(核质与内质网的胞浆面)；目的2)确定被切割的C-末端尾巴的命运；目的3)确定对ZmpSte24切割重要的Prelamin A内的识别序列，并确定Zmpste24如何使用纯化的酶来切割Prelamin A；以及目的4)研究未能切割Prelamin 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导致HGPS中Lamin A的毒性。耐人寻味的发现是由前层蛋白A的不完全处理引起的，这一发现强调了全面了解整个处理途径的重要性，我们在本提案中解决了这一问题。我们的研究将为孕激素紊乱的治疗选择提供洞察力。最近的研究发现，抑制ZmpSte24介导的Prelamin A的处理可能有助于HIV治疗引起的副作用，并可能与正常衰老的机制有关，这一发现加强了这项研究的意义。 与公共卫生相关：核结构蛋白Lamin A的突变会导致过早衰老障碍Hutchinson-Gilford Progeria综合征(HGPS)和一系列被称为“椎板病”的疾病。这个项目解决了有关Lamin A生物学的基本悬而未决的问题，包括它是如何在细胞内处理的，以及异常处理如何导致疾病，例如在HGPS中。我们的研究将为早衰障碍的潜在治疗选择提供洞察力，并可能揭示正常衰老过程的潜在机制。

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.