MECHANISMS RESPONSIBLE FOR GENOMIC INSTABILITY IN PREMATURE AGING LAMINOPATHIES

描述(申请人提供)：A-型层粘连蛋白是一种中间丝蛋白，它为连接染色质和调节核结构和功能的蛋白质复合体提供了支架。在LMNA基因突变与各种退行性疾病联系在一起后，人们对Lamins的兴趣增加了，这些疾病被广泛称为椎板病、癌症和衰老。A-型层蛋白的改变与疾病之间的因果关系以及所涉及的分子途径还知之甚少。我们的数据揭示了A型Lamins在维持端粒和稳定53BP1中的新功能，53BP1是DNA损伤反应途径中的中介。A-型层蛋白的丢失会导致端粒缩短、端粒染色质缺陷、DNA修复受损、基因组不稳定性增加，以及功能失调的端粒的非同源末端连接缺陷，这一过程需要53BP1。其中一些表型可能与53BP1缺乏症相一致。为了支持这一观点，将53BP1重组为A型Lamins缺陷细胞可以挽救DNA修复中的缺陷。阐明53BP1降解的分子机制可能为治疗提供新的靶点。我们的初步数据表明，53BP1被组织蛋白酶L和蛋白酶体降解。有趣的是，一个早衰症小鼠模型(Zmpste24-/-)被证明显著上调组织蛋白酶L，显示出低水平的53BP1，这表明放松对组织蛋白酶L介导的53BP1降解的调控可能是椎板病中的常见事件。重要的是，我们发现维生素D，已知的Cystatin D[8]激活剂，组织蛋白酶L的内源性抑制剂，可以挽救53BP1的水平，提供一种可能的策略来防止53BP1的降解，改善椎板病变的表型。需要检验的假设是，A型Lamins的改变导致组织蛋白酶L上调，导致53BP1蛋白的不稳定，DNA修复和端粒稳定性的缺陷，导致基因组不稳定和一些Lamin相关疾病的表型。我们的假设将在三个具体目标下进行检验。在目标1下，我们将阐明在椎板病变中导致53BP1失稳的分子机制，并评估针对这一途径和改善DNA修复的方法。在目标2下，我们将确定组织蛋白酶L/53BP1通路在椎板病变中解除调控对端粒稳定性的影响程度。在目标3中，我们将确定维生素D对组织蛋白酶L/53BP1通路的调节是否改善了体内Lamin相关疾病的病理生理学。这些研究不仅对阐明A型层粘连蛋白影响端粒功能和DNA修复的分子机制具有重要意义，而且对了解层粘连蛋白相关疾病，包括退行性椎板病、早衰综合征和癌症的病理生理学基础也具有重要意义。我们的研究结果可能转化为针对53BP1降解途径的新治疗策略的开发，以改善与这些疾病相关的表型。 与公共卫生相关：了解A型板层蛋白的细胞功能已经成为一个高度热门的主题，因为它们与一系列广泛称为椎板病的退行性疾病有关，也与癌症和衰老有关。A型椎板缺陷导致疾病的分子途径知之甚少。DNA损伤修复缺陷引起的基因组不稳定性被认为与椎板病的发病机制有关。这项建议旨在解开导致基因组不稳定性增加的A-型层蛋白丢失后改变的分子机制。此外，我们将评估针对这些分子机制的方法，以确定改善椎板病表型的治疗方法。同时，将进行原代细胞(体外)和小鼠椎板病变模型(体内)的研究。从这项研究中获得的知识将对开发治疗板层蛋白相关疾病的方法具有重要的潜在意义。

DESCRIPTION (provided by applicant): A-type lamins are intermediate filament proteins that provide a scaffold for tethering chromatin and protein complexes regulating nuclear structure and function. Interest in lamins increased after the association of mutations in the LMNA gene with a variety of degenerative disorders broadly termed laminopathies, cancer and aging. The causal relationship between alterations of A-type lamins and disease and the molecular pathways involved are poorly understood. Our data revealed novel functions for A-type lamins in the maintenance of telomeres and in the stabilization of 53BP1, a mediator in the DNA damage response pathway. Loss of A-type lamins leads to telomere shortening, telomeric chromatin defects, impaired DNA repair, increased genomic instability, and defects in the non-homologous end-joining of dysfunctional telomeres, a process that requires 53BP1. Some of these phenotypes would be consistent with 53BP1 deficiency. In support of this notion, reconstitution of 53BP1 into A-type lamins-deficient cells rescued defects in DNA repair. Elucidating molecular mechanism responsible for degradation of 53BP1 might provide novel targets for therapy. Our preliminary data indicates that 53BP1 is degraded by the protease Cathepsin L and by the proteasome. Interestingly, a mouse model of progeria (Zmpste24-/-) shown to markedly upregulate Cathepsin L, exhibits low levels of 53BP1, suggesting that deregulation of the Cathepsin L-mediated degradation of 53BP1 might be a common event in laminopathies. Importantly, we found that treatment with vitamin D, a known activator of Cystatin D [8], the endogenous inhibitor of Cathepsin L, rescues 53BP1 levels, providing a putative strategy to prevent 53BP1 degradation, ameliorating phenotypes of laminopathies. The hypothesis to be tested is that alterations in A-type lamins causing upregulation of Cathepsin L lead to destabilization of 53BP1 protein and defects in DNA repair and telomere stability, contributing to genomic instability and the phenotypes of some lamin-related diseases. Our hypothesis will be tested under three specific aims. Under Aim 1, we will elucidate the molecular mechanisms leading to destabilization of 53BP1 in laminopathies and evaluate ways to target this pathway and improve DNA repair. Under Aim 2, we will determine the extent to which deregulation of the Cathepsin L/53BP1 pathway in laminopathies affects telomere stability. Under Aim 3, we will determine whether regulation of the Cathepsin L/53BP1 pathway by vitamin D ameliorates the pathophysiology of lamin-related diseases in vivo. The studies performed here will be of significance not only for elucidating the molecular mechanisms by which A-type lamins impact on telomere function and DNA repair, but also for understanding the underlying basis of the pathophysiology of lamin-related diseases, including degenerative laminopathies, premature aging syndromes and cancer. The results of our study could translate into the development of novel therapeutic strategies that target 53BP1 degradation pathway to improve the phenotypes associated to these diseases. PUBLIC HEALTH RELEVANCE: Understanding the cellular functions of A-type lamins, structural components of the nucleus, has become a highly topical subject due to their implication in a number of degenerative disorders broadly named laminopathies, as well as in cancer and aging. The molecular pathways by which defects in A-type lamins contribute to disease are poorly understood. Genomic instability caused by defective repair of DNA damage has been proposed to contribute to the pathogenesis of laminopathies. This proposal aims to unravel molecular mechanisms that are altered upon loss of A-type lamins leading to increased genomic instability. In addition, we will evaluate ways to target these molecular mechanisms with the goal of identifying treatments that ameliorate the phenotypes of laminopathies. Both, studies with primary cells (in vitro) and with mouse models of laminopathies (in vivo) will be conducted. The knowledge obtained from this research will be of great possible significance for the development of therapeutic approaches to treat lamin-related diseases.