MECHANISMS RESPONSIBLE FOR GENOMIC INSTABILITY IN PREMATURE AGING LAMINOPATHIES

描述（由申请人提供）：A型核纤层蛋白是中间丝蛋白，其提供用于束缚染色质和调节核结构和功能的蛋白质复合物的支架。在LMNA基因突变与广泛称为核纤层蛋白病、癌症和衰老的各种退行性疾病相关联后，对核纤层蛋白的兴趣增加。A型核纤层蛋白的改变与疾病之间的因果关系和所涉及的分子途径知之甚少。我们的数据揭示了A型核纤层蛋白在维持端粒和稳定53 BP 1（DNA损伤反应途径中的介体）中的新功能。A型核纤层蛋白的缺失导致端粒缩短、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53 BP 1。这些表型中的一些与53 BP 1缺陷一致。为了支持这一观点，将53 BP 1重组到A型核纤层蛋白缺陷细胞中挽救了DNA修复中的缺陷。阐明53 BP 1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，53 BP 1被蛋白酶组织蛋白酶L和蛋白酶体降解。有趣的是，显示出显著上调组织蛋白酶L的早衰症小鼠模型（Zmpste 24-/-）表现出低水平的53 BP 1，表明组织蛋白酶L介导的53 BP 1降解的失调可能是核纤层蛋白病中的常见事件。重要的是，我们发现用维生素D（一种已知的胱抑素D的激活剂[8]，组织蛋白酶L的内源性抑制剂）治疗可以挽救53 BP 1水平，提供了一种预防53 BP 1降解的推定策略，改善了层粘连蛋白病的表型。待检验的假设是，A型核纤层蛋白的改变引起组织蛋白酶L的上调，导致53 BP 1蛋白的不稳定以及DNA修复和端粒稳定性的缺陷，从而导致基因组不稳定和一些核纤层蛋白相关疾病的表型。我们的假设将在三个具体目标下进行检验。根据目标1，我们将阐明导致53 BP 1在核纤层蛋白病中不稳定的分子机制，并评估靶向该途径和改善DNA修复的方法。在目标2下，我们将确定层粘连蛋白病中组织蛋白酶L/53 BP 1通路的失调影响端粒稳定性的程度。在目标3下，我们将确定维生素D对组织蛋白酶L/53 BP 1通路的调节是否改善体内层粘连蛋白相关疾病的病理生理学。本研究不仅对阐明A型核纤层蛋白影响端粒功能和DNA修复的分子机制具有重要意义，而且对理解核纤层蛋白相关疾病的病理生理学基础具有重要意义，包括退行性核纤层蛋白病、早衰综合征和癌症。我们的研究结果可以转化为针对53 BP 1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。

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.