Nuclear envelope dynamics in Hutchinson-Gilford progeria syndrome

哈钦森-吉尔福德早衰症(HGPS，OMIM 176670)是一种罕见的过早衰老障碍，由于心肌梗死或中风导致的平均年龄为14岁。LMNA基因的突变是导致HGPS的原因，HGPS属于椎板病家族。与HGPS相关的最常见的突变是LMNA基因第11外显子内G608G(GGC&GT；GGT)位置的新生杂合单碱基对替换。这种突变导致前层蛋白A羧基末端50个氨基酸的缺失；被截断的蛋白质称为孕激素。板层蛋白有助于维持核膜的形状和稳定性，并参与DNA复制和转录的调节。孕激素只经历一些正常的翻译后修饰，并永久保持法尼化。孕激素的毒性部分归因于其法尼基。在这一应用的第一轮中，我们证明了孕激素在有丝分裂过程中引起了时空偏差，特别是依赖于与核膜和核孔分布的适当结合的过程。我们为孕激素引起的细胞、基因组和代谢缺陷的增加提供了重要和关键的数据。以前关于孕激素的研究已经评估了它在间期的定位和活性，我们的研究跟踪了孕激素在整个有丝分裂过程中不可避免的参与，这一现象到目前为止被显著地忽视了。我们的方法揭示了几种蛋白质(核纤层蛋白、Emerin和SUN1)有丝分裂重新分布的显著和特定的延迟，包括在有丝分裂的最后阶段SUN1在内质网中的捕获和在HGPS细胞中的NE重组中核孔的延迟招募。这些变化伴随着染色质滞后的显著增加，这导致了双核细胞和基因组的不稳定。基于这些观察，我们推测SUN1在核膜上的关键位置及其与孕激素的异常相互作用直接与HGPS细胞核膜系统异常的产生有关。这会导致HGPS细胞的核形状、鼻咽癌定位、细胞动态平衡和功能的改变，从而导致HGPS细胞发生凋亡和过早衰老。为了验证这一假说，我们建议研究以下具体目标：(1)表征孕激素在细胞内的运输和核膜结合；(2)表征孕激素-SUN1相互作用及其对核孔分布和核完整性的必然影响。

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare, premature aging disorder that leads to death at an average age of 14 years, as a result of myocardial infarction or stroke. Mutations in the LMNA gene are responsible for HGPS, which belongs to the family of laminopathies. The most common mutation associated with HGPS is a de novo heterozygous single base pair substitution at position G608G (GGC>GGT) within exon 11 of the LMNA gene. This mutation results in the deletion of 50 amino acids at the carboxyl-terminal tail of prelamin A; the truncated protein is referred to as progerin. Lamins help maintain the shape and stability of the nuclear envelope and are involved in the regulation of DNA replication and transcription. Progerin undergoes only some of the normal posttranslational modifications and remains permanently farnesylated. The toxicity of progerin has been attributed, in part, to its farnesyl moiety.During the first round of this application, we demonstrated that progerin elicits spatiotemporal deviations in mitotic processes, particularly the processes that are dependent on proper association with the nuclear envelope and nuclear pore distribution. We contributed important and crucial data to the increasing list of cellular, genomic and metabolic defects caused by progerin. Previous studies on progerin have assessed its localization and activity during interphase, our study followed progerin throughout its inevitable participation in mitosis, a phenomenon that has been remarkably ignored to date. Our approach unraveled significant and specific delays in the mitotic redistribution of several proteins (nuclear lamins, emerin, and SUN1), including the trapping of SUN1 in the ER at the last stages of mitosis and delayed recruitment of nuclear pores in the re-assembling NE in HGPS cells. These changes were accompanied by a substantial increase in chromatin lagging, which caused binucleated cells and genomic instability.Based on these observations, we hypothesized that the critical positioning of SUN1 at the nuclear envelope and its aberrant interaction with progerin are directly implicated in the generation of an abnormal nuclear membrane system in HGPS cells. This consequently leads to alterations in the nuclear shape, NPC positioning, cellular homeostasis and function that drive HGPS cells to undergo apoptosis and premature senescence. To test this hypothesis, we propose to investigate the following Specific Aims: (1) characterize progerin intracellular trafficking and nuclear envelope association; and (2) characterize progerin-SUN1 interaction and its inevitable consequences on nuclear pore distribution and nuclear integrity.