突变前核纤层蛋白A对细胞衰老的作用

早老症(Hutchinson-Gilford progeria syndrome,HGPS)是一种极其罕见的遗传性疾病,它是由LMNA基因突变引起的,产生一个截短的lamin A 蛋白称为 progerin.核纤层蛋白异常A加工积累的 progerin 能够破坏核纤层的支架功能,替代正常蛋白质与其配体结合,导致细胞核畸形和早老表型.     

核层蛋白A前体的法尼基化与衰老

编码核层蛋白A(lamin A)的LMNA基因突变导致法尼基化的核层蛋白A前体(prelamin A)不能被进一步加工成成熟的核层蛋白A,从而导致一种Hutchinson-Gilford早老症综合征(Hutchinson-Gilford progeria syndrome,HGPS)。一种更严重的早老症——限制性皮肤病(restrictive dermopathy,RD),是由于缺失核层蛋白A前体加工过程中的剪切酶ZMPSTE24引起的。ZMPSTE24的缺失阻止了法尼基化的核层蛋白A前体不能正常加工成为成熟的核层蛋白A,同时导致法尼基化的核层蛋白A前体的堆积。在HGPS和RD病人的成纤维细胞中,发现法尼基化的核层蛋白A前体都定位在核膜,从而影响细胞核膜的完整性,并导致细胞核形的异常,进而导致衰老。最近研究表明经过法尼基酰转移酶抑制剂(farnesyltransferase inhibitor,FTI)处理后的细胞的核形异常减少。同时,FTI能够改善HGPS和RD小鼠的早老症状。本文就核层蛋白A前体的法尼基化对衰老的影响有关研究进展作一综述。     

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

LMNA基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss肌营养不良症(Emery-Dreifussmusculardystrophy,EDMD)、扩张型心肌病(dilatedcardiomyopathy,DCM)和儿童早老症(Hutchinson-Glifordprogeriasyndrome,HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNAKO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。     

A型核纤层蛋白的研究进展

A型核纤层蛋白由LMNA基因编码,为核纤层的主要成分,呈动态网状结构,位于核膜下层,起重要的机械支持作用,直接或间接与染色质相互作用,在维持染色质结构、转录、DNA复制和细胞凋亡等方面发挥重要作用.LMNA基因及其编码蛋白lamin A/C异常能引起一组人类遗传病,称为核纤层蛋白病.为深入了解A型核纤层蛋白的正常生理功能及其在相关核纤层蛋白病中的作用,本文就A型核纤层蛋白的结构分类、修饰组装、动力学、相互作用蛋白及相关核纤层蛋白病等方面进行综述.     

核纤层蛋白与核纤层病的研究进展

核纤层蛋白(lamin)是中间纤维蛋白家族的重要成员,其多聚体组成的网格状结构紧贴于核膜内侧,在维持细胞核的正常及有丝分裂过程中发挥着重要的作用。近年来,大量研究表明编码核纤层蛋白的基因尤其是lamin A编码基因(LMNA)突变会引起一系列的疾病,即核纤层病(lami-nopathy)。该文就核纤层蛋白和核纤层病的关系进行综述,有助于读者了解核纤层蛋白的重要性,也为核纤层病的治疗提供线索。     

体外细胞核组装中核纤层与核膜的关系

采用非洲爪蟾卵提取物非细胞体系,以外源Lambda DNA诱导细胞核的体外组装,以此实验模式为基础,研究了细胞核体外组装过程中核纤层的组装,结果表明核纤层蛋白参与细胞核的体外组装过程,核内骨架的组装与核纤层的组装在时间上是有序的,核内骨架的组装可能为核纤层的装配提供了先决条件.在非洲爪蟾卵提取物非细胞体系中加入抗核纤层蛋白抗体,抑制核纤层的正常装配过程,核膜组装发生异常.结果提示核纤层的组装与核膜的组装是密切相关的.     

高等哺乳动物LEM结构域蛋白家族的研究进展

在高等动物细胞开放式有丝分裂过程中,细胞核膜会发生高度有序的周期性去组装和装配的动态变化。近年的研究结果表明是LEM家族蛋白成员通过与BAF因子相互作用介导了内核膜、核纤层蛋白以及染色体之间的相互作用。LEM蛋白、核纤层蛋白以及BAF因子直接相互作用形成的三元复合体在结构与功能上是相互依赖的,在此结构与功能上组成的网络体系是形成细胞核的一些基本生物学过程的重要条件。该复合体在调控有丝分裂M期后期和末期染色体的正常分离、有丝分裂后核膜的重组装,细胞分裂间期细胞核及核膜形态维持,调控DNA复制和DNA损伤修复,调节基因表达和信号通路以及逆转录病毒感染等方面发挥着重要的生物学功能。并且LEM蛋白相关基因的异常对核纤层疾病和肿瘤的发生发展具有重要的影响。文章主要针对LEM蛋白家族成员的结构以及功能研究进展进行了详细的综述。     

早老素导致基因组不稳定的机制

基因组不稳定（genomic instability）是机体衰老的标志之一,也是儿童早老症（Hutchinson Gilford progeria syndrome, HGPS)患者细胞的典型特征。HGPS的发生与早老素（progerin)堆积密切相关,但早老素如何引起基因组不稳定尚缺乏系统性的阐述。基因组的结构稳定与DNA的正确复制、DNA损伤修复、端粒的维持和稳定以及表观遗传学修饰密切相关。本文主要讨论早老素在改变正常核纤层结构的基础上,通过影响相关通路关键蛋白质的水平或者定位,引起细胞内氧化应激增强、DNA复制应激和DNA损伤修复障碍,细胞DNA损伤增多和端粒的加速缩短,并在改变组蛋白甲基化和乙酰化方面导致基因组不稳定的机制。     

植物细胞核纤层的研究

应用细胞成分选择性抽提方法,结合非树脂包埋去包埋电镜技术显示悬浮培养的胡萝卜细胞和银杏雄性生殖细胞均具有核纤层结构,免疫印迹反应证明这两种细胞的核纤层由A型和B型核纤层蛋白组成;至少分别含有66ku,84ku和66ku,86ku多肽,免疫胶体金标记将这些蛋白定位在核周缘,光镜和电镜原位分子杂交显示植物细胞具有与动物细胞核纤层蛋白cDNA同源的序列存在;其mRNA分选的部位主要分布在靠近核膜周围的胞质部分,实验结果证明植物细胞确实存在核纤层.     

核纤层蛋白的研究进展

核纤层普遍存在于高等真核细胞的细胞核中,向外与内层核膜上的蛋白结合,向内与染色质的特定区段结合,其主要成分是核纤层蛋白。核纤层蛋白主要参与细胞核的形状和大小的维持、核膜的组织、DNA的复制及有丝分裂。近年来的研究表明,核纤层蛋白与许多人类疾病密切相关。目前,核纤层蛋白在人类的各种组织和细胞中已有比较系统的研究,并且呈组织特异性及发育时序性表达。本文将就核纤层的最新研究进展做一综述。     

Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.     

Aging and nuclear organization: lamins and progeria

The discoveries of at least eight human diseases arising from mutations in LMNA, which encodes the nuclear A-type lamins, have revealed the nuclear envelope as an organelle associated with a variety of fundamental cellular processes. The most recently discovered diseases associated with LMNA mutations are the premature aging disorders Hutchinson-Gilford progeria syndrome (HGPS) and atypical Werner's syndrome. The phenotypes of both HGPS patients and a mouse model of progeria suggest diverse compromised tissue functions leading to defects reminiscent of aging. Aspects of the diseases associated with disrupted nuclear envelope/lamin functions may be explained by decreased cellular proliferation, loss of tissue repair capability and a decline in the ability to maintain a differentiated state.     

Lamin A, farnesylation and aging

Lamin A is a component of the nuclear envelope that is synthesized as a precursor prelamin A molecule and then processed into mature lamin A through sequential steps of posttranslational modifications and proteolytic cleavages. Remarkably, over 400 distinct point mutations have been so far identified throughout the LMNA gene, which result in the development of at least ten distinct human disorders, collectively known as laminopathies, among which is the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). The majority of HGPS cases are associated with a single point mutation in the LMNA gene that causes the production of a permanently farnesylated mutant lamin A protein termed progerin. The mechanism by which progerin leads to premature aging and the classical HGPS disease phenotype as well as the relationship between this disorder and the onset of analogous symptoms during the lifespan of a normal individual are not well understood. Yet, recent studies have provided critical insights on the cellular processes that are affected by accumulation of progerin and have suggested that cellular alterations in the lamin A processing pathway leading to the accumulation of farnesylated prelamin A intermediates may play a role in the aging process in the general population. In this review we provide a short background on lamin A and its maturation pathway and discuss the current knowledge of how progerin or alterations in the prelamin A processing pathway are thought to influence cell function and contribute to human aging.     

Zmpste24基因缺失与早老症

衰老是一种生理完整性丧失,功能受损,疾病和死亡风险增加的过程。早老症（HGPS）是一种加速化的衰老疾病,是研究人类正常衰老理想的疾病模型。由LMNA基因突变产生prelamin AΔ50在细胞内累积是造成早老症的主要原因,早老症病人表现出寿命急剧缩短,老化特征明显的现象,例如脱发、皮下脂肪减少、骨质疏松以及早逝。 锌金属蛋白酶Zmpste24 是prelamin A加工成为成熟lamin A蛋白的关键酶。敲除Zmpste24基因的小鼠表现出与早老症高度一致的衰老表型,同时也存在非常相似的发病机制,如染色质异常、DNA损伤和干细胞功能缺失等。Zmpste24缺失小鼠作为典型的早老模型小鼠因其衰老周期短,衰老特征明显而获得广泛应用。本文总结了以Zmpste24缺失早老小鼠为模型取得的早老相关分子机制的研究进展,以及抗衰老策略的最新发现。     

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.     

Perturbation of wild-type lamin A metabolism results in a progeroid phenotype

Mutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of disease-linked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.     

The role of lamins and mutations of LMNA gene in physiological and premature aging

Lamins belong to type V intermediate filaments superfamily. They are the main structural constituencies of the nuclear lamina but they also influence on chromatin structure, regulation of gene expression, localization and probably protein degradation. Because lamins play many different roles within the cell, mutations in their genes can results in variety of pathological phenotypes. Mutations in LMNA gene are the cause of many different diseases, called laminopathies. Among laminopathies are muscle tissue diseases, adipose tissue diseases and also progerias, the premature aging syndromes. One of the progerias, which results from mutation in LMNA gene, is Hutchinson-Gilford progeria syndrome (HGPS). It seems that the same molecular mechanisms which are responsible for premature aging of cells of HGPS patients, are involved in physiological aging.     

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNAmutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.