FHA结构域调控蛋白GarA通过改变氨基酸组分调控基因组在UV和氧化胁迫下的稳定性

发布时间：2018-04-18 13:16

本文选题：FHA结构域 + 基因组稳定性　；参考：《西南大学》2014年硕士论文

【摘要】：结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的结核病严重威胁着人类健康,每年有上百万的人感染结核病,已经成为死亡率仅次于艾滋病的全球第二大传染病。在世界卫生组织WHO已经发布了最新的《2013年全球结核病报告》中指出：2012年新增结核病860万例,其中死亡人数到达了130万。其主要由于多重耐药菌和广泛耐药菌的出现,以及HIV的共感染,使得结核病形势更加严峻。因此,对结核病的致病菌——结核分枝杆菌的深入研究迫在眉睫。在各种环境胁迫下维持基因组的稳定性是生物生存和繁殖的关键。结核菌是人类重大传染疾病结核病的致病菌[1]。在分枝杆菌长期的进化过程中,又会遭遇各种环境压力的选择作用,比如：紫外线照射[2]。结核分枝杆菌为了在巨噬细胞和肉芽肿中存活,必须适应有氧化压力[3]。紫外线会使生物体DNA上两个相连的胸腺嘧啶形成胸腺嘧啶二聚体,使DNA链发生断裂,使复制酶无法工作,从而对生物体造成损伤[4]。氧化压力是生物在有氧环境生存必须面对的一种压力条件,通过影响Ca2+浓度引起DNA的损伤[5]。但是,在这么多不利条件下,结核分枝杆菌的基因组一直保持非常稳定[6],那么维持基因组的稳定性在分枝杆菌适应外界压力环境和侵染宿主细胞的过程中就显得非常重要。有关研究表明：分枝杆菌进化出了一系列的修复和耐受机制来抵抗UV给DNA带来的损伤。分析结核分枝杆菌基因组表明：其基因组含有DNA修复系统,比如：DNA损伤修复、核算切除修复(NER)、基础切除修复(BER)、重组修复、非同源末端连接、SOS修复等[7,8]。为了抵抗巨噬细胞和肉芽肿,结核分枝杆菌也具有抵抗活性氧调节的机制。在革兰氏阴性菌中,OxyR蛋白是抵抗氧化压力重要的组成成分[9]. OxyR是活性氧簇感应器,同时也是一个转录因子,包括去毒酶(过氧化氢酶和氢过氧化物酶)和alkyl氢过氧化物酶。虽然这几十年来对结核分枝杆菌的研究不断深入,维持基因组稳定性仍然有很多地方还有待研究。 FHA的全称为forkhead-associated domain,是叉头相关结构域,是许多调控蛋白的磷酸肽识别结构域。包含有FHA结构域的蛋白涉及许多细菌生长代谢的过程,包括细胞形态的调节、三型分泌系统、孢子形成、碳水化合物的储存、转运、信号转导和乙胺丁醇耐受以及致病菌与宿主之间的相互关系。在真核生物中,许多包含有FHA结构域的蛋白分布在细胞核,参与细胞周期检验点的形成和维持,DNA修复和转录调控。本文研究的GarA蛋白(MSMEG_3647)是典型的含有磷酸化识别FHA结构域的调控蛋白。有研究表明,未磷酸化GarA蛋白能与α-酮戊二酸脱羧酶(KGD)、依赖NAD的谷氨酸脱氢酶(GDH)、谷氨酸合酶的活性(GS)相结合并对其的活性产生影响[10]。在结核分枝杆菌中,GarA蛋白作为一个典型的FHA结构域调控蛋白怎么可能只参与代谢功能的调控昵?本文利用Himarl转座子插入突变库,成功筛选了本文筛选到了一株GarA (MSMEG_3647)蛋白突变的耻垢分枝杆菌M. smegmatis A GarA,其菌落形态与M. smegmatis mc2155相比明显变得光滑,单菌落形态减小、疏水性系数明显增高,生物膜形态也发生变化。这表明：GarA (MSMEG_3647)蛋白突变会引起细胞壁脂质成分的变化。我们的实验还证明了GarA (MSMEG_3647)蛋白突变的耻垢分枝杆菌M. smegmatis A GarA对紫外线照射耐受及对H202敏感,其可能是与基因组稳定性相关。我们还发现了菌体内游离氨基酸代谢及组分也发生了改变。本研究发现了一种UV和氧化压力应激的新型分子机制：GarA蛋白缺失会引起菌体内氨基酸组分含量变化,从而导致菌体对紫外线及氧化压力的耐受情况发生变化。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) caused by tuberculosis, a serious threat to human health, there are millions of people infected with TB each year, has become the world's second largest infectious disease mortality after AIDS. The WHO WHO has released the latest <2013 pointed out that the global TB report >: 2012 new 8 million 600 thousand cases of tuberculosis, including the death toll reached 1 million 300 thousand. It is mainly due to emergence of multiple drug resistant and extensively drug-resistant bacteria, and co infection of HIV and tuberculosis makes the situation even more severe. Therefore, further study of pathogenic bacteria of Mycobacterium tuberculosis tuberculosis disease is imminent.
Under various environmental stresses to maintain genome stability is the key to biological survival and reproduction. Mycobacterium tuberculosis is a major human infectious diseases, tuberculosis pathogenic bacteria [1]. in the evolutionary process of mycobacteria in the long history of the choice of the encounter various environmental stresses such as: ultraviolet irradiation [2]. of Mycobacterium tuberculosis in macrophages and granuloma in order to survive, must adapt to the oxidative stress [3]. ultraviolet rays will make organisms DNA two connected two thymine formation of thymine dimers, break the DNA strands, the replicase cannot work, thus opposite objects cause oxidative [4]. damage stress is in a condition of biological pressure oxygen environment must face, through the influence of the concentration of Ca2+ caused by injury [5]. DNA but, in so many adverse conditions, the Mycobacterium tuberculosis genome has remained very stable [6], then maintain gene The stability of the group is very important in the process of Mycobacterium adaptation to the external stress environment and the infection of the host cells.
The study showed that: Mycobacterium evolved a series of repair and tolerance mechanisms to resist the damage caused by the DNA to UV. The analysis showed that the genome of Mycobacterium tuberculosis genome contains DNA repair system, such as: DNA repair, accounting for excision repair (NER), base excision repair (BER), recombination repair, non homologous end joining, SOS repair and [7,8]. to resist macrophages and Mycobacterium tuberculosis granuloma, also has a mechanism against ROS regulation. In gram negative bacteria. The OxyR protein is an important component of resistance to oxidative stress [9]. OxyR is ROS sensors, as well as a transcription factor, including detoxification enzymes (hydrogen peroxide the enzyme and hydrogen peroxidase) and alkyl hydrogen peroxidase. Although these decades of research on Mycobacterium tuberculosis continues, the maintenance of genomic stability there are still many places have yet to be Research.
The full name of FHA is forkhead-associated domain, is related to structure of forkhead domain, is to identify the structure of phosphopeptide domain. Many proteins contain FHA domain proteins involved in the metabolism of bacteria growth in many processes, including the regulation of cell morphology, type three secretion system, spore formation, carbohydrate storage, transport, the relationship between signal transduction and ethambutol resistance and pathogens and the hosts. In eukaryotes, many contain FHA domain protein located in the nucleus, involved in the formation and maintenance of cell cycle checkpoints, DNA repair and transcriptional regulation.
This paper studies the GarA protein (MSMEG_3647) is a typical regulatory protein containing phosphorylated FHA recognition domain. Studies have shown that the non phosphorylated GarA protein with alpha ketoglutarate decarboxylase (KGD), NAD dependent glutamate dehydrogenase (GDH), the activity of glutamate synthase (GS) combined to produce effect of [10]. in Mycobacterium tuberculosis on the activity regulation of Nick how could GarA protein as a typical FHA domain proteins participate only in metabolic function? The Himarl transposon insertion mutant library, this paper successfully screened screened an GarA protein (MSMEG_3647) mutations in Mycobacterium smegmatis M. smegmatis A GarA, M. smegmatis mc2155 and its colony morphology obviously becomes smooth, reduce the single colony morphology, hydrophobicity coefficient increased obviously, biofilm morphology is changed. This indicated that GarA (MSMEG_3647) protein mutation will lead Changes in cell wall lipid composition. The results also show that GarA (MSMEG_3647) protein mutation in Mycobacterium smegmatis M. smegmatis A GarA on the ultraviolet radiation tolerant and sensitive to H202, which may be related to genomic stability. We also found the free amino acid metabolism in bacteria and its components have changed this study found. A new molecular mechanism of UV and oxidative stress: loss of GarA protein can cause bacteria in amino acid composition content change, which leads to the tolerance of bacterial UV and oxidative stress change.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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