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Kinetics, Regulation, And Mechanisms Of Biochemical Reactions

生化反应的动力学、调控和机制

基本信息

批准号：
7734932
负责人：
P. BOON Chock
金额：
$ 135.61万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734932
关键词：
1,2-diacylglycerol 3-methyladenine Acute Promyelocytic Leukemia Aging Alzheimer&apos s Disease Amino Acids Amyloid beta-Protein Amyloid beta-Protein Precursor Anions Apoptosis Attenuated B-Lymphocytes Behavior Binding Biochemical Reaction Biological Biological Assay Biological Process Bos taurus Catalysis Cattle Cell Aging Cell Line Cell Nucleus Cell membrane Cell physiology Cells Collaborations Data Detergents Development Diglycerides Disease Elevation Enzymes Etiology Excision Exhibits Familial Amyotrophic Lateral Sclerosis Family Free Radicals Generations Homeostasis Hydrogen Peroxide Hydrolysis In Vitro Inositol Inositol 1,4,5-Trisphosphate Kinetics Link MG132 Mediating Membrane Messenger RNA Metabolic Micelles Modeling Molecular Molecular Chaperones Molecular Weight Monitor Mouse Strains Mus National Institute of Diabetes and Digestive and Kidney Diseases Nuclear Numbers Onset of illness Oxidative Stress Pathway interactions Peroxidase Peroxidases Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipase Phospholipase C Physiological Play Population Post-Translational Protein Processing Process Production Proteasome Inhibitor Protein Overexpression Proteins Proteomics Quality Control RNA RNA marker Reactive Oxygen Species Regulation Reporting Research Research Personnel Rhodopsin Role Route Second Messenger Systems Signal Transduction Staging Stress Structure Superoxides TP53 gene Testing Tetanus Helper Peptide Thapsigargin Transducers Transfection Translating Translations Tube UBD protein Ubiquitin Ubiquitin Like Proteins base caspase-3 cell growth regulation design dimer electric field hydroethidine imaging probe in vivo inhibitor/antagonist macrophage member mimetics mouse model mutant oxidation peroxiredoxin polypeptide prevent pro-caspase-3 protein protein interaction second messenger size sugar transcription factor CHOP

项目摘要

Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Covalent modification of proteins by ubiquitin-like modifiers has been implicated to play a role in oxidative stress and in the regulation of diverse cellular processes. To elucidate the enzymatic pathways and identify their target proteins, a general proteomic approach was developed. It involved establishing stable HEK293 Tet-On cell lines for expressing ubiquitin-like modifier proteins and their mutants. We revealed: (a) Overexpressing SUMO-2/3 in HEK293 cells induced cellular senescence while overexpressing SUMO-1induced apoptosis, and its Cys-54 played an important role in regulating apoptosis.(b) Overexpression of FAT10 in HEK293 cells showed p53 and a number of high molecular weight proteins were FATylated, the latter were induced by TNF-a and significantly accumulated in the presence of proteasome inhibitor, MG132. FATylation of p53, the first FAT10 targeted protein identified, greatly enhanced its transcriptional activity. Furthermore, overexpressing FAT10 also led to a reduction in the size of Promyelocytic Leukemia Nuclear Bodies (PML-NBs) and altered their distribution in the nucleus. Our results suggest a dynamic mechanism for FAT10-mediated p53 activation and leads to the translocation of p53 into functional PML-NBs where p53 undergoes conformational change and activation. (ii) RNAs are highly susceptible to ROS-mediated oxidation. The mechanisms of RNA oxidation and their physiological consequences were studied. We previously showed that moderate oxidation of mRNA leads to production of dysfunctional polypeptides, due to translation errors. Using an mRNA-encoding bovine rhodopsin as a model, we investigated the biological impact of oxidized mRNA-induced translation errors on protein quality control. Our results demonstrate that (a) transfection of the in vitro oxidized rhodopsin mRNA into HEK293 cells led to an accumulation of high molecular weight rhodopsin derivatives, and (b) translation of the oxidized rhodopsin mRNA up-regulated the ER stress transducers, including ATF6 activation, elevation of CHOP transcription factor, phosphorylated eIF2, and ATF4 expression, as well as a moderate increase in caspase-3 activity. GC/MS analysis revealed that thapsigargin, an ER stress inducer, treated HEK293 cells exhibited a transient increase in cytosolic Ca(II) and induced cellular RNA oxidation. Thus, thapsigargin may, in part, exert its effect on ER stress via a mechanism mediated by oxidized RNA-induced translation errors. In addition, GC/MS analysis of oxidized RNA revealed that in vitro and in vivo oxidation of RNA yielded both oxidized base and abasic sugar derivatives. The latter provides an accurate marker for RNA oxidation. (iii). To continue our efforts to elucidate the mechanism of familial amyotrophic lateral sclerosis (FALS), we studied the effects of Cu(II) chaperone of SOD1 protein(CCS) on the aggregate formation of SOD1 mutants in cells. SOD1 (WT, A4V, G85R, or G93A) was overexpressed either by itself or co-expressed with CCS in HEK293 cells. Our data showed that overexpression of either A4V or G85R, but not WT, SOD1 led to the formation of detergent insoluble high molecular weight SOD1 derivatives. However, overexpression of CCS together with SOD1 mutants greatly attenuated aggregate formation. Co-expression of the CCS mutant(C244,246S), which cannot transfer Cu(II) to the SOD1, yielded the same effect, indicating that activity of SOD1 or CCS is not required for attenuating the aggregate accumulation. Inhibitor study showed that CCS was mostly degraded by macroautophagy pathway (inhibited by 3-methyladenine (3-MA)), and the detergent-soluble G85R, when co-expressed with CCS, was greatly elevated by the presence of 3-MA, indicating that CCS could protect G85R from degradation. However, 3-MA exhibited no effect on the detergent soluble A4V when co-expressed with CCS. These results suggest that CCS prevents aggregate formation by stabilizing SOD1 mutants through the activity independent protein-protein interaction. Overexpressed CCS and G85R (cannot bind Cu(II)) appeared to be degraded through the macroautophagy pathways. However, the Cu(II) containing SOD1s, including A4V and G93A, are not degraded in this fashion. Our results and those reported recently showing overexpression of CCS accelerates the disease onset in the G93A mouse model suggest that aggregates formation may not be the primary cause of FALS. (iv) Reversible protein glutathionylation plays a key role in cellular regulation and cell signaling. Sulfiredoxin (Srx), an enzyme catalyzing the reduction of Cys-sulfinic derivatives of peroxiredoxin (Prx), a family of peroxidases that catalyzes the removal of hydrogen peroxide and organic hydroperoxides. Prx1, the abundant and ubiquitously expressed member of 2 Cys Prx, exists in various oligomeric forms. The decamer or higher multimer of Prx I can function as a molecular chaperone, while its dimer possesses high peroxidase activity. We showed that Prx1can be multiply glutathionylated and Srx serves as a deglutathionylating enzyme specifically for 2-Cys Prxs. Glutathionylation shifted Prx I from its decameric structure to a population consisting mainly of dimers. Thus, glutathionylation converts Prx I from a molecular chaperone to a peroxidase enzyme. (v) Accumulation of ROS has been linked to Alzheimers disease mediated by Beta-amyloid peptides of 39 to 42 amino acid residues derived from the amyloid precursor protein, APP. Using the superoxide- and hydrogen-peroxide specific fluorescent indicators, hydroethidine and DCFH, we studied the increase in superoxide radical anions and hydrogen peroxide in SH-SY5Y cells stably overexpressed the mutant amyloid-Beta protein precursor and its fragments. We also monitored intracellular Ca(II)level. Our observations indicate that the dysregulation of Ca (II) homeostasis may play a role in APP mediated ROS generation. (vi) Using a mouse B cell line (FOX-NY) and a macrophage (J7444A.1) line from the same mouse strain as a model, we showed that externally applied moderate electric field induced PS externalization on the B cell membrane without procaspase-3 activation. We further showed that these electric field induced apoptosis mimetic B cells were recognized and cleared by the macrophages. (vii) Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerol and inositol 1,4,5-trisphosphate. Both products serve as second messengers that can initiate diverse processes like proliferation, differentiation, etc. We have been studying the mechanisms for PLC regulation with focus on PLC-alpha. The major difficulty in studying the catalysis of phospholipases derives from the fact that their substrates are embedded in membranes, or in detergent micelles in test tube assays. Consequently, kinetics of the phospholipase action are very complicated to analyze and not easy to translate into physiological occurrences. To better understand the behavior of PLC-n, we tried to breakdown the catalytic process into two manageable stages--interaction with bulk membranes and subsequent substrate recognition/hydrolysis. Study of the latter process requires soluble substrates, which are not available. We have designed a new PLC substrate, a inositol trisphosphate derivative conjugated with a fluorogenic group. Attempt for this synthesis was carried out by the Imaging Probe Development Center, NHLBI. The product obtained from IPDC was not pure, but its preliminary data confirmed its utility as anticipated. However, to obtain a pure product proved to be extremely difficult. We are now in the process of developing a new, efficient synthetic route in collaboration with William Trenkle, NIDDK.

代谢调节部分的研究人员集中在以下项目上：(i)泛素样修饰剂对蛋白质的共价修饰在氧化应激和多种细胞过程的调节中起作用。为了阐明酶促途径并鉴定其靶蛋白，开发了一种通用的蛋白质组学方法。它涉及建立稳定的HEK293 Tet-On细胞系，用于表达泛素样修饰蛋白及其突变体。我们发现(a)在HEK293细胞中过表达SUMO-2/3诱导细胞衰老，过表达sumo -1诱导细胞凋亡，其Cys-54在调节细胞凋亡中发挥重要作用。(b) HEK293细胞中FAT10的过表达显示p53和一些高分子量蛋白被FATylated，后者是由TNF-a诱导的，并在蛋白酶体抑制剂MG132存在下显著积累。作为第一个被发现的FAT10靶向蛋白，p53的脂酰化极大地增强了其转录活性。此外，过表达FAT10还导致早幼粒细胞白血病核小体（PML-NBs）的大小减小，并改变其在细胞核中的分布。我们的研究结果提示了fat10介导的p53激活的动态机制，并导致p53易位为功能性PML-NBs，其中p53经历构象变化和激活。（ii） rna对ros介导的氧化非常敏感。研究了RNA氧化的机制及其生理后果。我们之前的研究表明，mRNA的适度氧化会导致翻译错误导致功能失调多肽的产生。以编码mrna的牛视紫红质为模型，研究了氧化mrna诱导的翻译错误对蛋白质质量控制的生物学影响。我们的研究结果表明(a)体外氧化视紫红质mRNA转染HEK293细胞导致高分子量视紫红质衍生物的积累，(b)氧化视紫红质mRNA的翻译上调内质网应激转导，包括ATF6激活，CHOP转录因子升高，磷酸化的eIF2和ATF4表达，以及caspase-3活性的适度增加。GC/MS分析显示，内质网应激诱导剂thapsigargin处理的HEK293细胞表现出短暂的胞浆Ca（II）升高和诱导细胞RNA氧化。因此，该信号素可能在一定程度上通过氧化rna诱导的翻译错误介导的机制对内质网应激发挥作用。此外，氧化RNA的GC/MS分析表明，RNA的体外和体内氧化均产生氧化的碱基和碱性糖衍生物。后者提供了RNA氧化的准确标记。(3)。为了进一步阐明家族性肌萎缩性侧索硬化症（FALS）的发病机制，我们研究了SOD1蛋白Cu（II）伴侣蛋白（CCS）对细胞中SOD1突变体聚集形成的影响。SOD1 （WT、A4V、G85R或G93A）在HEK293细胞中单独过表达或与CCS共表达。我们的数据表明，过表达A4V或G85R，而不是WT， SOD1导致形成洗涤剂不溶性的高分子量SOD1衍生物。然而，CCS和SOD1突变体的过表达大大减弱了聚集体的形成。不能将Cu（II）转移到SOD1的CCS突变体（C244,246S）的共表达产生了同样的效果，这表明SOD1或CCS的活性不需要减弱聚集积累。抑制剂研究表明，CCS主要通过巨噬途径被降解（被3-甲基腺嘌呤（3-MA）抑制），当与CCS共表达时，洗涤剂可溶性G85R在3-MA的存在下显著升高，表明CCS可以保护G85R不被降解。然而，当3-MA与CCS共表达时，对洗涤剂可溶性A4V没有影响。这些结果表明，CCS通过不依赖于活性的蛋白质相互作用来稳定SOD1突变体，从而阻止聚集体的形成。过表达的CCS和G85R（不能结合Cu(II)）似乎通过巨噬途径被降解。然而，含有SOD1s的Cu(II)，包括A4V和G93A，不会以这种方式降解。我们的研究结果以及最近报道的在G93A小鼠模型中显示CCS过表达加速疾病发作的研究表明，聚集体的形成可能不是FALS的主要原因。（iv）可逆蛋白谷胱甘肽化在细胞调控和细胞信号传导中起关键作用。硫氧还蛋白（Srx），一种催化还原过氧化物还蛋白（Prx）的cys -亚亚基衍生物的酶，Prx是催化去除过氧化氢和有机氢过氧化物的过氧化物酶家族。Prx1是2cys Prx中丰富且普遍表达的成员，以多种低聚形式存在。Prx I的十聚体或更高的多聚体可以作为分子伴侣，而其二聚体具有较高的过氧化物酶活性。我们发现prx1可以被多次谷胱甘肽化，而Srx则是一种专门针对2-Cys Prxs的去谷胱甘肽化酶。谷胱甘肽化将prx1从十聚体结构转变为主要由二聚体组成的种群。因此，谷胱甘肽化将Prx I从分子伴侣转化为过氧化物酶。(v) ROS的积累与淀粉样蛋白前体蛋白APP衍生的39至42个氨基酸残基的β -淀粉样肽介导的阿尔茨海默病有关。利用超氧化物和过氧化氢特异性荧光指标氢乙胺和DCFH，我们研究了SH-SY5Y细胞中超氧化物自由基阴离子和过氧化氢的增加，这些细胞稳定地过度表达突变的淀粉样蛋白前体及其片段。同时监测细胞内Ca（II）水平。我们的观察表明，Ca （II）稳态失调可能在APP介导的ROS生成中起作用。（vi）利用小鼠B细胞系FOX-NY和巨噬细胞J7444A.1作为模型，我们发现外用中等电场诱导的PS外化作用在B细胞膜上没有procaspase-3激活。我们进一步发现这些电场诱导的凋亡模拟B细胞被巨噬细胞识别和清除。（vii）磷脂酶C （PLC）催化磷脂酰肌醇4,5-二磷酸（PIP2）水解生成二酰基甘油和1,4,5-三磷酸肌醇。这两种产物都是第二信使，可以启动增殖、分化等多种过程。我们一直在研究PLC调控的机制，重点是PLC-alpha。研究磷脂酶催化作用的主要困难是由于磷脂酶的底物在试管试验中被包裹在膜或洗涤剂胶束中。因此，磷脂酶作用的动力学分析非常复杂，也不容易转化为生理现象。为了更好地理解PLC-n的行为，我们试图将催化过程分解为两个可管理的阶段——与大块膜的相互作用和随后的底物识别/水解。后一过程的研究需要可溶底物，而这是不可用的。我们设计了一种新的PLC底物，一种带荧光基团共轭的肌醇三磷酸衍生物。NHLBI成像探针开发中心进行了这种合成的尝试。从IPDC获得的产品不纯，但其初步数据证实了其预期的效用。然而，要获得纯净的产品被证明是极其困难的。我们现在正在与NIDDK的William Trenkle合作开发一种新的，高效的合成路线。