Kinetics, Regulation, And Mechanisms Of Biochemical Reactions

代谢调节部分的研究人员主要关注以下项目：(i)氧化还原介导的蛋白谷胱甘肽酰化和泛素样修饰剂（ubl）共价修饰的研究。(a)可逆蛋白谷胱甘肽化，在细胞调节和细胞信号传导中起关键作用。过氧化物还氧蛋白（Prx）是一个过氧化物酶家族的成员，参与去除过氧化氢和有机氢过氧化物。Prx1是2-Cys Prx中最丰富、最普遍表达的成员，以多种低聚形式存在。已知Prx1在过度氧化时经历从过氧化物酶到分子伴侣的功能变化。功能变化是由低分子量低聚物到具有分子伴侣活性的高分子量复合物的结构变化引起的。我们之前已经证明Prx1可以在C52、83和173位点被谷胱甘肽化。在目前的研究中，我们揭示了Prx1的谷胱甘肽化将其寡聚物状态从天然十聚体转变为主要由二聚体组成的群体，并同时将其功能从分子伴侣转变为过氧化物酶。(b) ubl对蛋白质的共价修饰已被认为在氧化应激反应和多种细胞过程的调节中发挥作用。为了阐明酶促途径并鉴定其靶蛋白，我们建立了一个稳定的HEK293 Tet-On细胞系来过表达UBL修饰蛋白及其突变体。通过这些方法，我们确定p53是第一个fat10修饰蛋白。此外，在HEK293细胞中，过表达FAT10可以显著上调p53的转录活性，而在MEF和HeLa细胞中则没有，这表明FAT10修饰对p53的影响可能是细胞特异性的，有待进一步阐明。（ii） Cu， zn -超氧化物歧化酶（SOD1）基因错义突变发挥其功能获得性导致家族性肌萎缩性侧索硬化症（FALS）运动神经元变性的机制尚存争议。提出的机制之一涉及增强SOD1突变体聚集体的形成。然而，这一机制与最近的一项研究不一致，该研究表明，与G93A小鼠相比，双转基因小鼠过度表达G93A- sod1和CCS铜伴侣（G93A/CCS），但没有显示出sod1阳性包裹体，尽管它们出现了加速的FALS症状（PNAS 104,6072 - 7,2007）。我们发现，CCS与SOD1突变体（如A4V或G93A）在AAV 293和HEK 293细胞中的共表达表明，CCS通过促进SOD1成熟以产生活性和稳定的同二聚体来阻止SOD1-聚集体的形成。对于失活的sod1突变体，如G85R， CCS可以形成相对稳定的异源二聚体，如G85R- cu (I)-CCS。这种异二聚体，以及CCS本身，很容易降解，主要是通过巨噬途径。CCS的过表达减少了细胞中SOD1突变体的线粒体摄取，这与老龄G93A/CCS小鼠的趋势相反。我们还发现转位到线粒体的SOD1是无活性的。我们的研究结果和之前的结果表明，CCS介导的铜离子插入形成活跃的SOD1突变体或不活跃的异源二聚体，增强了SOD1产生自由基的活性，从而导致SOD1的脱金属及其随后在线粒体中的积累，在G93A/CCS小鼠模型中观察到。因此，SOD1突变体诱导FALS的主要原因可能发生在聚集形成之前。因此，通过Cu离子介导的反应产生的自由基可以解释次要事件，包括mRNA氧化、聚集形成、SOD1的脱金属及其随后的线粒体易位。（iii）多杀性巴氏杆菌毒素（PMT）是一种有效的丝裂原，已知可激活磷脂酶c - β -1、Jak-Stat和Rho激酶途径。这些激活过程的机制尚不清楚。我们发现PMT在血清饥饿的Swiss 3T3细胞中诱导蛋白质合成、ATP合成和细胞扩散。pmt诱导蛋白合成的机制部分通过g - α -q依赖性mTORC1激活进行。此外，pmt诱导的mTORC1激活通过MEK/ERK1/2途径进行。根据我们对pmt诱导细胞扩散和蛋白质合成的数据，提出了一种机制方案。这一发现表明PMT可能通过mTOR信号通路发挥其一些生物学作用，已知mTOR信号通路在细胞生长控制和人类肿瘤发生中起重要作用。（iv） rna极易氧化。研究了RNA氧化的机制及其生理后果。我们发现，由于翻译错误，mRNA的适度氧化导致其翻译保真度显著降低。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，氧化视紫红质mRNA的翻译上调了内质网应激传感器。(5)磷脂酶C （PLC）同工酶在细胞刺激后产生第二信使，在哺乳动物细胞信号转导中起关键作用。plc - γ的激活需要酪氨酸磷酸化，我们一直在研究其细节。在过去的一年中，建立了一个相对较大的PLC-gamma1片段的细菌表达系统，该系统包含了与调控相关的所有结构域，包括需要磷酸化的关键Tyr残基。这种细菌表达的蛋白是稳定的，最重要的是，能够在体外对特定的Tyr残基进行化学计量磷酸化。我们计划与NHLBI的LMB合作，确定该超模的解结构。此外，还建立了一个系统来监测PLC-gamma1在不同酪氨酸激酶的体外磷酸化情况。磷酸化动力学研究揭示了该反应的许多有趣的生化细节。此外，与NIDDK的William Trenkle一起，我们正在为PLC合成一种新的荧光底物，以促进其活性测定。（六）活性氧的产生与β -淀粉样蛋白介导的阿尔茨海默病有关。为了研究a - β诱导的毒性机制，我们建立了稳定的SH-SY5Y细胞系，过度表达淀粉样蛋白前体及其突变体。监测细胞过度表达突变型和野生型淀粉样蛋白引起的超氧化物和过氧化氢生成以及细胞质Ca（II）水平和caspase-3活性的变化。结果提示Ca（II）稳态失调可能是诱导β淀粉样蛋白前体过表达细胞凋亡的重要因素。（vii） CPPs是一类短肽，具有带正电的氨基酸重复序列。这些肽能够单独或结合到更大的蛋白质或DNA片段时穿过细胞和脂质膜。易位可能涉及多种内吞途径。最近一项利用脂质双层膜的研究表明，PPC可以形成大孔。然而，这一结果并不能解释为什么较小的分子会被阻止通过孔隙。为了进一步研究这些cpp介导的途径，我们研究了荧光标记的Arg-9进入囊泡的摄取。我们发现Arg-9被转运到脂质囊泡中，这支持了内吞途径不需要的观点。此外，我们发现一个较小的猝灭分子未能进入囊泡，这表明pcp介导的长寿命孔隙不太可能存在。这些结果表明，易位途径可能遵循地毯-孔隙模型。

Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Studies on redox-mediated protein glutathionylation and covalent modification by ubiquitin-like modifiers (UBLs). (a) Reversible protein glutathionylation, plays a key role in cellular regulation and cell signaling. Peroxiredoxin (Prx) is a member of a family of peroxidases that is involved in removing hydrogen peroxide and organic hydroperoxides. Prx1, the most abundant and ubiquitously expressed member of 2-Cys Prx, exists in various oligomeric forms. Prx1 is known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We have previously shown that Prx1 can be glutathionylated at C52, 83, and 173. In the current study, we revealed that glutathionylation of Prx1 shifted its oligomeric status from native decamers to a population consisting mainly of dimers, and concomitantly shifts its function from molecular chaperone to peroxidase. (b) Covalent modification of proteins by UBLs has been implicated to play a role in oxidative stress response and in regulation of diverse cellular processes. To elucidate the enzymatic pathways and identify their target proteins, we established a stable HEK293 Tet-On cell line to overexpress UBL modifier proteins and their mutants. Using these methods, we identified p53 as the first FAT10-modified protein. In addition, the transcriptional activity of p53 can be significantly up-regulated by overexpressing FAT10 in HEK293 cells, but not in MEF and HeLa cells, indicating that the effect of FAT10 modification on p53 could be cell specific and remains to be elucidated. (ii) The mechanism by which missense mutations of the Cu,Zn-superoxide dismutase (SOD1) gene exert their gain-of-function to cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS) is under debate. One of the proposed mechanisms involves enhanced formation of aggregates of SOD1 mutants. However, this mechanism is inconsistent with a recent study showing that dual transgenic mice over-expressing G93A-SOD1 together with the CCS copper chaperone (G93A/CCS) do not show SOD1-positive inclusions, although they developed accelerated FALS symptoms compared to G93A mice (PNAS 104, 6072-7, 2007). We showed that co-expression of CCS with SOD1 mutants, such as A4V or G93A, in AAV 293 and HEK 293 cells shows that CCS prevents the formation of SOD1- aggregates by facilitating the maturation of SOD1 to yield active and stable homodimers. For inactive SOD1mutants, such as G85R, CCS can form a relatively stable heterodimer, e.g., G85R-Cu (I)-CCS. This heterodimer, as well as CCS itself, are readily degraded, primarily via a macroautophagy pathway. Overexpression of CCS reduces mitochondrial uptake of SOD1 mutants in cells, which is opposite to the trend observed in aged G93A/CCS mice. We also found that SOD1 translocated to mitochondria is inactive. Our findings, together with previous results, indicate that CCS-mediated copper ion insertion to form active SOD1 mutant homodimer or inactive heterodimer enhances the free radical-generating activity of SOD1,which leads to demetallation of SOD1 and its subsequent accumulation in mitochondria as observed in the G93A/CCS mouse model. Thus, the primary cause of FALS induced by SOD1 mutants likely occurs prior to aggregate formation. Thus, free radical generation via Cu ion-mediated reactions could account for the secondary events, including mRNA oxidation, aggregate formation, and demetallation of SOD1 and its subsequent mitochondria translocation. (iii) Pasteurella multocida toxin (PMT), is a potent mitogen known to activate phospholipase C-Beta-1, Jak-Stat, and Rho kinase pathways. The mechanism of these activation processes is not well understood. We show that PMT induces protein synthesis, ATP synthesis, and cell spreading in serum-starved Swiss 3T3 cells. The mechanism of PMT-induced protein synthesis proceeds, in part, via a G-alpha-q-dependent activation of mTORC1. Furthermore, PMT-induced mTORC1 activation proceeded via the MEK/ERK1/2 pathway. Based on our data on PMT-induced cell spreading and protein synthesis, a mechanistic scheme was proposed. This finding reveals that PMT could exert some of its biological effects via the mTOR signaling pathway, known to play an essential role in cell growth control and in human tumorigenesis. (iv) RNAs are highly susceptible to oxidation. The mechanisms of RNA oxidation and their physiological consequences were studied. We showed that moderate oxidation of mRNA leads to a significant reduction in its translation fidelity 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 show translation of the oxidized rhodopsin mRNA up-regulated the ER stress transducers. (v) Phospholipase C (PLC) isozymes generate second messengers upon cell stimulation play key roles in signal transduction of mammalian cells. Activation of PLC-gamma requires its tyrosine phosphorylation, the details of which we have been studying. In the past year, a bacterial expression system of a relatively large fragment of PLC-gamma1 containing all of the domains implicated in the regulation, including key Tyr residues to be phosphorylated was established. This bacterially expressed protein is stable and, most importantly, able to be stoichiometrically phosphorylated on specific Tyr residues in vitro. In collaboration with the LMB of the NHLBI, we plan to determine the solution structure of this supramodule. In addition, a system was established to monitor in vitro phosphorylation by various tyrosine kinases of PLC-gamma1 at individual sites. Phosphorylation kinetic studies revealed many interesting biochemical details of this reaction. In addition, with William Trenkle of NIDDK, we are synthesizing a novel fluorogenic substrate for PLC to facilitate its activity assay. (vi) Production of ROS has been linked to Alzheimers disease mediated by beta-amyloid. To investigate the mechanism of toxicity induced by A-beta, we established stable SH-SY5Y cell lines to overexpress the amyloid-beta protein precursor and its mutants. Changes in superoxide and hydrogen peroxide generation as well as cytosolic Ca(II) levels and caspase-3 activity due to cells overexpressing mutant and wild-type amyloid-beta proteins were monitored. The results suggest that the dysregulation of Ca(II) homeostasis may be an important factor in the induction of apoptosis in cells overexpressing the amyloid-beta protein precursor. (vii) CPPs are a class of short peptides, with repeating sequences of positively charged amino acids. These peptides are capable of traversing the cell and lipid membranes either alone or when conjugated to much larger proteins or DNA fragments. Translocation may involve various endocytic pathways. A recent study using lipid bilayer membranes indicated that large pores could be formed by PPC. The result, however, could not explain why smaller molecules would be prevented passage via the pores. To further study these CPP-mediated pathways, we studied the uptake of fluorescent-tagged Arg-9 into vesicles. We found that Arg-9 was transported into lipid vesicles, supporting the notion that endocytic pathways were not required. Furthermore, we found that a smaller quencher molecule failed to enter into the vesicles, indicating that CPP-mediated long-lived pores are unlikely. These results suggest that the translocation pathway may follow a carpet-pore model.