Protein Disulfide Bond Formation in the Reducing Environment of Cytoplasm

细胞质还原环境中蛋白质二硫键的形成

• 批准号: 10439311
• 负责人: Sun Hee Yim
• 金额: $ 44.46万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439311
• 关键词: Academic Research Enhancement Awards; Address; Affinity; Aging; Alkylating Agents; Antibodies; Apoptosis; Binding; Binding Sites; Biochemical; Biological Assay; Biological Markers; Biological Models; Biology; Cardiovascular Diseases; Cell Culture System; Cell Nucleus; Cell physiology; Client; Co-Immunoprecipitations; Complex; Cysteine; Cytoplasm; Cytosol; Diabetes Mellitus; Disease; Disulfides; Endoplasmic Reticulum; Environment; Event; Extracellular Space; Glutathione; Homeostasis; Hydrogen Peroxide; In Vitro; Knock-out; Knockout Mice; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Methods; Mitochondria; Modeling; Modification; Molecular; Natural regeneration; Nature; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductase; Pathway interactions; Pattern; Peroxidases; Physiological; Play; Property; Protein Disulfide Isomerase; Proteins; Reagent; Reducing Agents; Regulation; Research; Role; Selenocysteine; Serum; Signal Transduction; Signaling Molecule; Spermatids; Spermatogenesis; Sulfhydryl Compounds; System; TXN gene; Testis; Texas; Tissues; United States National Institutes of Health; Universities; base; catalase; cell growth regulation; cell type; comparative; data tools; disulfide bond; enzyme activity; extracellular; fundamental research; genetic regulatory protein; glutathione peroxidase; human disease; in vivo; knockout animal; metabolome; mouse model; mutant; oligomycin sensitivity-conferring protein; oxidation; peroxiredoxin; placental mammal; response; undergraduate student

ABSTRACT Nearly every cellular process is controlled by thiol-based redox regulation. However, the mechanisms of targeted cysteine oxidation in the reducing environments of cytosol, mitochondria, and nucleus remain unclear. Hydrogen peroxide (H2O2) is known to induce oxidation of cysteines in proteins, but the precise physiological mechanisms are not fully understood. This mild cellular oxidant is metabolized by six mammalian peroxiredoxins (Prxs) as well as several glutathione peroxidases and catalases. Prxs are expressed in a cell type- or tissue-specific manner, and their reduced forms are regenerated by thioredoxin, other thiol oxidoreductases, or glutathione. Previous studies indicate that Prxs can directly interact with regulatory proteins and participate in cellular signaling. Recently, the endoplasmic reticulum specific PrxIV (erPrxIV) was shown to function in the pathway of disulfide bond formation by oxidizing cysteines in protein disulfide isomerase, which further oxidized client proteins in the endoplasmic reticulum; together protein disulfide isomerase and erPrxIV formed disulfides in cellular proteins. We identified a new form of the disulfide forming PrxIV, cPrxIV, located in the cytosol. In preliminary studies, we found that cPrxIV is highly expressed in elongating spermatids in testes and is conserved in placental mammals. We further characterized the properties and expression patterns of this protein and generated a knockout mouse model to study the physiological function of cPrxIV. By analogy to the endoplasmic reticulum form of this protein, we hypothesize that cPrxIV catalyzes disulfide bond formation in the cytosol and this function is particularly important during spermatogenesis. We propose to characterize cPrxIV and its role in disulfide bond formation and redox homeostasis. Specifically, we intend to (i) determine if cPrxIV participates in disulfide bond formation in vitro and in vivo; (ii) identify interacting client partners and a reductase for cPrxIV; and (iii) characterize the redox metabolome of PrxIV KO mice.

摘要 几乎每一个细胞过程都是由硫醇基氧化还原调节控制的。然而， 在胞质溶胶、线粒体和细胞核的还原环境中的靶向半胱氨酸氧化仍不清楚。 已知过氧化氢（H2 O2）诱导蛋白质中半胱氨酸的氧化，但是精确的生理学上的氧化是不可逆的。 机制尚未完全理解。这种温和的细胞氧化剂由六种哺乳动物代谢 过氧化物氧还蛋白（Prxs）以及几种谷胱甘肽过氧化物酶和过氧化氢酶。prx在细胞中表达 类型或组织特异性的方式，其还原形式是由硫氧还蛋白，其他硫醇再生 氧化还原酶或谷胱甘肽。以前的研究表明，Prxs可以直接与调控因子相互作用， 蛋白质并参与细胞信号传导。最近，内质网特异性PrxIV（erPrxIV）被发现是一种新的蛋白。 显示通过氧化蛋白质二硫化物中的半胱氨酸在二硫键形成途径中起作用 异构酶，其进一步氧化内质网中的客户蛋白; 异构酶和erPrxIV在细胞蛋白中形成二硫化物。我们发现了一种新的二硫化物形成形式 PrxIV，cPrxIV，定位于胞浆。在初步的研究中，我们发现cPrxIV在大肠杆菌中高度表达。 在睾丸中延长精子细胞，在胎盘哺乳动物中是保守的。我们进一步描述了 这种蛋白质的性质和表达模式，并产生了敲除小鼠模型，以研究 cPrxIV的生理功能。通过类比这种蛋白质的内质网形式，我们假设 cPrxIV催化胞质溶胶中二硫键的形成，并且这种功能在 精子发生我们建议表征cPrxIV及其在二硫键形成和氧化还原中的作用 体内平衡具体地，我们打算（i）确定cPrxIV是否参与二硫键的形成， 体外和体内;（ii）鉴定cPrxIV的相互作用客户伙伴和还原酶;和（iii）表征cPrxIV的 PrxIV KO小鼠的氧化还原代谢组。