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.

抽象的 几乎每个细胞过程都受到基于硫醇的氧化还原调节的控制。然而，其机制 细胞质、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 (H2O2) 会诱导蛋白质中半胱氨酸的氧化，但精确的生理学 机制尚未完全了解。这种温和的细胞氧化剂由六种哺乳动物代谢 过氧化还原蛋白 (Prxs) 以及几种谷胱甘肽过氧化物酶和过氧化氢酶。 Prxs 在细胞中表达 类型或组织特异性方式，其还原形式由硫氧还蛋白、其他硫醇再生 氧化还原酶，或谷胱甘肽。先前的研究表明 Prxs 可以直接与监管相互作用 蛋白质并参与细胞信号传导。最近，内质网特异性 PrxIV (erPrxIV) 被 显示通过氧化蛋白质二硫键中的半胱氨酸在二硫键形成途径中发挥作用 异构酶，进一步氧化内质网中的客户蛋白；一起蛋白质二硫键 异构酶和 erPrxIV 在细胞蛋白质中形成二硫键。我们发现了二硫键形成的新形式 PrxIV，cPrxIV，位于细胞质中。在初步研究中，我们发现cPrxIV在 延长睾丸中的精子细胞，并在胎盘哺乳动物中保守。我们进一步表征了 该蛋白质的特性和表达模式，并生成了敲除小鼠模型来研究 cPrxIV 的生理功能。通过类比该蛋白质的内质网形式，我们假设 cPrxIV 催化细胞质中二硫键的形成，这一功能在 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。具体来说，我们打算 (i) 确定 cPrxIV 是否参与二硫键的形成 体外和体内； (ii) 确定相互作用的客户伙伴和 cPrxIV 的还原酶； (iii) 描述 PrxIV KO 小鼠的氧化还原代谢组。