Department of Oral Biochemistry, Dental Science Research Institute, Medical Research Center for Biomineralization Disorders, School of Dentistry, Chonnam National University, 300 Yongbong-Dong, Buk-Ku, Gwangju, 500-757 Republic of Korea.
Department of Molecular Medicine, Graduate School, Chonnam National University, Gwangju, Republic of Korea.
Proteome Sci. 2015 Feb 5;13:4. doi: 10.1186/s12953-014-0061-2. eCollection 2015.
Peroxiredoxin V (Prdx V) plays a major role in preventing oxidative damage as an effective antioxidant protein within a variety of cells through peroxidase activity. However, the function of Prdx V is not limited to peroxidase enzymatic activity per se. It appears to have unique function in regulating cellular response to external stimuli by directing interaction with signaling protein. In this study, we identified Prdx V interacting partners in mouse kidney under hypoxic stress using immunoprecipitation and shotgun proteomic analysis (LC-MS/MS).
Immunoprecipitation coupled with nano-UPLC-MS(E) shotgun proteomics was employed to identify putative interacting partners of Prdx V in mouse kidney in the setting of hypoxia. A total of 17 proteins were identified as potential interacting partners of Prdx V by a comparative interactomics analysis in kidney under normoxia versus hypoxia. Dihydrolipoamide branched chain transacylase E2 (DBT) appeared to be a prominent candidate protein displaying enhanced interaction with Prdx V under hypoxic stress. Moreover, hypoxic kidney exhibited altered DBT enzymatic activity compared to normoxia. An enhanced colocalization of these two proteins under hypoxic stress was successfully observed in vitro. Furthermore, peroxidatic cysteine residue (Cys48) of Prdx V is likely to be responsible for interacting with DBT.
We identified several proteins interacting with Prdx V under hypoxic condition known to induce renal oxidative stress. In hypoxic condition, we observed an enhanced interaction of Prdx V and DBT protein as well as increased DBT enzymatic activity. The results from this study will contribute to enhance our understanding of Prdx V's role in hypoxic stress and may suggest new directions for future research.
过氧化物酶 V（Prdx V）作为一种有效的抗氧化蛋白，通过过氧化物酶活性在多种细胞中发挥重要作用，防止氧化损伤。然而，Prdx V 的功能不仅限于过氧化物酶酶活性本身。它似乎通过与信号蛋白的定向相互作用具有独特的调节细胞对外界刺激反应的功能。在这项研究中，我们使用免疫沉淀和鸟枪法蛋白质组学分析（LC-MS/MS）鉴定了低氧应激下小鼠肾脏中的 Prdx V 相互作用伙伴。
免疫沉淀与纳升超高效液相色谱-电喷雾串联质谱（LC-MS/E）鸟枪法蛋白质组学相结合，用于鉴定低氧条件下小鼠肾脏中 Prdx V 的潜在相互作用伙伴。通过比较正常氧与低氧条件下肾脏的相互作用组学分析，共鉴定出 17 种可能与 Prdx V 相互作用的蛋白。二氢硫辛酰胺支链转酰酶 E2（DBT）似乎是一种突出的候选蛋白，在低氧应激下与 Prdx V 的相互作用增强。此外，与正常氧相比，低氧肾脏表现出改变的 DBT 酶活性。在体外成功观察到这两种蛋白质在低氧应激下的增强共定位。此外，Prdx V 的过氧物酶半胱氨酸残基（Cys48）可能负责与 DBT 相互作用。
我们鉴定了几种在已知诱导肾氧化应激的低氧条件下与 Prdx V 相互作用的蛋白。在低氧条件下，我们观察到 Prdx V 和 DBT 蛋白之间的相互作用增强以及 DBT 酶活性增加。这项研究的结果将有助于增强我们对 Prdx V 在低氧应激中的作用的理解，并可能为未来的研究提供新的方向。
