Laboratory of Chemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):2011-6. doi: 10.1073/pnas.1321783111. Epub 2014 Jan 21.
Dose-dependent oxidative stress by the anthracycline doxorubicin (Dox) and other chemotherapeutic agents causes irreversible cardiac damage, restricting their clinical effectiveness. We hypothesized that the resultant protein oxidation could be monitored and correlated with physiological functional impairment. We focused on protein carbonylation as an indicator of severe oxidative damage because it is irreversible and results in proteasomal degradation. We identified and investigated a specific high-molecular weight cardiac protein that showed a significant increase in carbonylation under Dox-induced cardiotoxic conditions in a spontaneously hypertensive rat model. We confirmed carbonylation and degradation of this protein under oxidative stress and prevention of such effect in the presence of the iron chelator dexrazoxane. Using MS, the Dox-induced carbonylated protein was identified as the 140-kDa cardiac myosin binding protein C (MyBPC). We confirmed the carbonylation and degradation of MyBPC using HL-1 cardiomyocytes and a purified recombinant untagged cardiac MyBPC under metal-catalyzed oxidative stress conditions. The carbonylation and degradation of MyBPC were time- and drug concentration-dependent. We demonstrated that carbonylated MyBPC undergoes proteasome-mediated degradation under Dox-induced oxidative stress. Cosedimentation, immunoprecipitation, and actin binding assays were used to study the functional consequences of carbonylated MyBPC. Carbonylation of MyBPC showed significant functional impairment associated with its actin binding properties. The dissociation constant of carbonylated recombinant MyBPC for actin was 7.35 ± 1.9 μM compared with 2.7 ± 0.6 μM for native MyBPC. Overall, our findings indicate that MyBPC carbonylation serves as a critical determinant of cardiotoxicity and could serve as a mechanistic indicator for Dox-induced cardiotoxicity.
蒽环类药物阿霉素（Dox）和其他化疗药物的剂量依赖性氧化应激会导致不可逆的心脏损伤，从而限制了它们的临床疗效。我们假设可以监测到由此产生的蛋白质氧化，并将其与生理功能障碍相关联。我们专注于蛋白质羰基化为严重氧化损伤的指标，因为它是不可逆的，会导致蛋白酶体降解。我们鉴定并研究了一种特定的高分子量心脏蛋白，该蛋白在自发性高血压大鼠模型中，在阿霉素诱导的心脏毒性条件下，羰基化显著增加。我们在氧化应激下证实了这种蛋白质的羰基化和降解，并在存在铁螯合剂地拉佐嗪的情况下预防了这种效应。使用 MS，鉴定出阿霉素诱导的羰基化蛋白为 140kDa 心肌肌球蛋白结合蛋白 C（MyBPC）。我们使用 HL-1 心肌细胞和纯化的未标记的重组心脏 MyBPC 证实了 MyBPC 的羰基化和降解在金属催化的氧化应激条件下。MyBPC 的羰基化和降解与时间和药物浓度有关。我们证明了 MyBPC 经羰基化后，在阿霉素诱导的氧化应激下，经蛋白酶体介导降解。共沉淀、免疫沉淀和肌动蛋白结合实验用于研究羰基化 MyBPC 的功能后果。MyBPC 的羰基化与肌动蛋白结合特性相关，表现出明显的功能障碍。与天然 MyBPC 相比，羰基化重组 MyBPC 与肌动蛋白的解离常数为 7.35±1.9μM，而天然 MyBPC 为 2.7±0.6μM。总的来说，我们的研究结果表明，MyBPC 的羰基化是心脏毒性的关键决定因素，并且可以作为阿霉素诱导的心脏毒性的机制指标。
