Role of protein nitration in cisplatin mediated ototoxicity

描述（由申请人提供）：耳蜗蛋白的硝化表明氧化翻译后修饰可能导致灾难性后果，这取决于硝化蛋白的功能活性。我们已经报道了顺铂诱导的两个突出耳蜗带硝化的证据。这些蛋白的功能作用可以通过静纤毛和抗硝基酪氨酸的神经元染色以及耳保护药物阻断硝化的能力来推测。本研究的长期目标是了解蛋白质硝化在耳毒性中的作用，并发现更好的预防耳毒性的介入药物。本研究的具体目的是：1)确定顺铂诱导耳蜗蛋白的硝化作用及其与听力损失的相关性；2)确定主要硝化耳蜗蛋白的身份；3)利用抑制剂对硝化蛋白进行调控，并与耳毒性的预防相关。听觉脑干反应和畸变产物耳声发射将用于评估听力功能，而耳蜗图将用于评估形态学损伤。耳蜗中硝化蛋白的定量和定位将分别通过免疫印迹和免疫细胞化学与抗硝基酪氨酸抗体进行。硝化蛋白将通过凝胶电泳和MALDI-TOF质谱鉴定。与硝化相关的蛋白质组反应将使用抗体微阵列进行研究，并使用途径分析软件进行分析。本研究的结果将揭示耳蜗蛋白硝化在顺铂介导的耳毒性中的作用，并将有助于确定有效的治疗干预靶点。

DESCRIPTION (provided by applicant): Nitration of cochlear proteins indicates oxidative posttranslational modification that can lead to catastrophic consequences, depending upon the functional activity of the nitrated proteins. We have reported evidence for cisplatin-induced nitration of two prominent cochlear bands. A functional role for these proteins is suggested by sterocilia and neuronal staining with anti-nitrotyrosine and by the ability of an otoprotective drug to block nitration. The long term aim of this research is to understand the role of protein nitration in ototoxicity and discover better interventional drugs that prevent ototoxicity. The specific aims of this proposal are 1) to determine cisplatin-induced nitration of cochlear proteins and its correlation with hearing loss 2) to establish the identity the major nitrated cochlear protein(s) and 3) to manipulate the nitrated proteins using inhibitor and correlate with prevention of ototoxicity. Auditory brain stem response and distortion product otoacoustic emissions will be used to evaluate the hearing function, while cochleograms will be used to assess morphological damage. Quantification and localization of nitrated proteins in the cochlea will be done by immunoblotting and immunocytochemistry, respectively, with antibodies against nitrotyrosine. Nitrated proteins will be identified by gel electrophoresis followed by MALDI-TOF mass spectrometry. Proteomic responses associated with nitration will be investigated using antibody microarray and analyzed with pathway analysis software. The outcome of this study will reveal the role of cochlear protein nitration in cisplatin mediated ototxicity and will facilitate the identification of effective targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Ototoxicity is one among the major dose-limiting factors of the widely used antineoplastic drug cisplatin. Nitration of proteins is considered as an important oxidative posttranslational modification that can lead to serious alterations in protein function. This study will reveal the correlation between cisplatin induced nitration of cochlear proteins and associated loss of hearing function. Identification of the cochlear proteins nitrated by cisplatin treatment and analysis of accompanying cochlear proteomic responses, with and without inhibition of nitration, will add more clarity to the molecular mechanism underlying cisplatin ototoxicity. These in turn will facililitate the identification and development of highly effective therapeutic targets to prevent cisplatin mediated ototoxicity.