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的耳蜗带硝化的证据。这些蛋白质的功能作用是由sterocilia和神经元染色与抗硝基酪氨酸和耳保护药物的能力，以阻止硝化。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干预药物。本提案的具体目的是：1）确定顺铂诱导的耳蜗蛋白硝化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）使用抑制剂操纵硝化蛋白并与预防耳毒性相关。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损伤。将分别通过免疫印迹和免疫细胞化学，用硝基酪氨酸抗体对耳蜗中的硝化蛋白进行定量和定位。将通过凝胶电泳和随后的MALDI-TOF质谱法鉴定硝化蛋白。将使用抗体微阵列研究与硝化相关的蛋白质组学反应，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。 公共卫生相关性：耳毒性是广泛使用的顺铂的主要剂量限制因素之一。蛋白质的硝化被认为是一种重要的氧化翻译后修饰，可导致蛋白质功能的严重改变。本研究将揭示顺铂诱导的耳蜗蛋白质硝化与相关听力损失之间的相关性。通过顺铂治疗和伴随的耳蜗蛋白质组学反应的分析，有和没有抑制硝化的耳蜗蛋白质硝化的鉴定，将增加顺铂耳毒性的分子机制的清晰度。这些反过来又将有助于鉴定和开发高效的治疗靶点，以预防顺铂介导的耳毒性。

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.