Emadali Anouk, Metrakos Peter P, Kalantari Fariba, Boutros Tarek, Boismenu Daniel, Chevet Eric
Department of Surgery, McGill University, Montreal, Quebec, Canada.
Proteome Sci. 2007 Jan 2;5:1. doi: 10.1186/1477-5956-5-1.
Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies.
Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R.
Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.
缺血再灌注（I/R）在肝移植过程中会引发细胞代谢的显著重编程，并且可能与多种细胞蛋白磷酸化水平的改变有关。在过去二十年中，酪氨酸磷酸化在多种重要信号通路中发挥关键作用这一点变得清晰，并且它与多种疾病相关。因此，对肝移植过程中酪氨酸磷酸化蛋白质组进行功能分析具有重大生物学意义，很可能会促成新的药物靶点的发现，并为新的治疗策略提供依据。
利用在器官获取和移植早期阶段采集的肝活检样本，我们旨在描绘肝脏缺血再灌注期间酪氨酸磷酸化的整体模式。一种基于纯化酪氨酸磷酸化蛋白然后使用质谱进行鉴定的蛋白质组学方法，使我们能够鉴定出Nck-1（一种SH2/SH3衔接蛋白）作为缺血再灌注损伤的潜在调节因子。通过免疫印迹、细胞分级分离和免疫组织化学，我们证明在缺血再灌注时肝组织中Nck-1的磷酸化、表达和定位受到影响。此外，对移植过程中Nck-1结合伴侣的质谱鉴定还表明，在缺血再灌注期间Nck-1与肌动蛋白之间存在动态相互作用。
综上所述，我们的数据表明Nck-1可能在缺血再灌注诱导的肌动蛋白重组中发挥作用，此前报道这种重组对移植肝的肝细胞有害。因此，Nck-1可能是设计新的器官保存策略的理想靶点，这可能有助于减少再灌注后肝脏损伤并改善移植结果。
