The universally conserved ATPase as mediator of cellular stress response

YchF是一种功能未知、普遍保守的ATPase，与GTP酶一样被归类为翻译因子，但它优先水解三磷酸腺苷而不是GTP。大肠杆菌和克鲁兹锥虫YchF显示了核糖体结合，但到目前为止对蛋白质合成的可能影响尚不清楚。人类YchF同源物Ola1似乎不与核糖体结合，而是与氧化应激反应的翻译后调节和肿瘤的发展有关。后一种建议与观察到的Ola1在许多肿瘤中的上调是一致的。然而，Ola1功能的分子机制和可能的相互作用伙伴是完全未知的。我们关于这个高度保守的蛋白在大肠杆菌中功能的数据表明，YchF抑制氧化应激反应。抑制不是由主要氧化应激反应蛋白(OxyR，KatG)的转录或翻译下调引起的，而是由过氧化氢酶活性的直接抑制引起的(温克等人，2012年)。YchF与过氧化氢酶/过氧化物酶KatG、Kate和AhpCF之间的直接相互作用通过位点特异的交联法和质谱学方法得到证实。这些数据表明，YchF通过一种迄今未知的对主要应激蛋白的翻译后抑制来调节氧化应激反应。YchF的磷酸化/去磷酸化及其ATPase活性对于这一抑制功能似乎是重要的。我们的数据表明，E.coliYchF和人类Ola1具有明显非常相似的功能，并作为氧化应激反应的普遍保守的抑制因子。我们的目的是确定YchF介导的抑制作用的靶点，并揭示其潜在的分子机制。易于修饰的模式生物大肠杆菌和酿酒酵母将被用来确定YchF对氧化应激的相互作用组，并揭示YchF依赖的靶蛋白抑制的生化和分子框架。这包括对YchF在氧化应激中的表达和修饰的详细分析。考虑到Ola1可能参与肿瘤的发展和转移，我们计划一旦我们在大肠杆菌/酿酒酵母中建立了YchF功能的坚实框架，就将我们的分析扩展到人类细胞系。这一知识转让将是延期提案的主要重点。

YchF is a universally conserved ATPase of unknown function, which is classified as a member of the translation-factor like GTPases, although it preferentially hydrolyzes ATP over GTP. Ribosome binding was shown for E. coli and Trypanosoma cruzii YchF, but the possible impact on protein synthesis is unknown so far. The human YchF homologue Ola1 does not seem to bind to ribosomes and instead is implicated in the post-translational regulation of the oxidative stress respsonse and in tumour development. This latter proposal is in line with the observed up-regulation of Ola1 in many tumours. However, the molecular mechanisms of Ola1 function and possible interaction partners are entirely unknown. Our data on the function of this highly conserved protein in E. coli demonstrate that YchF inhibits the oxidative stress response. Inhibition is not caused by a transcriptional or translational down-regulation of major oxidative stress response proteins (OxyR, KatG), but rather by a direct inhibition of catalase activity (Wenk et al., 2012). A direct interaction between YchF and the catalases/peroxidases KatG, KatE and AhpCF was demonstrated by site-specific cross-linking approaches and by mass spectrometry. These data suggest that YchF regulates the oxidative stress response by a so far unknown post-translational inhibition of major stress proteins. Phosphorylation/ dephosphorylation of YchF and its ATPase activity appear to be important for this inhibitory function. Our data suggest that E. coli YchF and human Ola1 have apparently very similar function and act as universally conserved inhibitors of the oxidative stress response. Our aim is to identify the targets of YchF mediated inhibition and to disclose the underlying molecular mechanism. The easily amendable model organisms E. coli and S. cerevisiae will be employed to determine the YchF interactome in response to oxidative stress and to reveal the biochemical and molecular framework of YchF-dependent inhibition of target proteins. This includes the detailed analyses on YchF expression and modification in response to oxidative stress. Considering the postulated involvement of Ola1 in tumour-development and metastasis, we plan to extend our analyses to human cell lines, once we have established a solid framework of YchF function in E. coli/S. cerevisiae. This knowledge transfer will be the main focus of an extension proposal.