Biochemical Characterization of Yersinia Effector YopJ

描述（由申请人提供）：本申请是为了更新R01 AI056404-01题为“耶尔森菌效应物YopJ的生化表征”，该申请于2008年9月30日获得资助，目前剩余资金不足一年。细菌效应物YopJ通过阻止MKKs和IKKb的激活来抑制MAPK和NFkB信号通路。正如我们的进展报告中所述，我们发现细菌效应物YopJ是一种乙酰转移酶，它修饰MKKs激活环上的丝氨酸和苏氨酸残基，从而直接与这些激酶竞争并通过磷酸化阻止这些激酶的激活。本次资助的总体目标是阐明Yersinia， YopJ表达的致病效应物的生化机制，该效应物利用新发现的丝氨酸/苏氨酸乙酰化修饰修饰蛋白质，并探索这是真核生物使用的调节机制的可能性。我们预测这种活性是一种进化保守的调控机制。支持这一说法的证据来自另外四种耶尔森菌效应物的酶特性的发现：YopH，一种酪氨酸磷酸酶；YopE， GAP；YpkA，丝氨酸激酶；木瓜样蛋白酶。与病毒病原体一样，细菌病原体从宿主那里窃取关键活动，并对其进行修改以适应自己的需要。在鼠疫耶尔森氏菌的情况下，这些毒力因子促进病原体增殖，以宿主的致命代价。显然，研究这些效应物很重要，因为它们都对我们思考真核生物信号的方式产生了深远的影响。在这里，我们描述了我们将用于研究这种新发现的丝氨酸和苏氨酸残基的翻译后修饰的具体目的。在这些目标中，我们将使用我们在上一个资助期建立的酵母系统来分析YopJ如何与MKKs相互作用及其抑制机制。这些研究不仅将深入了解YopJ的抑制活性，还将了解调节MKK激活的机制。我们还将通过动力学研究来研究YopJ蛋白家族乙酰化底物的酶促机制。最后，我们将开发工具来研究这种新发现的翻译后修饰及其在真核信号传导中的作用，包括发现可能参与丝氨酸和苏氨酸残基乙酰化的真核酶以及这种翻译后修饰的可能逆转。鼠疫耶尔森氏菌是引起中世纪黑死病的传染因子，假结核耶尔森氏菌和小肠结肠炎耶尔森氏菌（两种密切相关的食源性病原体）是胃肠道疾病的致病因子。耶尔森菌产生的细菌效应物之一YopJ是一种32kD蛋白，具有阻断多种信号通路的显著能力，包括所有MAPK信号通路和NFkB信号通路，从而抑制宿主细胞的先天免疫反应，促进细胞死亡。这项申请是为了更新R01 AI056404-01授权，我们发现YopJ利用一种新的翻译后修饰来抑制信号传导。YopJ是一种丝氨酸和苏氨酸乙酰转移酶，它修饰MKKs激活环上的丝氨酸和苏氨酸残基，从而直接与上游激酶竞争并阻止上游激酶的磷酸化。提出的三个特定目标旨在利用遗传学和生物化学来研究这种新的抑制机制，并研究其在真核信号通路中的可能用途。

DESCRIPTION (provided by applicant): This application is for the renewal of the R01 AI056404-01 entitled "Biochemical Characterization of Yersinia Effector YopJ" that was funded from 9/30/03 1/31/08 and currently has less than a year of funding remaining. The bacterial effector YopJ inhibits MAPK and NFkB signaling pathways by preventing activation of MKKs and IKKb. As described in our Progress Report, we discovered that the bacterial effector YopJ is an acetyltransferase that modifies serine and threonine residues on the activation loop of MKKs, thereby, directly competing with and preventing activation of these kinases by phosphorylation. The overall goal of this grant is to elucidate the biochemical mechanism for the pathogenic effector expressed by Yersinia, YopJ that uses the newly discovered posttranslational modification of serine/threonine acetylation to modify proteins and to explore the possibility that this is a regulatory mechanism used by eukaryotes. We predict this activity is an evolutionarily conserved regulatory mechanism. Support for this claim comes from the discovery of the enzymatic properties of four other Yersinia effectors: YopH, a tyrosine phosphatase; YopE, a GAP; YpkA, a serine kinase; YopT, papain-like protease. Bacterial pathogens, as with viral pathogen's, usurp critical activities from their hosts and modified them to suit their own needs. In the case of Yersinia pestis, these virulence factors promote the pathogens proliferation at the deadly expense of the host. Clearly these effectors are important to study because all of them have had a profound impact on our way of thinking about eukaryotic signaling. Herein, we describe the Specific Aims that we will use to study this newly discovered posttranslational modification on serine and threonine residues. In these Aims we will use a yeast system that we established over the last funding period to analyze how YopJ interacts with MKKs and its mechanism of inhibition. These studies will provide insight into, not only the inhibitory activity of YopJ, but also the mechanisms that regulate MKK activation. We will also investigate the enzymatic mechanism used by the family of YopJ proteins to acetylate their substrates using kinetic studies. Finally, we will develop tools to study this newly discovered posttranslational modification and its role in eukaryotic signaling, including the discovery of eukaryotic enzymes that maybe involved in acteylation of serine and threonine residues and the possible reversal of this posttranslational modification.Yersinia pestis is the infectious agent that caused the Black Death in the Middle Ages, and Y. pseudotuberculosis and Y. enterocolitica (two closely related food borne pathogens) are causal agents of gastrointestinal disorders. One of the bacterial effectors produced by Yersinia, YopJ, is a 32kD protein that possesses the remarkable capacity to block multiple signaling pathways including all of the MAPK signaling pathways and the NFkB pathways, resulting in inhibition of the innate immune response and promotion of cell death in host cells. This application is for the Renewal of the R01 AI056404-01 grant where we discovered that YopJ utilizes a new type of posttranslational modification to inhibit signaling. YopJ is a serine and threonine acetyltransferase that modifies serine and threonine residues on the activation loop of MKKs, thereby, directly competing with and preventing phosphorylation by upstream kinases. The three Specific Aims proposed for the renewal of this grant are designed to use genetics and biochemistry to study this novel inhibitory mechanism and to investigate its possible use in eukaryotic signaling pathways.