Structural Dynamics of Src-Family Kinase Activation

描述(由申请人提供)： 作为关键的信号转导分子，src家族的不适当或不及时的激活导致了许多癌症和艾滋病的进展。多个内部调节元件协作将蛋白质保持在不活跃的构象中，直到需要激酶活性为止。其他蛋白(包括HIV Nef)的突变或结合可以取代SH3和/或SH2结构域(S)，使其从非活性构象中的调控位置移位，从而导致不适当的激活和异常的信号传递。目前还不清楚激活过程中的构象变化是什么。如果获得了这些变化的详细描述，就会对Src家族的激酶有更全面的了解，因此可能会有更多的策略来对抗疾病相关的激活。氢交换(HX)质谱学技术可以用来研究蛋白质的结构变化，因为氢交换对蛋白质构象的变化非常敏感。HXMS提供了处理少量蛋白质的前景，这是大多数以前对Src家族激酶进行生物物理分析所不可能实现的条件。为了确定在正常和生命诱导的激活过程中Src家族蛋白的构象变化，将实现三个特定的目标：(1)确定Src家族蛋白激活的构象要求。模拟完全不活跃和完全活跃的HCK形式将分别用HXMS探测并将结果进行比较，从而揭示在激活过程中经历构象变化的HCK区域。(2)确定病毒蛋白如何改变Src家族激酶的非活性构象。HXMS将用HXMS检测HIV Nef和Lck对HIVNef和Lck激活HCK的作用。当HCK/Lck结合时与游离时氢交换的变化有望揭示病毒蛋白如何以及在哪里诱导激活构象变化。蛋白质的区域：蛋白质的相互作用应该提出新的、可供选择的靶向治疗区域。(3)确定不同的Src家族成员是否对HIV Nef有独特的构象反应因为并不是所有的Src家族的激酶都被HIV Nef激活，因此将在HIV Nef存在的情况下比较几个家族成员的HX，以确定构象如何有助于某些家庭成员对Nef诱导的激活的“天然抵抗”。综上所述，这三个目标有望提供大量信息，说明构象变化在Src家族激酶激活中的作用，这是疾病预防中的明显靶点。

DESCRIPTION (provided by applicant): Improper or untimely activation of Src-family kinases, key signal transduction molecules, contributes to many cancers and to AIDS progression. Multiple internal regulatory elements cooperate to hold the proteins in an inactive conformation until kinase activity is required. Mutations or binding of other proteins (including HIV Nef) can displace the SH3 and/or SH2 domain(s) from regulatory positions in the inactive conformation, thereby leading to inappropriate activation and abnormal signalling. It is unclear what the conformational changes are during activation. If detailed descriptions of the changes were obtained, a more complete understanding of Src-family kinases would result, and therefore additional strategies to combat disease-related activation may follow. Hydrogen exchange (HX) mass spectrometry (MS) techniques can be used to investigate structural changes in proteins because hydrogen exchange is very sensitive to changes in protein conformation. HXMS offers the promise of working with small amounts of protein, conditions not possible with most previous biophysical analyses of Src-family kinases. To identify conformational changes in Src-family proteins during normal and vitally-induced activation, three specific aims will be accomplished: (1) Define the conformational requirements for Src-family kinase activation. Forms of Hck that model completely inactive and completely active will be separately probed with HXMS and the results compared, thereby revealing regions of Hck that undergo conformational changes during activation. (2) Determine how viral proteins alter the inactive conformation of Src-family kinases. Activation of Hck by HIV Nef and Lck by the Tip protein from Herpesvirus saimiri will be probed with HXMS. Changes in hydrogen exchange when Hck/Lck are bound versus when free are expected to reveal how and where the viral proteins induce activating conformational changes. Regions of protein:protein interaction should suggest new, alternative areas to target therapeutically. (3) Establish if various Src-famity members have unique conformational responses to HIV Nef Because all Src-family kinases are not activated by HIV Nef, HX will be compared for several family members in the presence of HIV Nef to determine how conformation contributes to the "natural resistance" of some family members towards Nef-induced activation. Taken together, these three Aims are expected to provide substantial information about the role of conformational changes in the activation of Src-family kinases, obvious targets in disease prevention.