Structural Dynamics of Src-Family Kinase Activation

描述（由申请人提供）： Src家族激酶（关键信号转导分子）的不当或不合时宜的激活导致许多癌症和艾滋病进展。多个内部调节元件合作以将蛋白质保持在非活性构象，直到需要激酶活性。其他蛋白质（包括HIV Nef）的突变或结合可将SH 3和/或SH 2结构域从非活性构象的调节位置置换，从而导致不适当的激活和异常信号传导。目前还不清楚在激活过程中的构象变化是什么。如果获得这些变化的详细描述，将导致对Src家族激酶的更完整的理解，因此可能会出现对抗疾病相关激活的其他策略。 氢交换（HX）质谱（MS）技术可用于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提供了与少量蛋白质一起工作的希望，这是大多数Src家族激酶的先前生物物理分析不可能的条件。为了鉴定Src家族蛋白在正常和生命诱导的活化过程中的构象变化，将实现三个具体目标：（1）定义Src家族激酶活化的构象要求。 将用HXMS分别探测模型完全失活和完全活性的Hck形式，并将结果进行比较，从而揭示Hck在活化期间经历构象变化的区域。(2)确定病毒蛋白如何改变Src家族激酶的非活性构象。将用HXMS探测HIV Nef对Hck的激活和来自松鼠猴疱疹病毒的Tip蛋白对Lck的激活。当Hck/Lck结合时与游离时氢交换的变化预计将揭示病毒蛋白如何以及在何处诱导活化构象变化。蛋白质相互作用的区域应该是新的、可替代的治疗靶点。(3)确定各种Src家族成员是否对HIV Nef具有独特的构象反应由于所有Src家族激酶均不被HIV Nef激活，因此将在存在HIV Nef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族成员对Nef诱导的激活的“天然抗性”。总之，这三个目标预计将提供大量的信息，构象变化的作用，Src家族激酶的激活，在疾病预防中的明显目标。

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.