Protein-protein interaction domains: targets for neuropharmacologic intervention

描述（由申请人提供）：细胞具有多种手段来整合它们所暴露的外部刺激成协调的细胞反应。在这些过程中，信号转导系统各组成部分之间的直接和间接相互作用都起着重要作用。介导蛋白质-蛋白质相互作用的序列基序的发现，加上蛋白质氨基酸序列数据的可用性，允许鉴定推定的蛋白质结合对。本授权申请中的研究源于我们对磷脂酰肌醇特异性磷脂酶C-y（PLC-y）和PLC-p同工酶一级结构内的氨基酸序列的认识，这些氨基酸序列符合有丝分裂原活化蛋白激酶（MAPK）的对接基序/所谓的D结构域的共有序列。我们的初步研究支持PLC-γ和PLC-β同工酶直接与MAPK相互作用的假设。在拟议的研究中，我们的目标是证实推定的D-结构域的鉴定，并开发可用于研究PLC-MAPK蛋白质-蛋白质相互作用在体内的作用的细胞可渗透的药理学工具。这项研究的长期目标是了解PLC-MAPK信号整合在心理健康中的作用。拟议的研究包括三个目标。目的1是确定鉴定的D-结构域序列是否在体外结合MAPK。目标2研究将评估鉴定的D结构域肽序列阻断MAPK与其底物之间体外相互作用的能力;此外，我们将确定用于促进目标3中D结构域肽跨膜递送的修饰是否改变其破坏MAPK-底物相互作用的能力。在目标3中，我们将量化肽到突触神经体的递送，突触神经体是来自大脑的密封囊泡的制备物。从拟议的研究中获得的结果将扩大我们对信号整合机制的理解，提供有关将复杂的细胞外刺激整合到协调的细胞反应中的方法的重要信息。此外，这些研究将提供有价值的药理学工具，可用于研究PLC-MAPK相互作用在各种细胞反应中的作用，包括可塑性，生长和分化。反过来，这将有助于我们理解正常和异常的细胞功能。因此，这些研究将对包括神经生物学、细胞生物学、生物化学和癌症生物学在内的几个研究领域产生重大影响。

DESCRIPTION (provided by applicant): Cells possess a variety of means to integrate the external stimuli to which they are exposed into coordinated cellular responses. Both direct and indirect interactions between the components of signal transduction systems play important roles in these processes. The discovery of sequence motifs that mediate protein- protein interactions, coupled with the availability of protein amino acid sequence data, allows for the identification of putative protein binding pairs. The studies in this grant application arose from our recognition of amino acid sequences within the primary structures of phosphatidylinositol-specific phospholipase C-y (PLC-y) and PLC-p isozymes that conform to consensus sequences of docking motifs/so-called D-domains, for mitogen-activated protein kinases (MAPKs). Our initial studies support the hypothesis that PLC-y and PLC-p isozymes directly interact with MAPKs. In the proposed studies we aim to substantiate the identification of the putative D-domains and to develop cell-permeable pharmacologic tools that can be used to study the role of PLC-MAPK protein-protein interactions in vivo. The long-term goal for this research is to understand the role of PLC-MAPK signal integration in mental health. The proposed studies consist of three aims. Aim 1 is to determine whether the identified D-domain sequences bind MAPKs in vitro. Aim 2 studies will assess the ability of the identified D-domain peptide sequences to block in vitro interactions between MAPKs and their substrates; in addition, we will determine whether modifications that will be used to promote transmembrane delivery of the D-domain peptides in Aim 3 alter their ability to disrupt MAPK-substrate interactions. In Aim 3 we will quantify the delivery of the peptides into synaptoneurosomes, a preparation of sealed vesicles derived from brain. The results obtained from the proposed studies will expand our understanding of mechanisms of signal integration, providing important information about the means through which complex extracellular stimuli are integrated into coordinated cellular responses. In addition, these studies will provide valuable pharmacologic tools that can be used for studying the role of PLC-MAPK interactions in various cellular responses, including plasticity, growth, and differentiation. This, in turn, will aid in our understanding of both normal and aberrant cell functioning. Thus, these studies will have significant impact on several research fields, including neurobiology, cell biology, biochemistry, and cancer biology.