STRUCTURE FUNCTION AND REGULATION OF GAP JUNCTION PROTEINS

我们的中心目标是了解缝隙连接的化学调节的分子基础及其在细胞内的作用。 对细胞行为的影响。该项目自年成立以来一直是计划项目赠款的一部分。 1990.最近，我们已经表明，直接的，pH值依赖的分子内的羧基之间的关联 末端（CT）结构域和Cx43的胞质环的第二半。我们认为特定的肽 可以被设计为干扰这种相互作用并调节Cx43调节。我们使用了高通量 噬菌体展示试验以鉴定与Cx43 CT结合的肽。我们发现Cx43 CT 优先与含有特定“RXP”基序的肽结合。几种RXP肽与Cx43 CT结合 具有微摩尔亲和力，导致Cx43 CT结构域的结构修饰并修饰Cx43通道 功能和调节。本提案的目标是进一步表征这些肽并寻找 类似的序列可以调节Cx43通道的功能。具体目标是：目标1。到 表征RXP/Cx43 CT复合物的高级结构。目标2）描述RXP的影响- 1和RXP-4对Cx43通道的调节作用。目标3）要确定调解的结构性限制， RXP肽与Cx43 CT的结合及其对Cx43通道的功能作用。我们将使用迭代 光谱方法（核磁共振、表面等离子体共振）的组合， 蛋白质组学方法（高通量偏向噬菌体展示;肽合成）和功能测定， 开发了一种结构-活性相关的肽，具有更高的亲和力和选择性的功能效果。 这些肽将有可能成为操纵Cx43通道及其调节的探针。这些 这些研究将有助于更好地了解连接蛋白在健康和疾病中的作用。

Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its implications on cell behavior. This project has been a part of the Program Project Grant since its inception in 1990. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl terminal (CT) domain and the second half of the cytoplasmic loop of Cx43. We propose that specific peptides can be designed to interfere with this interaction and modulate Cx43 regulation. We have used a highthroughput phage display assay to identify peptides that bind to Cx43CT. We found that Cx43CT preferentially bound to peptides containing a specific "RXP" motif. Several RXP peptides bind to Cx43CT with micromolar affinity, cause structural modifications in the Cx43CT domain and modify Cx43 channel function and regulation. It is the goal of this proposal to further characterize these peptides and search for analogous sequences which may modulate the function of Cx43 channels. The specific aims are: Aim 1.) To characterize the high-order structure of RXP/Cx43CT complexes. Aim 2.) To characterize the effects of RXP- 1 and RXP-4 on the regulation of Cx43 channels. Aim 3.) To identify the structural constraints mediating the binding of RXP peptides to Cx43CT and their functional effects on Cx43 channels. We will use an iterative combination of spectroscopic methods (Nuclear Magnetic Resonance, Surface Plasmon sonance), proteomics approaches (high-throughput biased phage display; peptide synthesis) and functional assays to develop a structural-activity correlate toward peptides with higher affinity and selective functional effects. These peptides will potentially be probes for the manipulation of Cx43 channels and their regulation. These studies will lead to a better understanding of the role of connexins in health and disease.