Protein-Protein Interactions in Melanosome Biogenesis

黑素体生物发生中的蛋白质-蛋白质相互作用

• 批准号: 6610184
• 负责人: Vijayasaradhi Setaluri
• 金额: $ 24.44万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610184
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; acyl carrier protein; biological signal transduction; biosynthesis; confocal scanning microscopy; endoplasmic reticulum; enzyme inhibitors; fatty acylation; immunofluorescence technique; melanins; melanocyte; melanosomes; membrane proteins; palmitates; phosphatidylinositol 3 kinase; pigmentation; plasmids; protein localization; protein protein interaction; protein sequence; protein structure function; protein transport; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant): Defects in protein sorting and vesicular trafficking processes are known to result in human hypopigmentary disorders often associated with storage pool deficiency. Although cytosolic proteins and their interactions with organelle proteins appear to play a role in these processes, cytosolic proteins involved in the regulation of melanosome biogenesis have not been identified. Understanding the regulation of trafficking of melanosomal proteins has implications also for understanding autoimmune pigmentary disorders and immune responses in melanoma. In this proposal, we address the roles of melanosomal membrane protein TRP1/gp75, cytosolic PDZ protein GIPC and signaling protein APPL in melanosome biogenesis. Although gp75, an abundant melanosomal protein, is thought to influence biogenesis of melanosomes, specific role(s) it plays in human skin pigmentation is not known. We reasoned that interaction of cytosolic proteins with the unique C-terminal sequences of gp75 may be important for functions of gp75. We identified a PDZ protein GIPC that interacts with the C-terminus of newly synthesized gp75 and with an adaptor protein APPL. APPL, in turn, binds protein kinase B/Akt. Our working hypothesis is that interaction of GIPC with gp75 influences trafficking of gp75 and provides a link between gp75 sorting and signaling pathways that regulate melanosome biogenesis. The following experiments are designed: 1) We will test the hypothesis that interaction of GIPC with gp75 C-terminus plays a role in gp75 trafficking to premelanosomes. Sorting of newly synthesized mutant gp75 proteins lacking PDZ-binding motif and melanosomal targeting signal will be investigated. 2) We will test the hypothesis that GIPC influences trafficking by palmitoylation of gp75. Palmitoylation of gp75 by GIPC and the role of palmitoylation in gp75 trafficking will be assessed using mutant GIPC lacking acyl carrier domain and mutant gp75 protein lacking palmitoylation acceptor site. 3) We will test the hypothesis that interaction of GIPC with gp75 provides a link, via APPL, between gp75 sorting and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We will utilize inhibitors of PI3 kinase and expression of mutant APPL proteins lacking Akt binding motif to evaluate the role of PI3 kinase/Akt signaling pathway in gp75 trafficking and melanosome biogenesis.

描述（由申请人提供）：已知蛋白质分类和囊泡运输过程的缺陷会导致人类不足疾病通常与储存池缺乏有关。尽管胞质蛋白及其与细胞器蛋白的相互作用似乎在这些过程中起作用，但尚未鉴定出参与调节黑色素体生物发生的胞质蛋白。了解黑色素蛋白的运输调节也对了解黑色素瘤中自身免疫性色素疾病和免疫反应也具有影响。在此提案中，我们解决了黑色素体膜蛋白TRP1/GP75，胞质PDZ蛋白GIPC和信号蛋白Appl在黑色素体生物发生中的作用。尽管GP75是一种丰富的黑色素体蛋白，被认为会影响黑色素体的生物发生，但它在人类皮肤色素沉着中扮演的特定作用尚不清楚。我们认为，胞质蛋白与GP75独特的C末端序列的相互作用对于GP75的功能可能很重要。我们确定了与新合成的GP75的C末端相互作用的PDZ蛋白GIPC，并与衔接蛋白APPL相互作用。 Appl反过来结合蛋白激酶B/AKT。我们的工作假设是，GIPC与GP75的相互作用会影响GP75的运输，并在调节黑色素体生物发生的GP75分类和信号传导途径之间提供了联系。设计了以下实验：1）我们将检验以下假设：GIPC与GP75 C末端的相互作用在GP75运输中起作用。将研究缺乏PDZ结合基序和黑色素体靶向信号的新合成突变体GP75蛋白的排序。 2）我们将检验以下假设：GIPC通过GP75的棕榈酰化影响贩运。 GIPC对GP75的棕榈酰化以及棕榈酰化在GP75贩运中的作用将使用缺乏酰基载体结构域的突变GIPC和缺乏棕榈酰化受体部位的突变体GP75蛋白进行评估。 3）我们将检验以下假设：GIPC与GP75的相互作用在GP75排序和磷脂酰肌醇3-激酶（PI3K）/AKT途径之间提供了链接。我们将利用PI3激酶的抑制剂以及缺乏AKT结合基序的突变型APP蛋白的表达来评估PI3激酶/AKT信号通路在GP75运输和黑色素体生物发生中的作用。