Protein-Protein Interactions in Melanosome Biogenesis

描述（由申请人提供）：已知蛋白质分选和囊泡运输过程中的缺陷会导致人类色素低下疾病，通常与储存池缺乏相关。虽然细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发挥作用，但参与调节黑素小体生物发生的细胞质蛋白尚未被确定。了解黑素体蛋白运输的调控也有助于理解自身免疫性色素紊乱和黑色素瘤的免疫反应。在本研究中，我们研究了黑素体膜蛋白TRP1/gp75、胞质PDZ蛋白GIPC和信号蛋白APPL在黑素体生物发生中的作用。虽然gp75是一种丰富的黑素体蛋白，被认为影响黑素体的生物发生，但它在人类皮肤色素沉着中所起的具体作用尚不清楚。我们推断胞质蛋白与gp75独特的c端序列的相互作用可能对gp75的功能很重要。我们发现了一个PDZ蛋白GIPC，它与新合成的gp75的c端和一个接头蛋白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的相互作用影响gp75的运输，并提供gp75分选和调节黑素小体生物发生的信号通路之间的联系。实验设计如下：1)我们将验证GIPC与gp75 c端相互作用在gp75转运到前黑素体中发挥作用的假设。将研究新合成的缺乏pdz结合基序和黑素体靶向信号的gp75突变蛋白的分选。2)我们将检验GIPC通过gp75棕榈酰化影响转运的假设。利用缺乏酰基载体结构域的突变体GIPC和缺乏棕榈酰化受体位点的突变体gp75，将评估gp75被GIPC棕榈酰化以及棕榈酰化在gp75运输中的作用。3)我们将验证GIPC与gp75的相互作用通过APPL在gp75分选与磷脂酰肌醇3-激酶(PI3K)/Akt通路之间提供联系的假设。我们将利用PI3激酶抑制剂和缺乏Akt结合基元的突变APPL蛋白的表达来评估PI3激酶/Akt信号通路在gp75转运和黑素小体生物发生中的作用。

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.