Protein-Protein Interactions in Melanosome Biogenesis

描述(由申请人提供)：已知蛋白质分类和囊泡运输过程中的缺陷会导致人类发育不良障碍，通常与储存池不足有关。尽管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了作用，但参与调节黑素小体生物发生的细胞质蛋白尚未被鉴定。了解黑色素体蛋白运输的调节对于理解黑色素瘤的自身免疫性色素紊乱和免疫反应也具有重要意义。在这项建议中，我们讨论了黑素体膜蛋白TRP1/gp75、胞浆PDZ蛋白GIPC和信号蛋白APPL在黑素小体生物发生中的作用。虽然gp75是一种丰富的黑素小体蛋白，被认为影响黑素小体的生物发生，但它在人类皮肤色素沉着中扮演的具体角色(S)尚不清楚。我们推测胞浆蛋白与gp75独特的C末端序列的相互作用对gp75的功能可能是重要的。我们鉴定了一个PDZ蛋白GIPC，它与新合成的gp75的C末端相互作用，并与一个接头蛋白APPL相互作用。APPL进而与蛋白激酶B/Akt结合。我们的工作假设是GIPC与gp75的相互作用影响gp75的运输，并在gp75分选和调节黑素小体生物发生的信号通路之间提供了联系。实验设计如下：1)我们将检验GIPC与gp75 C末端相互作用在gp75转运到黑素前体中的作用这一假设。新合成的缺乏PDZ结合基序和黑素体靶向信号的突变体gp75蛋白的筛选将被研究。2)我们将检验GIPC通过gp75棕榈酰化影响运输的假设。利用缺失酰基载体结构域的突变体GIPC和缺失棕榈酰化受体位点的突变体gp75蛋白，研究GIPC对gp75的棕榈酰化作用及其在gp75转运中的作用。3)我们将检验这样一种假设，即GIPC与gp75的相互作用通过APPL在gp75分选和磷脂酰肌醇3-激酶(PI3K)/Akt通路之间提供了联系。我们将利用PI3激酶的抑制剂和缺乏Akt结合基序的突变型APPL蛋白的表达来评估PI3激酶/Akt信号通路在gp75运输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.