MELANOSOME PROTEIN SORTING, FOLDING & ANTIGEN PROCESSING

黑素体蛋白质排序、折叠

• 批准号: 6384745
• 负责人: Michael S Marks
• 金额: $ 23.92万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384745
• 关键词: CD4 molecule; CD8 molecule; HeLa cells; MHC class I antigen; MHC class II antigen; T lymphocyte; affinity chromatography; antigen presentation; chemical binding; chimeric proteins; crosslink; endoplasmic reticulum; fluorescence microscopy; glycoproteins; immunoelectron microscopy; melanocyte; melanosomes; membrane proteins; molecular shape; monophenol monooxygenase; neoplastic cell culture for noncancer research; protein binding; protein folding; protein transport; western blottings

Tyrosinase and gp100 function in melanin biosynthesis and are resident integral membrane proteins of a tissue-specific, lysosome-like organelle called the melanosome. Failure of melanocytes to properly sort tyrosinase or gp100 to the melanosome results in developmental and pigment defects such as oculocutaneous albinism. Melanosomal sorting may also affect immune responses to melanoma; tyrosinase and gp100 are among the few human tumor-associated antigens recognized by CD4+, major histocompatibility complex (MHC) class II-restricted T cells. The mechanisms by which these proteins are sorted to melanosomes and the relationship between melanosomal and MHC class II antigen sorting pathways are poorly understood. We hypothesize that these two pathways utilize similar intracellular sorting mechanisms and that proper sorting is necessary for MHC class II-dependent antigen presentation. In addition, tyrosinase is retained within the endoplasmic reticulum (ER) and degraded by the proteasome under certain developmental conditions. Proteasomal degradation may enhance antigen presentation by MHC class I molecules. We hypothesize that tissue-specific activities regulate the folding and/or assembly of tyrosinase and affect antigen processing in melanic and non-melanic cells. The Specific Aims are: 1. To identify melanosomal sorting determinants within tyrosinase and gp100. Transfected cells will be analyzed morphologically for localization of full-length molecules with targeted mutations or of chimeric proteins containing isolated topologic domains derived from tyrosinase and gp100. Cellular proteins that interact with identified determinants will be characterized biochemically. 2. To characterize the determinants of tyrosinase retention within the endoplasmic reticulum of non-melanic cells. Cell type differences between melanic and non-melanic cells in the ER protein folding environment and in the assembly and stoichiometry of tyrosinase and chimeric proteins containing isolated tyrosinase topologic domains will be determined using biochemical and genetic criteria. 3. To determine whether sorting and folding affect tyrosinase (and gp100) recognition by CD4+ and CD8+ T lymphocytes, respectively. Melanosomal proteins will be colocalized with MHC molecules in melanoma and transfected non-melanic cells. The effect of altering the protein sorting and/or retention properties of tyrosinase and gp100 on recognition by specific T cell clones will be determined.

酪氨酸酶和gp100在黑色素生物合成中的作用 组织特异性溶酶体样细胞器的完整膜蛋白 被称为黑素小体。黑素细胞不能正确分类 酪氨酸酶或gp100对黑素小体的作用导致发育和 色素缺陷，如眼皮肤白化病。黑素小体分类 也可能影响对黑色素瘤的免疫反应；酪氨酸酶和gp100 在为数不多的由CD4+识别的人类肿瘤相关抗原中， 组织相容性复合体(MHC)II类限制性T细胞。这个 这些蛋白质被分选到黑素小体和 黑素瘤与MHC-II类抗原分选的关系 人们对这些途径知之甚少。我们假设这两条路径 利用类似的细胞内分选机制和适当的分选 是MHC II类依赖抗原提呈所必需的。在……里面 此外，酪氨酸酶保留在内质网(ER)内 并在一定的发育条件下被蛋白酶体降解。 蛋白酶体降解可能通过MHC类增强抗原提呈 I分子。我们假设组织特异性活动调节 酪氨酸酶的折叠和/或组装并影响抗原处理 在黑色素和非黑色素细胞中。具体目标是：1. 确定酪氨酸酶和gp100中的黑素体分选决定因素。 将对转基因细胞进行形态分析，以定位 具有靶向突变或嵌合蛋白的全长分子 包含源自酪氨酸酶和gp100的分离拓扑域。 与已确定的决定因素相互作用的细胞蛋白将是 以生化为特征的。2.确定决定因素的特征 酪氨酸酶在非黑色素患者内质网中的滞留 细胞。黑色素性细胞与非黑色素性细胞类型的差异 ER蛋白的折叠环境以及在组装和化学计量学中的作用 酪氨酸酶和含有分离酪氨酸酶的嵌合蛋白 拓扑域将使用生化和遗传方法确定 标准。3.确定排序和折叠是否影响酪氨酸酶 (和gp100)分别由CD4+和CD8+T淋巴细胞识别。 黑色素体蛋白将与MHC分子在黑色素瘤中共存 并将其转化为非黑色素细胞。改变蛋白质的效果 酪氨酸酶和gp100的分选和/或保留特性 将确定特定T细胞克隆的识别能力。