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MELANOSOME PROTEIN SORTING, FOLDING & ANTIGEN PROCESSING

黑素体蛋白质排序、折叠

基本信息

批准号：
6518598
负责人：
Michael S Marks
金额：
$ 24.64万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2004-06-30
项目状态：
已结题

项目摘要

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gp 100在黑色素生物合成中起作用，组织特异性溶酶体样细胞器的膜整合蛋白叫做黑素体。黑色素细胞无法正确分类酪氨酸酶或gp 100对黑素体的作用导致发育和色素缺陷，如眼皮肤白化病。黑素体分选还可能影响对黑色素瘤的免疫反应;酪氨酸酶和gp 100是在CD 4+识别的少数人肿瘤相关抗原中，主要是组织相容性复合体（MHC）II类限制性T细胞。的这些蛋白质被分选到黑素体中的机制，黑素小体与MHC Ⅱ类抗原分选的关系路径知之甚少。我们假设这两条通路利用类似的细胞内分选机制，是MHC II类依赖性抗原呈递所必需的。在此外，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。蛋白酶体降解可增强MHC类抗原提呈 I分子。我们假设组织特异性活动调节酪氨酸酶的折叠和/或组装并影响抗原加工在黑色素细胞和非黑色素细胞中。具体目标是：1。到鉴定酪氨酸酶和GP 100中的黑素体分选决定因素。将对转染的细胞进行形态学分析，以定位细胞。具有靶向突变的全长分子或嵌合蛋白含有衍生自酪氨酸酶和GP 100的分离的拓扑结构域。与确定的决定簇相互作用的细胞蛋白质将被具有生物化学特征。 2. 描述的决定因素酪氨酸酶滞留在非黑素细胞的内质网内细胞黑素细胞和非黑素细胞之间的细胞类型差异 ER蛋白的折叠环境以及在组装和化学计量中酪氨酸酶和含有分离的酪氨酸酶的嵌合蛋白拓扑结构域将使用生物化学和遗传学方法确定，的搜索. 3.为了确定排序和折叠是否影响酪氨酸酶 (and gp 100）分别被CD 4+和CD 8 + T淋巴细胞识别。黑色素体蛋白将与黑色素瘤中的MHC分子共定位和转染的非黑素细胞。改变蛋白质的作用酪氨酸酶和GP 100在将确定特异性T细胞克隆的识别。