MOLECULAR BIOLOGY FOR SYNTHESIS OF GIANT PIGMENT GRANULES IN CONGENITAR PIGMENTARY DISEASES

本研究通过对酪氨酸酶及其相关蛋白（trp -1,2）从高尔基体（TGN）到黑素体的囊泡转运靶信号的研究，以期发现先天性色素疾病中巨色素颗粒合成的生物学和分子机制。具体来说，本研究的重点是表征低分子量gtp结合蛋白Rab7的生物学作用。为了研究酪氨酸酶家族蛋白的囊泡运输需要含有Rab7的室室，我们通过重组腺病毒表达酪氨酸酶和TRPs，并分析了在Rab7显性阴性突变体（Rab7N125I）存在或不存在的情况下，它们在人类无色素黑色素瘤细胞中的定位。与单独感染Ad-HT相比，联合感染Ad-HT和TRP-1 （Ad-TRP-1）导致酪氨酸酶活性和黑色素生成增强。在ad - ht感染的细胞中，许多新合成的酪氨酸酶蛋白在整个细胞质的Igp85阳性溶酶体颗粒中共定位，而在Rab7N125I存在的细胞质远端区域，酪氨酸酶和Igp85蛋白的共定位明显减少。在ad -TRP-1感染的细胞中，在整个细胞质中检测到TRP-1，但未在溶酶体前体（早期内体）EEA-1颗粒中共定位。然而，当Rab7N125I存在时，TRP-1保留在eea -1阳性颗粒中。我们正在分析TRP-1和酪氨酸酶在黑素小体生物合成的囊泡运输中的功能域。

This study examines the target signals for the vesicular transport of tyrosinase and its related proteins (TRP-1,2) from Golgi (TGN) to melanosomes, in the hope to identify the biological and molecular mechanism involved in the synthesis of giant pigment granules in congenital pigmentary diseases. Specifically this study focused on characterization of the biological role of a low molecular weight GTP-binding protein, Rab7. To investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and TRPs by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal Igp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization tyrosinase and Igp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected cells, TRP-1 was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules.We are in the process of analyzing the functional domains of TRP-1 and tyrosinase in this vesicular transport of the melanosome biosynthesis.