Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients
Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients
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DOI：
10.1182/blood-2002-09-2789
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发表时间：
2003-04-01
期刊：
影响因子：
20.3
通讯作者：
de Saint Basile, G
中科院分区：
文献类型：
--
作者：
Ménasché, G;Feldmann, J;de Saint Basile, G
Rab27 a是Rab家族的一员，迄今为止是第一个与人类疾病（即Griscelli综合征2型）相关的成员。Rab27a基因的突变导致色素以及细胞毒性颗粒转运缺陷，解释了部分白化病和严重免疫紊乱的特征。它们为指定Rab蛋白的关键结构和功能残基提供了独特的机会。我们在这里表明，在2中观察到的α 4（Alal52Pro）或β 5（Leu130Pro）环中引入脯氨酸残基。这些自发突变体，显着影响鸟苷三磷酸（GTP）和鸟苷二磷酸（GDP）的核苷酸结合活性的Rab27 a，可能是通过破坏蛋白质折叠。第三个突变体Trp73Gly位于开关界面处的一个不变的疏水三联体内，并且先前显示在活性Rab3A中介导rabphilin3A效应器相互作用。Trp73Gly显示出与组成型活性突变体Gln78Leu相同的核苷酸结合和GTdR特性。然而，与Gln78Leu相反，Trp73Gly突变体构建体既不与Rab27a效应因子亲黑素相互作用，也不改变黑素体分布和细胞毒性颗粒胞吐作用。在73位引入的取代，包括Ras中存在的亮氨酸残基，并没有恢复Rab 27 a蛋白功能。两者合计，我们的研究结果表征了Rab蛋白的新的关键残基，并确定了Rab27a的Trp73残基作为与Rab27a的特异性效应物相互作用的关键位置，无论是在黑素细胞还是细胞毒性细胞中。
Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated With a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well it as cytotoxic granule transport defects, accounting for the partial albinism And severe immune disorder characteristics of this syndromes So far, 3 Rab27a missense mutations have been identified. they open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha4 (Alal52Pro) or beta5 (Leu130Pro) loop, observed in 2. of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within An invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding And GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.