Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin
Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin
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DOI：
10.1093/hmg/10.25.2899
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发表时间：
2001-12-01
影响因子：
3.5
通讯作者：
Sampson, JR
中科院分区：
文献类型：
--
作者：
Hodges, AK;Li, SW;Sampson, JR
由TSC 1和TSC 2基因编码的hamartin和tuberin的关键功能可能是紧密相连的。这些蛋白质直接相互作用，影响任一基因的突变导致结节性硬化症表型。然而，相互作用的hamartin和tuberin的区域尚未得到很好的定义，它们之间的相互作用和结节性硬化症的发病机制之间的关系还没有被探索。为了解决这些问题，一系列的hamartin和tuberin构建体被用来测定在酵母双杂交系统中的相互作用。Hamartin（氨基酸302-430）和tuberin（氨基酸1-418）彼此强烈相互作用。编码假定卷曲螺旋（氨基酸346-371）的区域是必需的，但不足以介导与hamartin的相互作用，因为还需要更多的N-末端残基。预测编码卷曲螺旋的错构蛋白（氨基酸719-998）区域能够寡聚化，但对于与块茎蛋白的相互作用并不重要。在结节性硬化症患者中发现的微妙的非截短突变位于错构蛋白（N198_F199delinsI;593- 595 delACT）或结节蛋白（G294 E和I365 del）的假定结合区域内，通过酵母双杂交测定和来自Cos 7细胞的全长蛋白质的免疫共沉淀评估，消除或显著降低了蛋白质的相互作用。与此相反，在与致病TSC 2突变相同的区域中的三个非致病性的tuberin错义多态性（R261 W，M286 V，R367 Q）没有。这些结果表明，相互作用的关键生长抑制功能的hamartin和tuberin的要求。
Critical functions of hamartin and tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302-430) and tuberin (amino acids 1-418) interacted strongly with one another. A region of tuberin encoding a putative coiled-coil (amino acids 346-371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719-998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593-595delACT) or tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and tuberin.