Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.
Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.
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DOI：
10.1021/bi3001853
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发表时间：
2012-05-08
期刊：
影响因子：
2.9
通讯作者：
Glover KJ
中科院分区：
文献类型：
--
作者：
Rieth MD;Lee J;Glover KJ
小窝蛋白-1是在小窝中发现的最重要的蛋白质，小窝是作为信号平台的质膜的细胞表面内陷。小窝蛋白-1跨膜结构域中的单点突变（脯氨酸132变为亮氨酸）对体内小窝形成具有有害影响，并且与各种疾病状态，特别是侵袭性乳腺癌有关。使用凝胶过滤色谱和分析超离心的组合，我们发现，一个全功能的结构小窝蛋白-1（Cav 162 -178）是一个单体的十二烷基磷酸胆碱胶束。相反，Cav 162 -178的P132 L突变体是二聚体。为了进一步探索P132 L突变体的二聚化，制备了各种截短的构建体（Cav 182 -178、Cav 196 -178、Cav 162 -136、Cav 182 - 136、Cav 196 -136），其揭示了在小窝蛋白-1的跨膜结构域（残基96-136）中发生寡聚化。为了表征突变体的结构，对Cav 196 -136 P132 L突变体进行了溶血肉豆蔻酰磷脂酰甘油中的溶液状态NMR实验。化学位移分析显示，与野生型相比，跨膜结构域中的螺旋2延长了4个残基（野生型，残基111 - 129;突变体，残基111-133），这对应于突变体的螺旋2中的额外转角。最后，Cav 162 -178的位置132处的点突变（P132 A、P132 I、P132 V、P132 G、P132 W、P132 F）揭示没有其它疏水性氨基酸可以保持Cav 162 -178的单体状态，这表明脯氨酸132在支持适当的小窝蛋白-1行为中是关键的。
Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo, and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation we found that a fully-functional construct of caveolin-1 (Cav162–178) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav162–178 was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav182–178, Cav196–178, Cav162–136, Cav182–136, Cav196–136) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96–136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav196–136 P132L mutant. Chemical shift analysis revealed that compared to the wild type, helix 2 in the transmembrane domain was lengthened by four residues (wild type, residues 111 to 129; mutant, residues 111–133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav162–178 (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav162–178 which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.