Phosphonocarboxylates inhibit the second geranylgeranyl addition by Rab geranylgeranyl transferase.
Phosphonocarboxylates inhibit the second geranylgeranyl addition by Rab geranylgeranyl transferase.
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DOI：
10.1074/jbc.m806952200
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发表时间：
2009-03-13
期刊：
影响因子：
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通讯作者：
Seabra MC
中科院分区：
文献类型：
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作者：
Baron RA;Tavaré R;Figueiredo AC;Błazewska KM;Kashemirov BA;McKenna CE;Ebetino FH;Taylor A;Rogers MJ;Coxon FP;Seabra MC
Rab geranylgeranyl transferase （RGGT）催化Rab gtpase中（通常）两个c端半胱氨酸的翻译后geranylgeranyl （GG）修饰。在这里，我们研究了双膦酸盐类似物在一个膦酸基团被羧酸盐（磷酸羧酸盐，PC）取代的Rab香叶基-香叶酰化反应的机理。使用的磷酸羧酸盐是先前报道的3-PEHPC和2-羟基-3-咪唑[1,2-a]吡啶-3-酰基-2-磷酸丙酸（(+)-3-IPEHPC），这是一种比IC50和Ki强25倍的相关化合物。（+）-3-IPEHPC对GG焦磷酸盐（GGPP）表现为混合型抑制剂，对Rab底物表现为非竞争性抑制剂。基于以下证据，我们提出膦羧酸盐仅阻止第二次GG转移到Rabs。首先，以单个半胱氨酸基序（如CAAX）结尾的Rab蛋白的香叶基化不受抑制剂的影响，无论是在体外还是在体内。其次，在Rab-CC蛋白上添加-AAX序列可以保护底物免受抑制剂的抑制。第三，我们直接证明，在(+)-3-IPEHPC存在的情况下，Rab-CC和Rab-CXC蛋白仅被一个GG修饰。（+）-3-IPEHPC的存在导致Rab n端半胱氨酸优先被修饰，这表明在RGGT催化中，半胱氨酸的香叶酰化顺序是优先的。我们的研究结果进一步表明，该抑制剂与RGGT上的ggpp结合位点不同。我们认为膦羧酸盐抑制剂与活性位点附近的GG-半胱氨酸结合位点结合，这是使单GG- rab在第二次GG添加时对齐所必需的。这些抑制剂可能代表了一种治疗rab介导疾病的新方法。
Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.