Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation.
Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation.
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DOI：
10.1093/hmg/ddp567
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发表时间：
2010-03-15
影响因子：
3.5
通讯作者：
Taylor JP
中科院分区：
文献类型：
--
作者：
McCray BA;Skordalakes E;Taylor JP
Rab gtp酶是一种分子开关，通过在活跃的gtp结合形式和不活跃的gdp结合形式之间循环，协调囊泡运输、成熟和融合。活性周期与GTP水解相耦合，并受到调节蛋白的严格控制。GTPase Rab7错义突变通过未知机制导致显性遗传性轴突变性，称为Charcot-Marie-Tooth型2B。我们展示了GTP结合的L129F突变体Rab7的2.8 Å晶体结构，显示了效应结合区和催化位点的正常构象，但核苷酸结合袋的改变预计会改变GTP的结合。通过广泛的生化分析，我们证明Rab7的疾病相关突变不会导致内在的GTPase缺陷，但允许不受调节的核苷酸交换导致过度激活和不依赖于水解的失活。与增强的活性一致，突变体Rab7与效应蛋白子集的相互作用显着增强。此外，动态成像显示突变的Rab7异常地保留在靶膜上。然而，我们发现突变体Rab7激活的增加被不受调节的、不依赖于GTP水解的膜循环所抵消。值得注意的是，疾病突变能够挽救gtpase缺陷突变体的膜循环。因此，我们证明了疾病突变将Rab7从通常由调节蛋白施加的空间和时间控制中分离出来，并不是通过获得新的毒性功能，而是通过对天然Rab7活性的错误调节导致疾病。
Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.
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影响因子：
9.8
作者：
Cardoso, C;Leventer, RJ;Ledbetter, DH
通讯作者：
Ledbetter, DH
影响因子：
4
作者：
Jäger, S;Bucci, C;Eskelinen, EL
通讯作者：
Eskelinen, EL
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
30.8
作者：
D'Adamo, P;Menegon, A;Toniolo, D
通讯作者：
Toniolo, D
DOI：
10.1083/jcb.131.6.1435
发表时间：
1995-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Feng Y;Press B;Wandinger-Ness A
通讯作者：
Wandinger-Ness A