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SmgGDS-607 Regulation of RhoA GTPase Prenylation Is Nucleotide-Dependent
Biochemistry ( IF 2.9 ) Pub Date : 2018-06-25 00:00:00 , DOI: 10.1021/acs.biochem.8b00567 Benjamin C. Jennings 1 , Alexis J. Lawton 1 , Zeinab Rizk 1 , Carol A. Fierke 1
Biochemistry ( IF 2.9 ) Pub Date : 2018-06-25 00:00:00 , DOI: 10.1021/acs.biochem.8b00567 Benjamin C. Jennings 1 , Alexis J. Lawton 1 , Zeinab Rizk 1 , Carol A. Fierke 1
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Protein prenylation involves the attachment of a hydrophobic isoprenoid moiety to the C-terminus of proteins. Several small GTPases, including members of the Ras and Rho subfamilies, require prenylation for their normal and pathological functions. Recent work has suggested that SmgGDS proteins regulate the prenylation of small GTPases in vivo. Using RhoA as a representative small GTPase, we directly test this hypothesis using biochemical assays and present a mechanism describing the mode of prenylation regulation. SmgGDS-607 completely inhibits RhoA prenylation catalyzed by protein geranylgeranyltransferase I (GGTase-I) in an in vitro radiolabel incorporation assay. SmgGDS-607 inhibits prenylation by binding to and blocking access to the C-terminal tail of the small GTPase (substrate sequestration mechanism) rather than via inhibition of the prenyltransferase activity. The reactivity of GGTase-I with RhoA is unaffected by addition of nucleotides. In contrast, the affinity of SmgGDS-607 for RhoA varies with the nucleotide bound to RhoA; SmgGDS-607 has a higher affinity for RhoA-GDP compared to RhoA-GTP. Consequently, the prenylation blocking function of SmgGDS-607 is regulated by the bound nucleotide. This work provides mechanistic insight into a novel pathway for the regulation of small GTPase protein prenylation by SmgGDS-607 and demonstrates that peptides are a good mimic for full-length proteins when measuring GGTase-I activity.
中文翻译：
SmgGDS-607 RhoA GTPase异戊烯化的调节是核苷酸依赖性的
蛋白质异戊二烯化涉及疏水性类异戊二烯部分与蛋白质C末端的连接。几个小的GTPases，包括Ras和Rho亚家族的成员，由于其正常和病理功能而需要异戊二烯化。最近的研究表明，SmgGDS蛋白在体内调节小型GTPases的异戊烯基化。使用RhoA作为代表性的小GTP酶，我们使用生化测定法直接测试了这一假设，并提出了描述异戊二烯调节模式的机制。SmgGDS-607在体外完全抑制蛋白Geranylgeranyltransferase I（GGTase-I）催化的RhoA异戊二烯化放射性标记掺入分析。SmgGDS-607通过与小GTPase的C末端尾部结合并阻止其进入（底物螯合机制），而不是通过抑制异戊二烯基转移酶活性来抑制异戊二烯基化。GGTase-I与RhoA的反应性不受核苷酸添加的影响。相反，SmgGDS-607对RhoA的亲和力随与RhoA结合的核苷酸而变化；与RhoA-GTP相比，SmgGDS-607对RhoA-GDP的亲和力更高。因此，SmgGDS-607的异戊二烯阻断功能受结合的核苷酸调节。这项工作为通过SmgGDS-607调节小GTPase蛋白异戊二烯化的新途径提供了机械学见解，并证明了当测量GGTase-I活性时，肽是全长蛋白的良好模拟物。
更新日期：2018-06-25
中文翻译：
SmgGDS-607 RhoA GTPase异戊烯化的调节是核苷酸依赖性的
蛋白质异戊二烯化涉及疏水性类异戊二烯部分与蛋白质C末端的连接。几个小的GTPases，包括Ras和Rho亚家族的成员，由于其正常和病理功能而需要异戊二烯化。最近的研究表明，SmgGDS蛋白在体内调节小型GTPases的异戊烯基化。使用RhoA作为代表性的小GTP酶，我们使用生化测定法直接测试了这一假设，并提出了描述异戊二烯调节模式的机制。SmgGDS-607在体外完全抑制蛋白Geranylgeranyltransferase I（GGTase-I）催化的RhoA异戊二烯化放射性标记掺入分析。SmgGDS-607通过与小GTPase的C末端尾部结合并阻止其进入（底物螯合机制），而不是通过抑制异戊二烯基转移酶活性来抑制异戊二烯基化。GGTase-I与RhoA的反应性不受核苷酸添加的影响。相反，SmgGDS-607对RhoA的亲和力随与RhoA结合的核苷酸而变化；与RhoA-GTP相比，SmgGDS-607对RhoA-GDP的亲和力更高。因此，SmgGDS-607的异戊二烯阻断功能受结合的核苷酸调节。这项工作为通过SmgGDS-607调节小GTPase蛋白异戊二烯化的新途径提供了机械学见解，并证明了当测量GGTase-I活性时，肽是全长蛋白的良好模拟物。
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