A fragment activity assay reveals the key residues of TBC1D15 GTPase-activating protein(GAP) in Chiloscyllium plagiosum
作者单位：Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and BiomedicineCollege of Life ScienceZhejiang Sci-Tech University
会议名称：《中国生物化学与分子生物学会第十二届全国会员代表大会暨2...》
会议日期：2018年
学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学]
关 键 词：TBC1D15 Rab-GAP activity evolution substrate specificity
摘 要：GTPase-activating proteins(GAPs) with a TBC(Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases.Here,we analyzed the Rab-GAP specificity of TBC1 D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity.Sequence alignment showed that five arginine residues of the TBC1 D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark.A fragment activity assay was conducted by altering the five residues of Shark TBC1 D15-GAP to arginine,and the corresponding arginine in TBC1 D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1 D15-GAP.Our data revealed that the residues of G28,K45,K119,K122 and K221 in the Shark TBC1 D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11.Mutation of the five residues significantly altered the Shark TBC1 D15-GAP activity.These results revealed that the substrate specificity of TBC1 D15 had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals.Collectively,the data support a different mechanism of Shark TBC1 D15-GAP in substrate selection,which imitates the function of Shark TBC1 D15-GAP in marine drug development.