选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离...选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3+]、[M2+]和[M+]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。展开更多
HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mam- malian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial ho- meostasis. PDZ dom...HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mam- malian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial ho- meostasis. PDZ domain is one of the most important protein-protein interaction modules and is in- volved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding proper- ties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P?3, which means that the P?3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.展开更多
文摘选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3+]、[M2+]和[M+]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。
基金the National Basic Research Program (Grant No. 2004CB520804)the National High Technology Research and Development Program (Grant No. 2006AA02Z308)+1 种基金the National Natural Science Foundation of China (Grant Nos. 30270657, 30230150, and 3037030)Beijing Natural Science Foundation (Grant No. 5072037)
文摘HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mam- malian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial ho- meostasis. PDZ domain is one of the most important protein-protein interaction modules and is in- volved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding proper- ties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P?3, which means that the P?3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.
