Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin
Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin
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DOI：
10.1074/jbc.m113.497917
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发表时间：
2013-10-18
影响因子：
4.8
通讯作者：
Fricker, Lloyd D.
中科院分区：
文献类型：
--
作者：
Berezniuk, Iryna;Lyons, Peter J.;Fricker, Lloyd D.
胞质羧肽酶5（CCP 5）是从微管蛋白切割C-末端和/或侧链氨基酸的酶亚家族的成员。基于涉及CCP 5在细胞系中过表达和用抗血清检测微管蛋白形式的研究，提出CCP 5选择性切割谷氨酰化微管蛋白的分支点。在本研究中，我们研究了纯化的CCP 5对合成肽以及可溶性和微管蛋白和紫杉醇稳定的微管的活性，使用抗血清和质谱的组合来检测产品。小鼠CCP 5从猪脑微管蛋白的侧链去除多个谷氨酸残基和分支点谷氨酸。此外，CCP 5从脱酪氨酸微管蛋白切除C-末端谷氨酸。该酶还从紫杉醇稳定的微管的侧链和C末端去除多个谷氨酸残基。CCP 5缩短并从对应于3-微管蛋白的C-末端区域的合成肽中除去侧链谷氨酸，而胞质羧肽酶1缩短侧链而不切割肽连接的残基。通过CCP 5裂解键的速率比去除单个连接的谷氨酸残基的速率慢得多。总的来说，我们的数据表明，CCP 5的功能作为一个双功能的脱谷氨酰胺酶裂解和连接的谷氨酸从微管蛋白。
Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble - and -tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain - and -tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated -tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of 3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' -linked residues. The rate of cleavage of linkages by CCP5 is considerably slower than that of removal of a single -linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both - and -linked glutamate from tubulin.