Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays.
Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays.
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DOI：
10.1006/abbi.2000.1874
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发表时间：
2000-07
影响因子：
3.9
通讯作者：
C. Rao-Naik;J. Chandler;B. McArdle;J. Callis
中科院分区：
文献类型：
--
作者：
C. Rao-Naik;J. Chandler;B. McArdle;J. Callis
利用针对泛素特异性蛋白酶（UBP）半胱氨酸（Cys）盒的寡核苷酸，从拟南芥花mRNA中鉴定出一种新的UBP（AtUBP 5）的cDNA。924个氨基酸的AtUBP 5包含所有UBP的特征区域，并且与哺乳动物UBP（Unp）总体上具有35%的同一性和53%的相似性，这是由于这些区域之外的额外显著相似性。AtUBP 5与两个未表征的拟南芥基因组序列具有48%的同一性和58%的相似性，但在UBP保守区之外与其他两个先前发表的拟南芥UBP AtUBP 3和AtUBP-4不同。使用体内大肠杆菌检测，这使得共同表达的GSTAtUBP和基板，我们表明，所有三个UBP是活跃的。AtUBP 5在活性位点半胱氨酸的N-末端没有311个氨基酸，或在Cys和His盒之间没有233个非保守氨基酸，或两者都没有，表明核心区域是足够的。在体内和体外测定中，GSTAtUBP 3、-4和-5表现出对特定Ub-Ub键的偏好，表明可及性和/或构象是重要的，并证明这些酶在裂解后裂解。由AtUBP 5 Cys盒与AtUBP 3氨基酸组成的嵌合UBP是活性的，并表现出AtUBP 3特异性，表明UBP的模块化性质和切割位点的特异性不由Cys盒决定。
A cDNA for a new ubiquitin-specific protease (UBP), AtUBP5, was identified from Arabidopsis thaliana flower mRNA using an oligonucleotide made against the conserved UBP cysteine (Cys) box. The 924-amino-acid AtUBP5 contains the regions characteristic of all UBPs and has 35% identity and 53% similarity overall to a mammalian UBP (Unp), resulting from additional significant similarity outside these regions. AtUBP5 has 48% identity and 58% similarity overall to two uncharacterized Arabidopsis genomic sequences but is distinct outside the UBP conserved regions from two other previously published Arabidopsis UBPs, AtUBP3 and -4. Using in vivo Escherichia coli assays, which allow co-expression of GSTAtUBPs and substrates, we show that all three UBPs were active. AtUBP5 was active without 311 amino acids N-terminal to the active site cysteine, or without 233 nonconserved amino acids between the Cys and His boxes, or without both, indicating the core region was sufficient. In in vivo and in vitro assays, GSTAtUBP3, -4, and -5 exhibited preference for specific Ub-Ub linkages, suggesting accessibility and/or conformation is important and demonstrating that these enzymes cleave post-translationally. A chimeric UBP consisting of the AtUBP5 Cys box with AtUBP3 amino acids was active and exhibited AtUBP3 specificity, indicating that the modular nature of UBPs and specificity for cleavage sites is not determined by the Cys box.