HDAC6 and Ubp-M BUZ Domains Recognize Specific C-Terminal Sequences of Proteins

被引:24
作者
Hard, Ryan L. [1 ,2 ]
Liu, Jiangxin [3 ]
Shen, Juan [1 ,2 ]
Zhou, Pei [3 ]
Pei, Dehua [1 ,2 ]
机构
[1] Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA
[2] Ohio State Univ, Dept Chem, Columbus, OH 43210 USA
[3] Duke Univ, Med Ctr, Dept Biochem, Durham, NC 27710 USA
基金
美国国家卫生研究院;
关键词
PARTIAL EDMAN DEGRADATION; BOUND PEPTIDES; UBIQUITIN; POLYUBIQUITIN; MOTIF; TRANSCRIPTION; PROTEOLYSIS; COMPLEX; MODULE; ROLES;
D O I
10.1021/bi101014s
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of I-I DAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and 11 DAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT 10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the H DAC6 BUZ domain with low micromolar K-D values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.
引用
收藏
页码:10737 / 10746
页数:10
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