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

Abstract

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, the 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 HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library which displayed 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/proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 12 and 22 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, Histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low μM KD 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-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.

Prostate tumour overexpressed-1 promotes tumourigenicity in human breast cancer via activation of Wnt/β-catenin signalling

“…Interestingly, this study revealed that PTOV1 represses expression of DKK1 by decreasing histone acetylation of the DKK1 promoter. However, we demonstrated that PTOV1 recruits HDACs to the DKK1 promoter via forming a complex with the HDACs, which is consistent with previous reports that PTOV1 is a binding partner of HDACs [22,24]. Based on our observations and other studies, we hypothesize that PTOV1 may coordinate with other factors, such as CBX7, to modulate the balance between DKK1 promoter histone acetylation and deacetylation, and thereby control the cellular expression of DKK1.…”

Deubiquitylases From Genes to Organism

“…However, it is possible that the ZnF-UBP domain also presents a protein interaction module for the 72 other human proteins which possess COOH-terminal di-Gly motifs. One interesting member of this list is histone H4, which could serve to recruit both USP3 and USP16 to histone complexes, where they have been proposed to deubiquitylate histone H2A (87,110,183 p53 activation, as embryonic development is extended in USP7/p53 double-knockout embryos (124,125).…”

Solid-phase synthesis of C-terminal peptide libraries for studying the specificity of enzymatic protein prenylation

“…2 and Scheme S2) is based on the pioneering work of Kania, Zuckermann and Marlowe 7 and others. 8,9 A four residue (BBLL) linker was initially added onto amine-functionalized solid surfaces followed by coupling to the γ -carboxyl group of Fmoc-Glu-ODmab; this resulted in the installation of an orthogonally (Dmab) protected acid that could be used for subsequent on-resin cyclization. Fmoc removal and coupling of an HMPA linker followed by ester bond formation produced a depsipeptide containing an acid-labile ester bond.…”