Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity

Abstract

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.

New insights into virulence mechanisms of rice pathogen Acidovorax avenae subsp. avenae strain RS-1 following exposure to ß-lactam antibiotics

“…The full-length hcp with BamH I and EcoR I restriction sites was amplified by the designed primer pair Pt-hcp, and cloned into pET-28a + to construct the expression plasmid pET-28a + - hcp , which was transferred into E. coli BL21 (DE3) for expression of Hcp-His fusion protein. Total proteins were extracted based on the method of Lin et al 43 . After purification over a Ni-NTA-Sepharose column (Bio Basic Inc., Canada), separation by a 15% SDS-PAGE gel and confirmation by western blot using Anti-6 × His Tag rabbit polyclonal antibody as described by Yang et al 44 , the Hcp-His fusion protein was used for the immunization of rabbits to obtain polyclonal antisera.…”

Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

“…In addition, the C-terminal region of MTF-1 contains a cysteine-rich motif (C-X-C-X-C-X-C) believed to be conserved in vertebrates and has been shown to be necessary for full metal induction [8]. Recent studies have also identified a conserved SUMO-interacting motif ((V/I-X-(V/I)-(V/I)) that immediately precedes the cysteine-rich domain and appears to play important roles in regulating the subcellular localization and transcriptional activity of MTF-1 [7, 43]. A truncated MTF-1 homologue in zebrafish lacking the cysteine-rich motif typically found in vertebrates has previously been reported [5].…”

“…Recent studies have demonstrated that sumoylation of MTF-1 suppresses its transcriptional activity [7] even though it does not alter the Zn-induced nuclear translocation or MRE-binding activity of MTF-1 [7, 43]. Although the exact function of sumoylated MTF-1 is not known, it is speculated that the SUMO moiety may interact with either transcriptional suppressors or activators to regulate DNA-binding activity [43]. While a mouse MTF-1 K627R mutant was no longer able to undergo sumoylation, it did not display any reduction in transcriptional activity compared to the wild-type MTF-1 [7].…”