Calcium Ion Responsive DNA Binding in a Zinc Finger Fusion Protein

Onoda, Akira; Arai, N; Shimazu, Naoto; Yamamoto, H.; Yamamura, Takeshi

doi:10.1021/ja052477m

Cited by 15 publications

(7 citation statements)

References 60 publications

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“…To further investigate the regulation of OmpA by SmpB, we monitored the fluorescence activities of the P ompA and EGFP translational fusion in the presence or absence of SmpB expression. It has been reported that divalent metal ion Mg 2+ or Ca 2+ stabilized the molecule conformation and affected interactions through neutralizing the negative charges ( Lusetti et al, 2003 ; Onoda et al, 2005 ; Ralec et al, 2017 ; Kim et al, 2018 ). Hence, divalent metal ion Mg 2+ or Ca 2+ was added and the results demonstrated all the cells maintained constant growth ( Figure 2A ), while the cells expressing SmpB showed the significant fluorescent enhancement when supplemented with 5 mM Mg 2+ compared with the controls ( Figure 2B ), signifying Mg 2+ may help to stabilize the binding of protein and DNA structure.…”

Section: Resultsmentioning

confidence: 99%

Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway

Liu

Chang

et al. 2022

Front. Microbiol.

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The small protein B (SmpB), previously acting as a ribosome rescue factor for translation quality control, is required for cell viability in bacteria. Here, our study reveals that SmpB possesses new function which regulates the expression of outer membrane protein A (ompA) gene as a transcription factor in Aeromonas veronii. The deletion of SmpB caused the lower transcription expression of ompA by Quantitative Real-Time PCR (qPCR). Electrophoretic mobility shift assay (EMSA) and DNase I Footprinting verified that the SmpB bound at the regions of −46 to −28 bp, −18 to +4 bp, +21 to +31 bp, and +48 to +59 bp of the predicted ompA promoter (PompA). The key sites C52AT was further identified to interact with SmpB when PompA was fused with enhanced green fluorescent protein (EGFP) and co-transformed with SmpB expression vector for the fluorescence detection, and the result was further confirmed in microscale thermophoresis (MST) assays. Besides, the amino acid sites G11S, F26I, and K152 in SmpB were the key sites for binding to PompA. In order to further develop peptide antimicrobial agents, the peptide aptamer PA3 was screened from the peptide aptamer (PA) library by bacterial two-hybrid method. The drug sensitivity test showed that PA3 effectively inhibited the growth of A. veronii. In summary, these results demonstrated that OmpA was a good drug target for A. veronii, which was regulated by the SmpB protein and the selected peptide aptamer PA3 interacted with OmpA protein to disable SmpB-OmpA signal pathway and inhibited A. veronii, suggesting that it could be used as an antimicrobial agent for the prevention and treatment of pathogens.

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Section: Resultsmentioning

confidence: 99%

Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway

Liu

Chang

et al. 2022

Front. Microbiol.

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“…The bands were quantified with FUJI FILM Science Lab 2001 Image Gauge software. The binding ratio of FLAG-p53 to target DNA was calculated using equation R = I b /( I b + I f ), where I b and I f are the intensities of FLAG-p53-bound and free DNA bands, respectively [50, 51]. …”

Section: Methodsmentioning

confidence: 99%

Evaluation of Zinc (II) chelators for inhibiting p53-mediated apoptosis

et al. 2013

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In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53-null cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

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“…The three‐dimensional structure of the peptide was calculated from the experimental constraints using the simulated annealing method with XPLOR (version 3.851)32. The calculation was performed according to a similar procedure described previously 33, 34. The structures and structural parameters were analyzed using PROCHECK‐NMR (Table 2)35.…”

Section: Methodsmentioning

confidence: 99%

Minimal motif peptide structure of metzincin clan zinc peptidases in micelles

Onoda

Suzuki

Ishizuka

et al. 2009

Journal of Peptide Science

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It is well known that the functions of metalloproteins generally originate from their metal-binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal-binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc-binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc-binding sequence His-Glu-Xaa-Xaa-His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin-1 and investigated the folding properties of the Trp-labeled peptides WAHEIAHSLGLFHA (STR-W1), AWHEIAHSLGLFHA (STR-W2), AHEIAHSLGWFHA (STR-W11), and AHEIAHSLGLFHWA (STR-W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp-incorporated peptides, STR-W1 and STR-W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR-W14 revealed that the conformation in the C-terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn-bound state of stromelysin-1 in crystal structures. The structural analyses of the qualitative Zn-binding properties of this motif peptide provide an interesting Zn-binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc-binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment.

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Calcium Ion Responsive DNA Binding in a Zinc Finger Fusion Protein

Cited by 15 publications

References 60 publications

Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway

Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway

Evaluation of Zinc (II) chelators for inhibiting p53-mediated apoptosis

Minimal motif peptide structure of metzincin clan zinc peptidases in micelles

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