Metalloid Reductase Activity Modified by a Fused Se0 Binding Peptide.
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ABSTRACT:
SUBMITTER: Butz ZJ
PROVIDER: S-EPMC7478903 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A glutathione reductase (GSHR)-like enzyme in Pseudomonas moraviensis stanleyae was previously implicated as underlying the bacterium's remarkable SeO3 2- tolerance. Herein, this enzyme is sequenced, recombinantly expressed, and fully characterized. The enzyme is highly adapted for selenodiglutathione substrates (K m = 336 μM) compared to oxidized glutathione (K m = 8.22 mM). The recombinant expression of this enzyme in the laboratory strains of Escherichia coli conveys a 10-fold increase in IC90 for SeO3 2-. Moreover, selenium nanoparticles are observed when the enzyme is overexpressed in the cells exposed to SeO3 2-, but not in the corresponding no-enzyme controls. The analyses of the structural homology models of the enzyme reveal changes in the parts of the enzyme associated with product release, which may underlie the Se substrate specialization. Combined, the observations of adaptation to Se reduction over oxidized glutathione reduction as well as the portability of this nanoparticle-mediated SeO3 2- tolerance into other cell lines suggest that the P. moraviensis GSHR may be better described as a GSHR-like metalloid reductase.
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Project description:Summary Oriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems. Graphical abstract Highlights • The four GBP sequences are genetically fused to catalytic subunit of GDHγα complex• The cofactor-surface interface was investigated with 3D models of fusion enzymes• The four systems exhibit diverse electrochemical results depending on GBP type Electrochemistry; Bio-electrochemistry; Materials science; Materials chemistry
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Project description:BackgroundThere is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery.ResultsBinding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE.ConclusionThis study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.
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