Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

Minard, Karyl I.; Carroll, Christopher A.; Weintraub, Susan T.; McAlister-Henn, Lee

doi:10.1016/j.freeradbiomed.2006.09.024

Cited by 12 publications

(10 citation statements)

References 60 publications

(86 reference statements)

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“…Evidence of Disulfide Bond Formation-We next sought to fluorescently label cysteines that participate in disulfide bonds (38,39). Proteins were reacted first with iodoacetamide (to block free sulfhydryls) and Alexa Fluor 680 succinimidyl ester (as a general fluorescent label) as described under "Experimental Procedures."…”

Section: Resultsmentioning

confidence: 99%

“…Disulfide Detection Assay-To determine whether cysteine residues introduced into the ⑀-toxin formed disulfide bonds, proteins were reacted with iodoacetamide, to block free sulfhydryls, followed by reduction and labeling of sulfhydryls with a fluorescent marker (38,39). Purified ⑀-toxin preparations (20 g) were incubated with iodoacetamide (2.7 mM final concentration, to block cysteines not participating in disulfide bonds) and Alexa Fluor 680 succinimidyl ester (0.5 mM final concentration, to fluorescently label the proteins) in the presence of 1% SDS and 4 M urea.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Pelish

McClain

2009

Journal of Biological Chemistry

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The Clostridium perfringens ⑀-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ⑀-toxin. Site-specific mutations were introduced into the gene encoding ⑀-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wildtype ⑀-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ⑀-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric poreforming toxins and provide the basis for developing new therapeutics for treating intoxication by ⑀-toxin.The Clostridium perfringens ⑀-toxin is one of the most potent bacterial toxins (1, 2). The ⑀-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3-6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7-9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ⑀-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10 -13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ⑀-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins a...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Pelish

McClain

2009

Journal of Biological Chemistry

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“…Patterns of DNA damage and protein oxidation were similar in the zwf1Δidp2Δ yeast mutant shifted to acetate medium and in the parental strain following a similar shift in the presence of exogenous hydrogen peroxide (3, 6). In the current study, we therefore examined changes in cellular NAD(H) levels following an acute exogenous challenge of the parental strain with hydrogen peroxide during growth in acetate medium.…”

mentioning

confidence: 89%

“…We showed that peroxidases including catalase were ineffective in removal of hydrogen peroxide. Instead, the decrease in viability of the zwf1Δidp2Δ mutant and associated increase in levels of intracellular oxidants correlated with damage to cellular macromolecules (3, 6), suggesting that Zwf1p and Idp2p have redundant functions in reducing NADP + to NADPH for thiol-based antioxidant systems needed to eliminate detrimental byproducts of endogenous oxidative metabolism.…”

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confidence: 99%

Pnc1p Supports Increases in Cellular NAD(H) Levels in Response to Internal or External Oxidative Stress

Minard

McAlister-Henn

2010

Biochemistry

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Following transfer from medium with fermentable glucose to medium with non-fermentable acetate as the carbon source, cellular levels of NAD(H) were found to increase ~two-fold in a parental yeast strain. Similar transfer of a mutant strain subject to endogenous oxidative stress under these conditions produced more dramatic increases in cellular levels of NAD(H), and elevations above parental levels were shown to be due to the nicotinimidase Pnc1p. Similar transient increases in NAD(H) levels observed in the parental strain following addition of exogenous hydrogen peroxide were also attributable to Pnc1p.

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“…G6PD-deficient human foreskin fibroblasts display slow growth and early onset of senescence [19]. A yeast model with co-disruption of major NADPH sources (G6PD and cytosolic IDH) undergoes growth inhibition and loses viability [20]. Whereas loss of G6PD or IDH alone fails to induce a growth defect, a NADPH compensatory mechanism at the cellular level may maintain NADPH homeostasis and warrant normal development.…”

Section: Introductionmentioning

confidence: 99%

IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans

Yang

Liu

et al. 2019

J Mol Med

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NADPH is a reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH-producing enzymes predisposes cells to growth retardation and demise. It was hypothesized that double deficiency of the NADPH-generating enzymes, GSPD-1 (Glucose-6-phosphate 1-dehydrogenase), a functional homolog of human glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and IDH-1 (isocitrate dehydrogenase-1) affect growth and development in the nematode, Caenorhabditis elegans ( C. elegans ). The idh-1 ; gspd-1 ( RNAi ) double-deficient C. elegans model displayed shrinkage of body size, growth retardation, slowed locomotion, and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by severe NADPH insufficiency by the idh-1 ; gspd-1 ( RNAi ) double-deficiency. The principal component analysis (PCA) points to a distinct metabolomic profile of idh-1 ; gspd-1 ( RNAi ) double-deficiency. Further metabolomic analysis revealed that NADPH-dependent and glutamate-dependent amino acid biosynthesis were significantly affected. The reduced pool of amino acids may affect protein synthesis, as indicated by the absence of NAS-37 expression during the molting process. In short, double deficiency of GSPD-1 and IDH-1 causes growth retardation and molting defects, which are, in part, attributed to defective protein synthesis, possibly mediated by altered amino acid biosynthesis and metabolism in C. elegans . Electronic supplementary material The online version of this article (10.1007/s00109-018-01740-2) contains supplementary material, which is available to authorized users.

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Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

Cited by 12 publications

References 60 publications

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Pnc1p Supports Increases in Cellular NAD(H) Levels in Response to Internal or External Oxidative Stress

IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans

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