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Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants.

Gross SM, Lehman SL - PLoS ONE (2013)

Bottom Line: Signaling by reactive oxygen species has emerged as a major physiological process.Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species.These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology, University of California, Berkeley, California, United States of America.

ABSTRACT
Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

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Cysteines unique to an isoform are well conserved in vertebrate evolution.A. Presence of unique cysteines in the rat isoforms, compared to their presence in human, rat chicken frog and zebrafish isoforms. Each row represents a cysteine that is found in only one of the isoforms of a rat myofilament protein. Columns represent species (evolutionary relationships are displayed at top). Red boxes indicate that the cysteine in the rat isoform is present in the isoform of the other species. B. Percent of histidine, cysteine, and methionine residues conserved across five vertebrate species (human, rat, chicken, frog, zebrafish) in fast and slow isoforms of TnI, TnC, RLC, and MLC. C. Percent of methionine, histidine and cysteine residues common to fast and slow isoforms of rat MHC, TnI, TnC, RLC and MLC relative to the total number in the fast isoform.
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pone-0069110-g002: Cysteines unique to an isoform are well conserved in vertebrate evolution.A. Presence of unique cysteines in the rat isoforms, compared to their presence in human, rat chicken frog and zebrafish isoforms. Each row represents a cysteine that is found in only one of the isoforms of a rat myofilament protein. Columns represent species (evolutionary relationships are displayed at top). Red boxes indicate that the cysteine in the rat isoform is present in the isoform of the other species. B. Percent of histidine, cysteine, and methionine residues conserved across five vertebrate species (human, rat, chicken, frog, zebrafish) in fast and slow isoforms of TnI, TnC, RLC, and MLC. C. Percent of methionine, histidine and cysteine residues common to fast and slow isoforms of rat MHC, TnI, TnC, RLC and MLC relative to the total number in the fast isoform.

Mentions: The conservation of cysteines between muscle types led us to ask when during vertebrate evolution each cysteine specific to one isoform emerged. Analysis of fish, frog, chicken, rat and human amino acid sequences revealed that nearly all isoform specific cysteines emerged early in vertebrate evolution, prior to the divergence of fish and amphibians (Fig. 2A). Among the isoform specific cysteines that emerged more recently, two were analyzed in further detail. Cysteine 134 of fTnI is absent from fish, frog, chicken, quail and turkey sequences, but is present in the zebra finch and chameleon sequences, indicating that not all birds and reptiles lack this site (Fig. S1B). Cys 63 of rat fast MLC1 is unusual across vertebrate evolution since it is expressed in some slow and some fast isoforms. The cysteine is found in the human ventricular isoform and rodent fast isoforms, but is absent from rodent ventricular isoforms and the human fast isoform. Analysis of additional vertebrate species shows that this cysteine is found in slow frog isoforms, but not in fast isoforms of frog, bird, or fish sequences (Supplementary Fig. S2).


Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants.

Gross SM, Lehman SL - PLoS ONE (2013)

Cysteines unique to an isoform are well conserved in vertebrate evolution.A. Presence of unique cysteines in the rat isoforms, compared to their presence in human, rat chicken frog and zebrafish isoforms. Each row represents a cysteine that is found in only one of the isoforms of a rat myofilament protein. Columns represent species (evolutionary relationships are displayed at top). Red boxes indicate that the cysteine in the rat isoform is present in the isoform of the other species. B. Percent of histidine, cysteine, and methionine residues conserved across five vertebrate species (human, rat, chicken, frog, zebrafish) in fast and slow isoforms of TnI, TnC, RLC, and MLC. C. Percent of methionine, histidine and cysteine residues common to fast and slow isoforms of rat MHC, TnI, TnC, RLC and MLC relative to the total number in the fast isoform.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716824&req=5

pone-0069110-g002: Cysteines unique to an isoform are well conserved in vertebrate evolution.A. Presence of unique cysteines in the rat isoforms, compared to their presence in human, rat chicken frog and zebrafish isoforms. Each row represents a cysteine that is found in only one of the isoforms of a rat myofilament protein. Columns represent species (evolutionary relationships are displayed at top). Red boxes indicate that the cysteine in the rat isoform is present in the isoform of the other species. B. Percent of histidine, cysteine, and methionine residues conserved across five vertebrate species (human, rat, chicken, frog, zebrafish) in fast and slow isoforms of TnI, TnC, RLC, and MLC. C. Percent of methionine, histidine and cysteine residues common to fast and slow isoforms of rat MHC, TnI, TnC, RLC and MLC relative to the total number in the fast isoform.
Mentions: The conservation of cysteines between muscle types led us to ask when during vertebrate evolution each cysteine specific to one isoform emerged. Analysis of fish, frog, chicken, rat and human amino acid sequences revealed that nearly all isoform specific cysteines emerged early in vertebrate evolution, prior to the divergence of fish and amphibians (Fig. 2A). Among the isoform specific cysteines that emerged more recently, two were analyzed in further detail. Cysteine 134 of fTnI is absent from fish, frog, chicken, quail and turkey sequences, but is present in the zebra finch and chameleon sequences, indicating that not all birds and reptiles lack this site (Fig. S1B). Cys 63 of rat fast MLC1 is unusual across vertebrate evolution since it is expressed in some slow and some fast isoforms. The cysteine is found in the human ventricular isoform and rodent fast isoforms, but is absent from rodent ventricular isoforms and the human fast isoform. Analysis of additional vertebrate species shows that this cysteine is found in slow frog isoforms, but not in fast isoforms of frog, bird, or fish sequences (Supplementary Fig. S2).

Bottom Line: Signaling by reactive oxygen species has emerged as a major physiological process.Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species.These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology, University of California, Berkeley, California, United States of America.

ABSTRACT
Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

Show MeSH
Related in: MedlinePlus