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Quantitative studies of caspase-3 catalyzed ?II-spectrin breakdown.

ABSTRACT: Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs)-in particular SBDPs of 150 kDa (SBDP150) and 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of ?II-spectrin model proteins by caspase-3 in detail with gel electrophoresis, fluorescence and mass spectrometry methods. Amongst the predicted and reported sites, our results show that caspase-3 cleaves after residues D1185 and D1478, but not after residues D888, D1340 and D1475. We also found that the cleavage at these two sites is independent of each other. It may be possible to inhibit one site without affecting the other site. Cleavage after residue D1185 in intact ?II-spectrin leads to SBDP150, and cleavage after D1478 site leads to SBDP120. Our results also show that the cleavage after the D1185 residue is unusually efficient, with a kcat/KM value of 40,000 M(-1) s(-1), and the cleavage after the D1478 site is more similar to most of the other reported caspase-3 substrates, with a kcat/KM value of 3000 M(-1) s(-1). We believe that this study lays out a methodology and foundation to study caspase-3 catalyzed spectrin breakdown to provide quantitative information. Molecular understanding may lead to better understanding of brain injuries and more precise and specific biomarker development.

SUBMITTER: Witek MA

PROVIDER: S-EPMC3786445 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown.

Witek Marta A MA Fung L W-M LW

Brain research 20130812

Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs)-in particular SBDPs of 150 kDa (SBDP150) and 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of αII- ...[more]

PMID: 23948103

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Project description:To determine if alpha II-spectrin breakdown products can be detected in the serum of neonates with congenital heart disease in the perioperative period.Prospective observational cohort study.Pediatric cardiac ICU in an urban tertiary care academic center.Neonates with congenital heart disease undergoing surgical repair or palliation.Serial blood sampling for measurement of 120 and 150 kDa spectrin breakdown products.Fourteen neonates with congenital heart disease undergoing cardiac surgery were evaluated. Nine infants underwent open-heart surgery and five underwent closed-heart surgery. Serum spectrin breakdown products were measured with sandwich enzyme-linked immunosorbent assay preoperatively and then 6, 24, 48, 72, and 96 hours following surgery. Brain imaging was obtained as part of routine clinical care in 12 patients preoperatively and six patients postoperatively. Six patients had normal preoperative imaging (three closed-heart surgery and three open-heart surgery), whereas six had evidence of neurologic injury prior to surgery (one closed-heart surgery and five open-heart surgery). Only one patient had a postoperative imaging study that lacked injury. All others demonstrated infarction or hemorrhage. Spectrin breakdown product 120 kDa significantly increased 24 hours after open-heart surgery compared to preoperative values and time-matched closed-heart surgery levels. Spectrin breakdown product 150 kDa significantly increased 6 hours after open-heart surgery compared to preoperative values. There was no significant change in spectrin breakdown products following closed-heart surgery. Peak spectrin breakdown products significantly increased following open-heart surgery compared to closed-heart surgery.Spectrin breakdown products can be detected in the serum of neonates with congenital heart disease in the perioperative period and levels increased to a greater degree in infants following open-heart surgery. These findings suggest that, in future work, serum spectrin breakdown products may potentially be developed as biomarkers for brain necrosis and apoptosis in infants with congenital heart disease.

Dataset Information

Quantitative studies of caspase-3 catalyzed ?II-spectrin breakdown.

Publications

Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown.

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