A novel calpastatin-based inhibitor improves postischemic neurological recovery

Department of Pathology, Henry Ford Hospital, 1 Ford Place, 5D, Detroit, MI 48202, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 06/2009; 385(1):94-9. DOI: 10.1016/j.bbrc.2009.04.141
Source: PubMed


Calpastatin, a naturally occurring protein, is the only inhibitor that is specific for calpain. A novel blood-brain barrier (BBB)-permeant calpastatin-based calpain inhibitor, named B27-HYD, was developed and used to assess calpain's contribution to neurological dysfunction after stroke in rats. Postischemic administration of B27-HYD reduced infarct volume and neurological deficits by 35% and 44%, respectively, compared to untreated animals. We also show that the pharmacologic intervention has engaged the intended biologic target. Our data further demonstrates the potential utility of SBDP145, a signature biomarker of acute brain injury, in evaluating possible mechanisms of calpain in the pathogenesis of stroke and as an adjunct in guiding therapeutic decision making.

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    • "It is thus reasonable to conclude that the high calpastatin found in the infected cells is responsible for the protective effects of the mycoplasma against Ab-toxicity. Inhibition of calpain activity by exogenous pharmacological means, including calpastatin-based inhibitors, has been considered for therapy of various diseases, in which calpain is involved (Anagli et al., 2009; Pietsch et al., 2010; Ray et al., 2002). Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases and neural damage produced by trauma. "
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    ABSTRACT: Mycoplasmas are frequent contaminants of cell cultures. Contamination leads to altered synthetic and metabolic pathways. We have found that contamination of neuroblastoma SH-SY5Y cells by a strain of Mycoplasma hyorhinis derived from SH-SY5Y cell culture (NDMh) leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease, calpain) in NDMh-infected cells. We have now examined effects of amyloid-β-peptide (Aβ) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aβ was toxic to clean cells, resulting in necrotic cell damage. Aβ treatment led to activation of calpain and enhanced proteolysis, cell swelling, cell membrane permeability to propidium iodide (PI) (without nuclear apoptotic changes), and diminished mitochondrial enzyme activity (XTT reduction). Aβ-toxicity was attenuated in the high calpastatin-containing NDMh-infected cells, as shown by inhibition of calpain activation and activity, no membrane permeability, normal cell morphology, and maintenance of mitochondrial enzyme activity (similar to attenuation of Aβ-toxicity in non-infected cells overexpressing calpastatin following calpastatin-plasmid introduction into the cells). By contrast, staurosporine affected both clean and infected cells, causing apoptotic damage (cell shrinkage, nuclear apoptotic alterations, caspase-3 activation and caspase-promoted proteolysis, without PI permeability, and without effect on XTT reduction). The results indicate that mycoplasma protects the cells against certain types of insults involving calpain. The ratio of calpastatin to calpain is an important factor in the control of calpain activity. Exogenous pharmacological means, including calpastatin-based inhibitors, have been considered for therapy of various diseases in which calpain is implicated. Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases.
    Full-text · Article · Mar 2011 · Neurochemistry International

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    ABSTRACT: The gene coding for glutathione S-transferase (GST) has been isolated from the Mytilus edulis hepatopancreas. Open reading frame analysis indicated that the M. edulis GST (meGST) gene encodes a protein of 206 amino acid residues with a calculated molecular mass of 23.68 kDa. The deduced amino acid sequence showed high sequence similarity with the sequence of the pi class GST. The meGST was expressed in Escherichia coli, and the recombinant meGST was purified by affinity chromatography and characterized. The recombinant meGST exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis with respect to CDNB as substrate gave a K(m) of 0.68 mM and a V(max) of 0.10 mmol/min per mg protein. The recombinant meGST had a maximum activity at approximately pH 8.5, and its optimum temperature was 39 degrees C. The predicted three-dimensional structure of the meGST revealed the N-terminal domain possesses a thioredoxin fold and the six helices of the C-terminal domain make a alpha-helical bundle. These features indicate that the meGST belongs to pi class GST.
    Preview · Article · Nov 2004 · Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology
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