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O-GlcNAcylation of αB-crystallin regulates its stress-induced translocation and cytoprotection

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Abstract

Under normal conditions, the ubiquitously expressed αB-crystallin functions as a chaperone. αB-crystallin has been implicated in a variety of pathologies, consistent with a build-up of protein aggregates, such as neuromuscular disorders, myofibrillar myopathies, and cardiomyopathies. αB-crystallins’ cardioprotection is partially attributed to its translocation and binding to cytoskeletal elements in response to stress. The triggers for this translocation are not clearly understood. In the heart, αB-crystallin undergoes at least three significant post-translational modifications: phosphorylation at ser-45 and 59 and O-GlcNAcylation (O-linked attachment of the monosaccharide β-N-acetyl-glucosamine) at thr-170. Whether phosphorylation status drives translocation remains controversial. Therefore, we evaluated the role of αB-crystallins’ O-GlcNAcylation in its stress-induced translocation and cytoprotection in cardiomyocytes under stress. Immunoblotting and precipitation experiments with anti-O-GlcNAc antibody (CTD110.6) and glycoprotein staining (Pro-Q Emerald) both demonstrate robust stress-induced O-GlcNAcylation of αB-crystallin. A non-O-GlcNAcylatable αB-crystallin mutant (αB-T170A) showed diminished translocation in response to heat shock and robust phosphorylation at both ser-45 and ser-59. Cell survival assays show a loss of overexpression-associated cytoprotection with the non-glycosylatable mutant to multiple stresses. While ectopic expression of wild-type αB-crystallin strongly stabilized ZsProSensor, a fusion protein rapidly degraded by the proteasome, the non-O-GlcNAcylatable version did not. Therefore, we believe the O-GlcNAcylation of αB-crystallin is a dynamic and important regulator of both its localization and function.

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Abbreviations

O-GlcNAc:

O linked N-acetyl glucosamine

OGT:

O-GlcNAc transferase

GCA:

O-GlcNAcase

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Acknowledgments

The authors are grateful to the Cardiovascular Institute and the Department of Physiology at Loyola University Chicago, Health Sciences Division for support of this project. A special acknowledgement is owed to the laboratories of Drs. Samarel and Mestril, for assistance with neonatal rat ventricular myocytes. The authors also thank Dr Mestril for helpful discussions. This project could not have been completed without the generous support of the Midwest Affiliate of the American Heart Association [SDG 0235411Z to JLM], the Potts Foundation, the Beck-Scanlon Cardiovascular Research Development Award, and the Marian and Ralph Falk Foundation.

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  1. Health Sciences Division, Department of Medicine, The Cardiovascular Institute, Loyola University Chicago, 2160 S 1st Ave, Maywood, IL, 60153, USA

    Vigneshwaran Krishnamoorthy, Anthony J. Donofrio & Jody L. Martin

  2. Health Sciences Division, Department of Cell and Molecular Physiology, Loyola University Chicago, 2160 S 1st Ave, Maywood, IL, 60153, USA

    Vigneshwaran Krishnamoorthy & Jody L. Martin

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  1. Vigneshwaran Krishnamoorthy
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Krishnamoorthy, V., Donofrio, A.J. & Martin, J.L. O-GlcNAcylation of αB-crystallin regulates its stress-induced translocation and cytoprotection. Mol Cell Biochem 379, 59–68 (2013). https://doi.org/10.1007/s11010-013-1627-5

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