Mutation of COOH-terminal lysines in overexpressed alpha B-crystallin abrogates ischemic protection in cardiomyocytes.
High levels of alpha B-crystallin are present in the cardiomyocyte, yet little is understood about the function and importance of this protein. Like many other small heat shock proteins, alpha B-crystallin forms large oligomeric complexes whose size can be regulated by posttranslational modifications. The size of these complexes can modify the function of the protein. A naturally occurring COOH-terminal mutant has many detrimental effects in the lens of the eye and altered oligomerization. Therefore, we mutated the two COOH-terminal lysines of alpha B-crystallin to glycines (K174/175G) and adenovirally mounted them to transduce cardiomyocytes. We analyzed the effect of this mutation on oligomerization, microtubular stabilization, and ischemic outcome. A nearly 45% downward shift in complex size was observed with the mutant by native PAGE followed by immunoblotting. The overexpressed protein no longer protected the tubulin cytoskeleton against ischemic stress by confocal analysis. The mutant caused a 30% increase in cytosolic enzyme release with ischemia compared with control, whereas a 33% decrease was associated with wild-type alpha B-crystallin overexpression. We conclude that the COOH terminus of alpha B-crystallin is crucial to its proper function.
Martin
Jody L
JL
Department of Physiology, Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois 60153, USA. jmart10@lumc.edu
Bluhm
Wolfgang F
WF
He
Huaping
H
Mestril
Ruben
R
Dillmann
Wolfgang H
WH
D000818:Animals; D002478:Cells, Cultured; D003459:Crystallins / Q000096:biosynthesis* / Q000235:genetics*; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008239:Lysine / Q000235:genetics*; D046911:Macromolecular Substances; D008870:Microtubules / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017202:Myocardial Ischemia / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D014161:Transduction, Genetic; D014404:Tubulin / Q000378:metabolism
D003459:Crystallins; D046911:Macromolecular Substances; D014404:Tubulin; D008239:Lysine