Project Grant R01AG088215

THE FUNCTION OF POST-TRANSLATIONAL MODIFICATIONS OF MYOSIN AND ITS CHAPERONE UNC-45 THAT OCCUR WITH ADULT AGING - PROJECT SUMMARY ONE OF THE UNFORTUNATE EFFECTS OF AGING IS THE LOSS OF MUSCLE MASS, STRENGTH, AND FUNCTION, TERMED "SARCOPENIA", WHEN IT IS NOT ASSOCIATED WITH AN UNDERLYING DISEASE. SARCOPENIA AFFECTS 40-50% OF INDIVIDUALS OVER 80 YEARS OF AGE, AND IS A MAJOR CONTRIBUTOR TO PHYSICAL DISABILITY, POOR QUALITY OF LIFE, AND DEATH AMONG THE ELDERLY. THE MOLECULAR MECHANISMS RESPONSIBLE FOR SARCOPENIA ARE NOT WELL-UNDERSTOOD. IN AGED MUSCLE, MYOSIN UNDERGOES CHEMICAL MODIFICATIONS THAT CORRELATE WITH ALTERATIONS IN ITS STRUCTURE AND DIMINISHED CONTRACTILE FUNCTION. GUIDED BY OUR PRELIMINARY STUDIES, WE HYPOTHESIZE THAT AGING LEADS TO ALTERED MYOSIN-CHAPERONE INTERACTIONS, AS A RESULT OF CHEMICAL MODIFICATIONS OF THESE PROTEINS THAT ACCUMULATE WITH AGE, ULTIMATELY CONTRIBUTING TO THE DIMINISHED MUSCLE MASS OF SARCOPENIA. THROUGH A UNIQUE COLLABORATION BETWEEN TWO EXPERT RESEARCH LABS, WE WILL TEST THIS HYPOTHESIS USING A NOVEL COMBINATION OF IN VITRO BIOCHEMICAL AND BIOPHYSICAL ASSAYS AND IN VIVO TECHNIQUES TO PROBE THE EFFECTS OF POST-TRANSLATIONAL MODIFICATIONS ON THE UNC-45/HSP90/MYOSIN SYSTEM DURING AGING. OUR PRELIMINARY DATA SHOW THAT DURING AGING OF ADULT C. ELEGANS THERE IS A TIME-DEPENDENT DECREASE IN THE STEADY STATE LEVELS OF THESE PROTEINS: FIRST HSP-90, THEN UNC-45 AND THEN MYOSIN. DURING AGING, WE ALSO OBSERVE A LOSS OF UNC-45 THAT IS CORRELATED WITH AN INCREASE IN PHOSPHORYLATION OF THE PROTEIN: WE HAVE IDENTIFIED SEVERAL SITES OF AGE-ASSOCIATED PTMS, INCLUDING A PHOSPHO-SERINE IN THE HSP-90 BINDING TPR REPEATS (S111) AND PHOSPHORYLATION OF TWO CONSERVED SERINES IN THE CHAPERONING DOMAIN OF UNC-45 (S659 AND S723). BASED ON OUR PRELIMINARY DATA, WE HYPOTHESIZE THAT AN IMPORTANT MECHANISM OF SARCOPENIA IS AGE-ASSOCIATED CHEMICAL MODIFICATIONS OF UNC-45 AND MYOSIN THAT REDUCE THE STABILITY AND/OR FUNCTION OF THESE CRUCIAL MUSCLE PROTEINS. IN THIS PROJECT, WE WILL USE A NOVEL COMBINATION OF BIOPHYSICAL AND IN VIVO TOOLS TO TEST THIS HYPOTHESIS AT MULTIPLE SCALES (PROTEIN TO MYOFIBRIL TO CELL TO ANIMAL). IN AIM 1, WE WILL TEST THE HYPOTHESIS THAT AGE-DEPENDENT PTMS OF UNC-45 REDUCE ITS STABILITY AND/OR CHAPERONE FUNCTION, THUS REDUCING THE ABILITY OF UNC- 45 TO REFOLD DAMAGED MYOSIN. IN AIM 2, WE WILL TEST THE HYPOTHESIS THAT AGE-DEPENDENT PTMS OF MYOSIN ALTER ITS STABILITY AND/OR MOTOR ACTIVITY. BY CREATING C. ELEGANS MUTANTS IN WHICH RESIDUES THAT NORMALLY UNDERGO POST- TRANSLATIONAL MODIFICATION CANNOT BE MODIFIED, WE WILL TEST THE HYPOTHESIS THAT SUCH CHANGES WILL RESULT IN DELAYED OR REDUCED SARCOPENIA. C. ELEGANS HAS PROVIDED VALUABLE INSIGHTS, TRANSLATABLE TO ALL ANIMALS, ON MYOFIBRIL ASSEMBLY AND MAINTENANCE, GENES RESPONSIBLE FOR LONGEVITY AND AGING, AND IS AN ESTABLISHED MODEL FOR STUDYING SARCOPENIA. OUR HYPOTHESES, IF CONFIRMED, WILL REPRESENT A NEW PARADIGM IN THE BIOLOGY OF SARCOPENIA, WITH POTENTIAL FOR NOVEL THERAPEUTIC APPROACHES, USING RATIONAL DRUG DESIGN TO TARGET DIFFERENT SPECIFIC ENZYMES (E.G., PROTEIN KINASES) TO PREVENT AGING-ASSOCIATED CHEMICAL MODIFICATIONS OF MYOSIN AND UNC-45, AND HENCE DELAYING THE ONSET OR REDUCING THE SEVERITY OF SARCOPENIA IN THE ELDERLY.