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Structure and proteolytic digestion of the intrinsically disordered C-terminal tail of cardiac troponin I

  • Author / Creator
    Zahran, Somaya
  • Intrinsically disordered regions (IDRs) are protein sequences that do not acquire a fixed 3-D configuration under physiologic conditions. Their solvent-exposed nature makes them susceptible to post-translational modifications like proteolysis, which makes them vital for cellular regulation. However, this can also make the production of these protein sequences in the lab challenging. One method of overcoming this difficulty is to direct their expression into inclusion bodies by attaching IDRs to fusion partners. We used a membrane protein, PagP, as a novel fusion partner to produce large IDRs, like the ones found in the cardiac troponin complex (cTn), from E. coli bacteria.cTn is part of the cardiac thin filament. It is comprised of three subunits: cTnI, the inhibitory subunit; cTnT, the tropomyosin binding subunit; and cTnC, the Ca2+ binding subunit. cTnI contains a well-structured core that is tightly bound to the other cTn subunits and two extended IDRs corresponding to its N- and C-terminal tails. The cTnI C-terminal tail plays a vital role in shutting off myocardial contraction by anchoring troponin-tropomyosin to a position on actin that blocks actin-myosin cross-bridging, but the structural details have not yet been fully elucidated. We were able to produce pure amounts of the cTnI C-terminal tail and study its structure alone and also when bound to actin. Our NMR structural studies revealed that while the tail is predominantly disordered, it does contain regions with helical propensity that become more ordered in the presence of actin.cTn leaks into the bloodstream in myocardial injury, along with other cardiac proteins. Clinical assays measuring cTnI and cTnT have shown superior sensitivity and specificity for cardiac tissue, establishing them as the current gold standard biomarkers for diagnosing myocardial infarction, that is, irreversible heart muscle death due to prolonged ischemia. The traditional view is that cTn release requires irreversible cardiac myocyte death. However, studies have documented cTn elevations in healthy volunteers after strenuous activity or with reversible cardiac injuries without detectable infarcts by radiologic imaging. In addition, cTn levels were found to poorly correlate with infarct size as measured by cardiac MRI. Thus, we and many others postulate that cTn can in fact be released by sub-lethal injuries. Moreover, since cell death leads to extensive activation of proteases, we further proposed that focal necrosis in myocardial infarction would lead to extensively proteolyzed cTnI, while less severe reversible cardiac injuries would release cTnI in a more intact form. We conducted a pilot study on 29 patients with a wide variety of underlying cardiac injuries and found this assertion to be true. cTnI was proteolyzed most consistently at its C-terminal tail with increasing severity of injury/infarct. Our results suggest that accounting for the extent of cTnI proteolysis could improve the correlation between serum cTnI concentrations and the severity of the underlying cardiac injury.Our studies on characterising the structure and function of the disordered C terminal tail of cTnI reveal a greater role for structural biology in overcoming current limitations of the cTn assay and enhancing its clinical utility.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-g4qb-pb56
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.