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Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I
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Background
Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal
tail, cTnI135-209, binds actin to shut off cardiac muscle contraction, whereas the competing
calcium-dependent binding of the switch region, cTnI146-158, by cardiac troponin C (cTnC) triggers
contraction. The N-terminal tail, cTnI1-37, regulates the calcium affinity of cTnC. cTnI is known to
be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two
intracellular proteases implicated in ischemia-reperfusion injury.
Methods
Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1-77 and cTnI135-209,
were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both
constructs using liquid chromatography-mass spectrometry (LC-MS) and solution NMR studies of
the C-terminal tail.
Results
cTnI135-209 is intrinsically disordered, though it contains three regions with helical
propensity (including the switch region) that acquire more structure upon actin binding. We
identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In
contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical
cTnI switch region is targeted by both proteases.
Conclusions
Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and
calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be
understood in terms of their interactions as probed by NMR studies.
General Significance
cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many
protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use
as gold standard biomarker of myocardial injury. -
- Date created
- 2019-01-01
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- Type of Item
- Article (Draft / Submitted)
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- License
- © 2019 Elsevier B.V. All rights reserved