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Abstract
The 13.5 kDa N-terminal part of the propeptide remains associated with mature cathepsin
C after proteolytic activation and excision of the activation peptide. This residual
pro-part, isolated from the recombinant enzyme, folds spontaneously and rapidly to
a stable, compact monomer with secondary structure and stable tertiary interactions.
Folding and unfolding kinetics of the residual pro-part with intact disulfides are
complex, and accumulation of transient intermediates is observed. The cleaved form
of the pro-part isolated from natural human cathepsin C also folds, suggesting that
the intact form comprises two folding domains. The linkages of the two disulfide bridges
have been established as 30-118 and 54-136 for the native enzyme. The native disulfide
bonds can be re-formed from the fully reduced and denatured state by oxidative refolding,
resulting in a domain that is spectroscopically indistinguishable from the original
refolded residual pro-part. Both disulfides are solvent-exposed and can be reduced
in the absence of denaturant. The reduced form retains most or all of the native tertiary
structure and is only approximately 2 kcal.mol(-1) less stable than the oxidized form.
It folds fast relative to the rate of biosynthesis, to the same conformation as the
oxidized form. Folding and disulfide formation are sequential. These results indicate
that the proenzyme folds sequentially in vivo and that the residual pro-part constitutes
a rapidly and independently folding domain that stabilizes the mature enzyme. It thus
fulfills the criteria required of an intramolecular chaperone. It may also be involved
in stabilizing the tetrameric structure of the mature enzyme.