No abstract

The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme. Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M. . These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity. Surprisingly, Lys ؊36 3 Glu, Ala ؊30 3 Thr and Pro respectively. The Ile ؊48 3 Thr mutant propeptide was unable to refold denatured subtilisin BPN and gave a 100-fold higher K i (118 ؋ 10 ؊9 M) than the wild-type propeptide. The N59-mer propeptide extending from Leu ؊19 to Met ؊78 was unable to function as a chaperone. Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra. The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme. This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide-subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme.

No abstract

Heterotopic ossification (HO), acquired or hereditary, endochondral or intramembranous, is the formation of true bone outside the normal skeleton. Since perivascular Gli1+ progenitors contribute to injury induced organ fibrosis, and CD133 is expressed by a variety of populations of adult stem cells, this study utilized Cre-lox based genetic lineage tracing to test the contribution to endochondral HO of adult stem/progenitor cells that expressed either Gli1 or CD133. We found that both lineages contributed broadly to different normal tissues with distinct patterns, but that only Gli1-creERT labeled stem/progenitor cells contributed to all stages of endochondral HO in a BMP dependent, injury induced, transgenic mouse model. Hedgehog (Hh) signaling was abnormal at endochondral HO lesion sites with increased signaling surrounding the lesion but diminished signaling within it. Thus, local dysregulation of Hh signaling participates in the pathophysiology of endochondral HO. However, unlike a previous report of intramembranous HO, systemic inhibition of Hh signaling was insufficient to prevent the initiation of the endochondral HO process or to treat the existing endochondral HO, suggesting that Hh participates in, but is not essential for endochondral HO in this model. This could potentially reflect the underlying difference between intramembranous and endochondral HO. Nevertheless, identification of this novel stem/precursor cell population as a HO-contributing cell population provides a potential drugable target.

The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone which is crucial for proper folding of the active enzyme. This nascent N-terminal propeptide is removed after completion of the folding process. Here we present a possible pathway by which intramolecular chaperones mediate protein folding. Using circular dichroism to analyze acid-denatured subtilisin we have identified a folding-competent state which can refold to an active conformation in the absence of the propeptide. Earlier work had shown that guanidine hydrochloridedenatured subtilisin was in a state incapable of folding in absence of its propeptide. Comparison of the foldingincompetent and folding-competent states indicates that refolding is facilitated by the presence of residual structure present only in the folding-competent state. The analysis further indicates that the propeptide is essential for inducing this state. Therefore the folding-competent state may lie on-or be in rapid equilibrium with an intermediate on-the folding pathway of subtilisin. In the absence of the propeptide, formation of such a state-and hence refolding-is extremely slow.A large number of proteases, both in prokaryotes and in eukaryotes, are synthesized as precursors with N-terminal prosegments which play a vital role in the folding pathway (1). Subsequently, these propeptides are cleaved proteolytically to generate active enzymes. Hence, proteins such as subtilisins (2, 3), a-lytic protease (4), and carboxypeptidase (5), when unfolded in guanidine hydrochloride (Gdn'HCl) solution, refold only in the presence of their propeptides. Using subtilisin E as a model system, work in our laboratory has unambiguously shown that the 77-amino acid propeptide plays a vital role in folding the 275 amino acids of the mature enzyme (6). Because the propeptide is covalently attached to the N terminus of the mature enzyme prior to its maturation, and because each propeptide molecule mediates the folding of one enzyme molecule (generally the corresponding molecule), the term intramolecular chaperones has been coined for such propeptides to distinguish them from chaperones (6-8). Concurrent with this proposition, it was shown that the 13-amino acid propeptide of bovine pancreatic trypsin inhibitor facilitated the in vitro folding pathway by acting as a tethered, solvent-accessible, intramolecular thiol-disulfide reagent (9). Recently, it was shown that the propeptide of a-lytic protease helped to overcome a kinetic block in the folding pathway (10). of subtilisin facilitates folding in a manner different from that of a-lytic protease.Earlier work in our laboratory showed that in vitro the propeptide from subtilisin E could refold not only subtilisin E but also subtilisin BPN' and subtilisin Carlsberg. All three of these subtilisins are highly homologous and have very similar three-dimensional structures. In the present work, we have taken advantage of the ability of the propeptide of subtilisin E to facilitate refolding of subtilisin B...