Functional Analysis of the Propeptide of Subtilisin E as an Intramolecular Chaperone for Protein Folding

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment. Tripeptidyl peptidase I (TPP I)2 is an acidic lysosomal hydrolase sequentially removing tripeptides en bloc from the N terminus of small, unstructured polypeptides (for a review, see Ref.1). The natural substrate(s) of the enzyme still remains elusive. TPP I is widely distributed in the human body, with a high expression level in brain tissue (2, 3). Naturally occurring mutations in TPP I underlie a devastating neurodegenerative disorder of early childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2) (4).On the basis of significant sequence homology and inhibitory studies, TPP I was assigned to a family of pepstatin-insensitive serine-carboxyl proteases recently renamed sedolisins (for a Like most proteolytic enzymes, TPP I is synthesized as an inactive precursor, zymogen (8, 9). The prepro-TPP I consists of a 19-amino acid (aa) signal peptide cleaved off when the newly forming polypeptide chain is inserted into the endoplasmic reticulum lumen, the 176-aa prosegment (or prodomain), and a 368-aa catalytic domain. As we demonstrated earlier, the removal of the prosegment from a purified TPP I proenzyme (pro-TPP I) occurs via an autocatalytic, intramolecular mechanism, whereby the mature enzyme does not significantly participate in its own generation (10). Efficient in vitro processing and autoactivation of the proenzyme is triggered by lowering the pH of the proenzyme solution to below 4.5. When pro-TPP I is activated at pH 4.5 and above, it is processed slowly and generates additional 6-and 14-aa N-terminal extensions in the newly formed polypeptide, rendering it enzymatically inactive (10). However, as we showed earlier, the presence of polyanionic compounds, such...

Section: Discussionsupporting

confidence: 54%

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Golabek

Dolzhanskaya

Walus

et al. 2008

“…ProD Can Inhibit Subtilisin Activity and Chaperone Folding of Denatured Subtilisin-It has been established that ProWT inhibits subtilisin through slow binding inhibition kinetics (29). Because the primary sequence and the amino acid composition between ProWT and ProD are significantly different, we examined whether ProD can inhibit the proteolysis of the synthetic substrate and whether this inhibition follows slow binding kinetics ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Folding Pathway Mediated by an Intramolecular Chaperone

Yabuta

Subbian

Oiry

et al. 2003

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or ''intramolecular chaperones'' to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wildtype propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined ␣؊␤ conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT⅐subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.Subtilisin E is an alkaline serine protease isolated from Bacillus subtilis (1). In vivo this protein is produced as pre-prosubtilisin (2), wherein the pre-region (signal peptide) facilitates protein secretion and the pro-region (propeptide) functions as an intramolecular chaperone that guides correct folding of subtilisin (2-6). Upon completion of folding, the propeptide is autoproteolytically removed (7). This is necessary because the propeptide is a potent inhibitor of subtilisin activity (4).Propeptide-mediated folding mechanisms exist in several unrelated protease families. However, there is no sequence conservation in propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a common mechanism of action (9). Subtilisin (2-6) and ␣-lytic protease (10 -13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models for the subtilase family that spans prokaryotes and eukaryotes. Amino acid sequence analysis of this family establishes that alth...

“…Initially, it functions as an intramolecular chaperone to facilitate folding of the precursor. Then, upon autocatalytic processing, it forms a complex with the mature protease, inhibiting its activity until degraded by the mature protease in the final step of maturation (23)(24)(25). Based on this example, we anticipated that pSspB would represent an inert zymogen.…”

Section: Maturation Of the Sspb Prepropeptide Requiresmentioning

confidence: 99%

Identification of a Novel Maturation Mechanism and Restricted Substrate Specificity for the SspB Cysteine Protease ofStaphylococcus aureus

Massimi¹,

Park²,

Rice³

et al. 2002

106

The SspB cysteine protease of Staphylococcus aureus is expressed in an operon, flanked by the sspA serine protease, and sspC, encoding a 12.9-kDa protein of unknown function. SspB was expressed as a 40-kDa prepropeptide pSspB, which did not undergo autocatalytic maturation. Activity of pSspB was reduced compared with 22-kDa mature SspB, but it was equivalent to mature SspB after incubation with SspA, which specifically removed the pSspB N-terminal propeptide. SspC abrogated the activity of pSspB when incubated in a 1:1 complex but had no effect on SspA or papain. Activity of the pSspB⅐SspC complex was restored when incubated with SspA, and SspC was cleaved by SspA but not pSspB. Thus, SspC maintains pSspB as an inert zymogen, and SspA is required for removal of the propeptide and inactivation of SspC. Like the papain protease family, SspB cleaved substrates with a hydrophobic amino acid at P2 but had a strong preference for arginine at P1. It did not cleave casein, serum albumin, IgG, or IgA, but it promoted detachment of cultured keratinocytes and cleaved fibronectin and fibrinogen at sites recognized by urokinase plasminogen activator and plasmin, respectively. It also processed high molecular weight kininogen in a manner resembling plasma kallikrein. Thus, SspB exhibits a novel maturation mechanism and mimics the specificity of plasma serine proteases.