Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.     

Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases

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 wild-type 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 alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. 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.     

General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease

Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide.     

Molecular cloning and expression in Escherichia coli of the extracellular endoprotease of Aeromonas caviae T-64, a pro-aminopeptidase processing enzyme(1).

PA protease (pro-aminopeptidase processing protease) activates the pro-aminopeptidases from Aeromonas caviae T-64 and Vibrio proteolytica by removal of their pro-regions. Cloning and sequencing of the PA protease gene revealed that PA protease was translated as a preproprotein consisting of four domains: a signal peptide; an N-terminal propeptide; a mature region; and a C-terminal propeptide. The deduced amino acid sequence of the PA protease precursor showed significant homology with several bacterial metalloproteases. Expression of the PA protease gene in Escherichia coli indicated that the N-terminal propeptide of the PA protease precursor is essential to obtain the active form of the protease. The N- and C-terminal propeptides of the expressed pro-PA protease were processed autocatalytically.     

Mutated intramolecular chaperones generate high-activity isomers of mature enzymes

The propeptide of carboxypeptidase Y precursor (proCPY) acts as an intramolecular chaperone that ensures the correct folding of the mature CPY (mCPY). Here, to further characterize the folding mechanism mediated by the propeptide, folding analysis was performed using a yeast molecular display system. CPYs with mutated propeptides were successfully displayed on yeast cell surface, and the mature enzymes were purified by the selective cleavage of mutated propeptides. Measurement of the activity and kinetics of the displayed CPYs indicated that the propeptide mutation altered the catalytic efficiency of mCPY. Although the mature region of the wild-type and mutant CPYs had identical amino acid sequences, the mCPYs from the mutant proCPYs had higher catalytic efficiency than the wild-type. These results indicate that proteins with identical amino acid sequence can fold into isomeric proteins with conformational microchanges through mutated intramolecular chaperones.     

Mechanism of Fine-tuning pH Sensors in Proprotein Convertases: IDENTIFICATION OF A pH-SENSING HISTIDINE PAIR IN THE PROPEPTIDE OF PROPROTEIN CONVERTASE 1/3*

The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a “gatekeeper” that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.     

Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases.

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (DeltaDeltaG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.     

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63?±?0.80 to 71.44?±?3.20?min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.     

Extracellular metalloproteases from bacteria

Bacterial extracellular metalloproteases (BEMPs) are a large group of metal-containing proteases secreted by heterotrophic bacteria. In this review, the diversity, structural characteristics, mechanisms of maturation, physiological roles, and applications of BEMPs are described. BEMPs are distributed among nine families of metalloproteases because of differences in primary sequences and structural characteristics. Until now, all of the BEMPs identified have been endoproteases harboring one catalytic Zn²⁺ in the active centers. BEMPs are usually synthesized as inactive zymogens with a propeptide that is covalently linked to and inhibits the catalytic domain. The removal of the propeptides of BEMPs is dependent on other proteases or an autocleavage process. The main physiological function of BEMPs is to degrade environmental proteins and peptides for bacterial heterotrophic nutrition. As extracellular proteases, BEMPs vary greatly in enzymology properties to adapt to their respective environments. BEMPs have been widely used in the food and pharmaceutical industries. In order to broaden the application of BEMPs, it is essential to explore novel BEMPs and apply gene/protein engineering to improve the production and properties of promising BEMPs.     

Propeptides Are Sufficient to Regulate Organelle-Specific pH-Dependent Activation of Furin and Proprotein Convertase 1/3

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64

PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding. It was revealed that the processing of the proform involved a stepwise autoprocessing mechanism. Firstly, the N-terminal propeptide was autocatalytically removed on completion of refolding and secondly, the C-terminal propeptide was autoprocessed after the degradation of the N-terminal propeptide. Both the N- and C-terminal propeptides existed as intact peptides after their successive removal, and they were subsequently degraded gradually. The degradation of the N-terminal propeptide appears to be the rate-limiting step in the maturation of the proform of PA protease.     

Human polyserase-2, a novel enzyme with three tandem serine protease domains in a single polypeptide chain

We have cloned a human cDNA encoding a new serine protease that has been called polyserase-2 (polyserine protease-2) because it is the second identified human enzyme with several tandem serine protease domains in its amino acid sequence. The first serine protease domain contains all characteristic features of these enzymes, whereas the second and third domains lack one residue of the catalytic triad of serine proteases and are predicted to be catalytically inactive. This complex domain organization is also present in the sequences of mouse and rat polyserase-2 and resembles that of polyserase-1, which also contains three serine protease domains in its amino acid sequence. However, polyserase-2 lacks additional domains present in polyserase-1, including a type II transmembrane motif and a low-density lipoprotein receptor A module. Enzymatic analysis demonstrated that both full-length polyserase-2 and its first serine protease domain hydrolyzed synthetic peptides used for assaying serine proteases. Nevertheless, the activity of the isolated domain was greater than that of the entire protein, suggesting that the two catalytically inactive serine protease domains of polyserase-2 may modulate the activity of the first domain. Northern blot analysis showed that polyserase-2 is expressed in fetal kidney; adult skeletal muscle, liver, placenta, prostate, and heart; and tumor cell lines derived from lung and colon adenocarcinomas. Finally, analysis of post-translational processing mechanisms of polyserase-2 revealed that, contrary to those affecting to the membrane-bound polyserase-1, this novel polyprotein is a secreted enzyme whose three protease domains remain as an integral part of a single polypeptide chain.     

Propeptide-Mediated Inhibition of Cognate Gingipain Proteinases

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism’s cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K_i values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.     

Processing of protealysin precursor

Protealysin, a protease previously described by us in Serratia proteamaculans, belongs to the group of thermolysin-like proteases (TLPs) that differ from classical TLPs by the precursor structural organization. The propeptide of protealysin precursor has no significant structural similarity to the propeptides of most TLPs. The functions of protealysin-like precursors and mechanisms of their action remain unclear. We studied the pathway of protealysin precursor processing in vitro using standard approaches: modification of the catalytic site and monitoring immobilized precursor maturation. The Glu(113) → Ala substitution inhibited the precursor maturation, which pointed to the autocatalytic processing. The mutant precursor exposure to active protealysin converted it to the mature enzyme, thus, indicating the intermolecular processing. Intermolecular processing of the mutant protein by other proteases such as thermolysin or subtilisin is also possible. The intact protealysin precursor was efficiently autoprocessed in solution but not after immobilization. These data indicate that the processing of protealysin precursor differs from that of classical TLPs. The protealysin propeptide is cleaved by an autocatalytic or heterocatalytic intermolecular mechanism and is most likely not removed intramolecularly.     

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.     

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.

Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome. The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings. Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1. Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly. We show here that the propeptides have both common and distinct functions in proteasome biogenesis. Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation. However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly. Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation. For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated. In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism.     

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N701/F721) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.