Function Of Propeptide Research Articles

Comparative modelling studies of fruit bromelain using molecular dynamics simulation.

Fruit bromelain is a cysteine protease accumulated in pineapple fruits. This proteolytic enzyme has received high demand for industrial and therapeutic applications. In this study, fruit bromelain sequences QIM61759, QIM61760 and QIM61761 were retrieved from the National Center for Biotechnology Information (NCBI) Genbank Database. The tertiary structure of fruit bromelain QIM61759, QIM61760 and QIM61761 was generated by using MODELLER. The result revealed that the local stereochemical quality of the generated models was improved by using multiple templates during modelling process. Moreover, by comparing with the available papain model, structural analysis provides an insight on how pro-peptide functions as a scaffold in fruit bromelain folding and contributing to inactivation of mature protein. The structural analysis also disclosed the similarities and differences between these models. Lastly, thermal stability of fruit bromelain was studied. Molecular dynamics simulation of fruit bromelain structures at several selected temperatures demonstrated how fruit bromelain responds to elevation of temperature.

Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system

Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide.

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall.

Four residues of propeptide are essential for precursor folding of nattokinase.

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

The Propeptides of VEGF-D Determine Heparin Binding, Receptor Heterodimerization, and Effects on Tumor Biology

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

Selectivity of propeptide-enzyme interaction in cathepsin L-like cysteine proteases

Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans-experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

Mass Spectrometry Reveals the Missing Links in the Assembly Pathway of the Bacterial 20 S Proteasome

The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.

Functions of propeptide parts in cysteine proteases.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca(++) -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.

Structure-function relationships in class CA1 cysteine peptidase propeptides.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.

Structure-function relationships in class CA1 cysteine peptidase propeptides.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.

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.

Novel Propeptide Function in 20 S Proteasome Assembly Influences β Subunit Composition

The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor beta-type subunits gather in position on an alpha-subunit ring, followed by the association of two half-proteasomes and beta-subunit processing. In vertebrates three additional beta-subunits (beta1i/LMP2, beta2i/MECL1, and beta5i/LMP7) can be synthesized and substituted for constitutive homologues (beta1/delta, beta2/Z, and beta5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six beta-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH(2)-terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.

Production of Mature Human Apolipoprotein A-I in a Baculovirus–Insect Cell System: Propeptide Is Not Essential for Intracellular Processing but May Assist Rapid Secretion

To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus–insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus–insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.

Autocatalytic Subunit Processing Couples Active Site Formation in the 20S Proteasome to Completion of Assembly

The eukaryotic 20S proteasome is responsible for the degradation of many cellular proteins, but how it is assembled and how its distinct active sites are formed are not understood. Like other proteasome subunits, the yeast Doa3 protein is synthesized in precursor form. We show that the N-terminal propeptide is required for Doa3 incorporation into the proteasome and, remarkably, that the propeptide functions in trans, suggesting it serves a chaperone-like function in proteasome biogenesis. Propeptide processing is not required for proteasome assembly but is needed for maturation of a specific subset of active sites. The likely nucleophile for these sites is provided by the N-terminal threonine of mature Doa3. Additional data indicate that precursor processing is autocatalytic and requires association of the two halves of the proteasome particle, thereby preventing formation of proteolytic sites until the central hydrolytic chamber has been sealed off from the rest of the cell.

Multi-step processing of procathepsin L in vitro

The proteolytic processes involved in the conversion of procathepsin L to cathepsin L on a negatively charged surface, dextran sulfate, were studied. Upon incubation for 30 min at 37°C, pH 5.5 with dextran-sulfate and dithiothreitol, purified procathepsin L showed maximal activation and, correspondingly, the complete conversion to the 30 kDa, single chain mature form of enzyme was observed. In contrast, incubation under the same conditions on ice rather than at 37°C for 30 or 60 min resulted in partial proteolysis to produce a 31 kDa form without a significant increase in activity. Amino terminal amino acid sequence analyses showed that the 30 kDa form obtained by incubation at 37°C corresponds to the purified form of mature cathepsin L with a 2 amino acid extension at the amino terminal, and that the 31 kDa form generated by incubation on ice possesses a 6 amino acid amino terminal extension, suggesting that the activation and processing of procathepsin L are different processes, and that 4 amino acid residues (Glu-Pro-Leu-Met) at the carboxyterminal in the propeptide function to prevent the activation of processed cathepsin L.

Pro‐peptide as an intermolecular chaperone: renaturation of denatured subtilisin E with a synthetic pro‐peptide

The amino-terminal pro-sequence consisting of 77 amino acid residues is required to guide the folding of secreted subtilisin E, a serine protease, into active, mature enzyme (ikemura et al., 1987). Furthermore, denatured subtilisin E can be folded to active enzyme in an intermolecular process with the aid of an exogenously added pro-subtilisin E, the active site of which was mutated (Zhu et al., 1989). In this report, we have synthesized the pro-peptide of 77 residues (corresponding to -1 to -77 in the sequence, where residue +1 is the N-terminal amino acid residue of the mature protein), and have found that it could intermolecularly complement the folding of denatured subtilisin E to active enzyme. Furthermore, we have found that the synthetic pro-peptide exhibits specific strong binding to the active mature enzyme by inhibiting it competitively at its active centre with an upper limit to a Ki of 5.4 x 10(-7). In contrast, synthetic pro-peptides corresponding to -44 to -77, -1 to -64 and -1 to -43 inhibited the enzyme with Ki values weaker by two orders of magnitude. The results indicate that the sequence extending from -1 to -77 is essential for specificity of interaction, perhaps generating a conformation that accounts for both roles found hitherto, i.e. specific binding to the active centre, and guiding of the refolding to active enzyme. Thus these results suggest that the pro-peptide functions as an intramolecular chaperone [corrected].

Mutational analysis of the glutamine phosphoribosylpyrophosphate amidotransferase pro-peptide.

Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region. These mutations affect processing and enzyme activities. Processing of the leader is dependent upon glutamic acid residues at positions -2 and -1 as well as Cys1. In addition, processing requires a pro-peptide longer than 3 residues. Function of the active site cysteine is dependent on pro-peptide processing. Enzyme purified from a pro-peptide deletion strain has activity and iron content that is comparable to the wild type. These results establish that the pro-peptide is not essential for enzyme maturation, but they leave unanswered the question of pro-peptide function.