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PubMed1997

In addition to SEC11, a newly identified gene, SPC3, is essential for signal peptidase activity in the yeast endoplasmic reticulum.

Among the three characterized subunits comprising the signal peptidase complex of the yeast Saccharomyces cerevisiae (Sec11p, Spc1p, and Spc2p), only Sec11p is essential for cell growth, signal peptide cleavage, and signal peptidase-dependent protein degradation. Here we report the cloning of the SPC3 gene encoding the homolog to mammalian signal peptidase subunit SPC22/23. We find that Spc3p is also required for cell growth and signal peptidase activity within the yeast endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2000

The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase.

Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme. First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.

Bacterial Outer Membrane Proteins↗
PubMed2003

Targeting presenilin-type aspartic protease signal peptide peptidase with gamma-secretase inhibitors.

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma-secretase complex that catalyzes intramembrane cleavage of beta-amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that gamma-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing Abeta generation without affecting other intramembrane-cleaving aspartic proteases.

Alzheimer Disease↗
PubMed2003

Genetic complementation in yeast reveals functional similarities between the catalytic subunits of mammalian signal peptidase complex.

Type I signal peptidases (SPs) comprise a family of structurally related enzymes that cleave signal peptides from precursor proteins following their transport out of the cytoplasmic space in eukaryotic and prokaryotic cells. One such enzyme, the mitochondrial inner membrane peptidase, has two catalytic subunits, which recognize distinct cleavage site motifs in their signal peptide substrates. The only other known type I SP with two catalytic subunits is the signal peptidase complex (SPC) in the mammalian endoplasmic reticulum. Here, we tested the hypothesis that, as with inner membrane peptidase catalytic subunits, SPC catalytic subunits exhibit nonoverlapping substrate specificity. We constructed two yeast strains without endogenous SP, one expressing canine SPC18 and the other expressing a truncation of canine SPC21 (SPC21 Delta N), which lacks 24 N-terminal residues that prevent expression of SPC21 in yeast. By monitoring a variety of soluble and membrane-bound substrates, we find that, in contrast to the tested hypothesis, SPC catalytic subunits exhibit overlapping substrate specificity. SPC18 and SPC21 Delta N do, however, cleave some substrates with different efficiencies, although no pattern for this behavior could be discerned. In light of the functional similarities between SPC proteins, we developed a membrane protein fragmentation assay to monitor the position of the catalytic sites relative to the surface of the endoplasmic reticulum membrane. Using this assay, our results suggest that the active sites of SPC18 and SPC21 Delta N are located 4-11 A above the membrane surface. These data, thus, support a model that SPC18 and SPC21 are functionally and structurally similar to each other.

Amino Acid Sequence↗
PubMed2004

Peptidase activity of the Escherichia coli Hsp31 chaperone.

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.

Adenosine Triphosphate↗
PubMed2004

The identification of residues that control signal peptidase cleavage fidelity and substrate specificity.

Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at -1 (P1) and small or larger aliphatic residues at the -3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used site-directed mutagenesis, mass spectrometric analysis, and in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile-144 residue. Changes of the Ile-144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the -1 residue in a double mutant in which both Ile-86 and Ile-144 were changed to an alanine. Also, alteration of the Ile-144 and Ile-86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage following a -1 Asn residue. This work shows that Ile-144 and Ile-86 contribute to the signal peptidase substrate specificity and that Ile-144 is important for the accuracy of the cleavage reaction.

Amino Acids↗
PubMed2006

An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.

Amino Acid Sequence↗
PubMed2006

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library.

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.

Asparagine↗
PubMedDate not supplied

Expression of PZ-peptidases by cultures of several pathogenic fungi. Purification and characterization of a collagenase from Trichophyton schoenleinii.

Peptidolytic activity was studied in the broken-cell extracts of 17 isolates of pathogenic fungi tested with phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (PZ-PLGPA) as a substrate. All the fungi studied except Candida spp., Cryptococcus neoformans and two actinomycetes hydrolyzed the substrate and therefore contained a so-called PZ-peptidase activity. Of all the positive strains, Trichophyton schoenleinii, a pathogenic fungus showed the highest activity and was therefore chosen as a source for PZ-peptidase purification. The four chromatographic steps, a 'negative' dye column, a 'positive' dye column, hydroxyapatite Ultrogel, and modified TSK (HW 55), gave a highly purified peptidase with a 12% overall yield. Inhibitor studies suggested that the 82 000 M(r) PZ-peptidase is a metalloproteinase. Moreover it cleaved native rat type I collagen. Partial peptide sequencing showed a strong sequence homology to regions of two metalloproteinases previously identified in the yeast Saccharomyces cerevisiae and in rat.

Amino Acid Sequence↗
PubMed1996

HLA-DM and MHC class II molecules co-distribute with peptidase-containing lysosomal subcompartments.

MHC class II molecules associate with peptides in the endocytic pathway. Different endosomal locations for peptide loading of class II molecules, varying from early endosomes (EE) to lysosomes, have been assigned on the basis of subcellular fractionation experiments. We have determined the intracellular location of HLA-DM, a molecule that supports peptide loading of class II molecules, by separating vesicles from the melanoma cell line Mel JuSo on the basis of buoying density and surface charge. In both fractionations, HLA-DM co-fractionated with a lysosomal compartment containing beta-hexosaminidase (beta-hex) activity and not with endosomes. Further analysis showed that HLA-DM mainly co-fractionated with a sub-lysosomal structure characterized by a relative low density and containing both pro- and mature cathepsin D and MHC class II molecules. Fluid phase markers first enter this compartment before entering high-density lysosomes that contain exclusively mature cathepsin D, some HLA-DM and no detectable MC class II molecules. Finally we determined the intracellular location of neutral and acidic peptidases. Whereas neutral peptidase activity was detected in the endoplasmic reticulum and/or plasma membrane fractions, acidic peptidase activity exclusively migrated at the position of HLA-DM containing lysosomal vesicles. Our results show that class II molecules co-migrate with HLA-DM, pro- and mature cathepsin D, beta-hex and acidic peptidase activity. HLA-DM, cathepsin d and class II molecules were not observed at the position of EE. Our data suggest that HLA-DM-mediated peptide loading of class II molecules occurs in a lysosomal subcompartment.

Cathepsin D↗
PubMed1996

Cell surface peptidases in human endometrium.

Three cell surface peptidases have been shown to be present in the human endometrium. Aminopeptidase N and neutral endopeptidase are detected on the endometrial stromal cells and decidual cells, while dipeptidyl peptidase IV is detected on the endometrial glandular cells and surface epithelium. As these cell surface peptidases can degrade a variety of biologically-active peptides including cytokines and growth factors, they are considered to be involved in the local metabolism of these molecules. In addition, recent studies have indicated that they are involved in local immune responses, cell attachment, and cellular maturation/ differentiation of endometrial cells, and suggest an important role of these endometrial cell surface peptidases in implantation processes.

Aminopeptidases↗
PubMed1987

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities.

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).

Atmospheric Pressure↗
PubMed1993

Presence of multiple forms of peptidase inhibitors in rat milk.

Peptides such as somatostatin (SS14), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factors (IGF-I and IGF-II) are present in breast milk from various species, and their significance in the developing gastrointestinal tract has been suggested. Our recent studies have indicated that rat milk soluble fraction (RMSF) protects SS14 in the gastrointestinal lumen by inhibiting in vitro the luminal peptidolysis. In the present studies, we have shown that RMSF inhibited in vitro degradation by midjejunal luminal flushings of suckling rats of 125I-labeled somatostatin 14[Tyr11], EGF, TGF alpha, IGF-I and IGF-II, as well as trypsin activity in vitro against benzoyl-L-arginyl-p-nitroanilide. The inhibitory factors present in the RMSF were further fractionated by gel filtration on Sephadex G100, ion-exchange chromatography on DEAE-Sephadex, and fast protein liquid chromatography (FPLC). Gel filtration of Sephadex G100 separated RMSF into three peaks of proteins: G1, G2, and G3; peptidase inhibitor activities were present exclusively in G1. Ion-exchange chromatography on DEAE-Sephadex column resolved peptidase inhibitory activity (G1) into three different peaks, D1, D2, and D3, eluted at sodium chloride concentrations of 0.05 M, 0.1 M, and 0.2 M, respectively. Further purification of D2 by FPLC resulted in a fraction rich in peptidase inhibitory activity, which was essentially free of trypsin inhibitory activity. Results indicate the presence of at least three peptidase inhibitors in rat milk, which may play a role in the protection of milk-borne peptides in the gastrointestinal lumen.

Animals↗
PubMed1999

The gene encoding the prepilin peptidase involved in biosynthesis of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli.

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.

Amino Acid Sequence↗
PubMed1985

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci.

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep. lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.

Cell Membrane↗
PubMed1995

The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues.

Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pIII. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Accl fragment on account of its hybridization with the DNA fragment containing the type IV pre-pilin leader-peptidase gene pilD/xcpA of Pseudomonas aeruginosa. Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre-pilin leader peptidases, XpsO is unique in not having the two conserved -CXXC-motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein-secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa, both the sensitivity against infection with the pilus-specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.

Amino Acid Sequence↗
PubMed1987

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin.

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.

Animals↗
PubMed1995

Identification of Trp300 as an important residue for Escherichia coli leader peptidase activity.

We previously reported that leader peptidase from Escherichia coli was extensively inactivated by reaction with N-bromosuccinimide with concomitant and selective modification of the Trp300 and Trp310 residues [Kim, Y.-T., Muramatsu, T. & Takahashi, K. (1995) J. Biochem. (Tokyo) 117, 535-544]. This indicated that one or both of these tryptophan residues are important for the activity of the enzyme. In order to define further the role of individual tryptophan residues in the activity of leader peptidase, site-directed mutagenesis studies were performed to replace each tryptophan residue with phenylalanine and/or alanine. The replacements of Trp20, Trp59, Trp261, Trp284, and Trp310 with phenylalanine hardly affected the enzyme activity toward a synthetic peptide substrate and the ability to complement the temperature sensitivity of the mutant leader peptidase in E. coli IT41. In contrast, the activity toward the synthetic substrate was significantly decreased by replacement of Trp300 with phenylalanine or alanine. The kcat values of the W300F and W300A mutant enzymes were reduced to 42% and 22%, respectively, of that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type enzyme. Moreover, the complementing ability in E. coli IT41 was lost (almost) completely when Trp300 was replaced with phenylalanine or alanine. These results strongly indicate that Trp300 in leader peptidase is important for the catalytic mechanism and/or the construction of the active site structure of the enzyme.

Base Sequence↗