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PubMed2006

Cloning, expression, and purification of functional Sec11a and Sec11b, type I signal peptidases of the archaeon Haloferax volcanii.

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.

Amino Acid Sequence↗

PubMed1998

Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM.

Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.

Amino Acid Sequence↗

PubMed2004

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line.

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.

Aprotinin↗

PubMed1979

Inactivation of two hyperactive LH-RH analogues by rat hypothalamic peptidases.

Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are beleived to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself; the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TUB)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptidase with increased biological activity.

Animals↗

PubMed1993

Epithelium removal and peptidase inhibition enhance relaxation of human airways to vasoactive intestinal peptide.

In this study we evaluated the role of epithelial versus subepithelial peptidases in the responses of isolated peripheral and central human airways to VIP. Human airways were obtained at thoracotomies (n = 8) and studied in organ baths. Intact or epithelium-denuded strips of central and peripheral airways were incubated with or without a cocktail of peptidase inhibitors containing phosphoramidon (2.5 micrograms/ml), leupeptin, aprotinin, captopril, soybean trypsin inhibitor (all 20 micrograms/ml), and bestatin (2.8 micrograms/ml). After precontraction with histamine (5 x 10(-6) M), cumulative concentration-response curves to VIP (10(-10) to 10(-7) M) were obtained. Both intact central and peripheral airways showed only minor relaxations to VIP irrespective of the precontraction level. Removal of the epithelium and addition of peptidase inhibitors additively increased the sensitivity (> 20-fold) and maximal response to VIP in both central and peripheral airways. We conclude that (1) VIP relaxes both central and peripheral human airways but only in the absence of epithelium and/or the presence of peptidase inhibitors, and (2) both epithelial and subepithelial peptidases are important in the inactivation of VIP in human airways.

Adult↗

PubMed1978

Effect of alloxan-induced diabetes on intestinal peptidases in the rat.

Intestinal transport of amino aicds, similar to sugar absorption, is enhanced in experimental diabetes. Because peptidases play a significant role in peptide digestion, we examined the effect of diabetes on intestinal peptidases. Leucyl-naphthylamidase and leucyl-glycine hydrolase (brush border peptidases) and prolyl-glycine hydrolase (cytosol peptidase) were assayed in the brush border and cytosol fraction in diabetic rats 7 days after alloxan administration. Mucosal weight, protein concentration, and total and specific activity of leucyl-naphthylamidase and leucyl-glycine hydrolase were significantly increased in diabetes in the brush border but not in cytosol fraction. By contrast, prolyl-glycine hydrolasw was not affected in cytosol fraction or brush border. These data indicate that brush border peptidases are increased in experimental diabetes. This adaptive response of the small intestinal mucosa is similar to disaccharidase elevation and alteration in the intestinal absorptive function which occurs in experimental diabetes.

Animals↗

PubMed1996

Peptidase inhibitor-induced antidiuresis mediated through angiotensin and opioid receptors in the rat hypothalamus.

We examined the effects on urine outflow rate after microinjections of thiorphan (a carboxypeptidase inhibitor) and bestatin (an aminopeptidase inhibitor) into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of anesthetized hydrated rats to determine the possible role of neuropeptides in the regulation of urine production. After individual microinjection of the peptidase inhibitors into the nuclei, only thiorphan at 100 nmol administered into the PVN significantly decreased the urine outflow rate. Two consecutive microinjections of the peptidase inhibitors at 100 nmol each into the nuclei induced potent antidiuresis. These effects after microinjections of the peptidase inhibitors into the PVN and SON were diminished by pretreatment with [Sar1,Ile8]angiotensin (ANG) II (an ANG II receptor antagonist) and naloxone (an opioid receptor antagonist) in the PVN and with [Sar1,Ile8]ANG II in the SON, respectively. A vasopressin (AVP) receptor antagonist, d(CH2)5-D-Tyr(Et)VAVP (i.v.), completely blocked the antidiuresis by microinjections of the peptidase inhibitors into both the nuclei. Urinary osmotic pressure was significantly increased by consecutive microinjections of the peptidase inhibitors into the PVN and SON. These results suggest that endogenously-released ANG II and opioid peptides in the PVN and ANG II in the SON regulate urine production mediated through increased AVP release.

Aminopeptidases↗

PubMed2004

Regulatory role of membrane-bound peptidases in the progression of gynecologic malignancies.

Membrane-bound peptidases play a key role in the control of growth, differentiation, and signal transduction of many cellular systems by degrading bioactive peptides. Thus, abnormal changes in their expression pattern and catalytic function result in altered peptide activation, which contributes to neoplastic transformation or progression. In this review, we describe our recent findings along with work from other groups on the expression and biological functions of membrane-bound peptidases in cancer, focusing on the regulatory roles of three peptidases, aminopeptidase A (APA), neutral endopeptidase (NEP) and placental leucine aminopeptidase (P-LAP), in the progression of gynecologic malignancies. APA, NEP and P-LAP are differentially expressed and localized in various gynecologic malignancies including cervical cancer, endometrial cancer, ovarian cancer and choriocarcinoma in a tumor-type specific pattern. The expression levels are up- or down-regulated depending on histological grade or disease progression. These peptidases play regulatory roles in tumor cell proliferation, invasion or angiogenesis via degradation/inactivation of target peptides such as angiotensin II, endothelin-1 and oxytocin, which act on cancer cells as stimulatory or inhibitory factors. Thus, membrane-bound peptidases may become not only a new diagnostic/prognostic marker, but also a novel molecular target for the treatment of gynecologic malignancies.

Disease Progression↗

PubMed2005

Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas.

AIM: Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors, and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals. METHODS: The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC. RESULTS: The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P<0.05). The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract. CONCLUSION: These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.

Adult↗

PubMed1999

Peptidase activity of beta-lactamases.

Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of beta-lactamases. The kcat/Km values were below 0.1 M(-1). s(-1), but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C beta-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-d-Ala-d-Ala dipeptides than with the diacetyl- and alpha-acetyl-l-Lys-d-Ala-d-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble dd-peptidases. A comparison between the beta-lactamases and dd-peptidases showed that it might be as difficult for a dd-peptidase to open the beta-lactam ring as it is for the beta-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

Bacillus↗

PubMed2000

The relative importance of cysteine peptidases in osteoarthritis.

OBJECTIVE: To assess the activity of cysteine peptidases in cultured human articular chondrocytes as well as in osteoarthritic (OA) cartilage and subchondral bone, and to interpret their relative importance in cartilage destruction and remodeling of the subchondral region. METHODS: Intracellular and secreted cysteine peptidase activity was measured in chondrocytes using fluorimetric assays, and enzymes were immunolocalized using monospecific antibodies. Enzyme histochemistry in normal and OA femoral heads was used to characterize enzymatic activity in full thickness samples containing cartilage and subchondral bone. The zonal distribution of cathepsin activity was measured in tissue slices of normal and OA femoral heads cut parallel to the joint surface, using fluorogenic substrates. RESULTS: Cathepsins B and L were localized by immunohistochemistry with lysosome-like structures in dedifferentiated chondrocytes. Free cysteine peptidase activity (i.e., not requiring prior activation), secreted and intracellularly stored by chondrocytes, was due to cathepsin B, while cathepsin L contributed a minor fraction of the total activity, and was seen only after activation at acidic pH. Histochemistry and activity measurements confirmed cathepsin B as the major, active cysteine peptidase in OA cartilage, particularly at sites where matrix neosynthesis took place. However, free cathepsin L and/or cathepsin K activity was found subchondrally in association with cathepsin B in osteophytes, in zones undergoing bone remodeling, and at sites of inflammation. CONCLUSION: Cathepsin B, not cathepsin L or cathepsin K, is a candidate for articular cartilage catabolism in OA. While cathepsin K is the major osteoclastic cysteine peptidase, cathepsin L and cathepsin B may also participate in the remodeling processes of bone as well as in bone erosion by inflammatory cells.

Adult↗

PubMed2003

Peptidases prevent mu-opioid receptor internalization in dorsal horn neurons by endogenously released opioids.

To evaluate the effect of peptidases on mu-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and alpha-neoendorphin, but not endomorphins or beta-endorphin. The omission of any one inhibitor abolished Leu-enkephalin-induced internalization, indicating that all three peptidases degraded enkephalins. Amastatin preserved dynorphin A-induced internalization, and phosphoramidon, but not captopril, increased this effect, indicating that the effect of dynorphin A was prevented by aminopeptidases and neutral endopeptidase. Veratridine (30 microm) or 50 mm KCl produced MOR-1 internalization in the presence of peptidase inhibitors, but little or no internalization in their absence. These effects were attributed to opioid release, because they were abolished by the selective MOR antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) and were Ca(2+) dependent. The effect of veratridine was protected by phosphoramidon plus amastatin or captopril, but not by amastatin plus captopril or by phosphoramidon alone, indicating that released opioids are primarily cleaved by neutral endopeptidase, with a lesser involvement of aminopeptidases and dipeptidyl carboxypeptidase. Therefore, because the potencies of endomorphin-1 and endomorphin-2 to elicit internalization were unaffected by peptidase inhibitors, the opioids released by veratridine were not endomorphins. Confocal microscopy revealed that MOR-1-expressing neurons were in close proximity to terminals containing opioids with enkephalin-like sequences. These findings indicate that peptidases prevent the activation of extrasynaptic MOR-1 in dorsal horn neurons.

Aminopeptidases↗

PubMed1977

[Pz peptidase activity in serum (author's transl)].

Synthetic PZ peptide, synthesized by Wünsch and Heidrich, was used as substrate to determine PZ peptidase activities in the sera of man, rabbit, guinea pig and mouse. The PZ peptidase from rabbit serum was purified 200 fold and characterized. The enzyme has an isoelectric point of 5.0 and pH optima at pH 7.2 and pH 7.9. Its behaviour on gel filtration points to a molecular weight of 60.000 dalton. The peptidase is inhibited by heavy metal ions, SH reagents and serum. Ca ions are EDTA are without any effect. In contrast to collagen peptidases from micro-organisms, the enzyme from rabbit serum does not attack native, but only denatured collagen. The results suggest that PZ peptidases participate in collagen breakdown by cleaving the collagen fragments which are released by the collagenases.

Adult↗

PubMed1990

A new look at Pz-peptidase.

Work with new, quenched fluorescence substrates of Pz-peptidase has led to the discovery that Pz-peptidase, soluble metallo-endopeptidase and endo-oligopeptidase A are either identical, or very closely related enzymes. Pz-peptidase is a metallo-endopeptidase with marked thiol-dependence. It has been confirmed that inhibitors of the type designed by Orlowski for soluble metallo-endopeptidase are effective tools in studying Pz-peptidase. There is little evidence to support the proposed role of Pz-peptidase in connective tissue matrix degradation, and the main function of the enzyme may be intracellular.

Amino Acid Sequence↗

PubMed1977

Intestinal brush border peptidases.

A brush-border-specific antiserum was raised in rabbits, with Triton X-100-solubilized brush border proteins from pig intestine being used as antigens. The antiserum was used in immunoelectrophoretic studies of brush border proteins solubilized with Triton X-100. Five immunoprecipitates were obtained which corresponded to microsomal aminopeptidase (EC 3.4.11.2), asparate aminopeptidase (EC 3.4.11.7), lactase (beta-galactosidase, EC 3.2.1.23), maltase (exo-1,4-alpha-glucosidase, EC 3.2.1.3) and sucrase-isomaltase (sucrose alpha-glucohydrolase, EC 3.2.1.48). A faint immunoprecipitate was also found for the glycylprolyl dipeptidyl peptidase (EC 3.4.14.-). The brush border proteins were solubilized on a large scale from a brush border membrane preparation by the use of Triton X-100; the peptidases obtained were homogeneous in size and had hydrophobic properties. By chromatography on columns of concanavalin A-Sepharose, hydroxyapatite, Ultrogel AcA 34, DEAE-cellulose and immunosorbent, gamma-glutamyl transpeptidase (gamma-glutamyl transferase, EC 2.3.2.2) and microsomal aminopeptidase were each isolated in separate fractions. Glycylprolyl dipeptidyl peptidase and asparate aminopeptidase were obtained in another fraction. Immunoelectrophoretic, inhibitor and chromatographic studies showed that the intestinal brush border peptidases are similar to the corresponding particulate peptidases obtained from other organs.

Amino Acids↗

PubMed1987

A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli.

Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-terminal leader sequences from many secreted and membrane proteins. Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase. We have characterized the topology of leader peptidase based on its sensitivity to protease digestion. Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively. We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger. Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K-protected fragment but does reduce the size of the trypsin-protected peptide. Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are removed from the protein, it must extend from the amino terminus to approximately residue 80. Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105. These data suggest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.

Amino Acid Sequence↗

PubMed1987

Evidence that "vaginal peptidase" is a bacterial gene product.

A peptidase has been described in vaginal samples, termed "vaginal peptidase." This enzyme has been proposed as a tissue specific marker for vaginal debris. We have explored the presence of this enzyme in vaginal swabs from alleged sexual assault victims and volunteer donors as well as bacterial cultures. These studies reveal that "vaginal peptidase" is composed of a family of peptidase isozymes that originate from several bacterial species. The characterization of "vaginal peptidase" as a tissue specific marker for vaginal debris is premature.

Bacteria↗

PubMed1987

The internal signal sequence of Escherichia coli leader peptidase is necessary, but not sufficient, for its rapid membrane assembly.

Leader peptidase of Escherichia coli, a protein of 323 residues, has three hydrophobic domains. The first, residues 1-22, is the most apolar and is followed by a polar region (23-61) which faces the cytoplasm. The second hydrophobic domain (residues 62-76) spans the membrane. The third hydrophobic domain, which has a minimal apolar character, and the polar, carboxyl-terminal two-thirds of the protein are exposed to the periplasm. Deletion of either the amino terminus (residues 4-50) or the third hydrophobic region (residues 83-98) has almost no effect on the rate of leader peptidase membrane assembly, while the second hydrophobic domain is essential for insertion (Dalbey, R., and Wickner, W. (1987) Science 235, 783-787). To further define the roles of these domains, we have replaced the normal, cleaved leader sequence of pro-OmpA and M13 procoat with regions containing either the first or second apolar domain of leader peptidase. The second apolar domain supports the translocation of OmpA or coat protein across the plasma membrane, establishing its identity as an internal, uncleaved signal sequence. In addition to this sequence, we now find that leader peptidase needs either the amino-terminal domain or the third hydrophobic domain to permit its rapid membrane assembly. These results show that, although a signal sequence is necessary for rapid membrane assembly of leader peptidase, it is not sufficient.

Base Sequence↗