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PubMed1998

A new signal peptidase gene from Streptomyces lividans TK21.

Using synthetic oligonucleotides derived from known signal peptidase genes and a multicopy plasmid as a vector, a signal peptidase gene (sipZ) from Streptomyces lividansTK21 has been cloned. The primary structure of the gene has been determined and the amino acid composition of the SipZ protein inferred. SipZ is 258 aa long and showed homology to other type I signal peptidases, containing like them an N-terminal transmembrane anchor. Alignment of SipZ with other known SPases allowed the identification of a conserved sequence of amino acids specific for Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1998

The Sip(Sli) gene of Streptomyces lividans TK24 specifies an unusual signal peptidase with a putative C-terminal transmembrane anchor.

Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.

Amino Acid Sequence↗
PubMed1983

Characterization of papaya peptidase A as an enzyme of extreme basicity.

Papaya peptidase A, a papain-like enzyme, has formerly been shown to contain a larger excess of basic amino acids and to have a higher isoelectric point than any of the other enzymes in the papaya latex. Determinations of the free electrophoretic mobility as a function of pH now establishes the isoelectric point of papaya peptidase A as 11.7 and that of succinylated papaya peptidase A as 3.8. Although the specific activity of the enzyme appears only slightly affected by the succinylation, the accompanying change in the charge/mobility ratio seems to indicate a relatively large conformational change upon succinylation.

Cysteine Endopeptidases↗
PubMed2002

Extracellular peptidase activity tunes motor pattern modulation.

We are examining how extracellular peptidase activity sculpts the peptidergic actions of modulatory projection neurons on rhythmically active neuronal circuits, using the pyloric circuit in the stomatogastric ganglion (STG) of the crab Cancer borealis. Neurally released peptides can diffuse long distances to bind to their receptors. Hence, different neurons releasing the same neuropeptide into the same neuropil may reach the same receptor complement. However, extracellular peptidases can limit neuropeptide diffusion and terminate its actions. Distinct versions of the pyloric rhythm are elicited by selective activation of different projection neurons, including those with overlapping sets of cotransmitters. Two of these projection neurons, modulatory commissural neuron 1 (MCN1) and the modulatory proctolin neuron (MPN), contain the neuropeptide proctolin plus GABA. MCN1 also contains Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). CabTRP Ia is not fully responsible for the distinct actions of MCN1 and MPN. Because there is aminopeptidase activity in the STG that terminates proctolin actions, we tested the hypothesis that the differences in the actions of MCN1 and MPN that are not mediated by CabTRP Ia result from the differential actions of aminopeptidase activity on proctolin released from these two projection neurons. We found that the pyloric circuit response to these two projection neurons becomes more similar when this aminopeptidase activity is blocked. This result supports the hypothesis that extracellular peptidase activity enables different projection neurons to use the same neuropeptide transmitter for eliciting distinct outputs from the same neuronal circuit.

Aminopeptidases↗
PubMed1998

Inhibitors of bacterial signal peptidases.

Signal peptidases are serine endoproteinases responsible for the proteolytic removal of N-terminal amino acid signal sequences from pre-secretory proteins in all cellular lifeforms including bacteria. The demonstrated essentiality of the enzymatic activity and the ubiquitous occurrence in bacteria, coupled with the significant molecular differences between bacterial signal peptidases and those of eukaryotes, define these enzymes as potential molecular targets for the development of novel antibacterial agents. Few compounds are known to inhibit bacterial signal peptidases and the most significant advance in SPase inhibition has been the discovery of penem systems as time dependent irreversible inhibitors. They are thought to act via acylation of the active site serine. SPases are only acylated by the 5S penem stereochemistry, a stereochemical preference mirrored in other azetidinone containing inhibitors. The implications of this is that the active site serine of SPases approach their substrate from the opposite side of the peptidic backbone to that of all other serine protease families whose structures are known. The activity of penems is significantly enhanced by the incorporation of a C6 hydroxyethyl substituent, thought to bind into the S1 pocket of the enzyme. Penem inhibition of SPases has been demonstrated in vitro, in isolated enzyme assays, and in vivo in pulse-chase assays.

Anti-Bacterial Agents↗
PubMed1999

Clearance of human brain natriuretic peptide in rabbits; effect of the kidney, the natriuretic peptide clearance receptor, and peptidase activity.

Although the synthetic version of the cardiac peptide human brain natriuretic peptide (hBNP) has demonstrated beneficial cardiovascular effects in clinical studies, little is known about mechanisms governing its elimination from the blood. This study measured the role of the kidney, the natriuretic peptide clearance (NP-C) receptor, and peptidase digestion on the elimination of synthetic hBNP from the plasma compartment of rabbits. The estimated plasma steady state resulting from a continuous i.v. infusion was achieved within 50 min and was related in a linear manner with the infusion rate of the drug. Complete restriction of kidney blood flow by bilateral suture-ligation of the renal arteries compared with sham-treated animals reduced the clearance of hBNP by approximately half (24 +/- 9 ml/min versus 47 +/- 14 ml/min, respectively, p <. 007). Pharmacological blockade of the NP-C receptor with a clearance receptor-specific analog of atrial natriuretic peptide increased in a statistically significant and dose-related manner the plasma steady-state level of hBNP during continuous i.v. infusion of hBNP (maximum effect of 1.9 +/- 0.3-fold, p <.01). The peptidase inhibitor phosphoramidon increased in a dose-related manner the plasma steady-state level of hBNP 1.7 +/- 0.4-fold during continuous i.v. infusion of hBNP in rabbits. These data suggest that the kidney, the NP-C receptor, and peptidases are all important in the elimination of hBNP from the plasma compartment.

Animals↗
PubMed1976

Intestinal peptidase activity following whole-body irradiation in mice.

Dipeptidase activity (Gly-L-Leucine, L. Leu-Glycine, Gly-L-Phe, L Phe-Gly) was measured in the cytoplasmic fraction of the small bowel mucosa of mice eating three synthetic, defined formula, diets or Purina Chow. The activity/milligram protein of all peptidases was found to be reduced seven days after the last of six weekly exposures to 400 rads (Cobalt 60). The magnitude of reduction varied from half the control value for L-Leu-Glycine peptidase to between one fourth and one fifth the control values for the other three peptidases. These findings may partly explain the improved tolerance to radiation in mice eating a diet containing amino acids (protein hydrolysate) instead of whole protein.

Animals↗
PubMed1990

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.

Amino Acid Sequence↗
PubMed1990

The effect of the typical peptidases (proteases) inhibitors on the degradation of Glp6 (125I) Tyr8SP6-11 hexapeptide in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain.

The main peptidase PN/cutting Tyr8-Gly9 or Gly9-Leu10 bond (of sequence Glp6-Phe7-Tyr8-Gly9-Leu10-MetNH2) seems to be, at least in part, cysteine type enzyme. Cutting of Phe7-Tyr8 bond with PC enzyme is apparently negligible. Further degradation of labelled PN products seems to be accomplished with PI, being serine enzyme at least in part. Metalloenzymes, including "enkephalinase", seem to be of minor importance in hexapeptide degradation, at least in its very low concentration. Some typical inhibitors enhance the degradation what might be explained assuming that products of action of one peptidase strongly inhibit the other peptidase's action. Namely, products of PN and PI seem to inhibit PC except the hippocampal synaptosomes where the opposite is true.

Animals↗
PubMed1991

Prediction of the site of signal peptidase cleavage in normal and variant human preproalbumin.

We describe the use of a template for the recognition site of signal peptidase to give score predictions for cleavage at normal and aberrant sites in human and chicken preproalbumin. As expected, the highest score obtained for human preproalbumin was at the normal cleavage site between -7 Ser and -6 Arg. The mutation in preproalbumin Kaikoura (-2 Arg----Cys) introduced an aberrant cleavage site in the propeptide between the new -2 Cys and -1 Arg. Although the new site had a lower template score (6.88) than the normal site (9.99), it is cleaved in vivo about five times faster. The presence of minor proportions of des-Asp and des-Asp-Ala-albumin in normal human plasma was not found to correlate with possible aberrant signal peptidase cleavage. These truncated albumin species, therefore, appear to be generated through cleavage of mature albumin in circulation. Chicken preproalbumin had only one probably signal peptidase cleavage site, the one utilised in vivo. Template scores should be regarded as qualitative predictions rather than definitive quantitative indicators.

Algorithms↗
PubMed1990

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase.

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.

Amino Acid Sequence↗
PubMed1979

[Inhibition of PZ-peptidases by serum (author's transl)].

The inhibition of two PZ-peptidases by several sera was studied with the aid of methods based on the PZ-peptidase assay of Wünsch and Heidrich Clostridiopeptidase A is inhibited by all sera, whilst the PZ-peptidase from the rabbit serum is inhibited only by heterologous sera. The inhibition is weak and cannot be compared with the strong serum inhibition shown by collagenases.

Animals↗
PubMed1990

Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site.

Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site. These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains. It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity. This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site. In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide. The maturation of the mutant precursor species expressed in vivo was examined. Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3. The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site. The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites. This appears to represent another example of redundant information contained within the signal peptide.

ATP-Binding Cassette Transporters↗
PubMed1989

Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays.

In eukaryotic cells ubiquitin is synthesized as a polyubiquitin protein or as a protein fused at the carboxyl terminus to other polypeptides. An enzyme activity, ubiquitin protein peptidase, has been proposed to process these precursors by cleaving the peptide bond between adjoining ubiquitin molecules or between ubiquitin and the fused peptides. Using the cleavage of a 35S-labeled yeast ubiquitin protein fused to a synthetic 38-residue peptide obtained by in vivo metabolic labeling in Escherichia coli in an expression system based on the interaction of bacteriophage T7 RNA polymerase and its promoter, it is possible to detect a processing activity in soluble yeast extract. The specificity of the cleavage suggests this activity could be the in vivo processing activity for various ubiquitin precursor proteins in yeast cells. A similarly labeled ubiquitin protein fused to one cysteine residue was also utilized to detect an activity capable of removing a single cysteine residue from ubiquitin in a soluble extract. Employing assays based on the cleavage of labeled ubiquitin protein fusions, a ubiquitin protein peptidase activity from Saccharomyces cerevisiae was purified about 15,000-fold to yield a protein mixture consisting of only a few protein species. The major protein band which comigrated with the activities in in vitro assays has an apparent molecular weight of 29,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two other protein species, about 20,000 and 10,000 in molecular weight, also comigrated with the in vitro activities throughout the purification procedure. Though our most purified protein fraction was shown to cleave various artificial ubiquitin protein fusions under our experimental conditions, it cannot cleave a ubiquitin dimer protein, suggesting the existence of functionally distinct ubiquitin protein peptidases. Our experimental protocol for preparing various labeled ubiquitin protein precursors provides a means to explore various processing enzymes existing in cells. The same protocol may also be adapted to prepare substrates for the study of other specific protein processing enzymes.

Amino Acid Sequence↗
PubMed1989

Uncoupling of co-translational translocation from signal peptidase processing in a mutant rat preapolipoprotein-A-IV with a deletion that includes the COOH-terminal region of its signal peptide.

In order to characterize the function of the COOH-terminal (c) region of eukaryotic signal peptides, a 14-amino acid long segment was deleted from a secreted rat liver and intestinal protein, preapolipoprotein-A-IV. This deletion spanned the c-region plus all potential signal peptidase cleavage sites. The functional consequences of this mutation were assessed using an in vitro transcription/translation/microsomal membrane processing system. Removal of these residues had no effect on interaction of the nascent preprotein with signal recognition particle as measured by a translational arrest assay. Although no signal peptidase cleavage of the mutant was detected, the efficiency of its co-translational translocation was similar to the wild type protein. A postinitiation translocation assay was utilized to compare the translocation capabilities of nascent wild type and mutant proteins as their chain lengths were progressively increased. No difference was detected between the two species suggesting that their initial conformations are functionally equivalent as measured by the translocation machinery. We conclude from these studies of preapolipoprotein-A-IV that (i) the efficiency of translocation is not dependent on signal peptidase cleavage and (ii) structural features present in the c-region of the signal peptide are not necessary for interaction with signal recognition particle or for subsequent targeting to, and translocation across, microsomal membranes.

Amino Acid Sequence↗
PubMed1989

Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase.

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.

Amino Acid Sequence↗
PubMed1989

A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein.

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (Böhni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.

Amino Acid Sequence↗
PubMed1985

Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane.

Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins. We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter. This strain requires arabinose for growth. When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein). These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K. However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane. Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane.

Arabinose↗