Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis

Cell-cell communication regulates many important processes in bacteria. Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis. The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide. To identify the protease(s) involved in production of the mature Phr signalling peptides, we developed assays for detecting cleavage of one of the B. subtilis Phr pentapeptides, CSF, from the proCSF precursor. Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF. Mutants lacking the three proteases that fit these criteria, subtilisin, Epr and Vpr, had a defect in CSF production. Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B. subtilis, PhrA, but they were not able to process the PhrE signalling peptide of B. subtilis, indicating that there are probably other unidentified proteases involved in Phr peptide production. Subtilisin, Epr and Vpr are members of the subtilisin family of proteases that are widespread in bacteria, suggesting that many bacterial species may be capable of producing Phr signalling peptides.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides

In Bacillus subtilis, extracellular peptide signaling regulates several biological processes. Secreted Phr signaling peptides are imported into the cell and act intracellularly to antagonize the activity of regulators known as Rap proteins. B. subtilis encodes several Rap proteins and Phr peptides, and the processes regulated by many of these Rap proteins and Phr peptides are unknown. We used DNA microarrays to characterize the roles that several rap-phr signaling modules play in regulating gene expression. We found that rapK-phrK regulates the expression of a number of genes activated by the response regulator ComA. ComA activates expression of genes involved in competence development and the production of several secreted products. Two Phr peptides, PhrC and PhrF, were previously known to stimulate the activity of ComA. We assayed the roles that PhrC, PhrF, and PhrK play in regulating gene expression and found that these three peptides stimulate ComA-dependent gene expression to different levels and are all required for full expression of genes activated by ComA. The involvement of multiple Rap proteins and Phr peptides allows multiple physiological cues to be integrated into a regulatory network that modulates the timing and magnitude of the ComA response.     

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.     

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

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.     

Functional analysis of the putative fusion domain of the baculovirus envelope fusion protein F

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F(1). The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

Biosynthesis and posttranslational processing of site-directed endoproteolytic cleavage mutants of pro-neuropeptide Y in mouse pituitary cells

Although pairs of basic amino acids are common endoproteolytic sites in prohormones, the enzymes responsible for these cleavages have not yet been characterized. To investigate the specificity of these endoproteases, cDNAs encoding pro-neuropeptide Y (pro-NPY) containing all four pairs of basic amino acids were expressed in AtT-20 cells. Pro-NPY was selected as a model substrate because it undergoes a single cleavage at the sequence -Lys-Arg- during posttranslational processing. AtT-20 cells, a mouse anterior pituitary corticotrope line, were selected because they synthesize pro-adrenocorticotropic hormone (pro-ACTH)/endorphin and cleave a well characterized subset of the eight pairs of basic amino acids in the precursor. Altered cDNAs encoding pro-NPY with -Arg-Arg-, -Arg-Lys-, or Lys-Lys- at the cleavage site were used to generate stable cell lines. The production of NPY and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, reversed-phase high performance liquid chromatography, ion-exchange high performance liquid chromatography, tryptic peptide mapping, and microsequencing. Direct amino acid labeling confirmed the identity of the pair of basic amino acids at the cleavage site. Even when the four pairs of basic amino acids were presented in the same structural context, the rate, extent, and type of cleavage was substrate-specific. Pro-NPY(-Arg-Arg-) was cleaved at a rate similar to that observed for the wild-type pro-NPY(-Lys-Arg-). In contrast, pro-NPY(-Arg-Lys-) was cleaved at a much lower rate, and pro-NPY (-Lys-Lys-) was cleaved very poorly. Following endoproteolytic cleavage, the pair of basic amino acids present did not alter the production of mature NPY with a COOH-terminal Tyr-NH2. While two of the three mutant pro-NPY molecules were processed to wild-type carboxyl-terminal peptide, the carboxyl-terminal peptide derived from pro-NPY(-Arg-Lys-) contained an amino-terminal lysine residue, indicating that biosynthetic endoproteolysis occurred in the middle or at the amino terminus of the pair of basic amino acid residues at the cleavage site. Expression of wild-type or mutant pro-NPY inhibited cleavages within the endogenous pro-ACTH/endorphin; poorly cleaved pro-NPY mutants (Lys in the second position of the cleavage site) were the most potent inhibitors of pro-ACTH/endorphin cleavage.     

Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro

A series of synthetic peptides representing authentic proteolytic cleavage sites of human rhinovirus type 14 were assayed as substrates for purified 3C protease. Competition cleavage assays were employed to determine the relative specificity constants (Kcat/Km) for substrates with sequences related to the viral 2C-3A cleavage site. Variable length peptides representing the 2C-3A cleavage site were cleaved with comparable efficiency. These studies defined a minimum substrate of 6 amino acids (TLFQ/GP), although retention of the residue at position P5 (ETLFQ/GP) resulted in a better substrate by an order of magnitude. Amino acid substitutions at position P5, P4, P1', or P2' indicated that the identity of the residue at position P5 was not critical, whereas substitutions at position P4, P1' or P2' resulted in substrates with Kcat/Km values varying over 2 orders of magnitude. In contrast to the 2C-3A cleavage site, small peptide derivatives representative of the 3A-3B cleavage site were relatively poor substrates, which suggested that residues flanking the minimum core sequence may influence susceptibility to cleavage. The 3C protease of rhinovirus type 14 was also capable of cleaving peptides representing comparable cleavage sites predicted for coxsackie B virus and poliovirus.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

Molecular analysis of Phr peptide processing in Bacillus subtilis

In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.     

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.     

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Complete amino acid sequence of the thioesterase domain of chicken liver fatty acid synthase

The complete amino acid sequence of thioesterase domain of chicken liver fatty acid synthase has been determined by sequencing peptides produced by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage. The thioesterase domain consists of 300 amino acid residues. All of the tryptic peptides of the thioesterase domain were isolated and sequenced, except the segment covered from position 109 to position 124. Peptides resulting from digestion by Staphylococcus aureus V8 protease and cyanogen bromide cleavage filled the missing part and overlapped the complete sequence of the entire thioesterase domain. The NH2 terminus of the thioesterase domain was determined to be lysine by sequencing the whole domain up to 20 residues while the COOH terminus was identified as serine through carboxyl peptidase Y cleavage. The active site of the thioesterase domain of chicken fatty acid synthase was suggested to be the serine on position 101 according to its homology with other serine-type esterases and proteases which have a common structure of -Gly-X-Ser-Y-Gly- with the variable amino acids X and Y disrupting the homology.     

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.     

Pentapeptides containing two dehydrophenylalanine residues ‐ synthesis,structural studies and evaluation of their activity towards cathepsin C

Synthesis, structural and biological studies of pentapeptides containing two ΔPhe residues (Z and E isomers) in position 2 and 4 in peptide chain were performed. All the investigated peptides adopted bent conformation and majority of them could exist as two different conformers in solution. Only pentapeptides, containing free N‐termini appeared to act as weak inhibitors of cathepsin C with the slow‐binding, competitive mechanism of inhibition, free acids being bound slightly better than their methyl esters. Results of molecular modeling suggested significant difference between peptides, depending of the type of amino acid residue in position 5 in peptide chain. Dehydropeptides containing Gly residue in this position may act as competitive slow‐reacting substrates and therefore exhibit inhibitory‐like properties. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

The extracellular transport signal of the Vibrio cholerae endochitinase (ChiA) is a structural motif located between amino acids 75 and 555

ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kan(r)), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.