Liane M. Mende-Mueller

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non‐covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non‐cytotoxic derivative PA103 exsA::Ω and several cytotoxic and non‐cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70‐kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70‐kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non‐cytotoxic strains or PA103 exsA::Ω. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70‐kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non‐cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co‐ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein.

Induction of the 17-kDa ubiquitin-like protein ISG15/UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons. The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus. Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation. The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin. Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly157-Gly158 peptide bond to generate a mature ISG15 carboxyl terminus. Irreversible inhibition of pro-ISG15 processing activity by thiol-specific alkylating agents and a pH rate dependence conforming to titration of a single group of pK a 8.1 indicate the 100-kDa enzyme is a thiol protease. Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein. As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope‐specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH2‐ and COOH‐terminal htt fragments of 20–100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63–64 kDa htt fragment detected by the NH2‐domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60–61 kDa band (detected by the NH2‐domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63–64 kDa and 60–61 kDa NH2‐domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66‐kDa, instead of the 63–64 kDa N‐domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH2‐ and COOH‐terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH2‐terminal htt fragments [detected with anti(1–17) serum] compared to controls. Cortex from HD and control brains showed similar NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48‐kDa COOH‐terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH2‐terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys‐Arg, Arg‐Arg, and Lys‐Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin‐containing peptides, peptide E and BAM‐22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM‐22P at the Lys‐Arg site between the two basic residues. The Arg‐Arg site of both peptide E and BAM‐22P was cleaved at the NH2‐terminal side of the paired basic residues to generate [Met]‐enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH2‐terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys‐Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH2‐terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.

Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases.

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same α-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, β-sheet and α-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([35S]Met)preproenkephalin. The 70‐kDa CG aspartic proteinase was purified by concanavalin A‐Sepharose, Sephacryl S‐200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val‐d‐Leu‐Pro‐Phe‐Val‐d‐Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys172‐Arg173 pair located at the COOH‐terminus of (Met)enkephalin‐Arg6‐Gly7‐Leu8, as assessed by peptide microsequencing. The importance of full‐length prohormone as substrate was demonstrated by the enzymes ability to hydrolyze 35S‐labeled proenkephalin and proopiomelanocortin and its inability to cleave tri‐ and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.

Avian liver 3-hydroxy-3-methylglutaryl-CoA synthase: Distinct genes encode the cholesterogenic and ketogenic isozymes

Full length cDNA (1.85 kb) coding for an avian liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase has been isolated and sequenced. The cDNA isolation relied on hybridization to a 32P-labeled oligonucleotide coding for a portion of the active site of HMG-CoA synthase. The identity of the avian liver cDNA was confirmed by comparison of the deduced amino acid sequence with experimentally determined protein sequence data generated upon isolation and analysis of several cysteine-containing tryptic peptides prepared from the purified ketogenic avian liver enzyme. Structural comparisons with the hamster enzyme also support this assignment. In liver, two distinct forms of HMG-CoA synthase exist to support cholesterogenic and ketogenic pathways, although this latter pathway accounts for most of the enzyme activity. In order to determine which isozyme is encoded by the isolated avian liver cDNA, the deduced amino acid composition, protein sequence, and pI have been compared with the corresponding protein chemistry data that were experimentally determined using the ketogenic enzyme. Results of these comparisons unambiguously indicate that the cDNA encodes the avian liver cholesterogenic enzyme. Observed differences between deduced and empirically determined sequence data rule out the possibility that differential splicing of a primary transcript derived from one gene can account for both isozymes.

Evaluation of Protein Sequencing Core Facilities: Design, Characterization, and Results from a Test Sample (ABRF-91SEQ)

Publisher Summary A continuing effort to inform investigators about the average capabilities of protein sequencing core laboratories has been mounted by the Association of Biomolecular Resource Facilities (ABRF) sequencing committee for the past several years. Samples with both specific design goals and well-characterized components were distributed to ABRF-member core facilities worldwide. The analysis of these data for STD-1, ABRF-89SEQ, and ABRF-90SEQ hasd provided valuable information to users regarding the performance of the average facility. In continuation of this effort, a test sample designated ABRF-91SEQ was distributed to 198 ABRF-member protein sequencing core facilities. This chapter describes the results from the analysis of the first 90 sets of data. The results of ABRF-91SEQ are consistent with and further support the conclusions reached previously. These results are based on a one time analysis of a sample with little background information provided. There were 19 responses with 100% accuracy and 26 responses with 90–99.9% accuracy.

PROTEIN SEQUENCING OF POST-TRANSLATIONALLY MODIFIED PEPTIDES and PROTEINS: DESIGN, CHARACTERIZATION AND RESULTS OF ABRF-92SEQ

Publisher Summary This chapter presents a study focusing on protein sequencing of posttranslationally modified peptides and proteins. In this study, the test sample, designated ABRF-92SEQ, was designed to evaluate the ability of core facilities to correctly identify two relatively common posttranslational modifications and other sequencing identification problems. The peptide was 37 amino acids long and contained a phosphoserine at residue 4 and hydroxyproline at residue 11. The peptide was phosphorylated at residue 4 utilizing the cAMP dependent consensus site. Seventy-three percent of facilities used glass fiber filters as a solid support for sequencing. The remainder used Porten peptide or GF membrane filters (13.5%), Sequelon arylamine (4.0%), or PVDF, Sequelon DITC membrane or CD matrix (all less than 2%). Sixty-seven percent of the facilities used polybrene as a pretreatment of their membranes, with 50% of these using 1–4 ABI FIL cycles prior to sequence analysis. Seventy percent of the facilities who responded used standard procedures for sequence analysis.

Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)

The preference of the ‘prohormone thiol protease’ (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro‐neuropeptide Y (pro‐NPY), prooplomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE‐Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro‐NPY. The processing of POMC by PTP was minimal. These results demonstrate PTPs preference for certain prohormone substrates.