scholarly journals Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

1999 ◽  
Vol 67 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Brenda A. Wilson ◽  
Virgilio G. Ponferrada ◽  
Jefferson E. Vallance ◽  
Mengfei Ho
Keyword(s):  
Amino Acid ◽  
Pasteurella Multocida ◽  
Mutational Analysis ◽  
Vero Cells ◽  
Full Length ◽  
Induced Response ◽  
Intracellular Activity ◽  
C Terminus ◽  
N Terminus ◽  
Activity Domain

ABSTRACT We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 inXenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using theXenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entiretoxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein inEscherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals Determination of the immunogenic region in the LipL32 protein of Leptospira

2018 ◽  
pp. 27-33
Author(s):  
Mohammad Iskandar Jumat ◽  
Kenneth Francis Rodrigues ◽  
Azlyna Laribe ◽  
Rashidah Mohammad ◽  
Timothy William ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Specificity ◽  
Full Length ◽  
Immunogenic Region ◽  
C Terminus ◽  
Initial Symptoms ◽  
N Terminus ◽  
Low Sensitivity

Leptospirosis is a zoonotic disease caused by the pathogenic species of Leptospira. The initial symptoms include fever, myalgia, nausea, skin rash, chills, and headache, which can be misdiagnosed. LipL32 is the highly conserved and abundant outer membrane protein (OMP) of Leptospira, which is used as an antigen in serodiagnostic assays. We used three in silico methods to predict the immunodominant regions in the full-length LipL32 protein. We identified three regions, namely the N-terminus (NrLipL32, amino acid sequence 20th-120th), intermediate (amino acid sequence 120th-150th), and C-terminus (CrLipL32, amino acid sequence 160th-260th) regions. The full-length protein and two larger fragments were cloned into the pET22b plasmid and expressed in Escherichia coli BL21 (DE3). The purified proteins were used as antigens in an ELISA to detect Leptospira-specific antibodies. The CrLipL32 ELISA showed the highest sensitivity for IgM (73.3%) and IgG (65%), followed by the full-length rLipL32 ELISA (IgM 68% and IgG 60%). The full-length rLipL32 ELISA showed high specificity (IgM 85% and IgG 75%), followed by the NrLipL32 ELISA (IgM 75% and IgG 60%). The intermediate fragment showed very low sensitivity (IgM 17% and IgG 2%). The sensitivity of the rLipL32 ELISA could be enhanced by adding other OMPs of Leptospira.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT 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::Kanr), 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.

scholarly journals DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides

1999 ◽  
Vol 342 (1) ◽  
pp. 7-12 ◽  
Author(s):  
Simon DOWLER ◽  
Richard A. CURRIE ◽  
C. Peter DOWNES ◽  
Dario R. ALESSI
Keyword(s):  
Amino Acid ◽  
Sh2 Domain ◽  
Ph Domain ◽  
Phosphorylated Proteins ◽  
High Affinity ◽  
C Terminus ◽  
Acid Protein ◽  
N Terminus ◽  
Src Homology ◽  
Amino Acid Protein

We have identified a novel 280 amino acid protein which contains a putative myristoylation site at its N-terminus followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus. It has been termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1). DAPP1 is widely expressed and exhibits high-affinity interactions with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not with other phospholipids tested. These observations predict that DAPP1 will interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

scholarly journals Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

2010 ◽  
Vol 432 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Emily R. Slepkov ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Phospholipase C ◽  
Bacterial Pathogen ◽  
Amino Acid Residues ◽  
C Terminus ◽  
N Terminus ◽  
Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

The Biochemical Characterization of Three Forms of Ku86 Protein in Multiple Myeloma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3416-3416
Author(s):  
Charles A. Gullo ◽  
Feng Ge ◽  
Geraline Cow ◽  
Gerrard Teoh
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Protein Kinase ◽  
Cell Lines ◽  
Proteasome Inhibition ◽  
Full Length ◽  
Genome Maintenance ◽  
C Terminus ◽  
N Terminus ◽  
Ku Proteins

Abstract Karyotypic analysis of tumor cells from patients with multiple myeloma (MM), as well as MM cell lines, frequently demonstrates numerous complex chromosomal abnormalities. Moreover, new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32), often heralds transformation to more aggressive MM. Since DNA double stranded break repair (DSBR) is important in mediating these processes, these data suggest that abnormalities in DSBR could ultimately lead to genomic instability, clonal evolution and disease progression in MM. Truncated variants of Ku86 protein (i.e. Ku86v) have previously been detected in 86% to 100% of freshly isolated patient MM cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA protein kinase (DNA-PK), we and others have been interested in the altered expression and function of Ku86v proteins in genome maintenance in MM. Although a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in B cells and the K562 chronic myeloid leukemia cell line, we now show using whole cell Western immunoblotting that the RPMI 8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as at least 2 forms of Ku86v - a C-terminus truncated 69 kDa variant Ku86 protein (Ku86v-N); and an N-terminus truncated 56 kDa Ku86v (Ku86v-C). Expression of full-length Ku86 and Ku86v proteins was confirmed using electrophoretic mobility shift assays (EMSA) that incorporate a Ku86-specific DNA probe. In contrast, Ku86v proteins were not detected in the non-MM K562 cell line, by neither whole cell Western blotting nor EMSA, as was previously reported. These data confirm that MM cell lines contained bona fide Ku86v proteins that were generated intracellularly. However, the expected shorter mRNA transcripts of Ku86v’s were not detected using Northern blotting, indicating that Ku86v’s could have been generated by enzymatic cleavage, i.e. post-translational modification, rather than by alternative splicing. Since protease digestion of DNA protein kinase (DNA-PK) and Ku proteins is enhanced by proteasome inhibition (i.e. bortezomib treatment) in MM cell lines; these data taken in aggregate further suggest that proteolytic enzymes that are capable of digesting Ku proteins are constitutively activated, and possibly accumulate and/or become further activated under proteasome inhibition in MM cells. In order to characterize the functional role for Ku86v, we demonstrate using EMSA that both full-length Ku86 and Ku86v-N, but not Ku86v-C, are capable of binding DNA. Since the DNA binding motifs of Ku86 are located in the N-terminus, and the functional domains are located in the C-terminus, these data support the notion that whilst Ku86v-N binds DNA, it is in fact incapable of regulating DNA repair. By contrast, although Ku86v-C does not bind DNA, it may be capable of regulating other biological processes. Accordingly, we demonstrate that Ku86v-C binds to CDK4, E2F-4, BAX, Bcl2 and p53; suggesting at least a possible role for Ku86v proteins in regulating the growth and survival of MM cells. In conclusion, this study confirms that MM cells generate at least 3 forms of Ku86 protein, and that the processes of genome maintenance and/or myelomagenesis could be functionally regulated by these abnormal Ku86v proteins.

scholarly journals Effects of l- and d-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication

2002 ◽  
Vol 366 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Bouchaib BAHBOUHI ◽  
Nathalie CHAZAL ◽  
Nabil Georges SEIDAH ◽  
Cristina CHIVA ◽  
Marcelo KOGAN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Envelope Glycoprotein ◽  
Cleavage Site ◽  
Molecular Level ◽  
Principal Cell ◽  
Inhibition Constant ◽  
Glycoprotein Precursor ◽  
C Terminus ◽  
N Terminus

The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1BRU gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35μM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70μM. The data show that only dec14d and dec14dcmk could inhibit HIV-1BRU, HIV-2ROD and SIVmac251 replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant Ki = 4.6μM, whereas the peptide dec14l preferentially inhibited furin with a Ki = 28μM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors.

scholarly journals Mutational Analysis of Quinolone Resistance Protein QnrB1

2013 ◽  
Vol 57 (11) ◽  
pp. 5733-5736 ◽  
Author(s):  
George A. Jacoby ◽  
Marian A. Corcoran ◽  
Debra M. Mills ◽  
Caitlin M. Griffin ◽  
David C. Hooper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Essential Amino Acids ◽  
Quinolone Resistance ◽  
Single Amino Acid ◽  
Protective Activity ◽  
N Terminus ◽  
Resistance Protein

ABSTRACTAlanine substitutions and selected deletions have been used to localize amino acids in QnrB essential for its protective activity. Essential amino acids are found at positions i and i−2in the pentapeptide repeat module and in the larger of two loops, where deletion of only a single amino acid compromises activity. Deletion of 10 amino acids at the N terminus is tolerated, but removal of 3 amino acids in the C-terminal dimerization unit destroys activity.

scholarly journals Modular Organization of the Phd Repressor/Antitoxin Protein

2004 ◽  
Vol 186 (9) ◽  
pp. 2692-2698 ◽  
Author(s):  
Jeremy Allen Smith ◽  
Roy David Magnuson
Keyword(s):  
Amino Acid ◽  
Genetic Structure ◽  
Modular Organization ◽  
Toxin Gene ◽  
Resolution Limit ◽  
C Terminus ◽  
N Terminus ◽  
Repressor Activity ◽  
Host Death

ABSTRACT The P1 plasmid addiction operon is a compact genetic structure consisting of promoter, operator, antitoxin gene (phd), and toxin gene (doc). The 73-amino-acid antitoxin protein, Phd, has two distinct functions: it represses transcription (by binding to its operator) and it prevents host death (by binding and neutralizing the toxin). Here, we show that the N terminus of Phd is required for repressor but not antitoxin activity. Conversely, the C terminus is required for antitoxin but not repressor activity. Only a quarter of the protein, the resolution limit of this analysis, was required for both activities. We suggest that the plasmid addiction operon is a composite of two evolutionarily separable modules, an operator-repressor module and an antitoxin-toxin module. Consideration of similar antitoxin proteins and their surroundings indicates that modular exchange may contribute to antitoxin and operon diversity.

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