scholarly journals A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 142-148 ◽  
Author(s):  
Brian M. Forster ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Positive Charge ◽  
Bacterial Pathogen ◽  
Histidine Residue ◽  
Amino Acid Residues ◽  
Intracellular Growth ◽  
N Terminus ◽  
Membrane Bound

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

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.

scholarly journals Rapid resensitization of ASIC2a is conferred by three amino acid residues in the N-terminus

IBRO Reports ◽  
2019 ◽  
Vol 6 ◽  
pp. S427-S428
Author(s):  
Jae Seung Lee ◽  
Hae-Jin Kweon ◽  
Byung-Chang Suh ◽  
Hyosang Lee
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
N Terminus

scholarly journals Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Susan M. Mitchell ◽  
Morven Graham ◽  
Xinran Liu ◽  
Ralf M. Leonhardt
Keyword(s):  
Amino Acid ◽  
Pigment Cell ◽  
Specific Protein ◽  
Single Amino Acid ◽  
Amyloid Formation ◽  
Amino Acid Residues ◽  
Proteolytic Fragment ◽  
The Core ◽  
N Terminus ◽  
In Cis

AbstractThe pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

scholarly journals The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A

2006 ◽  
Vol 80 (22) ◽  
pp. 11343-11354 ◽  
Author(s):  
Haekyung Lee ◽  
Ying Liu ◽  
Edison Mejia ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Decisive Role ◽  
Amino Acid Residues ◽  
Subgenomic Replicon ◽  
Hydrophobic Domain ◽  
Virus Rna ◽  
Membrane Bound

ABSTRACT Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

scholarly journals Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

2015 ◽  
Vol 57 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Kenshi Watanabe ◽  
Makoto Ohno ◽  
Masahiro Taguchi ◽  
Seiji Kawamoto ◽  
Kazuhisa Ono ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residues ◽  
Fatty Acid Desaturases ◽  
Front End ◽  
Membrane Bound

scholarly journals A Molecular Genetic Dissection of the Evolutionarily Conserved N Terminus of Yeast Rad52

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 549-562
Author(s):  
Uffe H Mortensen ◽  
Naz Erdeniz ◽  
Qi Feng ◽  
Rodney Rothstein
Keyword(s):  
Dna Damage ◽  
Amino Acid ◽  
Dna Binding ◽  
Molecular Genetic ◽  
Mitotic Recombination ◽  
Amino Acid Residues ◽  
Γ Irradiation ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Γ Ray

Abstract Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of γ-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient γ-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair γ-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of γ-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by γ-irradiation.

scholarly journals Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity

2000 ◽  
Vol 352 (2) ◽  
pp. 251-255 ◽  
Author(s):  
Petra PRIJATELJ ◽  
Alenka ČOPIČ ◽  
Igor KRIŽAJ ◽  
Franc GUBENŠEK ◽  
Jože PUNGERČAR
Keyword(s):  
Amino Acid ◽  
Phospholipase A2 ◽  
Binding Affinity ◽  
Positive Charge ◽  
Basic Amino Acid ◽  
Terminal Region ◽  
Single Site ◽  
Amino Acid Residues ◽  
Multiple Site ◽  
Charge Reversal

The positive charge concentrated at the C-terminal region of ammodytoxin (Atx) A, which is involved in presynaptic toxicity, has been reversed. A six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E, where K108N = Lys108 → Asn etc.) was prepared, in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones. The mutant was approximately 30-fold less lethal, but still neurotoxic. Consistent with this, its binding affinity for the neuronal receptors decreased by only a factor of five. Additionally, a single-site mutant of AtxA was prepared, with substitution at only one position (K127T) out of six mutated in the six-site mutant. Its toxicity indicated that most, if not all, of the six mutated residues partially contribute to the decreased lethality of the multiple-site mutant.

scholarly journals Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney

1987 ◽  
Vol 241 (1) ◽  
pp. 229-235 ◽  
Author(s):  
P E Butler ◽  
M J McKay ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Degradation Products ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Mouse Kidney ◽  
Amino Acid Residues ◽  
Peptide Bonds ◽  
Membrane Bound ◽  
Terminal Amino

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S–S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

scholarly journals Rapid resensitization of ASIC2a is conferred by three amino acid residues in the N terminus

2019 ◽  
Vol 151 (7) ◽  
pp. 944-953
Author(s):  
Jae Seung Lee ◽  
Hae-Jin Kweon ◽  
Hyosang Lee ◽  
Byung-Chang Suh
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transmembrane Region ◽  
Acid Sensing Ion Channels ◽  
Electrophysiological Responses ◽  
N Terminus ◽  
Key Residues

Acid-sensing ion channels (ASICs), sensory molecules that continuously monitor the concentration of extracellular protons and initiate diverse intracellular responses through an influx of cations, are assembled from six subtypes that can differentially combine to form various trimeric channel complexes and elicit unique electrophysiological responses. For instance, homomeric ASIC1a channels have been shown to exhibit prolonged desensitization, and acid-evoked currents become smaller when the channels are repeatedly activated by extracellular protons, whereas homomeric or heteromeric ASIC2a channels continue to respond to repetitive acidic stimuli without exhibiting such desensitization. Although previous studies have provided evidence that both the desensitization of ASIC1a and rapid resensitization of ASIC2a commonly require domains that include the N terminus and the first transmembrane region of these channels, the biophysical basis of channel gating at the amino acid level has not been clearly determined. Here, we confirm that domain-swapping mutations replacing the N terminus of ASIC2a with that of ASIC2b result in de novo prolonged desensitization in homomeric channels following activation by extracellular protons. Such desensitization of chimeric ASIC2a mutants is due neither to internalization nor to degradation of the channel proteins. We use site-directed mutagenesis to narrow down the relevant portion of the N terminus of ASIC2a, identifying three amino acid residues within the N terminus (T25, T39, and I40) whose mutation is sufficient to phenocopy the desensitization exhibited by the chimeric mutants. A similar desensitization is observed in heteromeric ASICs containing the mutant subunit. These results suggest that T25, T39, and I40 of ASIC2a are key residues determining the rapid resensitization of homomeric and heteromeric ASIC2a channels upon proton activation.

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