Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from Neisseria meningitidis

2016 ◽  
Vol 26 (5) ◽  
pp. 312-319 ◽  
Author(s):  
Neha Sharma ◽  
Nanda G. Aduri ◽  
Anna Iqbal ◽  
Bala K. Prabhala ◽  
Osman Mirza
Keyword(s):  
Neisseria Meningitidis ◽  
Peptide Sequence ◽  
Peptide Transport ◽  
Ligand Specificity ◽  
Oligopeptide Transporter ◽  
Gene Encoding ◽  
Peptide Residue ◽  
C Terminus ◽  
Substrate Peptide ◽  
Living Organisms

Peptide transport in living organisms is facilitated by either primary transport, hydrolysis of ATP, or secondary transport, cotransport of protons. In this study, we focused on investigating the ligand specificity of the <i>Neisseria meningitidis</i> proton-coupled oligopeptide transporter (NmPOT). It has been shown that the gene encoding this transporter is upregulated during infection. NmPOT conformed to the typical chain length preference as observed in prototypical transporters of this family. In contrast to prototypical transporters, it was unable to accommodate a positively charged peptide residue at the C-terminus position of the substrate peptide. Sequence analysis of the active site of NmPOT displayed a distinctive aromatic patch, which has not been observed in any other transporters from this family. This aromatic patch may be involved in providing NmPOT with its atypical preferences. This study provides important novel information towards understanding how these transporters recognize their substrates.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell surface glycoprotein

1993 ◽  
Vol 106 (1) ◽  
pp. 31-43 ◽  
Author(s):  
X.R. Wu ◽  
T.T. Sun
Keyword(s):  
Transmembrane Domain ◽  
Core Protein ◽  
Surface Glycoprotein ◽  
Peptide Sequence ◽  
Type I ◽  
Apical Surface ◽  
Bladder Epithelium ◽  
Cell Surface Glycoprotein ◽  
Signal Peptide Sequence ◽  
C Terminus

Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells.

scholarly journals Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models

2017 ◽  
Vol 13 ◽  
pp. 2442-2457 ◽  
Author(s):  
Vladimir Kubyshkin ◽  
Nediljko Budisa
Keyword(s):  
Chemical Reactivity ◽  
Hydrolytic Stability ◽  
Ethyl Esters ◽  
Peptide Sequence ◽  
Molecular Scaffolds ◽  
Biologically Relevant ◽  
C Terminus ◽  
Ester Moiety ◽  
Peptide Models ◽  
Ph Range

Fluorinated moieties are highly valuable to chemists due to the sensitive NMR detectability of the 19F nucleus. Fluorination of molecular scaffolds can also selectively influence a molecule’s polarity, conformational preferences and chemical reactivity, properties that can be exploited for various chemical applications. A powerful route for incorporating fluorine atoms in biomolecules is last-stage fluorination of peptide scaffolds. One of these methods involves esterification of the C-terminus of peptides using a diazomethane species. Here, we provide an investigation of the physicochemical consequences of peptide esterification with partially fluorinated ethyl groups. Derivatives of N-acetylproline are used to model the effects of fluorination on the lipophilicity, hydrolytic stability and on conformational properties. The conformational impact of the 2,2-difluoromethyl ester on several neutral and charged oligopeptides was also investigated. Our results demonstrate that partially fluorinated esters undergo variable hydrolysis in biologically relevant buffers. The hydrolytic stability can be tailored over a broad pH range by varying the number of fluorine atoms in the ester moiety or by introducing adjacent charges in the peptide sequence.

scholarly journals Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Laura Dean Heckman ◽  
Maria H Chahrour ◽  
Huda Y Zoghbi
Keyword(s):  
Transcriptional Repression ◽  
Neurological Disorder ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Gene Encoding ◽  
C Terminus ◽  
Mecp2 Duplication Syndrome ◽  
Negative Effect ◽  
Duplication Syndrome

Loss of function of the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2) causes the progressive neurological disorder Rett syndrome (RTT). Conversely, duplication or triplication of Xq28 causes an equally wide-ranging progressive neurological disorder, MECP2 duplication syndrome, whose features overlap somewhat with RTT. To understand which MeCP2 functions cause toxicity in the duplication syndrome, we generated mouse models expressing endogenous Mecp2 along with a RTT-causing mutation in either the methyl-CpG binding domain (MBD) or the transcriptional repression domain (TRD). We determined that both the MBD and TRD must function for doubling MeCP2 to be toxic. Mutating the MBD reproduces the null phenotype and expressing the TRD mutant produces milder RTT phenotypes, yet both mutations are harmless when expressed with endogenous Mecp2. Surprisingly, mutating the TRD is more detrimental than deleting the entire C-terminus, indicating a dominant-negative effect on MeCP2 function, likely due to the disruption of a basic cluster.

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

scholarly journals Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types

1996 ◽  
Vol 317 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Linda HOWARD ◽  
Xiaohong LU ◽  
Sally MITCHELL ◽  
Susan GRIFFITHS ◽  
Paul GLYNN
Keyword(s):  
Snake Venom ◽  
Transmembrane Helix ◽  
Cell Types ◽  
Bovine Brain ◽  
Peptide Sequence ◽  
Cdna Libraries ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Mammalian Cell Lines ◽  
C Terminus

A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603–21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin–metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin–metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metalloprotease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (Mr 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin–metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.

scholarly journals Histone H1 Is Dispensable for Methylation-Associated Gene Silencing in Ascobolus immersusand Essential for Long Life Span

2000 ◽  
Vol 20 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Jose L. Barra ◽  
Laïla Rhounim ◽  
Jean-Luc Rossignol ◽  
Godeleine Faugeron
Keyword(s):  
Gene Silencing ◽  
Life Span ◽  
Sequence Similarity ◽  
Histone H1 ◽  
Peptide Sequence ◽  
Micrococcal Nuclease ◽  
Gene Encoding ◽  
H1 Histone ◽  
Mutant Strains ◽  
Long Life

ABSTRACT A gene encoding a protein that shows sequence similarity with the histone H1 family only was cloned in Ascobolus immersus. The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail. By constructing an artificial duplication of this gene, namedH1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process. This resulted in the complete loss of the Ascobolus H1 histone. Mutant strains lacking H1 displayed normal methylation-associated gene silencing, underwent MIP, and showed the same methylation-associated chromatin modifications as did wild-type strains. However, they displayed an increased accessibility of micrococcal nuclease to chromatin, whether DNA was methylated or not, and exhibited a hypermethylation of the methylated genome compartment. These features are taken to imply thatAscobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing. Mutant strains lacking histone H1 reproduced normally through sexual crosses and displayed normal early vegetative growth. However, between 6 and 13 days after germination, they abruptly and consistently stopped growing, indicating that Ascobolus H1 histone is necessary for long life span. This constitutes the first observation of a physiologically important phenotype associated with the loss of H1.

scholarly journals Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves

1999 ◽  
Vol 338 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Kazuya YOSHIMURA ◽  
Yukinori YABUTA ◽  
Masahiro TAMOI ◽  
Takahiro ISHIKAWA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Ascorbate Peroxidase ◽  
Differential Splicing ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
C Terminus ◽  
Mrna Variants ◽  
Alternatively Spliced ◽  
Splicing Efficiency ◽  
Spinach Leaves

We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795–800]. To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves. As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified. The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein. Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12. The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX. The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo. The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3´-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes.

scholarly journals AnkB, a Periplasmic Ankyrin-Like Protein inPseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

2000 ◽  
Vol 182 (16) ◽  
pp. 4545-4556 ◽  
Author(s):  
Michael L. Howell ◽  
Eyad Alsabbagh ◽  
Ju-Fang Ma ◽  
Urs A. Ochsner ◽  
Martin G. Klotz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Gene Encoding ◽  
Rnase Protection ◽  
C Terminus ◽  
Hydrophobic Amino Acids ◽  
Increased Sensitivity ◽  
Membrane Spanning

ABSTRACT In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. TheankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location ofankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ∼65% α-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katBand ankB are part of a small operon whose transcription is induced dramatically by H2O2, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of anankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H2O2, phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.

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