Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Biometals ; 33(4-5): 169-185, 2020 10.
Article in English | MEDLINE | ID: mdl-32648080

ABSTRACT

Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models. The protein interacts specifically with DNA at binding sites designated as 'Fur boxes'. In the present study, we have investigated the interaction between Fur from the fish pathogen Aliivibrio salmonicida (AsFur) and its target DNA using a combination of biochemical and in silico methods. A series of target DNA oligomers were designed based on analyses of Fur boxes from other species, and affinities assessed using electrophoretic mobility shift assay. Binding strengths were interpreted in the context of homology models of AsFur to gain molecular-level insight into binding specificity.


Subject(s)
Aliivibrio salmonicida/chemistry , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Repressor Proteins/metabolism , Aliivibrio salmonicida/metabolism , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , DNA, Bacterial/chemistry , Models, Molecular , Protein Binding , Repressor Proteins/chemistry
2.
FEBS Open Bio ; 9(10): 1674-1688, 2019 10.
Article in English | MEDLINE | ID: mdl-31420950

ABSTRACT

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat-labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128-Tyr129-Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides.


Subject(s)
Disulfides/chemistry , Exoribonucleases/chemistry , Exoribonucleases/genetics , Metagenome/genetics , Chromatography, Gel , Computational Biology , Crystallography, X-Ray , Disulfides/metabolism , Exoribonucleases/metabolism , Humans , Models, Molecular , Protein Conformation
3.
Extremophiles ; 23(3): 305-317, 2019 May.
Article in English | MEDLINE | ID: mdl-30826937

ABSTRACT

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterized the temperature optima and thermal stabilities of three minimal Lig E-type ATP-dependent DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig, and Par-Lig, show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely cold-adapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins. A comparative approach based on sequence comparison and homology modeling indicates that the cold-adapted features of Vib-Lig may be ascribed to differences in surface charge rather than increased local or global flexibility which is consistent with the contemporary emerging paradigm of the physical basis of cold adaptation of enzymes.


Subject(s)
Acclimatization/physiology , Cold Temperature , DNA Ligases , Gram-Negative Bacteria , Models, Molecular , DNA Ligases/chemistry , DNA Ligases/genetics , DNA Ligases/metabolism , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/genetics
4.
J Med Chem ; 59(7): 2918-27, 2016 Apr 14.
Article in English | MEDLINE | ID: mdl-26982623

ABSTRACT

Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.


Subject(s)
Antineoplastic Agents/pharmacology , Oligopeptides/blood , Oligopeptides/pharmacology , Animals , Antineoplastic Agents/blood , Blood Proteins , Cell Line, Tumor/drug effects , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Dogs , Drug Discovery , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Drug Stability , Half-Life , Humans , Mice, Inbred BALB C , Peptides/chemistry , Peptides/pharmacology , Rats
5.
Dev Comp Immunol ; 33(4): 638-45, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19063917

ABSTRACT

Type I interferons (IFNs) play a crucial role in innate immune responses against virus infections in vertebrates. Two IFNs (IFNa1 and IFNa2) have previously been cloned from Atlantic salmon. In the present work a polyclonal antiserum, which was generated against salmon IFNa1 was used to study its production in cells by immunoblot detection and neutralization of antiviral activity. The antiserum was first confirmed to detect and neutralize the antiviral activity of recombinant salmon IFNa1 produced in HEK293 cells. The antiserum also detected IFNa1 and neutralized 95-98% of the antiviral activity in supernatants of poly I:C stimulated salmon TO cells. This suggests that IFNa1/IFNa2 are the major IFNs produced by poly I:C stimulated TO cells. The antiserum neutralized most of the IFN activity in poly I:C stimulated head kidney leucocytes from three of five individuals, but in stimulated leucocytes from the other two individuals only 75% of the antiviral activity was neutralized. This shows that although IFNa1/IFNa2 are major IFNs secreted by poly I:C stimulated leucocytes, these cells can also produce additional molecules with IFN-like activity.


Subject(s)
Interferon Type I/immunology , Interferon-alpha/immunology , Leukocytes/immunology , Salmo salar/immunology , Animals , Antibodies/immunology , Cell Line , Cloning, Molecular , Cytopathogenic Effect, Viral/immunology , Humans , Infectious pancreatic necrosis virus/immunology , Interferon Type I/isolation & purification , Interferon-alpha/drug effects , Leukocytes/drug effects , Leukocytes/virology , Poly I-C/immunology , Poly I-C/pharmacology , Recombinant Proteins , Salmo salar/virology , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...