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A widespread peroxiredoxin-like domain present in tumor suppression- and progression-implicated proteins.
Get PDFBackground
Peroxide turnover and signalling are involved in many biological phenomena relevant to human diseases. Yet, all the players and mechanisms involved in peroxide perception are not known. Elucidating very remote evolutionary relationships between proteins is an approach that allows the discovery of novel protein functions. Here, we start with three human proteins, SRPX, SRPX2 and CCDC80, involved in tumor suppression and progression, which possess a conserved region of similarity. Structure and function prediction allowed the definition of P-DUDES, a phylogenetically widespread, possibly ancient protein structural domain, common to vertebrates and many bacterial species.
Results
We show, using bioinformatics approaches, that the P-DUDES domain, surprisingly, adopts the thioredoxin-like (Thx-like) fold. A tentative, more detailed prediction of function is made, namely, that of a 2-Cys peroxiredoxin. Incidentally, consistent overexpression of all three human P-DUDES genes in two public glioblastoma microarray gene expression datasets was discovered. This finding is discussed in the context of the tumor suppressor role that has been ascribed to P-DUDES proteins in several studies. Majority of non-redundant P-DUDES proteins are found in marine metagenome, and among the bacterial species possessing this domain a trend for a higher proportion of aquatic species is observed.
Conclusions
The new protein structural domain, now with a broad enzymatic function predicted, may become a drug target once its detailed molecular mechanism of action is understood in detail
Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine
Get PDFEtheno () adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate -adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of -adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (C.G. Cupples, J.H. Miller. Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345-5349) which allowed to monitor Lac+ revertants, the latter arose by GCAT or GCTA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in E. coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of C proceeds via a relatively stable intermediate, 3,N4--hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA+, alkB+, and mug+ controls. Considering the levels of CAA-induced Lac+ revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of C and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs C and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and εC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein
Reversion of argE3 to Arg+ in Escherichia coli AB1157 – an informative bacterial system for mutation detection
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This review concerns reversion of the argE3(ochre) nonsense mutation to prototrophy in E. coli AB1157 strain as an informative system for mutation detection. Strain AB1157 bears the argE3(ochre), hisG4(ochre) and thr-1(amber) mutations, and the supE44 amber suppressor on its chromosome. The Arg+ phenotype can be restored by (i) any base substitution at the argE3 site that changes the nonsense UAA codon to any sense nucleotide triplet or to UAG recognized by the supE44 amber suppressor, or (ii) suppressor mutations enabling the reading of the UAA nonsense codon. The argE3→Arg+ reversion-based system enables (i) determination of the spontaneous or induced mutation level; (ii) determination of base substitutions (suppressor analysis); (iii) examination of transcription-coupled repair (TCR) since targets for DNA damage are situated on the transcribed or coding strand of DNA; (iv) detection of mutations resulting from single stranded DNA damage. This review focuses on studies carried out since the early 1990s till now with the application of the AB1157-based mutation detection system. Recently, the system has been used to obtain new data on the processes of methyl methanesulfonate-induced mutagenesis and DNA repair in E. coli alkB– mutants
Recent Advances in Molecular Toxicology of Cadmium and Nickel
Get PDFCadmium (Cd) and nickel (Ni) are two toxic elements which are widespread in the human environment, but less recognized as hazardous by the general public. Herein, we describe molecular mechanisms of their toxicity towards humans, in the context of general chemical and toxicological properties of these metals. Following the introductory remarks, the routes of exposure are outlined. The next chapter covers the health hazards posed by cadmium and nickel with the main stress placed on diseases like cadmium induced nephropathy, reproductive disorders due to cadmium exposure, cadmium related COPD and cadmium carcinogenesis. In respect to nickel, acute toxicity, nickel allergy and nickel carcinogenicity were described. This overall description provides the basis for a detailed account of molecular mechanisms of cadmium and nickel toxicity. They include the involvement of metallothioneins and their role in the transport of Cd(II) ions, and the role of oxidative damage and DNA repair inhibition in cadmium carcinogenesis. The final issue covered in respect to molecular mechanisms of cadmium toxicity is its influence on cellular junctions. Molecular mechanisms of nickel toxicity are divided into subjects of nickel allergy and several mechanisms related to its carcinogenicity. The discussion is completed by the presentation of nickel and cadmium interactions with zinc fingers as a possible common ground of their molecular toxicity
Carbon-13 NMR Relaxation Study of the Internal Dynamics in Cyclodextrins in Isotropic Solution
Get PDFTranscription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors
Get PDFPoly(A) polymerase I (PAP I), encoded by the
pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through inXuencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three �70-dependent (p1, pB, and p2) and two �S-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative � factors by these eVectors of the stringent response might occur according to both passive and active models
Hydrogen peroxide-mediated induction of the Shiga toxinconverting lambdoid prophage ST2-8624 in Escherichia coli O157:H7
Get PDFShiga toxin-producing Escherichia coli (STEC) may cause bloody diarrhea and
hemorrhagic colitis, with sometimes severe complications. Because genes coding
for Shiga toxins are located on lambdoid prophages, effective toxin production
occurs only after prophage induction. However, although agents that effectively
induce prophage l (a paradigm of the family of lambdoid phages) under
laboratory conditions, such as UV irradiation or DNA replication inhibitors, are
well known, it is unlikely that such factors are present in human intestine infected
with STEC. In this report, we demonstrate that induction of a Shiga toxinconverting
prophage in its host (E. coli O157:H7) occurs not only in the presence
of DNA-interfering antibiotics (mitomycin C and norfloxacin) but also under
conditions of oxidative stress [following treatment with hydrogen peroxide
(H2O2)]. Under these conditions, we observed not only effective prophage
induction but also expression of the reporter gene (replacing the original stx2
gene). In the light of previously published reports, indicating that oxidative stress
conditions might occur during colonization of human intestine by enteric bacteria,
and that neutrophil-produced H2O2 can increase production of the Shiga toxin in
a clinical isolate of STEC, these results suggest that oxidative stress may be one of
the agents responsible for stimulating the pathogenicity determinants of STEC,
leading to induction of Shiga toxin-converting prophages in these bacteria
Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair.
Get PDFMethylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants
Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.
No full textPreviously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological applications of Ni(II)-dependent peptide bond hydrolysis
