RESUMO
Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas Fúngicas/genética , Mutação , Nucleosídeo Desaminases/genética , Leveduras/enzimologia , Motivos de Aminoácidos , Animais , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Nucleosídeo Desaminases/química , Nucleosídeo Desaminases/metabolismo , Leveduras/química , Leveduras/genéticaRESUMO
Yeast chaperon Hsp104 is known as a protein which is able to dissociate aggregates of the heat damaged proteins and prion aggregates into smaller pieces or monomers. In our work the effects of Hsp104 on the PrP-GFP and GFP proteins have been analyzed. The PrP-GFP protein forms the high molecular weight aggregates, whereas GFP is unable to aggregate in yeast cell. We have shown that Hsp104 regulates the amount of PrP-GFP and GFP in yeast cells and direction of chaperone action depends on promoter controlling production of these proteins. The overproduction of Hsp104 increases the amount of PrP-GFP and GFP proteins when the corresponding genes are under control of CUP1 promoter. In contrast, the overproduction of Hsp104 decreases the amount of PrP-GFP and GFP is case of their expression under control of GPD promoter. The effects of Hspl04 are not related with any changes in mRNA content of the genes under investigation and with ability of the proteins to form aggregates. Thus, the functions of this chaperon are not restricted by dissociation of the protein aggregates. Our data show that Hsp104 regulates the gene expression on the posttranscriptional level.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Choque Térmico/genética , Metalotioneína , Camundongos , Chaperonas Moleculares/genética , Proteínas da Gravidez/biossíntese , Proteínas da Gravidez/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.
