RESUMO
The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin alpha. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.
Assuntos
Núcleo Celular/enzimologia , Citidina Desaminase/metabolismo , Sinais de Localização Nuclear , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Citidina Desaminase/química , Citidina Desaminase/genética , DNA Complementar , Escherichia coli/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de AminoácidosAssuntos
Edição de RNA , Desaminase APOBEC-1 , Sequência de Aminoácidos , Animais , Apolipoproteínas B/química , Apolipoproteínas B/genética , Sítios de Ligação , Citidina Desaminase/química , Citidina Desaminase/genética , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Neoplasias/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
ApoB RNA-editing enzyme (APOBEC-1) is a cytidine deaminase. Molecular modeling and mutagenesis show that APOBEC-1 is related in quaternary and tertiary structure to Escherichia coli cytidine deaminase (ECCDA). Both enzymes form a homodimer with composite active sites constructed with contributions from each monomer. Significant gaps are present in the APOBEC-1 sequence, compared to ECCDA. The combined mass of the gaps (10 kDa) matches that for the minimal RNA substrate. Their location in ECCDA suggests how APOBEC-1 can be reshaped to accommodate an RNA substrate. In this model, the asymmetrical binding to one active site of a downstream U (equivalent to the deamination product) helps target the other active site for deamination of the upstream C substrate.