RESUMO
YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA- and RNA-dependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acid-long YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin- and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.
Assuntos
Dano ao DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Glicina/metabolismo , Humanos , Camundongos , Mutação , Células NIH 3T3 , Coelhos , Células Tumorais Cultivadas , Ubiquitina/metabolismoRESUMO
Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.
Assuntos
Escherichia coli/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Thermus thermophilus/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/isolamento & purificação , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Thermus thermophilus/químicaRESUMO
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.
Assuntos
DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Euryarchaeota/genética , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Reagentes de Ligações Cruzadas , DNA Topoisomerases Tipo I/metabolismo , DNA Arqueal/química , DNA Arqueal/ultraestrutura , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Euryarchaeota/química , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Concentração Osmolar , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Alinhamento de Sequência , Análise de Sequência , SoftwareRESUMO
Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.
