RESUMO
Plant cold shock domain proteins (CSDPs) are DNA/RNA-binding proteins. CSDPs contain the conserved cold shock domain (CSD) in the N-terminal part and a varying number of the CCHC-type zinc finger (ZnF) motifs alternating with glycine-rich regions in the C-terminus. CSDPs exhibit RNA chaperone and RNA-melting activities due to their nonspecific interaction with RNA. At the same time, there are reasons to believe that CSDPs also interact with specific RNA targets. In the present study, we used three recombinant CSDPs from the saltwater cress plant (Eutrema salsugineum) - EsCSDP1, EsCSDP2, EsCSDP3 with 6, 2, and 7 ZnF motifs, respectively, and showed that their nonspecific interaction with RNA is determined by their C-terminal fragments. All three proteins exhibited high affinity to the single-stranded regions over four nucleotides long within RNA oligonucleotides. The presence of guanine in the single- or double-stranded regions was crucial for the interaction with CSDPs. Complementation test using E. coli BX04 cells lacking four cold shock protein genes (ΔcspA, ΔcspB, ΔcspE, ΔcspG) revealed that the specific binding of plant CSDPs with RNA is determined by CSD.
Assuntos
Brassicaceae , Proteínas e Peptídeos de Choque Frio , Proteínas de Plantas , RNA de Plantas , Motivos de Aminoácidos , Brassicaceae/química , Brassicaceae/genética , Brassicaceae/metabolismo , Proteínas e Peptídeos de Choque Frio/química , Proteínas e Peptídeos de Choque Frio/genética , Proteínas e Peptídeos de Choque Frio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios Proteicos , RNA de Plantas/química , RNA de Plantas/genéticaRESUMO
Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-kappaB has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used 32P-labeled 17/19 bp nucleotide DNA duplex containing an NF-kappaB recognition site (kappaB-site) in which one of internucleotide phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 32P-labeled DNA duplex with NF-kappaB in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3 -end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-alpha promoted partial translocation of the DNA reagent into the nucleus.
Assuntos
Antineoplásicos/metabolismo , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Anticorpos/metabolismo , Antineoplásicos/química , Sequência de Bases , Núcleo Celular/metabolismo , Neoplasias do Colo/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Células HCT116 , Humanos , Immunoblotting , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Subunidades Proteicas/metabolismo , Rodaminas/metabolismo , Rodaminas/farmacologiaRESUMO
Metronidazolum decreased diffusion mobility of membrane surface water. It is possible, that Wuks's effect represents the molecular mechanism underlying this phenomenon. We suppose, that protective action of the derivatives of the nitroimidazole on cell lysis is related to changes of the diffusion mobility of the surface water.