RESUMO
The human heme-regulated eIF2α kinase, also called the human heme-regulated inhibitor (hHRI) is significantly up-regulated particularly at the level of translation during stress. In this report we show that during lead-stress, the regulation of hHRI mRNA translation is mediated through its 5'-untranslated region (UTR) that interacts with specific trans-acting factors. Further, vimentin has been identified as one of the trans-acting factors that contribute to this regulation.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Vimentina/metabolismo , eIF-2 Quinase/genética , Regiões 5' não Traduzidas , Linhagem Celular , Genes Reporter , Humanos , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Compostos Organometálicos/farmacologia , Ligação Proteica , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Regulação para Cima , Vimentina/fisiologia , eIF-2 Quinase/metabolismoRESUMO
In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.
Assuntos
Domínio Catalítico , Biossíntese de Proteínas , eIF-2 Quinase/química , eIF-2 Quinase/metabolismo , Acrilamida/química , Animais , Clonagem Molecular , Guanidina/farmacologia , Concentração de Íons de Hidrogênio , Peso Molecular , Desdobramento de Proteína/efeitos dos fármacos , Coelhos , Análise Espectral , eIF-2 Quinase/genética , eIF-2 Quinase/isolamento & purificaçãoRESUMO
The spacing between ribosome binding site (RBS) and AUG is crucial for efficient overexpression of genes when cloned in prokaryotic expression vectors. We undertook a brief study on the overexpression of genes cloned in Escherichia coli expression vectors, wherein the spacing between the RBS and the start codon was varied. SDS-PAGE and Western blot analysis indicated a high level of protein expression only in constructs where the spacing between RBS and AUG was approximately 40 nucleotides or more, despite the synthesis of the transcripts in the representative cases investigated.
Assuntos
Códon de Iniciação , Proteínas Recombinantes/genética , Ribossomos/química , Ribossomos/metabolismo , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1)=1.13ns and tau(2)=4.74ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.
