RESUMEN
In the original publication, the given name of the last author was incorrectly displayed as the name must read: Katsuyoshi Masuda.
RESUMEN
Human αB-crystallin (HSPB5) is frequently modified post-translationally by UV radiation, oxidation, and age-associated processes, which complicates functional analyses of the protein using natural sources. Thus, determining the biological function of HSPB5 at the molecular structure level requires unmodified protein. Here, we employed an Escherichia coli cell-free protein synthesis system to prepare unmodified, functionally active human HSPB5. An S30 extract prepared from E. coli strain BL21 (DE3) was used for HSPB5 synthesis. The efficacy of protein synthesis was assessed by monitoring influencing factors, such as the concentrations of Mg2+ and other reaction mixture constituents, and by evaluating batch and/or dialysis synthesis systems. Chaperone-like activity of synthesized HSPB5 was assayed using alcohol dehydrogenase (ADH) under thermal stress. The amount of HSPB5 synthesized using the cell-free system depended significantly on the concentration of Mg2+ in the reaction mixture. Use of condensed S30 extract and increased levels of amino acids promoted HSPB5 production. Compared with the batch system, HSPB5 synthesis was markedly increased using the dialysis system. The construction vector played a critical role in regulating the efficacy of protein synthesis. HSPB5 synthesized using the cell-free system had a native molecular mass, as determined by mass spectrometry analysis. The co-presence of synthesized HSPB5 suppressed heat-associated denaturation of ADH. Human HSPB5 synthesized using the cell-free system thus retains functional activity as a molecular chaperone.
Asunto(s)
Sistema Libre de Células , Cadena B de alfa-Cristalina/biosíntesis , Escherichia coliRESUMEN
Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5'-TGGAAAGGACCAG-3') and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP600125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5'-flanking region of Osp94 gene contains a hypertonicity sensitive cis -acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.