The induction mechanism of the molecular chaperone HSP70 in the gastric mucosa by Geranylgeranylacetone (HSP-inducer)

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Abstract

To elucidate the induction mechanism of HSP70 by geranylgeranylacetone (GGA), we investigated GGA specific binding proteins using a GGA-affinity column. Alteration of chaperone activity of HSP70 and binding affinity of HSP70 to heat shock factor-1 (HSF-1) was evaluated in the presence or absence of GGA. The binding domain of HSP70 to GGA was also analyzed. A 70-kDa protein eluted by 10 mM GGA from the GGA-affinity column was identical to constitutively expressed HSP70 on immunoblotting. GGA-binding domain of HSP70 was C-terminal of the protein as peptide-binding domain (HSP70C). The chaperone activity of HSP70 and recombinant HSP70C was suppressed by GGA. Furthermore, dissociation of the HSP70 from HSF-1 was observed in the presence of GGA. GGA preferentially binds to the C-terminal of HSP70 which binds to HSF-1. After dissociation of HSP70, free HSF-1 could acquire the ability to bind to HSE (the promoter region of HSP70) gene.

Section snippets

Plasmid or phagemid construction

A full-length human HSP70 cDNA was kindly provided by Dr. Richard Morimoto, Northwestern University (Evanston, Ill, U.S.A.). The polymerase chain reaction-amplified BamHI–HindIII fragments encoding the cDNA sequence, the N-terminal domain of HSP70 (residues 1–380) or the C-terminal domain of HSP70 (residues 381–640), were inserted into the digested pQE31 plasmid vector (Quiagen, Hilden, Germany). The N- or C-terminal domain of HSP70 was overexpressed in Escherichia coli M15 [pREP4] cells as an

GGA-affinity column chromatography

We investigated GGA-binding proteins using a GGA-affinity column. In general, the affinity column chromatography is defined as follows: a purified molecule is reversibly adsorbed on the ligand which is fixed to the matrix [25], [26]. To avoid nonspecific binding proteins, GGA-affinity columns were thoroughly washed with conditioning buffer. Proteins were eluted with 10 mM GGA from GGA-affinity column or control mock column, and eluants were detected on SDS/PAGE. Although no protein bands were

Discussion

GGA, an anti-ulcer drug, was first introduced as a nontoxic inducer of molecular chaperone HSP70 in rat gastric mucosa [14]. However, the induction mechanisms of HSP70 by GGA remain to be elucidated. In the present study, to address the questions, we investigated the specific GGA-binding proteins using a GGA-affinity column. The specific binding protein with a 70-kDa molecular mass was constitutively expressed molecular chaperone HSP70 (known as HSP73). The purified wild-type HSP70 also showed

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research to H.I. (Exploratory Research: No. 16651056) and M.O. (No. 17590610) from the Japanese Ministry of Education, Science, Sports and Culture.

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