Rabbit Anti-Human HINT1 (a.a. 41-126) Polyclonal antibody

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase ( LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 ( hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate ( AIPA) over tryptamine adenosine phosphoramidate monoester ( TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient ( k(cat)/K-m values) than the V97E and V97D mutants, respectively. Adenylation of wildtype, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K-m values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency ( k(cat)/K-m) of acylated AMP hydrolysis, but not maximal catalytic turnover ( k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.

The aim of this study was to investigate the methylation status of fragile histidine triad (FHIT) and the effects of FHIT on cell growth and cyclin D1 expression in hepatoma cells. The total proteins from the human hepatoma cell lines HepG2, Hep3B and Huh7 were collected and the expression levels of FHIT were analyzed. The methylation status in the promoter region of FHIT in the hepatoma cells was measured using methylation-specific polymerase chain reaction (PCR). The HepG2, Hep3B and Huh7 cells were subsequently treated with 5-aza-2 '-deoxycytidine (5-azadc) and the restoration of FHIT expression was then examined. A p-hemagglutinin (HA)-FHIT plasmid was constructed and used to transfect the HepG2 cells, and the inhibitory effects of the transfection on cell growth were then assessed. In addition, HepG2 cells were cotransfected with the pHA-FHIT plasmid and a cyclin D1 luciferase reporter plasmid, and the effects of FHIT on the activity of cyclin D1 transcription factor were analyzed using a luciferase assay. FHIT was observed to be expressed at a low level in Hep3B and HepG2 cells; however, it was expressed at a relatively high level in Huh7 cells. The promoter region of FHIT in the Hep3B and HepG2 cells was partially methylated, and 5-azadc treatment induced an increased expression of FHIT. The increased expression of FHIT inhibited the growth of HepG2 cells. Cotransfection with the pHA-FHIT plasmid significantly inhibited the transcriptional activity of the cyclin D1 promoter and decreased the expression of cyclin D1 in HepG2 cells. In conclusion, FHIT was partially methylated in the HepG2 and Hep3B hepatoma cells. The overexpression of FHIT inhibited cell growth and decreased the expression of cyclin D1 in HepG2 cells.