In a previous study, we identified the human counterpart of murine kinesin superfamily member 4 (KIF4), a microtubule-based motor protein [Oh, Hahn, Torrey, Shin, Choi, Lee, Morse and Kim (2000) Biochim. Biophys. Acta 1493, 219–224]. As an initial step to understand the function(s) of human KIF4, its subcellular localization in HeLa cells was examined by using immunocytochemical and subcellular fractionation methods, and it was found that most KIF4 is localized in the nucleus. Since murine KIF4 is known to transport cytoplasmic vesicles, dominant nuclear localization of the human counterpart was somewhat surprising. Subsequent subnuclear fractionation revealed predominant association of KIF4 with the nuclear matrix. These results clearly indicate that human KIF4 is, at least, a nuclear protein. In further confirmation of this conclusion, the hexapeptide PKLRRR (amino acids 773–778) in the molecule was found to function as a nuclear localization signal. During the mitotic phase of the cell cycle, human KIF4 was associated with the chromosomes, suggesting that human KIF4 might be a microtubule-based mitotic motor, with DNA as its cargo.

In a previous study, we identified the human counterpart of murine kinesin superfamily member 4 (KIF4), a microtubule-based motor protein [Oh, Hahn, Torrey, Shin, Choi, Lee, Morse and Kim (2000) Biochim. Biophys. Acta 1493, 219-224]. As an initial step to understand the function(s) of human KIF4, its subcellular localization in HeLa cells was examined by using immunocytochemical and subcellular fractionation methods, and it was found that most KIF4 is localized in the nucleus. Since murine KIF4 is known to transport cytoplasmic vesicles, dominant nuclear localization of the human counterpart was somewhat surprising. Subsequent subnuclear fractionation revealed predominant association of KIF4 with the nuclear matrix. These results clearly indicate that human KIF4 is, at least, a nuclear protein. In further confirmation of this conclusion, the hexapeptide PKLRRR (amino acids 773-778) in the molecule was found to function as a nuclear localization signal. During the mitotic phase of the cell cycle, human KIF4 was associated with the chromosomes, suggesting that human KIF4 might be a microtubule-based mitotic motor, with DNA as its cargo.

The inhibition of tyrosinase has attracted considerable attention for potential medicinal and cosmetic applications, as well as in agriculture. This study investigated the inhibition effects of thiol-associated Cu(2+) chelators and deduced a strategy for designing and/or selecting tyrosinase inhibitors. Among the several compounds tested, dithioglycerine (DTGC) was selected for further experiments on the inhibition kinetics on tyrosinase. Different types of tyrosinases derived from mushroom and from the transient overexpression in HEK293 cells were tested individually. The results showed that DTGC significantly inhibited human tyrosinase in a complex manner (slope-parabolic mixed-type inhibition), which was comparable to mushroom tyrosinase. The affinity of DTGC affinity to human tyrosinase was evaluated by setting up a K(i slope) equation. The results suggest that a Cu(2+) chelator modified with thiol groups has potential as a whitening agent. In addition, a strategy for designing and/or selecting tyrosinase inhibitors that target the active enzyme site was also suggested.

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Tyrosinase (monophenol, o-diphenol : oxygen oxidoreductase, EC 1.14.18.1) is a binuclear copper enzyme, which is fairly ubiquitously distributed throughout organisms, and is known to play an important role in melanin synthesis. [1][2][3][4] Tyrosinase has been demonstrated to catalyze the hydroxylation of phenols to catechols, as well as the oxidation of catechols to quinones. The active site of tyrosinase consists of two copper ions, each coordinated by histidine residues within the active sites. These two coppers are known to be essential for the catalytic activities of this enzyme.5) The binuclear copper active sites exist in all tyrosinases, regardless of source. 6,7) Therefore, the chelation of copper ions in this enzyme by flavonoid compounds or any other agents has been targeted as a strategy for the attenuation of tyrosinase activity, an objective relevant to cosmetic concerns, darkening problems in agricultural products, and certain medicinal reasons. [8][9][10][11][12][13][14] Melanin pigments are synthesized by melanocytes, and are deposited throughout the epidermis. This process determines skin color. [15][16][17][18] Tyrosinase is involved in two different ratelimiting steps in melanin synthesis; namely, the hydroxylation of tyrosine to 3,4-dihyroxyphenylalanine (DOPA), and the oxidation of DOPA to dopaquinone.19) The abnormal accumulation of melanin pigment can induce hyperpigmentations, such as freckles, melasma, and senile lentigines. These symptoms have responded, in the past, to treatment with depigmenting agents, including hydroquinone, ascorbic acid derivatives, retinoids, arbutin, and kojic acid. In particular, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is a copper-chelator, which has been used as a cosmetic agent in skin-whitening formulations. 20) However, safer and more effective depigmenting agents are clearly needed, as kojic acid is not only carcinogenic, but its associated whitening effects are also somewhat weak. 21,22) We have, in previous studies, applied several copper-specific chelating agents to mushroom tyrosinase, in an effort to screen an effective inhibitor, with the strategy of copper chelation at the active enzyme site.23) Among these compound, ammonium tetrathiomolybdate (ATTM) has been shown to exert a significant inhibitory effect, as compared to the effect of other tested substances. In this study, we have attempted to characterize the effects of ammonium tetrathiotungstate (ATTT) on tyrosinase. ATTT has been identified as an effective decoppering reagent for copper storage-related diseases in both rats and sheep. 24,25) However, the kinetics of its inhibition in copper-containing enzymes has been the subject of only minimal exploration. Thus, we have attempted to evaluate the inhibition kinetics of tyrosinase by ATTT. Our kinetic studies revealed that ATTT was capable of the inactivation of tyrosinase in vitro. We have also verified the inhibitory effects of ATTT on tyrosinase in human melanocyte cells, as compared with those evidenced by other known inhibi...

Abbreviations: HDAC, histone deacetylases; KRAB, Krüppel-associated box; PIAS1, protein inhibitor of activated STAT1; TIF1β, transcriptional intermediary factor 1β; TSA, trichostatin A; ZNF133, zinc finger protein 133 AbstractZinc finger protein 133 (ZNF133) is composed of a Krüppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.