Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking.

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity.

Zinc 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.

Inhibitory effect of ammonium tetrathiotungstate on tyrosinase and its kinetic mechanism.

Tyrosinase requires two copper ions at the active site, in order to oxidize phenols to catechols. In this study, the inhibitory effect of the copper-chelating compound, ammonium tetrathiotungstate (ATTT), on the tyrosinase activity was investigated. ATTT was determined to inactivate the activity of mushroom tyrosinase, in a dose-dependent manner. The kinetic substrate reaction revealed that ATTT functions as a kinetically competitive inhibitor in vitro, and that the enzyme-ATTT complex subsequently undergoes a reversible conformational change, resulting in the inactivation of tyrosinase. In human melanin-producing cells, ATTT evidenced a more profound tyrosinase-inhibitory effect than has been seen in the previously identified tyrosinase inhibitors, including kojic acid and hydroquinone. Our results may provide useful information for the development of whitening agent.

Complex inhibition of tyrosinase by thiol-composed Cu2+ chelators: a clue for designing whitening agents.

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.

Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate.

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.

Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution.

The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.

A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase.

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.

Aggregation and folding of recombinant human creatine kinase.

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.