Zinc finger protein HZF1 promotes K562 cell proliferation by interacting with and inhibiting INCA1.

Previously, we characterized a zinc finger protein gene HZF1 (ZNF16) and demonstrated that it played a significant role in the erythroid and megakaryocytic differentiation of K562 cells by knockdown of the gene. In this study, we examined the effect of HZF1 on the proliferation and apoptosis of K562 cells and identified the possible mechanism for this effect. By lentivirus-mediated gene transfer, we obtained stable K562 transductants with HZF1 overexpression (K562/WPXL-HZF1) and stable control transductants (K562/WPXL). Significantly rapid cell amplification was observed in K562/WPXL-HZF1 cells compared to K562/WPXL cells. The cell cycles of the two transductants were analyzed and the results demonstrated that HZF1 overexpression promoted the S to G2/M phase transition. Additionally, we found that the overexpression of HZF1 slightly inhibits the apoptosis of K562 cells induced by sodium arsenate. Furthermore, using a yeast two-hybrid (Y2H) system we identified the HZF1-interacting proteins and screened 29 potential binding partners of HZF1. Using a co-immunoprecipitation (Co-IP) assay, we confirmed the interaction between HZF1 and the inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1), and proved that this interaction leads to the inhibition of INCA1 function, which rescued the activity of CDK2 inhibited by INCA1. In conclusion, our results identified novel functions of the HZF1 gene and revealed a possible mechanism through which HZF1 affects K562 cell proliferation.

Identification of the trans-activation domain and the nuclear location signals of human zinc finger protein HZF1 (ZNF16).

We previously characterized a C2H2-type zinc finger protein HZF1 (ZNF16) and demonstrated its important role in erythroid and megakaryocytic differentiation. This protein was located in nucleus. In this study, we first approved that HZF1 solely could activate lacZ reporter gene in yeast host Y190. This self-activation phenomenon together with structure and distribution of HZF1 suggested it as a potential transcription factor. By the auto-activation experiments and the luciferase reporter system and deletion mutation analysis, we further located the trans-activation domain at amino acid residences 49-197 within the non-zinc finger region of HZF1. An acidic residue-rich subregion (amino acids 49-105) was important for the trans-activation effect, but it could not function independently. By deletion mutation analysis, we also identified three nuclear location signals, which were located in the regions of amino acids 255-280, 328-360, and 460-490, respectively, and all of them within the zinc finger region.

Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization.

Organophosphorus hydrolase is able to hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. In this work, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Pichia pastoris at a high expression level (approximately 5.5 g/L) using 3 L high-cell-density fermentation. The expression level is higher than those produced in other expression systems. The results of the SDS-PAGE and the Western blot analyses showed a major 36 kDa polypeptide band, which was the same size as that from the original bacteria, Pseudomonas pseudoalcaligenes C2-1. The expressed enzyme was recovered from the culture supernatant and purified by a single-step purification procedure with a recovery rate of 72.78%. The main physiochemical features of the recombinant OPHC2, including its optimum temperature and pH for the reaction, its temperature and pH stability, and its sensitivity to some metal ions and chemical reagents, were also characterized. With methyl parathion as a substrate, the optimum temperature and pH for enzyme activity are 65 degrees C and pH 9.0, respectively. It also shows good thermal and pH stability.