Department of Host-Defense Biochemistry
Reserches in our laboratory are focused on the molecular regulations of the phagocyte NADPH oxidase system that generates active oxygens essential for killing invading microbes.
Members
- Visiting Professor Tomoyuki Maekawa
- Assistant Professor Yoshito Fujii
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Activities
Selective expression mechaisms for gp 91phox, a core component of the oxidase.
The phagocyte NADPH oxidase is composed of membrane-integrated flavocytochrome b 558 containing gp 91phox and p 22phox, and cytosolic components such as p 47phox and p 67phox. Upon phagocytotic stimulation, cytosolic factors move to membrane to make an active complex with the flavocytochrome. Based on genetic analysis of CYBB , the gene encoding gp 91phox, in a patient with chronic granulomatous disease (CGD), we found that PU.1, a transcriptional activator, bound to gp 91phox promoter position centered to bp -53 was important for the expression of gp 91phox in neutrophils, monocytes, and B lymphocytes, but not in eosinophils. On the other hand, GATA-1 bound to a position centered to bp -98 had an important role in the expression of the gene in eosinophils.
Analysis of NADPH oxidase.
We are receiving blood samples to check whether the patients are CGD or not by testing genomic DNA and protein. And also we check mRNA to detect the length because there are skipping mechanisms to avoid pre-mature stop.
A novel GT-mismatch binidng protein.
In the analysis of a cis -element of gp 91phox promoter, we found a novel GT-mismatched DNA binding protein. An addition of unlabelled competitor homoduplex with G/C at bp -177, generated, but not erased, a strong 'supershifted' band in EMSA using the labelled probe with A/T at bp -177. Newly paired heteroduplex with the unlabelled upper stand with G at bp -177 and the labelled lower stand with T at the same position recruited nGTBP. This protein strictly required TRTGNB (R=purine, N=any base, B=not adenine, G paired with T) and 14-mer or longer for binding. G can be replaced by deaminated A, namely, hypoxanthine, suggesting deaminated C-6 is essential for nGTBP binding. Deamination of nucleotide bases are increased by high temperature and the repair of deaminated portions of DNA would be more important in tropical area than other areas. Transitions appreciably occured more at TRTRNB sites than at other sites in tumor supressor protein p 53 exons, suggesting this particular sites were fragile in tumor-prone cells. Relative frequency of esophageal cancer due to transitions at p 53 non-GpG sites in Brazilian mate-drinkers was relatively higher than that in world-wide patients. Cloning and purification of this nGTBP are now urgent issues in our laboraatory.
