Laboratory of Biochemistry

Thiopurine agents such as 6-mercaptopurine, azathiopurine, and 6-thioguanine are used as antitumor and immunosuppressive agents. These drugs are used for many diseases including leukemia and autoimmune diseases, because they are relatively cheap and effective. However, it is well established that a certain population of patients show severe side effects including leukopenia and alopecia. Recent genome-wide studies clarified that the mutations at p.Arg139Cys in the NUDT15 gene strongly correlate with the severe side effects caused by thiopurine agents. Therefore, the genetic test detecting the p.Arg139 codon is useful to determine the amount of thiopurine agents. However, it is still very difficult to determine the appropriate amounts of thiopurine agents even for patients with the p.Arg139 codon wild type. This could be because of mutations at different positions in the NUDT15 genes or the expression level of the NUDT15 protein. Therefore, in addition to the genetic test at p.Arg139 codon, the methods detecting the NUDT15 activity would be useful for the determination of appropriate amounts of thiopurine agents. This study aims to establish the detection methods for the NUDT15 enzymatic activity in human blood samples. We successfully detected the NUDt15 activity in the extracts derived from peripheral blood mononuclear cells (PBMC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To use this method in clinics, we are now trying to improve the methods for using the method in clinical practice.  

We have identified histone chaperones that regulate the chromatin structure. Recently, we showed that histone chaperone NPM1 directly binds to a transcription factor NFkB and regulates its DNA binding activity. Because NFkB is a central transcription factor in inflammatory responses and immune responses, it is presumed that NPM1 is also involved in these processes. In addition to NFkB, our research has suggested that other transcription factors are under the control of NPM1. We are currently studying the interaction between histone chaperones and transcription factors, to elucidate the biological significance of this interaction.

Nucleolus is well observed in growing cells under a light microscope and its primary function is to synthesize ribosomes, which are protein translation machines. Ribosomes are consisting of 4 ribosomal RNA (rRNAs) and about 80 ribosomal proteins. Among 4 rRNAs, 18S, 5.8S, and 28S rRNAs are synthesized in the nucleolus by RNA polymerase I as a single precursor RNA. Long precursor rRNA is successively processed by various proteins and RNAs. Ribosomal proteins are assembled on the rRNAs co-transcriptionally or after appropriate processing, although the detailed mechanism of the ribosomal protein assembly process is unknown. Given that growing cells consume a huge number of ribosomes, ribosome synthesis activity is correlated with the cell growth rate. Therefore, highly malignant cells show abnormally large nucleolus and the nucleolar abnormalities are sometimes used as a diagnostic marker of transformed cells. In addition, it should be noted that ribosome biogenesis is the most energy-consuming reaction in the cells and therefore the ribosome biogenesis activity is very sensitive to the cellular energy status. The functions of the nucleolus to sense the energy level of the cells are also closely related to cellular aging. We are currently trying to elucidate the molecular mechanism of the nucleolar formation to contribute to understanding cellular aging.

Regulation of gene expression is tightly controlled by various steps such as transcription, splicing, and nuclear export of mRNA, and their disruption can cause cancer. Some histone chaperones are expressed as mutant forms in cancer cells and are involved in oncogenesis. We are analyzing leukemogenesis mechanisms mediated by such proteins (Saito et. al, MCB, 2016). Transgenic (Tg) mice expressing the fusion gene product of TAF-I, a histone chaperone, showed suppression of hematopoietic differentiation and enlarged spleens. We are interested in clarifying at the molecular level what mechanism caused the suppression of differentiation.
 

Long human genomic DNA (about 2 m in human cells) is packed in the cell nucleus with 10 micrometers in diameter. To allow this compaction, chromatin structure plays a critical role. A Nucleosome consisting of 4 core histones, H2A, H2B, H3, and H4, and 147 base pairs of double-stranded DNA is a repeating unit of chromatin. Linker histones bind to the linker region of DNA between nucleosomes and help to form a high-order chromatin structure. We have identified and studied the functions of a group of proteins termed histone chaperones, which assemble and disassemble chromatin structures to allow gene expression and genomic DNA replication. Our research goal is to elucidate the molecular mechanisms by which chromatin structure is assembled and disassembled by focusing on the interaction between histone chaperones and histones including histone variants. In addition, histone chaperons identified in our lab are involved in sperm chromatin dynamics during spermatogenesis and fertilization. We are also interested in clarifying the molecular mechanism by which sperm chromatin is reconstructed during spermatogenesis and

It has been well established that abnormal signaling causes various diseases including cancer and metabolic syndromes. Therefore, it is very important to understand how the extracellular stimuli are properly transduced to cells. To uncover the full view of the signal transduction pathway, we are focusing on a well-established protein post-translational modification, phosphorylation and de-phosphorylation. By combining 2-dimentional gel electrophoresis and mass spectrometry analyses, we are trying to visualize the signal transduction pathways.

We are trying to develop novel anti-influenza virus drugs in collaboration with the lab of Professor Tomoda at Kitasato University. Influenza viruses use the Cap structure from host mRNA as a primer of viral mRNA synthesis. The viral RNA-dependent RNA polymerase complex mediates to endonucleoritically cleave mRNAs containing the Cap structure (Cap-snatching). Because host cells do not have this endonuclease activity, the viral nuclease activity is a good target for drug discovery. We originally established the assay system to monitor the Cap-snatching by the viral RNA polymerase complex. Using this assay system, we are now trying to identify molecules that inhibit viral mRNA synthesis.