Tomohiro Hamasaki

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease.

ISG20 is an interferon‐induced antiviral exoribonuclease that acts on single‐stranded RNA and also has minor activity towards single‐stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5′‐monophosphate (UMP) at 1.9 Å resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh‐group DNases, the ε subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2′‐OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.

Efficacy of a novel class of RNA interference therapeutic agents.

RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.

Functional peptide nanocarriers for delivery of novel anti-RelA RNA interference agents as a topical treatment of atopic dermatitis.

Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA(®) and PnkRNA(®), which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA(®) and PnkRNA(®). The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions.

A Simple Search of TM Segments in Polytopic Membrane Protein Using Matrix - Assisted Laser DesorptionIonization Time - of - Flight Mass Spectrometry

Using both high performance liquid chromatography (HPLC) and amino acid sequencing (AAS), we previously analyzed band 3 TM peptide-segments that make up the transmembrane protein structure. However, the HPLC/AAS combination method was highly time-consuming. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain accurate molecular weight information for proteins/peptides simply and sensitively. We applied the MALDI-TOF mass spectrometry technique to search for TM segments in membrane proteins. In combination with trypsin cleavages after alkali treatments (pH12 or 13) and sample preparation using organic solvents for MALDI-TOF mass spectrometry, we determined the TM segments of band 3 and glycophorin A in erythrocyte membrane. The method can be applied to other polytopic membrane proteins in erythrocyte membrane.