Eugene Hayato Morita

Identification of a damaged-DNA binding domain of the XPA protein

The XPA (xeroderma pigmentosum group A) protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV- or chemical carcinogen-damaged DNA, suggesting that it is involved in the recognition of several types of DNA damage during nucleotide excision repair processes. Here we identify a DNA binding domain of the XPA protein. The region of the XPA protein responsible for preferential binding to DNA damaged by UV or cis-diammine-dichloroplatinum(II) (cisplatin) is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif. CD (circular dichroism) measurements of the MF122 protein showed that it has a helix-rich secondary structure, suggesting that it is a discretely folded, functional mini-domain. The MF122 protein should be useful for structural investigation of the XPA protein and of its interaction with damaged DNA.

A wheat germ cell-free system is a novel way to screen protein folding and function

For high‐throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell‐free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15N‐labeled proteins and obtained 1H‐15N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and 1H‐15N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell‐free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell‐free protein synthesis system to high‐throughput protein structural analysis.

Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein.

Background: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV‐ or chemically damaged DNA lesions, and triggers the nucleotide excision repair process. This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA. Its primary structure suggests that this DNA binding domain may contain a zinc‐finger motif. To gain a more detailed insight into this zinc‐finger motif, we have measured the 113Cd‐NMR spectra of the DNA binding domains derived from the wild‐type and mutant XPA proteins.

Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Background: Elongation factor G of Escherichia coli is sensitive to oxidation. Results: Elongation factor G is inactivated via the formation of an intramolecular disulfide bond. Conclusion: Elongation factor G is a critical target during oxidative damage to the translation system. Significance: Oxidation of elongation factor G suggests a novel mechanism for the redox regulation of translation. Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mm H2O2 resulted in the complete loss of translational activity. The inactivation of EF-G by H2O2 was attributable to the oxidation of two specific cysteine residues, namely, Cys114 and Cys266, and subsequent formation of an intramolecular disulfide bond. Replacement of Cys114 by serine rendered EF-G insensitive to oxidation and inactivation by H2O2. Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

TTP at Ser245 Phosphorylation by AKT is Required for Binding to 14-3-3

Transferrin receptor trafficking protein (TTP) is a key molecule for selective internalization of the transferrin receptor (Tf-R) through endocytic protein complexes. To identify the proteins that directly regulate TTP, we performed a yeast two-hybrid analysis and identified 14-3-3, which can modulate the activation state of target proteins. Subsequent analyses demonstrated that TTP directly binds to multiple 14-3-3 isotypes via its Ser(245) residue (Ser(246) in human) and that these proteins are associated at the plasma membrane. Ser(245) was also found to be a substrate for AKT and the resulting Ser(245) phosphorylation induced the TTP-14-3-3 interaction. Exposure to hydrogen peroxide rapidly enhanced this association in an ovarian cell line. These results suggest that TTP Ser(245) is the principal target for the modulation of this protein via the AKT signalling cascade.

A molecular insight into Darwin's "plant brain hypothesis" through expression pattern study of the MKRN gene in plant embryo compared with mouse embryo.

MKRN gene family encodes zinc ring finger proteins characterized by a unique array of motifs (C3H, RING and a characteristic cys-his motif) in eukaryotes. To elucidate the function of the MKRN gene and to draw an analogy between plant root apical meristem and animal brain, we compared the gene expression pattern of MKRN in plant seeds with that of mouse embryo. The spatio-temporal expression of MKRN in seeds of pea and rice was performed using non radioactive mRNA in situ hybridization (NRISH) with DIG and BIOTIN labeled probes for pea and rice embryos respectively. Images of MKRN1 expression in e10.5 whole mount mouse embryo, hybridized with DIG labeled probes, were obtained from the Mouse Genome Database (MGD). MKRN transcripts were expressed in the vascular bundle, root apical meristem (RAM) and shoot apical meristem (SAM) in pea and rice embryos. The spatial annotation of the MKRN1 NRISH of whole mount mouse embryo shows prominent localization of MKRN1 in the brain, and its possible expression in spinal cord and the genital ridge. Localization of MKRN in the anterior and posterior ends of pea and rice embryo suggests to the probable role it may have in sculpting the pea and rice plants. The expression of MKRN in RAM may give a molecular insight into the hypothesis that plants have their brains seated in the root. The expression of MKRN is similar in functionally and anatomically analogous regions of plant and animal embryos, including the vascular bundle (spinal cord), the RAM (brain), and SAM (genital ridge) thus paving way for further inter-kingdom comparison studies.

Letter to the Editor: Backbone NMR assignments of a cyanobacterial transcriptional factor, SmtB, that binds zinc ions

In Synechococcus PCC7942, thesmt locus is responsible for tolerance to zinc and cadmium. This was verified by deletion of thesmt locus, which caused a reduction in zinc/cadmium tolerance (Turner et al., 1993). In thesmtlocus, there are two divergently transcribed genes, smtAandsmtB. ThesmtAgene encodes class II metallothionein (56 amino acid residues) (Shi et al., 1992), and thesmtBgene encodes the repressor of smtA transcription (122 amino acid residues) (Morby et al., 1993). In the absence of heavy metal ions, the transcription of smtA is repressed on binding of SmtB to the 100 bp operator-promoter region lying between the smtAand smtBgenes (Erbe et al., 1995), while the transcription is stimulated by trace amounts of heavy metal ions (especially zinc and cadmium). This stimulation is thought to be caused by inhibition of the complex formation between SmtB and the recognition DNA sequence, as a result of the heavy metal ion binding to SmtB (Erbe et al., 1995). It was found that SmtB predominantly forms a dimer and binds two zinc ions per subunit (Kar et al., 1997). Mutation work has suggested candidates for the amino acid residues ligating zinc ions (Turner et al., 1996). Recently, the crystal structure of the SmtB dimer was solved by X-ray crystallographic analysis and it was found that SmtB has a helix-turn-helix motif that might bind to the recognition DNA sequence (Cook et al., 1998). Furthermore, based on the results

Expression pattern of PsAPY1 during apical hook development in pea

Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) catalyzes hydrolysis of nucleoside tri- and di-phosphates to nucleoside monophosphates and orthophosphates. In the present study, the spatio-temporal expression of an apyrase gene (PsAPY1) in pea (Pisum sativum L. var. Alaska), was investigated during early stages of apical hook development using nonradioactive mRNA in-situ hybridization. During the formation of apical hook; at 45 hours after sowing (HAS), expression of PsAPY1 was obvious in epidermis and vascular bundle. By 60 HAS, the apical hook was completely formed. At this stage, transcript accumulation became higher than at the previous stage and expression was also visible in the cortex tissues of the developing hook. However, at 78 HAS, the curvature of the hook was reduced and hook was in the process of opening. At this time, expression of PsAPY1 was visible in all the above-mentioned tissues although the level of expression was slightly lower than at the previous stage (60 HAS). Apical hook formation provides a unique mechanism of protection for delicate shoot meristem in dicot plants. Its establishment is orchestrated by differential elongation rates of cells within the structure. The expression pattern of a gene provides essential information concerning the likely appearance and localization of its encoded protein and this helps to understand the mechanism of development of plant cells and tissues. Higher expression of PsAPY1 during the process of hook development indicates its essential role in the process of formation and maintenance of hook curvature and thus aids in protection of delicate shoot meristem.

Improved protein overexpression and purification strategies for structural studies of cyanobacterial metal-responsive transcription factor, SmtB from marine Synechococcus sp. PCC 7002.

There are structural and functional differences in SmtB homologs, metal-responsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains. The structure of SmtB from freshwater Synechococcus sp. strain PCC 7942 is elucidated with nuclear magnetic resonance (NMR) and crystallography techniques. But knowledge about the functioning of SmtB homologs from marine species is limited till date. To enable NMR spectroscopic studies for investigating structural and functional aspects, modified protocols with higher yields of isotopically labeled SmtB, from marine species like Synechococcus sp. PCC 7002 are essential. In this study, smtB gene was cloned from genome of Synechococcus sp. PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system. Further, the protocol for large-scale production, isotope labeling with 15N, and purification of recombinant SmtB in E. coli BL21(DE3)/pLysS cells was developed. Purified recombinant protein was successfully used for NMR spectroscopy experiments. These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp. PCC 7002.

MKRN expression pattern during embryonic and post-embryonic organogenesis in rice (Oryza sativa L. var. Nipponbare)

Rice MKRN is a member of the makorin RING finger protein gene (MKRN) family, which encodes a protein with a characteristic array of zinc-finger motifs conserved in various eukaryotes. Using non-radioactive in situ hybridization, we investigated the spatio-temporal gene expression pattern of rice MKRN during embryogenesis, imbibition, seminal and lateral root development of Oryza sativa L. var. Nipponbare. MKRN expression was ubiquitous during early organogenesis in the embryo along the apical–basal and radial axes. The expression of MKRN decreased during embryonic organ elongation and maturation compared to early embryogenesis, but increased again during imbibition. Tissue-specific and position-dependent MKRN expression was found during embryonic and post-embryonic root and shoot development. Meristematic cells ubiquitously expressed MKRN transcripts, while differentiating cells showed a gradual reduction and termination of MKRN expression. Interestingly, during post-germination MKRN expression was prominent and continued in the metabolically active, differentiated companion cells of the phloem. The differential expression pattern was observed both in the differentiating and differentiated cells. Also, MKRN was expressed in the various developmental stages of the lateral root primordia and the cells surrounding them. Expression of MKRN was also observed after periclinal division of the presumptive pericycle founder cells. The MKRN expression pattern during development of various growth stages suggests an important role of makorin RING finger protein gene (MKRN) in embryonic and post-embryonic organogenesis in both apical–basal and radial developmental axes of rice.