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Biomedical subjects

Hisashi Hirano

Publications and source records attributed to Hisashi Hirano.

At least 19 recordsLinked to original sources
PubMed2006

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma.

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.

Adenocarcinoma, Clear Cell↗
PubMed2006

A novel approach and protocol for discovering extremely low-abundance proteins in serum.

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.

Blood Proteins↗
PubMed2006

Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cells.

Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is approximately 680,000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits.

Amino Acid Sequence↗
PubMed2005

Identification of a new class of pistil-specific proteins of Petunia inflata that is structurally similar to, but functionally distinct from, the self-incompatibility factor HT.

Pollen-pistil interactions are thought to involve a wide variety of intercellular recognition events controlled by diverse proteins and other molecules. One of the best characterized interactions is the S-RNase-based gametophytic self-incompatibility (GSI) system found in Solanaceae, Rosaceae and Scrophulariaceae. Although the S specificity of the pistil and the pollen in these families is determined by the S locus-encoded proteins S-RNase and SLF/SFB, respectively, these proteins alone are not sufficient for operation of the GSI reaction. Other factors are also required and are classified into three groups. To date, the only known factor is the pistil-expressed small asparagine-rich protein HT-B in three solanaceous genera Nicotiana, Lycopersicon and Solanum. HT-B is a Group 2 factor that is required for pollen rejection but do not affect S-RNase expression; factors in the other groups have not yet cloned. Here, we identified a new class of HT-like proteins in the style of Petunia inflata and named it HTL. Through alternative splicing, it was found that two isolated homologous HTL cDNAs, HTL-A and HTL-B, derived from a single gene. Like HT-B, HTL showed pistil-specific accumulation as well as significant sequence similarity to HT including conserved cystein residues at the C-terminal region and a signal peptide for extracellular localization. However, unlike HT-B, HTL lacked an asparagine-rich domain. Thus, it represents a new class of HT proteins. To determine whether HTL is involved in GSI function, RNA silencing constructs for HTL-A and HTL-B were introduced into self-incompatible P. inflata. Although several transgenic lines showed no detectable levels of both HTL-A and HTL-B transcripts, they retained normal GSI function and produced large fruits upon compatible pollination. This suggests that since silencing of the HTL gene alone is not sufficient to affect reproductive physiology, the gene is functionally distinct from the GSI factor HT-B.

Flowers↗
PubMed2005

The role of glycosylation in the function of a 48-kDa glycoprotein from carrot.

Carrot extracellular dermal glycoprotein (EDGP) may play an important role in plant defense systems and in signal transduction. Our experiments show that differences in pI values of EDGP isoforms are caused by differences in amino acid sequence and not by heterogeneity in phosphorylation. The binding affinity of native EDGP for a 4-kDa hormone-like peptide from soybean was approximately one-third that of deglycosylated EDGP, and deglycosylation of EDGP caused complete loss of its ability to inhibit xyloglucan-specific endo-beta-1,4-glucanase. Experiments using tunicamycin-treated carrot cell cultures showed that glycosylation is essential for correct EDGP folding and secretion, and that tunicamycin does not affect EDGP gene transcription.

Cells, Cultured↗
PubMed2004

Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry.

The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.

Histidine↗
PubMed2004

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean.

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.

Alanine↗
PubMed2004

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein.

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.

Amino Acid Sequence↗
PubMed2004

Mass spectrometric analysis of expression of ATPase subunits encoded by duplicated genes in the 19S regulatory particle of rice 26S proteasome.

The 26S proteasome consisting of a 20S proteasome and a pair of 19S regulatory particles (RP) plays important roles in degradation of the ubiquitinated protein in eukaryotic cells. The RP consists of six different ATPase subunits and, at least, 11 non-ATPase subunits. In rice, we previously identified duplicated genes encoding four ATPase subunits, OsRpt1, OsRpt2, OsRpt4, and OsRpt5. In this study, the genomic sequences of all rice ATPase subunits were identified from the rice genome database and the genomic structure of ATPase subunit genes was determined. The rice RP was purified, and the ATPase subunit isoforms encoded by three pairs of duplicated genes, OsRpt2a/OsRpt2b, OsRpt4a/OsRpt4b, and OsRpt5a/OsRpt5b, were identified in RP by using electrospray ionization quadrupole time-of-flight mass spectrometry. The relative amounts and the expression patterns of these ATPase subunit isoforms in the bran were found to be different from those of the callus, suggesting the presence of multiform 19S regulatory particles engaged in the tissue-specific protein metabolism.

Adenosine Triphosphatases↗
PubMed2004

Technical aspects of functional proteomics in plants.

Since the completion of genome sequences of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. This analysis is achieved by separation and identification of proteins, determination of their function and functional network, and construction of an appropriate database. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis, nano-liquid chromatography and mass spectrometry, have rapidly been achieved. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. These techniques have provided the possibility of high-throughput analysis of function and functional network of proteins in plants. However, to cope with the huge information emerging from proteome analyses, more sophisticated techniques and software are essential. The development and adaptation of such techniques will ease analyses of protein profiling, identification of post-translational modifications and protein-protein interaction, which are vital for elucidation of the protein functions.

Databases, Protein↗
PubMed2003

N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome.

The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain.

Acetylation↗
PubMed2003

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.

Databases, Protein↗
PubMed2003

Wheat proteomics: relationship between fine chromosome deletion and protein expression.

To explore the relationship between fine chromosome deletion and protein expression in common wheat, the changes in protein composition of wheat seed proteome were investigated by using chromosome 1B. A momosomic alien chromosome addition line of common wheat was used to produce the fine deletion lines. Endosperm and embryo proteins were separated by two-dimensional gel electrophoresis (2-DE) and visualized by staining with Commassie Brilliant Blue, and gel images were analyzed with a computer assisted image analyzer. For the first time, fine gene locations of a few endosperm and embryo proteins were identified on the chromosome 1B. These proteins with their specific gene location on the chromosome can be used as protein markers in breeding programs for quality of wheat proteins. To identify wheat seed proteins and to understand their expression in relation to chromosome deletion, the feasibility of a new analytical approach based on isotope coded affinity tag labeling (ICAT) of peptides in tryptic digests followed by electrospray ionization mass spectrometry has been described. Simplification of the complex tryptic digest prior to mass spectral analysis was performed by treating the samples with light and heavy ICAT labeling reagents. A clear separation of peptide fragment containing the light and heavy reagents was achieved in mass spectral analysis. Out of the 14 peptides detected by mass fragment analysis of the euploid, four were down-regulated, nine up-regulated and one did not show any change due to the terminal deletion of chromosome 1B. Selected peptide fragments were subjected to tandem mass spectrometry analysis for sequence information and the resulting sequence information was submitted to databases for protein identification. Of the five proteins submitted, four were identified as alpha-amylase inhibitor, alpha-amylase/subtilisin inhibitor precursor, proteasome subunit alpha-type 7 and 1,4 alpha-glucan-D-maltohydrolase. With this approach it is possible to identify wheat seed proteins and to understand their expression, which have been reported to be difficult by 2-DE due to cosynthesis of proteins by genes from three genomes, A, B and D.

Chromosome Deletion↗
PubMed2003

Proteome analysis of diploid, tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression.

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.

Chromosome Aberrations↗
PubMed2003

A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes.

Previously, we isolated a 4-kDa peptide capable of binding to a 43-kDa receptor-like protein and stimulating protein kinase activity of the 43-kDa protein in soybean. Both of them were found to localize in the plasma membranes and cell walls. Here, we report the physiological effects of 4-kDa peptide expressed transiently in the cultured carrot and bird's-foot trefoil cells transfected with pBI 121 plasmid containing the 4-kDa peptide gene. At early developmental stage, the transgenic callus grew rapidly compared to the wild callus in both species. Cell proliferation of in vitro cultured nonembryogenic carrot callus was apparently affected with the 4-kDa peptide in the medium. Complementary DNAs encoding the 4-kDa peptide from mung bean and azuki bean were cloned by PCR and sequenced. The amino-acid sequences deduced from the nucleotide sequences are homologous among legume species, particularly, the sites of cysteine residues are highly conserved. This conserved sequence reflects the importance of intradisulfide bonds required for the 4-kDa peptide to perform its function. Three dimensional structure of the 4-kDa peptide determined by NMR spectroscopy suggests that this peptide is a T-knot scaffold containing three beta-strands, and the specific binding activity to the 43-kDa protein and stimulatory effect on the protein phosphorylation could be attributed to the spatial arrangements of hydrophobic residues at the solvent-exposed surface of two-stranded beta-sheet of 4-kDa peptide. The importance of these residues for the 4-kDa peptide to bind to the 43-kDa protein was indicated by site-directed mutagenesis. These results suggest that the 4-kDa peptide is a hormone-like peptide and the 43-kDa protein is involved in cellular signal transduction of the peptide.

Amino Acid Sequence↗
PubMed2003

Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein.

Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation. As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction. However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood. We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein. Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein. To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed. The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants. Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide. Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.

Albumins↗