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1.  Actin and Vimentin proteins with N-terminal deletion detected in tumor bearing rat livers induced by intraportal-vein injection of Ha-ras transfected rat liver cells 
The introduction of the tumorigenic v-Ha-ras oncogene-transformed rat liver epithelial cells (WBras), which is deficient in gap junctional intercellular communication (GJIC), into F344 rats, induces significant formation of hepatocellular tumors. GJIC plays a major role in maintaining tissue homeostasis. Using this in vivo tumor model system, we used 2-dimensional electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second dimension to globally identify proteins that are uniquely expressed in the livers of WBras-treated rats as compared to the sham control. Immunoblotting was used to identify Ras and Connexin43, which were the positive and negative marker proteins, respectively, of the introduced WBras cells. As predicted, immunoblotting indicated that the whole liver of tumor-bearing animals exhibited a decreased level of Connexin43 and an increased level of Ras. Connexin43 and GJIC were expressed and functional in normal liver, but not in the tumor. In addition to these two markers, an additional four proteins exhibited decreased levels and two proteins exhibited increased levels in the livers of tumor bearing animals. N-Terminal sequencing analysis was used to identify these proteins which were glucose-regulated protein 78, two isoforms of heat shock protein 60, and the β-chain of ATP synthase for the down regulated proteins, and β-Actin with a 46 amino acid deletion from its N-terminus and Vimentin with a 71 amino acid deletion from its N-terminus for the up regulated proteins. These data offer potentially new markers of liver tumorigenicity, particularly Vimentin.
doi:10.1002/ijc.24229
PMCID: PMC2680316  PMID: 19199359
gap junctional intercellular communication; Vimentin; liver; ras oncogene; tumor
2.  Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A from Geobacillus collagenovorans MO-1 
Pz peptidase A has been cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits thimet oligopeptidase and neurolysin.
Pz peptidase A is an intracellular M3 metallopeptidase found in the thermophile Geobacillus collagenovorans MO-1 that recognizes collagen-specific tripeptide units (Gly-Pro-Xaa). Pz peptidase A shares common reactions with mammalian thimet oligopeptidase (TOP) and neurolysin, but has extremely low primary sequence identity to these enzymes. In this work, Pz peptidase A was cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits TOP and neurolysin. The crystals belong to space group P21, with unit-cell parameters a = 56.38, b = 194.15, c = 59.93 Å, β = 106.22°. This is the first crystallographic study of an M3 family peptidase–PPI complex.
doi:10.1107/S174430910700334X
PMCID: PMC2330125  PMID: 17277461
Pz peptidase A; M3 metallopeptidases; collagen degradation; Geobacillus collangenovorans MO-1
3.  Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1 
Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.
doi:10.1107/S1744309106047543
PMCID: PMC2225360  PMID: 17142913
proline-specific aminopeptidase; Aneurinibacillus sp. strain AM-1; thermophiles
4.  Characteristic Features in the Structure and Collagen-Binding Ability of a Thermophilic Collagenolytic Protease from the Thermophile Geobacillus collagenovorans MO-1 
Journal of Bacteriology  2006;188(18):6572-6579.
A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in β-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.
doi:10.1128/JB.00767-06
PMCID: PMC1595469  PMID: 16952949
5.  Two Thimet Oligopeptidase-Like Pz Peptidases Produced by a Collagen- Degrading Thermophile, Geobacillus collagenovorans MO-1 
Journal of Bacteriology  2005;187(12):4140-4148.
A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.
doi:10.1128/JB.187.12.4140-4148.2005
PMCID: PMC1151727  PMID: 15937176
6.  Identification of a Helix-Turn-Helix Motif of Bacillus thermoglucosidasius HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor 
Journal of Bacteriology  2003;185(1):381-385.
In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.
doi:10.1128/JB.185.1.381-385.2003
PMCID: PMC141899  PMID: 12486078
7.  Cooperative Regulation of DOG2, Encoding 2-Deoxyglucose-6-Phosphate Phosphatase, by Snf1 Kinase and the High-Osmolarity Glycerol–Mitogen-Activated Protein Kinase Cascade in Stress Responses of Saccharomyces cerevisiae 
Journal of Bacteriology  2000;182(18):5121-5126.
We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Δ mutant, while the derepression of DOG2 was not observed in a snf1Δ mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Δ, hog1Δ, and snf1Δ. However, the osmotic induction was completely abolished in both the snf1Δ pbs2Δ mutant and the snf1Δ hog1Δ mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol–mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.
PMCID: PMC94660  PMID: 10960096

Results 1-7 (7)