Hyung Jin Cha

The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas.

AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.

Structural Double-Mutant Cycle Analysis of a Hydrogen Bond Network in Ketosteroid Isomerase from Pseudomonas Putida Biotype B.

KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.

Rapamycin inhibits both motility through down-regulation of p-STAT3 (S727) by disrupting the mTORC2 assembly and peritoneal dissemination in sarcomatoid cholangiocarcinoma

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase.

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two‐state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two‐state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X‐ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7 Å in 0 M urea, 17.3 Å in 5.2 M urea, and 25.1 Å in 7 M urea by SAXS. The size of KSI in 5.2 M urea was significantly decreased compared with those in 0 M and 7 M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2 M urea is distinctly different from native and fully‐unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using 1H NMR spectroscopy and Kratky plot supported the existence of a partially‐folded form of KSI which is distinct from those of native and fully‐unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.

Structure of Putrescine Aminotransferase from Escherichia Coli Provides Insights Into the Substrate Specificity Among Class III Aminotransferases.

YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5′-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzymes substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.

Structural Insights of the Nucleotide-Dependent Conformational Changes of Thermotoga maritima MutL Using Small-Angle X-ray Scattering Analysis

MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule.

15N NMR Relaxation Studies of Y14F Mutant of Ketosteroid Isomerase : The Influence of Mutation on Backbone Mobility

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis.

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.

Apoptosis-related mRNA expression profiles of ovarian cancer cell lines following cisplatin treatment

OBJECTIVE The aim of this study was to identify apoptosis-related genes of ovarian cancer cell lines following cisplatin treatment. METHODS We used IC(50) values and fluorescence-activated cell sorting analysis to compare cell death in 2 ovarian cancer cell lines, namely, SKOV-3 and OVCAR-3, upon treatment with cisplatin. Moreover, the change in transcriptional levels of apoptosis-associated genes was measured with a dendron-modified DNA microarray. RESULTS The protein levels for the up-regulated genes in each cell line were validated to identify the molecules that may determine the cellular behavior of cisplatin resistance. Eight genes were over-expressed in the 2 cell lines. The cisplatin-induced up-regulation of DAD1 in transcriptional and protein levels contributed to the cisplatin resistance of OVCAR-3, and the up-regulation of FASTK and TNFRSF11A in SKOV-3 resulted in its higher sensitivity to cisplatin than that of OVCAR-3. CONCLUSION In the present study, we have identified a set of genes responsible for apoptosis following cisplatin treatment in ovarian cancer cell lines. These genes may give information about the understanding of cisplatin-induced apoptosis in ovarian cancer.