Metallothionein 1 h tumour suppressor activity in prostate cancer is mediated by euchromatin methyltransferase 1.

Metallothioneins (MTs) are a group of metal binding proteins thought to play a role in the detoxification of heavy metals. Here we showed by microarray and validation analyses that MT1h, a member of MT, is down-regulated in many human malignancies. Low expression of MT1h was associated with poor clinical outcomes in both prostate and liver cancer. We found that the promoter region of MT1h was hypermethylated in cancer and that demethylation of the MT1h promoter reversed the suppression of MT1h expression. Forced expression of MT1h induced cell growth arrest, suppressed colony formation, retarded migration, and reduced invasion. SCID mice with tumour xenografts with inducible MT1h expression had lower tumour volumes as well as fewer metastases and deaths than uninduced controls. MT1h was found to interact with euchromatin histone methyltransferase 1 (EHMT1) and enhanced its methyltransferase activity on histone 3. Knocking down of EHMT1 or a mutation in MT1h that abrogates its interaction with EHMT1 abrogated MT1h tumour suppressor activity. This demonstrates tumour suppressor activity in a heavy metal binding protein that is dependent on activation of histone methylation.

Interaction of CSR1 with XIAP reverses inhibition of caspases and accelerates cell death.

Cellular Stress Response 1 (CSR1) is a tumor suppressor gene that is located at 8p21, a region that is frequently deleted in prostate cancer as well as a variety of human malignancies. Previous studies have indicated that the expression of CSR1 induces cell death. In this study, we found that CSR1 interacts with X-linked Inhibitor of Apoptosis Protein (XIAP), using yeast two-hybrid screening analyses. XIAP overexpression has been found in many human cancers, and forced expression of XIAP blocks apoptosis. Both in vitro and in vivo analyses indicated that the C-terminus of CSR1 binds XIAP with high affinity. Through a series of in vitro recombinant protein-binding analyses, the XIAP-binding motif in CSR1 was determined to include amino acids 513 to 572. Targeted knock-down of XIAP enhanced CSR1-induced cell death, while overexpression of XIAP antagonized CSR1 activity. The binding of CSR1 with XIAP enhanced caspase-9 and caspase-3 protease activities, and CSR1-induced cell death was dramatically reduced on expression of a mutant CSR1 that does not bind XIAP. However, a XIAP mutant that does not interact with caspase-9 had no impact on CSR1-induced cell death. These results suggest that cell death is induced when CSR1 binds XIAP, preventing the interaction of XIAP with caspases. Thus, this study may have elucidated a novel mechanism by which tumor suppressors induce cell death.

Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation.

Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

Integrin alpha 7 interacts with high temperature requirement A2 (HtrA2) to induce prostate cancer cell death.

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down-regulation and/or mutations of integrin alpha7 (ITGA7) in prostate cancer, liver cancer, soft tissue leiomyosarcoma, and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two-hybrid analysis revealed that the C-terminus of ITGA7 interacts with high temperature requirement A2 (HtrA2), a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, whereas down-regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site-directed protease-null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears to protect against cell death, since depleting laminin beta2 with a small-interfering RNA significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

Construction of a 3D model of nattokinase, a novel fibrinolytic enzyme from Bacillus natto. A novel nucleophilic catalytic mechanism for nattokinase.

A three-dimensional structural model of nattokinase (NK) from Bacillus natto was constructed by homology modeling. High-resolution X-ray structures of Subtilisin BPN' (SB), Subtilisin Carlsberg (SC), Subtilisin E (SE) and Subtilisin Savinase (SS), four proteins with sequential, structural and functional homology were used as templates. Initial models of NK were built by MODELLER and analyzed by the PROCHECK programs. The best quality model was chosen for further refinement by constrained molecular dynamics simulations. The overall quality of the refined model was evaluated. The refined model NKC1 was analyzed by different protein analysis programs including PROCHECK for the evaluation of Ramachandran plot quality, PROSA for testing interaction energies and WHATIF for the calculation of packing quality. This structure was found to be satisfactory and also stable at room temperature as demonstrated by a 300ps long unconstrained molecular dynamics (MD) simulation. Further docking analysis promoted the coming of a new nucleophilic catalytic mechanism for NK, which is induced by attacking of hydroxyl rich in catalytic environment and locating of S221.

Cloning, expression, characterization and phylogenetic analysis of arginine kinase from greasyback shrimp (Metapenaeus ensis).

Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent K(m) was 2.33+/-0.1 and 1.59+/-0.2 mM for ATP and l-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 degrees C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).

A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization.

A novel serine protease with fibrinolytic activity named CSP was purified from the culture supernatant of the fungus Cordyceps sinensis, a kind of Chinese herbal medicine. Analysis of the purified enzyme by SDS-PAGE indicated that CSP was a single polypeptide chain with an apparent molecular weight of 31 kDa, and N-terminal sequencing revealed that the first ten amino acid residues of the enzyme were Ala-Leu-Ala-Thr-Gln-His-Gly-Ala-Pro-Trp-. When casein was used as a substrate, the proteolytic activity of CSP reached its maximum at pH 7.0 and 40 degrees C. The effect of chemical agents on the enzyme activity indicated that CSP is a serine protease with a free cysteine residue near the active site. It hydrolysed fibrinogen, fibrin and casein with a high efficiency, while hydrolysing bovine serum albumin (BSA) and human serum albumin (HSA) to a lesser extent. CSP was found to be a plasmin-like protease, but not a plasminogen activator, and it preferentially cleaved the A alpha chain of fibrinogen and the alpha-chain of fibrin. Therefore, the extracellular protein CSP may represent a potential new therapeutic agent for the treatment of thrombosis.