Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach.

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-⌬C) was solved in an oligomeric state. The molecular structure of BMRF1-⌬C shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-⌬C, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-⌬C exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-⌬C associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-⌬C probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-⌬C is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.The Epstein-Barr virus (EBV), 4 a human herpesvirus harboring a 172-kbp dsDNA genome, is associated with several B-cell and epithelial cell malignancies and can choose between two alternative life cycles, latent and lytic infection (1). The EBV genomes are replicated as circular plasmid molecules, using the cellular replication machinery of the host in the latent phase of the viral life cycle. On the other hand, after the induction of lytic viral replication, the EBV genome is amplified 100 -1,000-fold by the viral replication machinery. The replication intermediates are large head-to-tail concatemers resulting from rolling-circle DNA replication initiated from oriLyt (2). The EBV replication machinery consists of seven viral gene products (3) as follows: the BZLF1 protein, an oriLytbinding protein; the BALF5 protein, a DNA polymerase catalytic subunit; the BMRF1 protein, a polymerase processivity factor; the BALF2 protein, a single-stranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, putative helicase, primase, and helicase-primase-associated proteins, respectively. It has been suggested that all of the proteins, except for the BZLF1 protein, work together at replication forks to synthesize the leading and lagging strands of the concatemeric EBV genome (2). The EBV DNA polymerase holoenzy...

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

e The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca 2؉ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca 2؉ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca 2؉ was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca 2؉ chelation, in comparison with the thermal melting point (T m ). The melting point changed from 54 to 49°C by the Ca 2؉ chelation, and it was restored to 54°C by the addition of excess Ca 2؉ . These results indicated that the interdomain flexibility and the domain arrangement of fulllength collagenase H are reversibly regulated by Ca 2؉ .