A norovirus protease structure provides insights into active and substrate binding site integrity

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.     

NFATc1 Expression as a Prognosticator in Urothelial Carcinoma of the Upper Urinary Tract

We recently found that NFATc1, a member of the NFAT family and a key regulator of the immune response, could induce bladder carcinogenesis and cancer progression. In this study, we immunohistochemically stained for NFATc1 in upper urinary tract urothelial carcinoma (UUTUC) specimens and paired nonneoplastic urothelial tissues. NFATc1 was positive in 51 [52%; 40 (40%) weak (1+), 9 (9%) moderate (2+), and 2 (2%) strong (3+)] of 99 UUTUCs, which was significantly higher than in benign urothelium [30 (36%) of 83; 28 (34%) weak and 2 (2%) moderate] (0 vs 1+/2+/3+, P = .038; 0/1+ vs 2+/3+, P = .023). There were no significant associations between NFATc1 expression pattern and tumor grade or pT stage. However, the positive rates of NFATc1 expression tended to be higher in renal pelvic tumors (60%) than in ureteral tumors (42%; P = .080) as well as in pN+ tumors (75%) than in pN0 tumors (49%; P = .089). Kaplan-Meier and log-rank tests revealed that moderate (2+) to strong (3+) NFATc1 expression correlated with lower progression-free survival (P = .032) and cancer-specific survival (P = .005) rates in the 99 cases. Patients with high (2+/3+) NFATc1 muscle-invasive tumor (n = 9) also had a significantly higher risk of cancer-specific mortality (P = .021) compared to those with low (0/1+) NFATc1 muscle-invasive tumor (n = 53). Thus, compared with nonneoplastic urothelium, a significant increase in the expression of NFATc1 in UUTUC was seen, implying the involvement of NFATc1 signals in the development of UUTUC. The current results further suggest that NFATc1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.     

Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.     

Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.     

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.