Binding of S-layer homology modules from Clostridium thermocellum SdbA to peptidoglycans

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.     

Essentiality of a newly identified carbohydrate-binding module for the function of CelB (BH0603) from the alkaliphilic bacterium Bacillus halodurans

CelB (BH0603) from Bacillus halodurans is a modular glycoside hydrolase with a family 5 catalytic module, an immunoglobulin-like module, and module PfamB of unknown function. The recombinant PfamB module bound to Avicel and was essential for CelB hydrolytic function. We propose that module PfamB be designated a new carbohydrate-binding module.     

Phagocytosis of N-acetyl-D-glucosamine particles, a Th1 adjuvant, by RAW 264.7 cells results in MAPK activation and TNF-alpha, but not IL-10, production

A practical and highly effective Th1 adjuvant should induce Th1 cytokines (IL-12, IL-18, and TNF-alpha) but not the Th2 cytokine IL-10, an inhibitor of Th1 responses. In this study, phagocytosis of N-acetyl-d-glucosamine polymer (chitin) particles by RAW 264.7 cells, a murine macrophage-like cell line, resulted in phosphorylation of MAPK (p38, Erk 1/2, and JNK), and production of relatively high levels of TNF-alpha and COX-2 with increased PGE(2) release. Similar results were observed in response to oligonucleotides with CpG motifs, mycobacterial components and endotoxin. However, these bacterial components also induced a large amount of IL-10. Chitin particles, in contrast, failed to induce detectable levels of IL-10, although the production of high levels of PGE(2) and TNF-alpha and the activation of MAPK's are potentially positive signals for IL-10 production. Thus, our results indicate that chitin particles act as a unique Th1 adjuvant for macrophages without inducing increased production of IL-10.     

Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer

Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.     

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.     

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human β-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24?h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.     

Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome-associated proteins

Dysfunction of the basement membrane protein QBRICK provokes Fraser syndrome, which results in renal dysmorphogenesis, cryptophthalmos, syndactyly, and dystrophic epidermolysis bullosa through unknown mechanisms. Here, we show that integrin α8β1 binding to basement membranes was significantly impaired in Qbrick-null mice. This impaired integrin α8β1 binding was not a direct consequence of the loss of QBRICK, which itself is a ligand of integrin α8β1, because knock-in mice with a mutation in the integrin-binding site of QBRICK developed normally and do not exhibit any defects in integrin α8β1 binding. Instead, the loss of QBRICK significantly diminished the expression of nephronectin, an integrin α8β1 ligand necessary for renal development. In vivo, nephronectin associated with QBRICK and localized at the sublamina densa region, where QBRICK was also located. Collectively, these findings indicate that QBRICK facilitates the integrin α8β1-dependent interactions of cells with basement membranes by regulating the basement membrane assembly of nephronectin and explain why renal defects occur in Fraser syndrome.     

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.