Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease

Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-sigma(E) protein RseA as an essential step in transmembrane signal transduction in the sigma(E) extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.     

Understanding mechanisms governing protein-protein interactions from synthetic binding interfaces

Recent advances in methodologies and design of combinatorial library selection have enabled comprehensive characterization of sequence space for protein-protein interaction interfaces and generation of fully synthetic binding interfaces. By exhaustively introducing and quantitatively analyzing mutations in natural interfaces, new insights into their molecular architecture and plasticity have emerged. Minimalist combinatorial libraries based on a restricted amino acid code have produced synthetic interfaces that rival natural ones using a different set of rules. A two amino acid code composed of just tyrosine and serine in the context of antibody CDR loops is sufficient to produce high affinity and specific interactions with different classes of protein targets. Structural analyses highlight the dominant role of Tyr in forming productive interactions and demonstrate the dominance of conformational diversity over chemical diversity in producing na?ve binding interfaces. Synthetic binding proteins are beginning to be used as a powerful crystallization tool to attack important structural biology problems that are recalcitrant to crystallization using traditional methods.     

Legionella pneumophila infection induces programmed cell death,caspase activation,and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

Background

Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.

Methods

Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.

Results

The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.

Conclusion

Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Aromatic claw: A new fold with high aromatic content that evades structural prediction

We determined the NMR structure of a highly aromatic (13%) protein of unknown function, Aq1974 from Aquifex aeolicus (PDB ID: 5SYQ). The unusual sequence of this protein has a tryptophan content five times the normal (six tryptophan residues of 114 or 5.2% while the average tryptophan content is 1.0%) with the tryptophans occurring in a WXW motif. It has no detectable sequence homology with known protein structures. Although its NMR spectrum suggested that the protein was rich in β‐sheet, upon resonance assignment and solution structure determination, the protein was found to be primarily α‐helical with a small two‐stranded β‐sheet with a novel fold that we have termed an Aromatic Claw. As this fold was previously unknown and the sequence unique, we submitted the sequence to CASP10 as a target for blind structural prediction. At the end of the competition, the sequence was classified a hard template based model; the structural relationship between the template and the experimental structure was small and the predictions all failed to predict the structure. CSRosetta was found to predict the secondary structure and its packing; however, it was found that there was little correlation between CSRosetta score and the RMSD between the CSRosetta structure and the NMR determined one. This work demonstrates that even in relatively small proteins, we do not yet have the capacity to accurately predict the fold for all primary sequences. The experimental discovery of new folds helps guide the improvement of structural prediction methods.     

Monobodies and other synthetic binding proteins for expanding protein science

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in‐depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.     

Effects of mycorrhizal fungi on plant populations

We discuss four potentially important interactions between mycorrhizal fungi and populations of plants. First, vesicular-arbuscular mycorrhizal colonization has been shown to increase reproduction (via both male and female functions) and offspring survival, and thus it can increase population size, at least in the short term. This is undoubtedly important to wild plant species and especially to those whose success depends on high rates of reproduction such as early successional annuals. Second, the positive response in growth and reproduction to vesicular-arbuscular mycorrhizal colonization may be inversely related to plant population density. All else being equal, this would tend to stabilize the density of natural plant populations over time. It may also explain why positive responses to mycorrhizal inoculation of dense crops are rare. Third, vesicular-arbuscular mycorrhizal fungi can increase inequality in size and reproduction among plants within a population. Mycorrhizal fungi may thus exaggerate the genetic overrepresentation in the next generation of the most robust individuals in the current generation. Fourth, established mycorrhizal plants may serve as important sources of inoculum for initially nonmycorrhizal, conspecific seedlings. This may affect regeneration, and could contribute to patchy distributions of species within the community.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.     

Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily. Presence of two cystatin domains in the N-terminal region

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1-112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113-225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.