PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.     

Screening Marine Fungi for Inhibitors of the C4 Plant Enzyme Pyruvate Phosphate Dikinase: Unguinol as a Potential Novel Herbicide Candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C₄ plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C₄ plant but no effect on a model C₃ plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.     

Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

Background  

The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA) is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET) uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2), a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function.     

C17 prevents inflammatory arthritis and associated joint destruction in mice

C17 was first described about ten years ago as a gene expressed in CD34+ cells. A more recent study has suggested a role for C17 in chondrogenesis and development of cartilage. However, based on sequence analysis, we believe that C17 has homology to IL-2 and hence we present the hypothesis that C17 is a cytokine possessing immune-regulatory properties. We provide evidence that C17 is a secreted protein preferentially expressed in chondrocytes, hence in cartilage-rich tissues. Systemic expression of C17 in vivo reduces disease in a collagen antibody-induced arthritis model in mice (CAIA). Joint protection is evident by delayed disease onset, minimal edema, bone protection and absence of diverse histological features of disease. Expression of genes typically associated with acute joint inflammation and erosion of cartilage or bone is blunted in the presence of C17. Consistent with the observed reduction in bone erosion, we demonstrate reduced levels of RANKL in the paws and sera of mice over-expressing C17. Administration of C17 at the peak of disease, however, had no effect on disease progression, indicating that C17's immune-regulatory activity must be most prominent prior to or at the onset of severe joint inflammation. Based on this data we propose C17 as a cytokine that s contributes to immune homeostasis systemically or in a tissue-specific manner in the joint.     

Herd-level risk factors associated with tuberculosis breakdowns among cattle herds in England before the 2001 foot-and-mouth disease epidemic

A case-control study of the factors associated with the risk of a bovine tuberculosis (TB) breakdown in cattle herds was undertaken within the randomized badger culling trial (RBCT). TB breakdowns occurring prior to the 2001 foot-and-mouth disease epidemic in three RBCT triplets were eligible to be cases; controls were selected from the same RBCT area. Data from 151 case farms and 117 control farms were analysed using logistic regression. The strongest factors associated with an increased TB risk were movement of cattle onto the farm from markets or farm sales, operating a farm over multiple premises and the use of either covered yard or 'other' housing types. Spreading artificial fertilizers or farmyard manure on grazing land were both associated with decreased risk. These first case-control results from the RBCT will be followed by similar analyses as more data become available.     

Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.