Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Type I interferon is required for T helper (Th) 2 induction by dendritic cells

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN‐I) in enabling this process. An IFN‐I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN‐I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1^?/? mice were incapable of initiating Th2 responses in vivo. These data demonstrate for the first time that the influence of IFN‐I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.     

The structural basis of novel endosome anchoring activity of KIF16B kinesin

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.     

The origin of C1A-C2 interdomain interactions in protein kinase Calpha

The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.     

The molecular basis of the differential subcellular localization of FYVE domains

This study systematically analyzed the structural and mechanistic basis of the regulation of subcellular membrane targeting using FYVE domains as a model. FYVE domains, which mediate the recruitment of signaling and membrane-trafficking proteins to phosphatidylinositol 3-phosphate-containing endosomes, exhibit distinct subcellular localization despite minor structural variations within the family. Biophysical measurements, cellular imaging, and computational analysis of various FYVE domains showed that the introduction of a single cationic residue and a hydrophobic loop into the membrane binding region of the FYVE domains dramatically enhanced their membrane interactions. The results indicated that there is a threshold affinity for endosomal localization and that endosomal targeting of FYVE domains is sensitive to small changes in membrane affinity about this threshold. Collectively these studies provide new insight into how subcellular localization of FYVE domains and other membrane targeting domains can be regulated by minimal structural and environmental changes.     

Clustering protein sequences with a novel metric transformed from sequence similarity scores and sequence alignments with neural networks

Background  

The sequencing of the human genome has enabled us to access a comprehensive list of genes (both experimental and predicted) for further analysis. While a majority of the approximately 30000 known and predicted human coding genes are characterized and have been assigned at least one function, there remains a fair number of genes (about 12000) for which no annotation has been made. The recent sequencing of other genomes has provided us with a huge amount of auxiliary sequence data which could help in the characterization of the human genes. Clustering these sequences into families is one of the first steps to perform comparative studies across several genomes.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.