Gotowa bibliografia na temat „DgkA Proteins”

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.

Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) α and ζ act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGKα and DGKζ to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGKζ, indicating functional differences between these proteins. DGKζ kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGKζ responsiveness. We used GFP-DGKζ and -DGKα chimeras to assess translocation dynamics during IS formation. Only GFP-DGKζ translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGKζ with DAG consumption at the IS. This study highlights a DGKζ-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.

Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase orin mesomethod, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing thein mesostructure of the integral membrane diacylglycerol kinase (DgkA) fromEscherichia coliis reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGKζ, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGKζ also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGKζ and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGKζ in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGKζ, which controls local accumulation of DAG that would otherwise activate RasGRP.

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKζ initiates RhoGDI release and Rac1 activation. In DGKζ-deficient fibroblasts PAK1 phosphorylation and Rac1–RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKζ, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKζ stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKζ, and PA to the activation of Rac1: the PA generated by DGKζ activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

Abstract The mechanism promoting exacerbated immune responses in allergy and autoimmunity as well as those blunting the immune control of cancer cells are of primary interest in medicine. Diacylglycerol kinases (DGKs) are key modulators of signal transduction, which blunt diacylglycerol (DAG) signals and produce phosphatidic acid (PA). By modulating lipid second messengers, DGK modulate the activity of downstream signaling proteins, vesicle trafficking and membrane shape. The biological role of the DGK α and ζ isoforms in immune cells differentiation and effector function was subjected to in deep investigations. DGK α and ζ resulted in negatively regulating synergistic way basal and receptor induced DAG signals in T cells as well as leukocytes. In this way, they contributed to keep under control the immune response but also downmodulate immune response against tumors. Alteration in DGKα activity is also implicated in the pathogenesis of genetic perturbations of the immune function such as the X-linked lymphoproliferative disease 1 and localized juvenile periodontitis. These findings suggested a participation of DGK to the pathogenetic mechanisms underlying several immune-mediated diseases and prompted several researches aiming to target DGK with pharmacologic and molecular strategies. Those findings are discussed inhere together with experimental applications in tumors as well as in other immune-mediated diseases such as asthma.

SummaryDiacylglycerol kinases (DGKs) are thought to attenuate diacylglycerol signals by converting diacylglycerol to phosphatidic acid. The nine mammalian diacylglycerol kinases that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. The activity of DGKs is regulated by mechanisms that can modify their access to diacylglycerol, affect their activity, or alter their ability to bind to other proteins. Although little is known of the specific function of DGKs in platelets, they likely influence actin reorganization and other signaling events requiring diacylglycerol.

We report here that the anterograde transport from the endoplasmic reticulum (ER) to the Golgi was markedly suppressed by diacylglycerol kinase δ (DGKδ) that uniquely possesses a pleckstrin homology (PH) and a sterile α motif (SAM) domain. A low-level expression of DGKδ in NIH3T3 cells caused redistribution into the ER of the marker proteins of the Golgi membranes and the vesicular-tubular clusters (VTCs). In this case DGKδ delayed the ER-to-Golgi traffic of vesicular stomatitis virus glycoprotein (VSV G) and also the reassembly of the Golgi apparatus after brefeldin A (BFA) treatment and washout. DGKδ was demonstrated to associate with the ER through its C-terminal SAM domain acting as an ER-targeting motif. Both of the SAM domain and the N-terminal PH domain of DGKδ were needed to exert its effects on ER-to-Golgi traffic. Kinase-dead mutants of DGKδ were also effective as the wild-type enzyme, suggesting that the catalytic activity of DGK was not involved in the present observation. Remarkably, the expression of DGKδ abrogated formation of COPII-coated structures labeled with Sec13p without affecting COPI structures. These findings indicate that DGKδ negatively regulates ER-to-Golgi traffic by selectively inhibiting the formation of ER export sites without significantly affecting retrograde transport.

Proteins play a central role in all the biological processes. The immense diversity in protein structures and functions despite similar underlying composition is intriguing. The work presented in this thesis aims to provide a deeper understanding of protein structure-function relationships. It describes some techniques that were developed in order to probe these relationships. Chapter 1 provides a general introduction to the topics discussed in the thesis. Chapter 2 focuses on an important aspect of protein structures, the cavities. Although proteins are composed of regular arrangements of secondary structures, namely, α helices and β sheets, there are some irregularities. The packing density is not uniform throughout the protein resulting into formation of cavities. The role of cavities has been previously probed using mutagenesis studies. The mutations designed to fill the cavities were observed to improve stability and cavity creating mutations adversely affected the stability. Cavities are thus reported to be important contributors to stability. In chapter 2, we refine and benchmark a method for prediction of protein cavities based on molecular dynamics simulations. The insights derived from the mutagenesis studies provide some basic understanding of substitution preferences in proteins. The exposed non-active site positions are more tolerant to mutations whereas, the buried positions are not. Introducing cavity filling and disulfide mutations in proteins have been demonstrated to improve stability. Engineering protein variants with improved stability has immense applications in biology. In chapter 3, we discuss an important application, i.e., immunogen designing. Surface glycoproteins of several viruses exhibit two conformations, namely the metastable prefusion conformation and the highly stable postfusion conformation adopted during the fusion of the virus with the host cell membrane. Stable immunogens exhibiting the prefusion conformation are promising candidates for subunit vaccines. In chapter 3, we discuss immunogen designing for the respiratory syncytial virus (RSV). In addition to stabilized mutants, temperature sensitive (Ts) mutants are another class of engineered proteins. The Ts mutants exhibit reduced activity levels above the permissive temperature. They have been extensively used in developmental biology. Ts mutants are excellent tools to modulate protein expression levels in cells. A model for prediction of Ts positions was developed previously. This model exploits residue hydrophobicity to infer residue burial in the structure solely based on the protein sequence. In the current work, we improved the accuracy of the model by incorporating structural information in the model. Chapter 4 describes the development and benchmarking of a server for the prediction of Ts mutants (TSpred). The TSpred server suggests a stereochemically diverse set of mutations at the putative buried positions which are would produce destabilization to different extents and at least one of them is likely to be Ts. The TSpred predictions were employed for designing live attenuated vaccine candidates for RSV. The work thus far elaborates on factors contributing to protein stability and application of that information for rationally designing mutants to modulate protein structure and function. In order to gain a deeper understanding of the role of each protein residue in its function, simultaneous analysis of multiple mutants is essential. Site saturation mutagenesis techniques generate all nineteen mutations at each residue position of the protein and the mutant function is linked to a phenotypic readout like cell viability or binding to a ligand. The mutant libraries are deep sequenced using one of the available platforms like Illumina, SOLiD, 454 and Ion torrent, and analysed to estimate the relative proportions of each of the mutants in the library. Automated programs are necessary to analyse the large amount of data generated after deep sequencing. A pipeline for analysis of data generated from the Illumina sequencing platform is discussed in chapter 5. A mutational sensitivity measure denoted as MSseq was previously derived for the Controller of Cell division or Death B protein (CcdB). The values of the MSseq parameter reflect mutant activity. The active or inactive phenotypes of various mutants were analysed as a function of residue burial. Additional insights about substitution preferences at buried positions were gained from this analysis. In addition to residue burial, the substitution preferences varied with the physico-chemical nature and the size of the wild type (WT) and the mutant side chains. The active site of the CcdB protein could be inferred based on the trends in the mutational sensitivity values. We quantitated these effects and developed a model, detailed in chapter 6, for prediction of the mutant phenotypes using a fraction of the CcdB mutational data. The model was observed to perform better than two other machine learning based predictors, SNAP2 and SuSPect. Chapter 7 describes an additional application of the mutational sensitivity data. By analysing the population distribution of the MSseq values, an empirical parameter, RankScore, was previously derived for each residue in CcdB. RankScore can be interpreted as a weighted average of the MSseq values. RankScore was found to correlate well with residue depth which measures the extent of burial of a residue. As the residue depths in the native structure correlated well (r = 0.6) with the RankScores, the residue depths in native-like models would also correlate well with RankScores. Based on this principle, native-like models could be distinguished from low quality decoys. In the analysis reported in chapter 7, we examine this methodology using decoy datasets for ~200 proteins. We also consider additional information like predicted secondary structure and model quality score to achieve better model discrimination. Studies thus far describe analyses performed using single protein mutants. Furthermore, information about residue interactions is also important. During the course of evolution, as the maintenance of specific interactions is essential for protein function, residues participating in such interactions are either conserved or varied in a correlated manner. Several computational models analysing such correlations among mutations are available. Experimental techniques are also available for identification of the spatially proximal residues. In chapter 8, we analyse various computational programs using CcdB and Diacylglycerol kinase A (DgkA) proteins and the results are then compared with the available experimental data. Overall little overlap was observed between the predictions based on the natural sequence co-variation and the experimental data. Both the computational and experimental approaches can be applied in conjugation as they provide complementary information. The analyses described in the current work would provide useful guidelines for rational design of mutations to modulate protein stability. This has important implications in immunogen designing. The tools developed as part of the current work can be applied for (i) rational designing of Ts mutants, (ii) the analysis of site saturation libraries, (iii) calculation or prediction of substitution preferences, (iv) structure prediction using correlated mutations as constraints, or (v) protein model discrimination. A small appendix section is also included in the thesis. Synonymous mutations with differential phenotypes were observed in our deep sequenced library. In order to analyse them further, we performed a multiple sequence alignment and analysed codon frequencies at different positions. However, only preliminary results are available and those are included in Appendix I section of this thesis.