Severine I. Gharbi

Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse.

DGKα and DGKζ negatively regulate the DAG/RasGRP1/Ras pathway in T cells. Study of the specific contribution of each isoform to DAG metabolism during immune synapse formation by use of a combination of RNAi and videomicroscopy techniques identifies DGKζ as mainly responsible for DAG consumption at the immunological synapse.

Translocation dynamics of sorting nexin 27 in activated T cells.

Sorting nexin 27 (SNX27) belongs to the sorting nexin family of proteins, which participate in vesicular and protein trafficking. Similarly to all sorting nexin proteins, SNX27 has a functional PX domain that is important for endosome binding, but it is the only sorting nexin with a PDZ domain. We identified SNX27 as a partner of diacylglycerol kinase ζ (DGKζ), a negative regulator of T cell function that metabolises diacylglycerol to yield phosphatidic acid. SNX27 interacts with the DGKζ PDZ-binding motif in early/recycling endosomes in resting T cells; however, the dynamics and mechanisms underlying SNX27 subcellular localisation during T cell activation are unknown. We demonstrate that in T cells that encounter pulsed antigen-presenting cells, SNX27 in transit on early/recycling endosomes polarise to the immunological synapse. A fraction of SNX27 accumulates at the mature immunological synapse in a process that is dependent on vesicular trafficking, binding of the PX domain to phosphatidylinositol 3-phosphate and the presence of the PDZ region. Downmodulation of expression of either SNX27 or DGKζ results in enhanced basal and antigen-triggered ERK phosphorylation. These results identify SNX27 as a PDZ-containing component of the T cell immunological synapse, and demonstrate a role for this protein in the regulation of the Ras–ERK pathway, suggesting a functional relationship between SNX27 and DGKζ.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.

Redundant and specialized roles for diacylglycerol kinases α and ζ in the control of T cell functions

Two isoforms of the enzyme that attenuates signaling by the second messenger diacylglycerol regulate multiple properties of T lymphocytes. Gloss T lymphocytes patrol the body continuously, and they use antigen-specific T cell receptors (TCRs) to scan and detect foreign peptides on the surface of infected or cancerous cells. When the TCR recognizes a foreign antigen, it sends signals that activate the T lymphocytes, enabling them to help other immune cells or to directly kill those cells that are infected or have become cancerous. T lymphocytes must discriminate correctly between self and foreign antigens because any failure to do so could lead them to destroy healthy cells, as occurs in autoimmune diseases, or to allow tumors to grow. The diacylglycerol kinases (DGKs) are enzymes that inhibit some of the signals needed for T cell activation; by characterizing them, we can better understand the mechanisms by which T lymphocytes distinguish between self and foreign antigens. This Review, which contains 5 figures and 105 references, highlights how the study of DGKs and the signals that they regulate is important because it will help scientists learn how to manipulate T cell responses for the treatment of diseases. The diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG)–mediated signals by catalyzing the conversion of DAG to phosphatidic acid. In T lymphocytes, the antigen-stimulated generation of DAG links signal strength to the intensity and duration of signaling by the Ras–extracellular signal–regulated kinase (ERK) and protein kinase C (PKC)–dependent pathways. The generation of DAG at the plasma membrane of T cells lies at the core of the mechanisms that delimit T cell functions. DGKα and DGKζ are the two main isoforms that are found in T cells, and several approaches define their precise contribution to T cell responses. Each of these isoforms has specialized and redundant functions that limit the intensity of DAG-regulated signals downstream of antigenic stimulation. This ability, which in normal T cells contributes to maintaining homeostasis and function, is exploited by tumors to evade immune surveillance. Modification of DGK activity offers new perspectives for the therapeutic manipulation of T cell functions for treatment of autoimmune pathologies, or for overcoming tumor-induced T cell tolerance. Precise knowledge of the mechanisms that sustain DGK isoform–specific regulation in T lymphocytes is indispensable for the development of new tools for pharmacological intervention.

Diacylglycerol kinase ζ: at the crossroads of lipid signaling and protein complex organization.

Diacylglycerol (DAG) and phosphatidic acid (PA) are lipids with unique functions as metabolic intermediates, basic membrane constituents, and second-signal components. Diacylglycerol kinases (DGK) regulate the levels of these two lipids, catalyzing the interconversion of one to the other. The DGK family of enzymes is composed of 10 isoforms, grouped into five subfamilies based on the presence of distinct regulatory domains. From its initial characterization as a type IV DGK to the generation of mouse models showing its importance in cardiac dysfunction and immune pathologies, diacylglycerol kinase ζ (DGKζ) has proved an excellent example of the critical role of lipid-metabolizing enzymes in the control of cell responses. Although the mechanism that regulates this enzyme is not well known, many studies demonstrate its subtle regulation and its strategic function in specific signaling and as part of adaptor protein complexes. These data suggest that DGKζ offers new opportunities for therapeutic manipulation of lipid metabolism.

Spanish human proteome project: dissection of chromosome 16

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

Discriminating Between the Paracellular and Transcellular Routes of Diapedesis

Leucocyte transendothelial migration (TEM) or diapedesis is pivotal in leucocyte trafficking during the inflammatory and immune responses. The endothelium plays an active role in this process, triggering an array of signalling pathways and reorganizing its cytoskeleton and membrane to facilitate leucocyte TEM. Diapedesis can occur between endothelial cells (paracellular) or through individual endothelial cells (transcellular). This latter route accounts for up to 30% of the total diapedesis in certain endothelial cell types in vitro. Mechanisms underlying both routes of diapedesis have been subjected to intense investigation during recent years. Here we describe a method to discriminate between the paracellular and the transcellular routes of diapedesis in vitro. The method is based on a transmigration assay of human T lymphoblasts through TNF-alpha-stimulated human primary endothelial monolayers, a triple fluorescence labelling of F-actin, the adhesion receptor ICAM-1 and the junctional protein beta-catenin and a subsequent acquisition of z-stacks of high-resolution confocal sections.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

Transient PKCα shuttling to the immunological synapse is governed by DGKζ and regulates L-selectin shedding

Summary Considerable evidence indicates that diacylglycerol (DAG) generation at the immunological synapse (IS) determines T cell functions by regulating the duration and amplitude of Ras/ERK signals. The exact mechanism by which DAG regulates Ras/ERK activation downstream of the T cell receptor (TCR) nonetheless remains poorly understood. Here we characterize PKC&agr; as a previously unrecognized component of the machinery that translates cell receptor occupancy into Ras/ERK-propagated signals. We show transient translocation of PKC&agr; to the IS, mediated by DAG generation at the contact area. Diacylglycerol kinase (DGK)&zgr; negatively regulated PKC&agr; translocation kinetics, whereas PKC&agr; activity limited its own persistence at the IS. Coordinated activation of DGK&zgr; and PKC&agr; in response to antigen recognition regulated the amplitude and duration of Ras/ERK activation; this in turn mediated early processes of T cell surface proteolysis such as L-selectin shedding. Analysis of DGK&zgr;-deficient mice further showed that increased DAG signaling is translated to downstream elements of this pathway, as reflected by enhanced PKC&agr;-dependent L-selectin shedding. We propose that early activation of a DAG–PKC&agr; axis contributes to the mechanisms by which antigen affinity translates into TCR biological responses.