Andrés Alonso

Loss of the VHR dual-specific phosphatase causes cell-cycle arrest and senescence.

Protein tyrosine phosphatases regulate important processes in eukaryotic cells and have critical functions in many human diseases including diabetes to cancer. Here, we report that the human Vaccinia H1-related (VHR) dual-specific protein tyrosine phosphatase regulates cell-cycle progression and is itself modulated during the cell cycle. Using RNA interference (RNAi), we demonstrate that cells lacking VHR arrest at the G1–S and G2–M transitions of the cell cycle and show the initial signs of senescence, such as flattening, spreading, appearance of autophagosomes, β-galactosidase staining and decreased telomerase activity. In agreement with this notion, cells lacking VHR were found to upregulate p21Cip–Waf1, whereas they downregulated the expression of genes for cell-cycle regulators, DNA replication, transcription and mRNA processing. Loss of VHR also caused a several-fold increase in serum-induced activation of its substrates, the mitogen-activated protein (MAP) kinases Jnk and Erk. VHR-induced cell-cycle arrest was dependent on this hyperactivation of Jnk and Erk, and was reversed by Jnk and Erk inhibition or knock-down. We conclude that VHR is required for cell-cycle progression as it modulates MAP kinase activation in a cell-cycle phase-dependent manner.

4-trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid, are potent inhibitors of nuclear factor κB activation

The effect of two derivatives of salicylate, 2‐hydroxy‐4‐trifluoromethylbenzoic acid (HTB) and 2‐acetoxy‐4‐trifluoromethylbenzoic acid (triflusal), on the activation of NF‐κB elicited by tumour necrosis factor‐α (TNF‐α) on human umbilical vein endothelial cells (HUVEC) was tested. The expression of the mRNA of vascular cell adhesion molecule‐1 (VCAM‐1) was studied as an example of a gene the expression of which is regulated by NF‐κB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF‐κB. The calculation of the IC50 values showed ∼amp;2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. Comparison of the potency of these compounds on VCAM‐1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36–43% inhibition at the same concentration. Inhibition of NF‐κB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose‐dependent inhibition of nitrite production by the L‐arginine pathway via iNOS. IC50 values for this effect were 1.13±0.12 mM (triflusal), 1.84±0.34 (HTB), 6.08±1.53 mM (aspirin) and 9.16±1.9 mM (salicylate). These data indicate that the incorporation of a 4‐trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF‐κB activation, VCAM‐1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo‐oxygenase.

Costimulation of dectin-1 and DC-SIGN triggers the arachidonic acid cascade in human monocyte-derived dendritic cells.

Inflammatory mediators derived from arachidonic acid (AA) alter the function of dendritic cells (DC), but data regarding their biosynthesis resulting from stimulation of opsonic and nonopsonic receptors are scarce. To address this issue, the production of eicosanoids by human monocyte-derived DC stimulated via receptors involved in Ag recognition was assessed. Activation of FcγR induced AA release, short-term, low-grade PG biosynthesis, and IL-10 production, whereas zymosan, which contains ligands of both the mannose receptor and the human β-glucan receptor dectin-1, induced a wider set of responses including cyclooxygenase 2 induction and biosynthesis of leukotriene C4 and IL-12p70. The cytosolic phospholipase A2 inhibitor pyrrolidine 1 completely inhibited AA release stimulated via all receptors, whereas the spleen tyrosine kinase (Syk) inhibitors piceatannol and R406 fully blocked AA release in response to immune complexes, but only partially blocked the effect of zymosan. Furthermore, anti-dectin-1 mAb partially inhibited the response to zymosan, and this inhibition was enhanced by mAb against DC-specific ICAM-3-grabbing nonintegrin (SIGN). Immunoprecipitation of DC lysates showed coimmunoprecipitation of DC-SIGN and dectin-1, which was confirmed using Myc-dectin-1 and DC-SIGN constructs in HEK293 cells. These data reveal a robust metabolism of AA in human DC stimulated through both opsonic and nonopsonic receptors. The FcγR route depends on the ITAM/Syk/cytosolic phospholipase A2 axis, whereas the response to zymosan involves the interaction with the C-type lectin receptors dectin-1 and DC-SIGN. These findings help explain the distinct functional properties of DC matured by immune complexes vs those matured by β-glucans.

KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases

Potassium channel tetramerization domain (KCTD) proteins contain a bric‐a‐brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage‐gated potassium channels. Some BTB‐domain‐containing proteins have been shown recently to participate as substrate‐specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty‐two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post‐transcriptionally in peripheral blood lymphocytes stimulated through the T‐cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N‐terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate‐specific adaptor for cullin3‐based E3 ligases.

Lipopolysaccharides of Brucella abortus and Brucella melitensis Induce Nitric Oxide Synthesis in Rat Peritoneal Macrophages

ABSTRACT Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucellain humans.

The extended human PTPome: a growing tyrosine phosphatase family.

Tyr phosphatases are, by definition, enzymes that dephosphorylate phospho‐Tyr (pTyr) from proteins. This activity is found in several structurally diverse protein families, including the protein Tyr phosphatase (PTP), arsenate reductase, rhodanese, haloacid dehalogenase (HAD) and His phosphatase (HP) families. Most of these families include members with substrate specificity for non‐pTyr substrates, such as phospho‐Ser/phospho‐Thr, phosphoinositides, phosphorylated carbohydrates, mRNAs, or inorganic moieties. A Cys is essential for catalysis in PTPs, rhodanese and arsenate reductase enzymes, whereas this work is performed by an Asp in HAD phosphatases and by a His in HPs, via a catalytic mechanism shared by all of the different families. The category that contains most Tyr phosphatases is the PTP family, which, although it received its name from this activity, includes Ser, Thr, inositide, carbohydrate and RNA phosphatases, as well as some inactive pseudophosphatase proteins. Here, we propose an extended collection of human Tyr phosphatases, which we call the extended human PTPome. The addition of new members (SACs, paladin, INPP4s, TMEM55s, SSU72, and acid phosphatases) to the currently categorized PTP group of enzymes means that the extended human PTPome contains up to 125 proteins, of which ~ 40 are selective for pTyr. We set criteria to ascribe proteins to the extended PTPome, and summarize the more important features of the new PTPome members in the context of their phosphatase activity and their relationship with human disease.

Coupling of C3bi to IgG inhibits the tyrosine phosphorylation signaling cascade downstream Syk and reduces cytokine induction in monocytes

The effect of coupling C3bi to immunoglobulin G (IgG) immune complexes (IC) on their ability to produce protein tyrosine phosphorylation and activation of the mitogen‐activated protein kinase (MAPK) and the Akt/protein kinase B (PKB) routes was assessedin human monocytes. Cross‐linking Fc receptors for IgG activated the protein tyrosine kinase Syk, phospholipases Cγ1 and Cγ2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the γ‐chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their β‐glucan content mimic the effect of fungi, produced a limited increase of tyrosine‐phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein‐1 (MCP‐1)/CC chemokine ligand 2 (CCL2), interleukin (IL)‐1β, and eotaxin‐2/CCL24, which were not observed with C3bi‐coated IC. Zymosan induced the expression of tumor necrosis factor α (TNF‐α), TNF‐β, IL‐10, IL‐6, and MCP‐2/CCL8, whereas the cytokine signature of C3bi‐coated zymosan also included interferon‐inducible protein 10/CXC chemokine ligand 10, platelet‐derived growth factor‐BB, and I‐309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.

Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

Platelet-activating factor : the effector of protein-rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis

1 The involvement of platelet‐activating factor (PAF) in immune complex‐induced/polymorphonuclear‐mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats. 2 Extravasation of protein‐rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed. 3 Treatment of rats with either UR‐12460 or BB‐823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein‐rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN. 4 Inhibition of NO production with both NG‐mono methyl‐l‐arginine and NG‐nitro‐l‐arginine failed to prevent protein‐rich plasma extravasation. 5 The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls. 6 Addition of 10 nm PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals. 7 Treatment with either UR‐12460 or BB‐823 prior to the induction of RPA produced an almost complete inhibition of NO production. 8 Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme. 9 Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR‐12460‐treated rats. 10 These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA, and also that PAF is implicated in the triggering of long‐term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS.

Proline-serine-threonine phosphatase interacting protein 1 inhibition of T-cell receptor signaling depends on its SH3 domain

Proline‐serine‐threonine phosphatase interacting protein 1 (PSTPIP1) is an adaptor protein associated with the cytoskeleton that is mainly expressed in hematopoietic cells. Mutations in PSTPIP1 cause the rare autoinflammatory disease called pyogenic arthritis, pyoderma gangrenosum, and acne. We carried out this study to further our knowledge on PSTPIP1 function in T cells, particularly in relation to the phosphatase lymphoid phosphatase (LYP), which is involved in several autoimmune diseases. LYP–PSTPIP1 binding occurs through the C‐terminal homology domain of LYP and the F‐BAR domain of PSTPIP1. PSTPIP1 inhibits T‐cell activation upon T‐cell receptor (TCR) and CD28 engagement, regardless of CD2 costimulation. This function of PSTPIP1 depends on the presence of an intact SH3 domain rather than on the F‐BAR domain, indicating that ligands of the F‐BAR domain, such as the PEST phosphatases LYP and PTP‐PEST, are not critical for its negative regulatory role in TCR signaling. Additionally, PSTPIP1 mutations that cause the pyogenic arthritis, pyoderma gangrenosum and acne syndrome do not affect PSTPIP1 function in T‐cell activation through the TCR.