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Biomedical subjects

Jan Tavernier

Publications and source records attributed to Jan Tavernier.

At least 19 recordsLinked to original sources
PubMed2007

The C-terminus of CIS defines its interaction pattern.

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein-protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.

Animals↗
PubMed2006

Nasal IL-5 levels determine the response to anti-IL-5 treatment in patients with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is characterized by an eosinophilic inflammation and high IL-5 levels. OBJECTIVES: Antagonizing the effect of IL-5 is a potential new treatment strategy in patients with nasal polyps. METHODS: In a double-blind, placebo-controlled, randomized, 2-center safety and pharmacokinetic study, 24 subjects with bilateral nasal polyps were randomized to receive a single intravenous infusion of reslizumab, a humanized anti-human IL-5 mAb, at 3 mg/kg or 1 mg/kg or placebo. We evaluated the safety and pharmacokinetics of reslizumab, and biologic activity was assessed by means of endoscopic evaluation of polyp size, symptoms, peripheral eosinophil counts, peripheral and local IL-5 levels, eotaxin levels, and eosinophil cationic protein levels. RESULTS: We demonstrated that a single injection of reslizumab up to 3 mg/kg is safe and well tolerated. Blood eosinophil numbers and concentrations of eosinophil cationic protein were reduced up to 8 weeks after treatment in serum and nasal secretions. Individual nasal polyp scores improved only in half of the treated patients for 4 weeks. Responders had increased IL-5 concentrations in nasal secretions at baseline compared with nonresponders, and logistic regression analysis revealed that increased nasal IL-5 levels (>40 pg/mL) predict the response to anti-IL-5 treatment. CONCLUSION: A single injection of anti-IL-5 reduces the size of nasal polyps for 4 weeks in half of the patients, and nasal IL-5 levels predict the response to anti-IL-5 treatment. CLINICAL IMPLICATIONS: Intravenous administration of a humanized anti-human IL-5 mAb is safe and reduces the size of nasal polyps in half of the patients.

Adolescent↗
PubMed2006

Functional cross-modulation between SOCS proteins can stimulate cytokine signaling.

SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct cross-modulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.

Animals↗
PubMed2006

A complex interaction pattern of CIS and SOCS2 with the leptin receptor.

Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.

Animals↗
PubMed2006

Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex.

The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.

Amino Acid Sequence↗
PubMed2006

Techniques: new pharmacological perspectives for the leptin receptor.

The function of leptin, initially confined to its role in energy homeostasis and obesity, has now expanded to the regulation of reproduction, glucose homeostasis, bone formation, wound healing and the immune system. Both stimulation and inhibition of the molecular target of leptin, the leptin receptor (LR), might find applications in disease treatment. Recent advances in the understanding of LR activation mechanisms have led to the design of LR antagonists. Several assays have been developed for the screening and evaluation of LR ligands. Both the extracellular and the intracellular domains of the LR are potential drug targets. The bioluminescence resonance energy transfer technique can be used to screen for compounds that target the extracellular part of the LR, and we propose that the novel reverse mammalian protein-protein interaction trap technique can be used to screen compounds that affect intracellular aspects of LR signalling. These assays can be easily adapted to other pharmacologically relevant receptors.

Animals↗
PubMed2006

MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells.

Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.

Blotting, Western↗
PubMed2006

Review: Negative regulation of leptin receptor signalling.

Leptin was discovered as an adipostat, regulating body weight by balancing food intake and energy expenditure. Recently, leptin emerged as a pleiotropic cytokine. It plays a substantial role in a wide spectrum of other functions including immune regulation, bone formation and fertility. Leptin signalling is under tight control. Aberrations of this stringent control system may be implicated in a variety of pathologies. Here, we review the various mechanisms that control cellular leptin receptor signalling.

Animals↗
PubMed2005

Mapping of the interface between leptin and the leptin receptor CRH2 domain.

Despite the impact of the leptin system on body weight and other physiologic processes, little is known about the binding of leptin to its receptor. The extracellular domain of the leptin receptor consists of two cytokine receptor homology (CRH) domains separated by an immunoglobulin-like domain, and followed by two juxtamembrane fibronectin type III modules. The CRH2 domain functions as a high-affinity binding site for leptin, and we previously demonstrated interaction with helices A and C of leptin. In this work, we constructed a homology model for the leptin/CRH2 complex and performed a detailed mutation analysis of the CRH2/leptin interface. Using both cell-based and in vitro binding assays using the isolated CRH2 domain, we show the critical role of hydrophobic interactions between Leu 13 and Leu 86 of leptin and Leu 504 in CRH2 in leptin binding and signalling. This binding pattern closely resembles the interaction of other four-helix bundle long chain cytokines with the CRH domain of their cognate receptors.

Amino Acid Sequence↗
PubMed2005

Leptin receptor activation depends on critical cysteine residues in its fibronectin type III subdomains.

The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.

Animals↗
PubMed2005

A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach.

Signaling via the erythropoietin receptor (EpoR) depends on the interaction of several proteins with phosphorylated tyrosine-containing motifs in its cytosolic domain. Detailed mapping of these interactions is required for an accurate insight into Epo signaling. We recently developed a mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based 2-hybrid method that operates in intact Hek293-T mammalian cells. As baits, we used intracellular segments of the EpoR containing 1 or 2 tyrosines. Several known signaling molecules, including cytokine-inducible SH2-containing protein (CIS), suppressor of cytokine signaling-2 (SOCS2), phosphatidylinositol 3'-kinase (PI3-K), phospholipase C-gamma (PLC-gamma), and signal transducer and activator of transcription 5 (STAT5) were used as prey. We also extended the MAPPIT method to enable interaction analysis with wild-type EpoR. In this relay MAPPIT approach, instead of using isolated EpoR fragments as bait, we used the full-length EpoR itself as a "receptor bait." Finally, we introduced MAPPIT in the erythroleukemic TF-1 cell line, which is a more natural setting of the EpoR. With these strategies several known interactions with the EpoR were analyzed and evidence for new interactions was obtained.

Amino Acid Motifs↗
PubMed2005

Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53.

Humans↗
PubMed2005

Immunostimulatory potential of hepatitis B nucleocapsid preparations: lipopolysaccharide contamination should not be overlooked.

The nucleocapsid of hepatitis B virus (HBV) allows insertions of heterologous peptides and even complete proteins. Because of its outstanding capacity to induce B-cell, T-helper and cytotoxic T-cell responses, this structure is considered to be an important instrument for future vaccine development. Most of the evidence for the unique immunogenic qualities of nucleocapsids has been generated in mice, which are not natural hosts of HBV. Moreover, most nucleocapsid preparations used in these studies were produced in a recombinant manner in Escherichia coli. Such preparations have been shown to contain lipopolysaccharide (LPS). Not unexpectedly, it is shown here that contaminating LPS, rather than the nucleocapsid structure itself, is responsible for the activation of human antigen-presenting cells. Careful examination of the literature dealing with the immunogenicity of HBV nucleocapsids suggests that the possible presence of LPS has been largely ignored or underestimated in several studies. This raises doubts on some of the underlying mechanisms that have been proposed to explain the unique immunogenicity of the HBV nucleocapsid.

Cells, Cultured↗
PubMed2005

Pleiotropy of leptin receptor signalling is defined by distinct roles of the intracellular tyrosines.

The leptin receptor (LEPR) is a class I cytokine receptor signalling via both the janus kinase/signal transducer and activator of transcription (JAK/STAT) and the MAP kinase pathways. In addition, leptin has been shown previously to activate AMP-activated kinase (AMPK) in skeletal muscle. To enable a detailed analysis of leptin signalling in pancreatic beta cells, LEPR point mutants with single or combined exchanges of the three intracellular tyrosines were expressed in HIT-T15 insulinoma cells. Western blots with activation state-specific antibodies recognizing specific signalling molecules revealed that the wild-type receptor activated STAT1, STAT3, STAT5 and ERK1/2 but failed to alter the phosphorylation of AMPK. Each of the three intracellular tyrosine residues in LEPR exhibited different signalling capacities: Tyr985 was necessary and sufficient for leptin-induced activation of ERK1/2; Tyr1077 induced tyrosyl phosphorylation of STAT5; and Tyr1138 was capable of activating STAT1, STAT3 and STAT5. Consistent results were obtained in reporter gene assays with STAT3 or STAT5-responsive promoter constructs, respectively. Furthermore, the sequence motifs surrounding the three tyrosine residues are conserved in LEPR from mammals, birds and in a LEPR-like cytokine receptor from pufferfish. Mutational analysis of the box3 motif around Tyr1138 identified Met1139 and Gln1141 as important determinants that define specificity towards the different STAT factors. These data indicate that all three conserved tyrosines are involved in LEPR function and define the pleiotropy of signal transduction via STAT1/3, STAT5 or ERK kinases. Activation and inhibition of AMPK appears to require additional components of the signalling pathways that are not present in beta cells.

Amino Acid Sequence↗
PubMed2004

Soluble interleukin-5 receptor alpha is increased in acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: During chronic obstructive pulmonary disease (COPD) exacerbations (AE-COPD), an influx of eosinophils into the bronchial mucosa has been described. Eosinophilic cationic protein (ECP) and soluble interleukin-5 receptor alpha (sIL5Ralpha) are secreted by eosinophils and increased in eosinophilic airway diseases. METHODS: We studied ECP and sIL5Ralpha expression in patients with COPD compared to healthy controls and smokers and investigated a possible association to viral exacerbations of COPD. Expression of sIL5Ralpha in serum was analyzed by ELISA and ECP by the Uni-Cap system. Induced sputum from patients with COPD was analyzed for six different respiratory viruses by nested PCR. RESULTS: ECP and sIL5Ralpha were significantly elevated in AE-COPD subjects (n = 54) compared to healthy controls (n = 11, p = 0.018). Furthermore, there was a significant increase in sIL5Ralpha, but not in ECP, in 30 patients with virus-associated AE-COPD compared to smokers without COPD (n = 16) and healthy controls. The increase in FEV(1) after resolution of the AE-COPD correlated with the decrease in sIL5Ralpha (r = 0.269, p = 0.034). CONCLUSIONS: sIL5Ralpha is increased in AE-COPD and not affected by smoking like ECP. sIL5Ralpha is increased in patients with virus-associated AE-COPD compared to smokers and controls. Concentrations of sIL5Ralpha mirror changes in the clinical status and lung function. These data support the involvement of eosinophils in acute exacerbations of COPD.

Blood Proteins↗
PubMed2004

Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

ATP Binding Cassette Transporter 1↗
PubMed2004

Mapping of the leptin binding sites and design of a leptin antagonist.

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

Amino Acid Sequence↗