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Hypothalamic leptin receptor (LR) signaling regulates body weight by balancing food intake and energy expenditure. It is well established that the human LR undergoes ectodomain shedding, but little is known about the fate of the remaining cytosolic domain. This study demonstrates that regulated intramembrane proteolysis (RIP) releases the LR intracellular domain (LR ICD), which translocates to the mitochondria where it binds to SOCS6. This LR ICD-SOCS6 interaction stabilizes both proteins on the mitochondrial outer membrane and requires a functional BC box in SOCS6 for mitochondrial association and a central motif in the LR ICD for SOCS6 binding. The LR ICD prevents CCCP-induced mitochondrial depolarization and mitophagy as shown by lowered Parkin translocation and p62 accumulation. Strict regulation of mitochondrial dynamics in the hypothalamus is known to be essential for body weight homeostasis. This is the first study showing that the LR can directly modulate mitochondrial biology. [ABSTRACT FROM AUTHOR]

In this short review, we summarize how insights into the structure and dynamics of interferon-receptor complex assembly and activation guided the design of a novel class of engineered type I interferons that combine a largely lost potency on non-targeted cells with high activity on targeted cells. These novel interferons are expected to exhibit lower systemic toxicities compared to other interferon therapy modalities and could open avenues to revive these cytokines for the treatment of patients suffering of cancer and viral infections. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor (TLR) signalling is initiated by the recruitment of one or more adaptor proteins to the activated receptor complex. At present, four of these proteins are identified, namely MyD88, Mal, Trif and Tram and their selective usage by different TLRs in part accounts for TLR-specific transcriptional responses. Recent findings described unique biochemical properties for each of these TIR-domain containing adaptors and revealed that these adapters are subjected to post-translational modification. We used mammalian protein–protein interaction trap (MAPPIT), a two-hybrid technique that functions in a mammalian cell context, to study the molecular interactions downstream of TLR activation. We demonstrate pathway walking from TLR4 to IRAK-1 and identified Mal as a bridging adaptor, linking MyD88 to the activated TLR4. [Copyright &y& Elsevier]

After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential. [ABSTRACT FROM AUTHOR]

Abstract: Besides the transcription-promoting role of histone acetyltransferases (HATs) and the transcription-delimiting function of histone deacetylases (HDACs) through histone acetylation and deacetylation respectively, HATs and HDACs also regulate the activity of several non-histone proteins. This includes signal transducers and activators of transcription (STATs), key proteins in cytokine signaling. Unlike Tyr phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by HATs and HDACs appears multifaceted and far more complex than initially imagined. Our review focuses on the latest trends and novel hypotheses to explain differential context-dependent STAT regulation by complex posttranslational modification patterns. We chart the knowledge on how STATs interact with HATs and HDACs, and additionally bring a transcriptional regulatory and gene-set specific role for HDACs in the picture. Indeed, a growing amount of evidence demonstrates, paradoxically, that not only HAT but also HDAC activity can be required for STAT-dependent transcription, in a STAT subtype- and cell type-dependent manner. Referring to recent reports, we review and discuss the various molecular mechanisms that have recently been proposed to account for this peculiar regulation, in an attempt to shed more light on the difficult yet important question on how STAT specificity is being generated. [Copyright &y& Elsevier]

Abstract: Many processes in a cell depend on protein–protein interactions (PPIs) and perturbations of these interactions can lead to diseases. Comprehensive knowledge of PPI networks will not only give us information on how the cell is organized, but will also provide new drug targets. Current binary PPI networks are mainly generated by high-throughput yeast two-hybrid. Due to the small overlap of these maps, it has long been assumed that these maps are of low quality containing many false positives. However, by using an orthogonal two-hybrid method, MAPPIT (mammalian protein–protein interaction trap), these maps were shown to be of high quality suggesting that the limited overlap is likely due to low sensitivity and not to low specificity. [Copyright &y& Elsevier]

A diverse series of mammalian two-hybrid technologies for the detection of protein–protein interactions have emerged in the past few years, complementing the established yeast two-hybrid approach. Given the mammalian background in which they operate, these assays open new avenues to study the dynamics of mammalian protein interaction networks, i.e. the temporal, spatial and functional modulation of protein–protein associations. In addition, novel assay formats are available that enable high-throughput mammalian two-hybrid applications, facilitating their use in large-scale interactome mapping projects. Finally, as they can be applied in drug discovery and development programs, these techniques also offer exciting new opportunities for biomedical research. [Copyright &y& Elsevier]

Activation of STAT proteins by cytokines is initiated by their Src homology 2 domain-mediated association with phosphotyrosine residues from the cytoplasmic domain of a receptor. Here, we show that the C terminus of the interleukin-22 receptor (IL-22R) recruits in a tyrosine-independent manner the coiled-coil domain of STAT3. Mutation of all IL-22R cytoplasmic tyrosines did not abolish activation of STAT3, in contrast to that of STAT1 and STAT5. Coimmunoprecipitation and glutathione S-transferase pulldown experiments showed that the coiled-coil domain of STAT3 is constitutively associated with the C-terminal part of IL-22R, and a chimeric STAT3-STAT5 protein containing the coiled-coil domain of STAT3 could be activated by this tyrosine-independent mechanism. Deletion of the C-terminal part of IL-22R dramatically decreased its ability to activate STAT3 and to mediate IL-22 activity in cell lines, demonstrating that preassociation of STAT3 with this cytokine receptor, independent from the interaction between the Src homology 2 domain and phosphotyrosines, is required for its full activity. [ABSTRACT FROM AUTHOR]

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown ofUBDin human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine- induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1 in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells. [ABSTRACT FROM AUTHOR]

Summary Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC′-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains. [ABSTRACT FROM AUTHOR]

The timely orchestration of multiple signalling pathways is crucial for the integrity of an organism and therefore tightly controlled. Gab family proteins coordinate signal transduction at the plasma membrane (PM) by acting as docking platforms for signalling components involved in MAP kinase (MAPK), PI3 kinase (PI3K), phospholipase C (PLC) and Rho family GTPase signalling. The interaction with these components as well as the targeting of the docking platform to the PM underlies complex spatial and temporal regulatory mechanisms. Deregulated Gab1 activation and membrane binding have been observed in some haematopoietic malignancies and solid tumours, thereby contributing, for example, to the development of Philadelphia chromosome-negative myeloproliferative neoplasms and certain lung cancers. Previously, we could demonstrate that the presence of PIP3 in the PM, which is increased in many cancer cells, is not sufficient for constitutive Gab1 membrane recruitment. In addition, MAPK-dependent phosphorylation of Gab1 at serine 552 (Ser552) is vital for Gab1 membrane binding. Here, we confirm our hypothesis that in the absence of MAPK activity an intrinsic part of Gab1 prevents binding to PIP3 at the PM. This epitope of Gab1, which encompasses Ser552, interacts directly with the Gab1 PH domain. Two arginines located in positions + 4 and + 8 of Ser552 are essential for the interaction with the PH domain, as well as for the inhibition of membrane recruitment of unphosphorylated Gab1. Ser552 phosphorylation is dispensable in respective arginine to alanine mutants of Gab1. Gab1 recruitment to the PM is highly dynamic and continuous PI3K and MAPK activities are both essential for sustained Gab1 membrane localisation. Our data document the existence of a sophisticated and robust control mechanism that prevents Gab1 translocation and signalling complex assembly after the activation of either MAPK or PI3K alone. [ABSTRACT FROM AUTHOR]

Background: Cytokine-induced IRE1α activation regulates adaptive/pro-apoptotic endoplasmic reticulum (ER) stress transition in type 1 diabetes (T1D). Results: The IRE1α-binding protein NMI modulates JNK activation and apoptosis in beta cells. Conclusion: NMI induction is a novel negative feedback mechanism regulating ER stress and apoptosis in cytokine-exposed beta cells. Significance: Clarifying cytokine modulation of ER stress may provide information to protect beta cells in early T1D. [ABSTRACT FROM AUTHOR]

Summary: Leptin activates its cognate receptor (LR) to regulate body weight and metabolically costly processes, such as reproduction and immune responses. Despite such benevolent pleiotropy, leptin-mediated signaling has been implicated in autoimmune diseases and breast cancer, thereby rejuvenating interest in leptin antagonism. We present comparative biochemical and structural studies of the LR ectodomain (LRecto) in complex with wild-type and antagonist leptin variants. We show that high-affinity binding of leptin to the cytokine receptor homology 2 domain of LRecto primes interactions with the Ig-domain (LRIg) of another leptin-bound LRecto to establish a quaternary assembly. In contrast, antagonist leptin variants carrying mutations at the LRIg binding site only enable binary complexes with LRecto. Acetylation of free cysteines in LRecto also abrogates quaternary complexes, suggesting a functional role for intrareceptor disulfides. We propose a revised conceptual framework for LR activation whereby leptin activates predimerized LR at the cell surface to seed higher order complexes with 4:4 stoichiometry. [Copyright &y& Elsevier]

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-KB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal-/- mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-de-pendent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/ Trif-dependent TLR4 signaling. [ABSTRACT FROM AUTHOR]

Background & Aims: Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. Methods: The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. Results: The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. Conclusions: Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells. [Copyright &y& Elsevier]

Abstract: The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow. [Copyright &y& Elsevier]

Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex. [ABSTRACT FROM AUTHOR]

Abstract: Leptin, a 16-kDa protein that is mainly secreted by adipocytes, plays a protective role in many cell types. It has been shown that leptin acts in the central and peripheral immune system to protect thymocytes. Cytosolic phospholipase A2 (cPLA2) is an enzyme that can specifically initiate the release of arachidonic acid (AA) to produce eicosanoids, which regulate inflammation and immune responses. Our previous work has shown that leptin is important to prevent apoptosis of thymocytes. However, the role of cPLA2 is still unclear, and the precise mechanism also remains to be elucidated. In this work, we demonstrated that leptin inhibited the LPS-induced toxicity and apoptosis of thymocytes. Western blot and RT-PCR showed that leptin led to a reduction of cPLA2 activity and mRNA level, as well as caspase-3 cleavage. Moreover, we found that leptin could decrease the activation of p38 MAPK. Accordingly, we pre-treated apoptotic thymocytes with the p38 MAPK inhibitor, SB203580 and observed an effect similar to the leptin alone treated group. SB203580 also suppressed expression of cPLA2 and cleavage of caspase-3. Based on these results, we suggest that leptin could attenuate LPS-induced apoptotic injury in mouse thymocyte cells, mainly through the p38/cPLA2 signalling pathway. The study of the regulatory role of leptin in LPS-induced thymocyte apoptosis can help to explain the role of leptin in the immune system and may provide a novel treatment option in cases of severe trauma, infection, shock, organ failure and autoimmune disease caused by thymic atrophy. [Copyright &y& Elsevier]

Abstract: In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON® expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. [Copyright &y& Elsevier]

Toll-like receptor signaling requires interactions of the Toll/ IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4- TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization. [ABSTRACT FROM AUTHOR]

Abstract: Enteral administration of lipid-enriched nutrition effectively attenuates inflammation via a cholecystokinin (CCK)-mediated vagovagal anti-inflammatory reflex. Cholecystokinin release and subsequent activation of the vagus are dependent on chylomicron formation and associated with release of additional gut peptides. The current study investigates the intestinal processes underlying activation of the CCK-mediated vagal anti-inflammatory pathway by lipid-enriched nutrition. Rats and mice were subjected to hemorrhagic shock (HS) or endotoxemia, respectively. Prior to the experimental procedures, animals were fasted or fed lipid-enriched nutrition. Pluronic L-81 (L-81) was added to the feeding to investigate involvement of chylomicron formation in activation of mesenteric afferent fibers and the immune-modulating potential of lipid-enriched nutrition. Ob/Ob mice and selective receptor antagonists were used to study the role of leptin, glucagon-like peptide 1 and peptide YY in activation of the nutritional reflex. Electrophysiological analysis of mesenteric afferents in mice revealed that lipid-enriched nutrition-mediated neural activation was abrogated by L-81 (P<.05). L-81 blunted the beneficial effects of lipid-enriched nutrition on systemic inflammation and intestinal integrity in both species (all parameters, P<.01). Ob/Ob mice required a higher dose of nutrition compared with wild-type mice to attenuate plasma levels of TNF-α and ileum-lipid binding protein, a marker for enterocyte damage (both P<.01), suggesting a higher stimulation threshold in leptin-deficient mice. Administration of a glucagon-like peptide 1-receptor antagonist, but not leptin or peptide YY antagonists, suppressed the effects of lipid-enriched nutrition. These data indicate that chylomicron formation is essential and activation of the glucagon-like peptide 1-receptor is involved in activation of the nutritional anti-inflammatory pathway by lipid-enriched nutrition. [Copyright &y& Elsevier]

Abstract: MAPPIT (mammalian protein–protein interaction trap) is a two-hybrid interaction mapping technique based on functional complementation of a type I cytokine receptor signaling pathway. Over the last decade, the technology has been extended into a platform of complementary assays for the detection of interactions among proteins and between chemical compounds and proteins, and for the identification of small molecules that interfere with protein–protein interactions. Additionally, several screening approaches have been developed to broaden the utility of the platform. In this review we provide an overview of the different components of the MAPPIT toolbox and highlight a number of applications in interactomics, drug screening and compound target profiling. [Copyright &y& Elsevier]

Abstract: The high mutation rate of Human Immunodeficiency Virus (HIV) leads to the rapid derivation of compound-resistant virus strains and thus necessitates the identification and development of compounds with alternative mode of actions. MAPPIT (MAmmalian Protein–Protein Interaction Trap) is a highly efficient tool to study protein–protein interactions in intact human cells and is applied to study the dimerization process of the HIV reverse transcriptase complex. Highly specific signals for the p66/p51 and p66/p66 interactions could readily be detected. Specificity was established further by introducing mutations in either subunit. Treatment with efavirenz resulted in an increased MAPPIT signal, with an EC50 value of 64nM for the p66/p51 interaction, and allowed detection of the p51/p51 homodimerization, confirming the context-dependent asymmetric contribution of both subunits. These results show that MAPPIT can be used as a novel screening tool for anti-HIV compounds in intact human cells. [Copyright &y& Elsevier]

Abstract: The suppressors of cytokine signalling (SOCS) box is a structural domain found at the C-terminus of over 70 human proteins. It is usually coupled to a protein interaction module such as an SH2 domain in case of SOCS proteins, a family of modulators of cytokine signaling. The SOCS box participates in the formation of E3 ligase complexes, marking activated cytokine receptor complexes for proteasomal degradation. A similar mechanism was recently uncovered for controlling SOCS activity itself, since SOCS2 was found to enhance the turnover of other SOCS proteins. The SOCS box can also add unique features to individual SOCS proteins: it can function as an adaptor domain as was demonstrated for SOCS3, or as a modulator of substrate binding in case of CIS. In this review we discuss these multiple roles of the SOCS box, which emerges as a versatile module controlling cytokine signaling via multiple mechanisms. [Copyright &y& Elsevier]

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-sheet formed by β-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 Ieptin·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 α-receptor, and forms the center of binding site 1. The 2:4 hexameric leptin·LR complex offers a rational explanation for mutagenesis studies and residue conservation. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 receptot 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. [ABSTRACT FROM AUTHOR]

In a previous characterization of the ABCA subfamily (of the ATP-binding cassette (ABC) transporters, we 1 identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters (Peelman, F., Labeur, C., Vanloo, B., Roosbeek, S., Devaud, C., Duverger, N., Denefle, P., Rosier, M., Vandekerckhove, J., and Rosseneu, M. (2003) J. Mol. BioL 325, 259–274). 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. [ABSTRACT FROM AUTHOR]

We identified putative protein kinase CK2 phosphorylation sites in the cytoplasmic R1 and R2 domains, downstream of nucleotide binding domains (NBD) of the ABCA1 transporter, and tested their functional significance. In vitro, the recombinant ABCA1 NBD1+R1 domain, and not the NBD2+R2 domain, was phosphorylated by CK2. We identified T1242, T1243 and S1255 as the phosphorylated residues in the R1 domain. In HEK-293 cells, ABCA1 flippase activity, apolipoproteins AI and AII binding to the cell, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine residues. Mutants mimicking threonine phosphorylation had activities close to that of WT ABCA1. Our data therefore suggest that protein kinase CK2 might regulate the transport activity of ABCA1 in vivo. [Copyright &y& Elsevier]

Background Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Methods Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Results Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4–5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Conclusion Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin. [ABSTRACT FROM AUTHOR]