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The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1–Ccz1–Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy–Inturned–Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy–Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation.

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) and AlphaFold2 modelling to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα (domains 1-2) with GP130 (domains 1-3) to initiate signalling. While both receptors contact the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα and Domain 1 of GP130. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. For this purpose, we here developed a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. We engineered two monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies (NB) for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enabled us to quantitatively resolve the stoichiometry and dynamics of the interferon-g (IFNg) receptor signaling complex in the plasma membrane of living cells by multicolor single molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we unambiguously identify ligand-induced receptor homo-and heterodimerization. Detailed analysis by multicolor single-molecule co-tracking and quantitative single-molecule FRET revealed transient assembly of IFNγ-receptor heterotetramers in the plasma membrane with a structural architecture as observed for the isolated ectodomains in vitro.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with Systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

Summary Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses., Graphical Abstract, Highlights • CDK8 regulates IL-6-mediated STAT3 S727 phosphorylation in primary human T cells • CDK8 controls STAT3 activity by limiting its resident time at gene loci • CDK8 inhibition increases IL-6-mediated Th17 differentiation, How IL-6 elicits its immune pleiotropic activities is not fully understood. Martinez-Fabregas et al. show that CDK8 represses IL-6-mediated transcription by limiting STAT3 resident time at the gene loci. By regulating CDK8 expression levels, immune cells can adapt their responses to STAT3-activating cytokines.

Significance TPO is a cytokine that signals through the receptor MPL (or TPO-R), and is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). TPO signaling is deregulated in essential thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R ECD as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial agonism, and that show decoupling of the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET., Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect “tuning” downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL)) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable, both experimentally and clinically. We have developed a series of surrogate protein-ligands for TPO-R, in the form of diabodies, that homodimerize the TPO-R on the cell surface in different geometries, in effect ‘tuning’ downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism and antagonism, thus decoupling the dual functions of TPO/TPO-R. Using scRNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in Essential Thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.Significance StatementThe TPO cytokine, which signals through its receptor c-MPL (or TPO-R), is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). Its signaling is deregulated in Essential Thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial to antagonism, thus decoupling the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET.

Interleukin-10 (IL-10) is a dimeric cytokine with both immunosuppressive and immunostimulatory activities; however, IL-10-based therapies have shown only marginal clinical benefits. Here, we explored whether the stability of the IL-10-receptor complex contributes to the immunomodulatory potency of IL-10. We generated an IL-10 mutant with enhanced affinity for its IL-10Rβ receptor using yeast surface display. Compared to the wild-type cytokine, the affinity-enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signalling complexes and triggered greater STAT1 and STAT3 activation in human monocytes and CD8(+) T cells. These effects in turn led to more robust induction of IL-10-mediated gene expression programs at low ligand concentrations in both human cell subsets. IL-10-regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and in CD8(+) T cell exhaustion. At non-saturating doses, IL-10 did not induce key components of its gene expression program, which may explain its lack of efficacy in clinical settings. Our engineered IL-10 variant showed a more robust bioactivity profile than that of wild-type IL-10 at low doses in monocytes and CD8(+) T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with wild-type IL-10. Our study provides insights into how IL-10-receptor complex stability fine-tunes IL-10 biology and opens new opportunities to revitalise failed IL-10 therapies.

Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10Rβ receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.

Activated by interaction Cytokines trigger immune responses when they bind to their cognate receptors. Class I cytokine receptors rely on the associated Janus kinase 2 (JAK2) to initiate signal transduction. There has been debate over whether activation involves ligand-induced dimerization of these receptors or ligand-induced conformational change of preformed dimers. Wilmes et al. imaged cytokine receptors in the plasma membranes of live human cells by single-molecule fluorescence microscopy and observed ligand-induced dimerization. They found that the JAK2 pseudokinase domains contribute to dimerization and that hyperactive JAK2 mutants promote dimerization, consistent with the model that dimerization triggers activation. Science , this issue p. 643

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

International audience; Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

Cytokines activate downstream signaling networks via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered variants of IL-6 with different affinities to the gp130 receptor chain to investigate how cytokine receptor binding kinetics influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes, from minimal to full agonist, and induced biased signaling, with changes in receptor binding kinetics affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This, in turn, leads to a graded gene expression response and substantial differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Cytokines comprise a large family of secreted ligands that are critical for the regulation of immune homeostasis. Cytokines initiate signaling via dimerization or oligomerization of the cognate receptor subunits, triggering the activation of the Janus Kinases (JAKs)/ signal transducer and activator of transcription (STATs) pathway and the induction of specific gene expression programs and bioactivities. Deregulation of cytokines or their downstream signaling pathways are at the root of many human disorders including autoimmunity and cancer. Identifying and understanding the mechanistic principles that govern cytokine signaling will, therefore, be highly important in order to harness the therapeutic potential of cytokines. In this review, we will analyze how biophysical (ligand-receptor binding geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters shape the cytokine signalosome and cytokine functional pleiotropy; from the initial cytokine binding to its receptor to the degradation of the cytokine receptor complex in the proteasome and/or lysosome. We will also discuss how combining advanced protein engineering with detailed signaling and functional studies has opened promising avenues to tackle complex questions in the cytokine signaling field.

Janus kinase (JAK2)V617F is the most common mutation found in patients with Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPNs). The discovery of this mutation over 15 years ago revolutionised MPN diagnosis and inspired the development of JAK inhibitors as new therapeutic interventions. However, despite extensive structural and biophysical studies using JAK2 domains in isolation, the exact molecular mechanisms of JAK2V617F activation remains elusive. We have previously demonstrated that expression of the thrombopoietin (TPO) receptor, MPL, which interacts directly with JAK2, is essential for disease development in a mouse model of a JAK2V617F-positiveMPN (Blood 2014 124:3956-3963). Using total internal reflection fluorescence (TIRF) microscopy, we visualized MPL interaction dynamics in live cells on single molecule level. Effective cell surface MPL fluorescence labelling and dual-color imaging allowed us to determine the level of MPL dimerization under various experimental conditions. Using this assay, we clearly established that MPL is monomeric at physiologically relevant receptor densities. However, TPO stimulation results in significant dimerization of MPL (>50%) and an equilibrium between monomers and dimers. This counters the current dogma that MPL exists at the membrane as a pre-formed dimer. Strikingly, we found that JAK2V617F shifts this monomer-dimer equilibrium leading to significant TPO-independent MPL dimerization providing a novel mechanistic model of oncogenic JAK2 activation. To highlight the role of ligand-independent receptor dimerization in JAK2 activation, we compared three groups of autoactivating mutations in the PK domain covering the FERM-SH2 (FS2)-PK linker region (Group I), residues in the proximity of the αC helix (Group II) and at the autoinhibitory PK-TK interface (Group III). Consistent MPL dimerization was only observed for mutations in groups I and II. Mutations in these groups both localize to a potential homomeric PK/PK interface that has been implicated as a switch of JAK activation. Using MD simulations, we also found that the FERM domain of JAK2 strongly interacts with the inner leaflet of the lipid bilayer of the plasma membrane via a single hydrophobic residue (L224) surrounded by several positively charged residues that allows the region to act as a membrane anchor. This tight coupling to the membrane enforces an appropriate orientation between the JAKs within the receptor dimers required for optimal intermolecular PK/PK interaction that is critical for receptor dimerization. To interfere with membrane anchoring, we introduced a negative charge in this position (L224E). Strikingly, ligand-independent MPL dimerization and activation by JAK2V617F was dramatically reduced upon introducing L224E, supporting the vital importance of L224 for orienting JAK2 at the membrane to allow productive PK-PK interactions. Here, we demonstrate that JAK2V617F mutation acts by altering and strengthening the intermolecular interactions involving the PK/PK dimerization interface. In essence, these mutations drive cytoplasmic stabilization of receptor-JAK dimers, bypassing extracellular stabilization of dimers via cytokine binding. These results provide critical and entirely novel mechanistic insights into signal initiation in MPNs and readdress the roles of receptor-associated proteins. Disclosures Hubbard: Ajax Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder.

Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to “tune” signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

International audience; Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Single-molecule tracking and spatial stochastic modeling reveal receptor dimer stabilization by nanoscale confinement zones., The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription., Endocytosis of the type I interferon receptor (IFNAR) is required for JAK/STAT signalling. Here the authors show that the internalized IFNAR1 and IFNAR2 subunits are differentially sorted by the retromer complex at the early endosome and this controls JAK/STAT signalling and gene transcription.

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.

Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

Tracking the motion of individual proteins on the surface of live cells has contributed considerably towards unveiling the functional organization of proteins in the plasma membrane. Individual proteins labeled with quantum dots (QDs) can be imaged over long time periods with ultrahigh spatial and temporal resolution, yielding powerful information on the spatiotemporal dynamics of proteins at the plasma membrane in live cells. A key challenge for the application of QDs is to site-specifically attach proteins to the surface of these nanoparticles in a stoichiometric manner without affecting protein function. Several procedures for rendering surfaces of QDs biocompatible have been described, thus reducing non-specific binding and protein denaturation on the QD surface. However, functionalized biocompatible QDs used to target cell surface proteins generally result in multipoint attachment to the target proteins because the number of functional groups on the QDs is very difficult to control. Such multiply functionalized QDs induce clustering of target proteins on the cell surface, biasing not only lateral diffusion but also the functional properties of these proteins. As a consequence, increased endocytosis has been observed upon binding of QDs functionalized with multiple epidermal grow factor (EGF) molecules to cell-surface EGF receptors. Stochastic functionalization of multiple reactive sites on the QD offers the choice of obtaining only a minor fraction of the QDs with a single functional group, or a significant fraction of QDs with multiple functional groups. Preparation of homogeneous, monofunctional QDs currently relies on electrophoretic purification, which has been achieved only for very small QDs. These compact QDs are designed with very thin surface coatings, which have the disadvantage of showing relatively strong non-specific interactions. Most approaches for targeting proteins using QDs in live cells are based on biotin–streptavidin interactions, which form quasi-irreversible complexes. For multiplexed, generic labeling of proteins on the cell surface, further targeting strategies are required. We have recently described tris(hydroxymethyl)methylamine–nitrilotriacetic acid (Tris-NTA) moieties for highly specific and stable attachment of fluorophores and other functional units to histidine-tagged proteins in vitro and on the surface of live cells. The lifetime of Tris-NTA complexes with His-tagged proteins is in the order of several hours, which is well-suited to medium-term single molecule tracking applications. Herein, we have attempted to control the functionalization degree of QDs with Tris-NTA by means of electrostatic repulsion. We devised a bottom-up coupling chemistry based on a novel Tris-NTA derivative (1; Figure 1a), which comprises a thiol-terminated hepta(ethylene glycol) linker. This compound was generated in situ by reduction of the disulfide-linked dimer (1a ; see the Supporting Information, Scheme S1) and coupled to commercially available polymer-coated and amine-functionalized QDs by means of a hetero-bifunctional cross-linker (Figure 1b). Covalently attachment of 1 to surfaces modified with maleimide-functionalized polyethylene glycol (PEG) polymer brush and specific immobilization of His-tagged proteins was confirmed by label-free detection (Supporting Information, Figure S1). To control the degree of functionalization with Tris-NTA on the QD surface, the reaction of 1 with surface maleimide groups was performed at low ionic strength. Under these conditions, all QDs were reacted with Tris-NTA, as confirmed by an increase in negative charges detected by anion exchange chromatography and agarose gel electrophoreses (Figure 1c,d). These assays indicated relatively monodisperse electrostatic properties after coupling of 1, despite the fact that it was reacted at a large excess (660 mm of compound 1 to 1 mm QD). Coupling of 1 at higher ionic strength yielded QDs with a substantially higher degree of functionalization, as confirmed by a further shift of the signals both in anion exchange chromatography and agarose gel electrophoresis (Figure 1c,d). To characterize the functional properties of Tris-NTAcoupled QDs, binding to immobilized hexahistidine (H6) [*] Dr. C. You, S. Wilmes, O. Beutel, S. L chte, Y. Podoplelowa, F. Roder, C. Richter, T. Seine, D. Schaible, Prof. Dr. J. Piehler Division of Biophysics, Universit t Osnabr ck Barbarstrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-969-2262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

Quantitative single-molecule receptor dimerization assays show dimerization of IFNAR1 and IFNAR2 upon IFN treatment, and reveal the limiting role of IFNAR1 binding affinity in complex assembly and the regulatory role of USP18., Type I interferons (IFNs) activate differential cellular responses through a shared cell surface receptor composed of the two subunits, IFNAR1 and IFNAR2. We propose here a mechanistic model for how IFN receptor plasticity is regulated on the level of receptor dimerization. Quantitative single-molecule imaging of receptor assembly in the plasma membrane of living cells clearly identified IFN-induced dimerization of IFNAR1 and IFNAR2. The negative feedback regulator ubiquitin-specific protease 18 (USP18) potently interferes with the recruitment of IFNAR1 into the ternary complex, probably by impeding complex stabilization related to the associated Janus kinases. Thus, the responsiveness to IFNα2 is potently down-regulated after the first wave of gene induction, while IFNβ, due to its ∼100-fold higher binding affinity, is still able to efficiently recruit IFNAR1. Consistent with functional data, this novel regulatory mechanism at the level of receptor assembly explains how signaling by IFNβ is maintained over longer times compared with IFNα2 as a temporally encoded cause of functional receptor plasticity.

Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions in vitro. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization.

Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.

International audience; Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-bytargeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient's whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of >120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. Disclosures No relevant conflicts of interest to declare.

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine–receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.

Type I Interferons (IFN) are key cytokines in the regulation of the innate immune response. All IFNs bind to a shared cell surface receptor comprised of two subunits, IFNAR1 and IFNAR2. Together with IFN the receptors assemble to hetero-trimeric “ternary” complexes which initiate phosphorylation cascades through the intracellularly associated Janus-Kinases Jak1 and Tyk2. Detailed structure–function analysis of IFNs has established that the dynamics and affinities of IFN interactions with the receptor subunits play a critical role for regulating signalling specificities. Here we have explored the IFN-induced assembly of the ternary signalling complex at physiological expression level by dual colour single molecule imaging. To this end, we employed highly specific and efficient orthogonal posttranslational labelling approaches with photostable organic fluorophores combined with TIRF-microscopy. By single molecule co-localization and co-tracking analysis we have mapped lateral distribution and diffusion of the two receptor subunits with very high spatial and temporal resolution in living cells. Thus, we were able to monitor IFN-induced assembly, co-diffusion and dissociation of individual ternary signalling complexes in the plasma membrane. Systematic modulation of the binding affinities towards the receptor subunits revealed that binding affinity of IFNα2 towards IFNAR1 is optimized for efficient ternary complex formation at physiological receptor surface concentrations. Interestingly, the negative feedback regulator ubiquitin-specific protease 18 (USP18) reduces receptor dimerization by IFNα2, but not IFNβ, which binds to IFNAR1 with higher affinity. Strikingly, this USP18-dependent loss of 2D affinity could be mimicked by truncating the receptor’s intracellular domains. These observations suggest that ternary complexes are stabilized by intracellular interactions of Jak1 and Tyk2 and that these interactions are interfered by USP18, thus selectively abrogating signalling by IFNs with low affinity towards IFNAR1.

Systemic toxicity currently prevents full clinical application of type I interferons (IFNs) for the treatment of cancers and viral infections. Although the use of IFNs fused to antibodies resulted in a 10-fold increased activity towards targeted cells, off-target side effects experienced by the patients are expected to remain a major obstacle. We present a novel strategy to engineer IFN α with high in vitro and in vivo activity towards cells expressing a specific surface marker while having an up to 1000-fold reduced activity towards non-relevant cells. The concept lies in the use of immunocytokines based on IFN mutants with strongly reduced binding affinity for the IFN receptor. Such a targeted IFN recovers almost maximally its antiviral activity uniquely on the targeted cells and acts to selected cell populations in mice with targeting efficiencies up to 3 orders of magnitude. This novel concept holds promise to revitalize the clinical potential of IFN α and other cytokines.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8 + T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8 + T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations: molecular dynamics simulations files Final structures of the simulated systems, as well as allsimulation files (Gromacs 5.1.4 format) and protein structures needed to carry out the simulations in Ref. [1]. The first part of the file name indicates the simulation system according to Table S4 in Ref [1]. Files include: -initial structures and end structures (.pdb and _end.pdb, respectively), -run input files (.tpr) -checkpoint files (.cpt) -simulation parameter files (.mdp) -system topology file (.top) -topology files included in the system topology file (.itp) [1] Wilmes S. et al., Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations, Science, 2019. (Submitted)