TLR7 gain-of-function genetic variation causes human lupus.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Small molecule drug discovery targeting the JAK-STAT pathway.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway functions as a central hub for transmitting signals from more than 50 cytokines, playing a pivotal role in maintaining hematopoiesis, immune balance, and tissue homeostasis. Dysregulation of this pathway has been implicated in various diseases, including immunodeficiency, autoimmune conditions, hematological disorders, and certain cancers. Proteins within this pathway have emerged as effective therapeutic targets for managing these conditions, with various approaches developed to modulate key nodes in the signaling process, spanning from receptor engagement to transcription factor activation. Following the success of JAK inhibitors such as tofacitinib for RA treatment and ruxolitinib for managing primary myelofibrosis, the pharmaceutical industry has obtained approvals for over 10 small molecule drugs targeting the JAK-STAT pathway and many more are at various stages of clinical trials. In this review, we consolidate key strategies employed in drug discovery efforts targeting this pathway, with the aim of contributing to the collective understanding of small molecule interventions in the context of JAK-STAT signaling. We aspire that our endeavors will contribute to advancing the development of innovative and efficacious treatments for a range of diseases linked to this pathway dysregulation.

The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo.

Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.

The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution.

Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15N relaxation parameters of backbone amides, without need for the protein structure being available.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4ß1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

Dissecting the molecular control of Interleukin 6 signaling using the M1 cell line.

Interleukin 6 is the classical member of the IL-6 family of cytokines which triggers activation of the JAK/STAT signaling cascade in cells. IL-6 is a pleiotropic cytokine that acts on many cell types and plays a critical role in immune responses, inflammation, and haematopoiesis. Our understanding of the molecular mechanisms governing IL-6 signaling has been aided by numerous studies of this signal transduction pathway, including those utilising the M1 cell line. Here we discuss the studies that we and others have undertaken using the M1 line to examine IL-6 inducible genes, particularly those targets that acts as negative regulators of signaling. Finally, we present a model for the current understanding of the IL-6 signaling pathway at a structural and mechanistic level.

Persistence of myelofibrosis treated with ruxolitinib: biology and clinical implications.

Activation of JAK-STAT signaling is one of the hallmarks of myelofibrosis, a myeloproliferative neoplasm that leads to inflammation, progressive bone marrow failure, and a risk of leukemic transformation. Around 90% of patients with myelofibrosis have a mutation in JAK2, MPL, or CALR: so-called 'driver' mutations that lead to activation of JAK2. Ruxolitinib, and other JAK2 inhibitors in clinical use, provide clinical benefit but do not have a major impact on the abnormal hematopoietic clone. This phenomenon is termed 'persistence', in contrast to usual patterns of resistance. Multiple groups have shown that type 1 inhibitors of JAK2, which bind the active conformation of the enzyme, lead to JAK2 becoming resistant to degradation with consequent accumulation of phospho-JAK2. In turn, this can lead to exacerbation of inflammatory manifestations when the JAK inhibitor is discontinued, and it may also contribute to disease persistence. The ways in which JAK2 V617F and CALR mutations lead to activation of JAK-STAT signaling are incompletely understood. We summarize what is known about pathological JAK-STAT activation in myelofibrosis and how this might lead to future novel therapies for myelofibrosis with greater disease-modifying potential.

Suppression of cytokine signaling by SOCS3: characterization of the mode of inhibition and the basis of its specificity.

Janus kinases (JAKs) are key effectors in controlling immune responses and maintaining hematopoiesis. SOCS3 (suppressor of cytokine signaling-3) is a major regulator of JAK signaling and here we investigate the molecular basis of its mechanism of action. We found that SOCS3 bound and directly inhibited the catalytic domains of JAK1, JAK2, and TYK2 but not JAK3 via an evolutionarily conserved motif unique to JAKs. Mutation of this motif led to the formation of an active kinase that could not be inhibited by SOCS3. Surprisingly, we found that SOCS3 simultaneously bound JAK and the cytokine receptor to which it is attached, revealing how specificity is generated in SOCS action and explaining why SOCS3 inhibits only a subset of cytokines. Importantly, SOCS3 inhibited JAKs via a noncompetitive mechanism, making it a template for the development of specific and effective inhibitors to treat JAK-based immune and proliferative diseases.

Physiochemical Characterization and Stability of Lipidic Cubic Phases by Solution NMR.

Lipidic inverse bicontinuous cubic phases (LCPs), formed via the spontaneous self-assembly of lipids such as monoolein, have found increasing applications in the stabilization and crystallization of integral membrane proteins for structural characterization using X-ray crystallography. Their use as effective drug release matrices has also been demonstrated. Nuclear magnetic resonance (NMR) spectroscopy, both solution and solid state, has previously been employed for the characterization of LCPs and related systems. Herein, we report a number of novel features of solution NMR for probing the fundamental composition and structural properties of monoolein-based LCPs. These include (1) more complete assignments of both 1H and 13C chemical shifts, (2) direct quantification of hydration level in LCPs using one-dimensional (1D) 1H NMR, and (3) monitoring longer-term stability of LCPs and evaluating alterations introduced into standard LCPs at the submolecular level.

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα).

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Measuring translational diffusion of 15N-enriched biomolecules in complex solutions with a simplified 1H-15N HMQC-filtered BEST sequence.

Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of 15N-enriched biomolecules using a 1H-15N HMQC-filtered band-selective excitation short transient (BEST) sequence as an alternative to the previously described SOFAST-XSTE sequence. The results demonstrate that accurate translational diffusion coefficients of 15N-labelled peptides and proteins can be obtained using this alternative 1H-15N HMQC-filtered BEST sequence which is implementable on NMR spectrometers equipped with probes fitted with a single-axis field gradient, including most cryoprobes dedicated to bio-NMR. The sequence is of potential use for direct quantification of protein or peptide translational diffusion within complex systems, such as in mixtures of macromolecules, crowded solutions, membrane-mimicking media and in bicontinuous cubic phases, where conventional sequences may not be readily applicable due to the presence of intense signals arising from sources other than the protein or peptide under investigation.

Cortical Layer Inversion and Deregulation of Reelin Signaling in the Absence of SOCS6 and SOCS7.

Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.

Inhibition of IL-6 family cytokines by SOCS3.

IL-6 a multi-functional cytokine with important effects in both inflammation and haematopoiesis. SOCS3 is the primary inhibitor of IL-6 signalling, interacting with gp130, the common shared chain of the IL-6 family of cytokines, and JAK1, JAK2 and TYK2 to control both the duration of signalling and the biological response. Recent biochemical and structural studies have shown SOCS3 binds to only these three JAKs, all of which are associated with IL-6 signalling, and not JAK3. This specificity is determined by a three residue "GQM" motif in the kinase domain of JAK1, JAK2 and TYK2. SOCS3 binds to JAK and gp130 simultaneously, and inhibits JAK activity in an ATP-independent manner by partially occluding the kinase's substrate binding groove with its kinase inhibitory region. We therefore propose a model in which each of gp130, JAK and SOCS3 are directly bound to the other two, allowing SOCS3 to inhibit IL6 signalling with high potency and specificity.

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5.

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.

The molecular regulation of Janus kinase (JAK) activation.

The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.