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Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFβ1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them,SH2B3- a negative regulator of cytokine and growth factor receptor signaling – harbors rare coding variants in over 5% of SLE patients. Here we show that unlike the variant found exclusively in healthy controls, mostSH2B3rare variants found in lupus patients are predominantly hypomorphic alleles. Generation of two mouse lines carrying variants orthologous to those found in patients revealed SH2B3 is important to limit the numbers of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role forSH2B3in human B cell tolerance and lupus risk.SummaryZhanget al. reveal a role for hypomorphic SH2B3 in lupus risk. The study shows rare and damaging variants identified in lupus patients enable breach of B cell immune tolerance checkpoints and suggests involvement for dysregulated IL-4R signaling and BAFF-R expression.

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 ofSocs1results 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 thein vivoimpact 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-R105AandSocs1-F59Amice 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.

LNK is a member of the SH2B family of adaptor proteins and is a non-redundant regulator of cytokine signalling. Cytokines are secreted intercellular messengers that bind to specific receptors on the surface of target cells to activate the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling pathway. Activation of the JAK-STAT pathway leads to proliferative and often inflammatory effects, and so the amplitude and duration of signalling are tightly controlled. LNK binds phosphotyrosine residues to signalling proteins downstream of cytokines and constrains JAK-STAT signalling. Mutations in LNK have been identified in a range of haematological and inflammatory diseases due to increased signalling following the loss of LNK function. Here, we review the regulation of JAK-STAT signalling via the adaptor protein LNK and discuss the role of LNK in haematological diseases.

Suppressor Of Cytokine Signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

SUMMARYEpithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT (P-EMT) and mesenchymal states, each of which are associated with cancer progression, invasive capabilities and ultimately metastasis. We have employed a lineage traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that have undergone P-EMT and complete EMT (C-EMT). Using an unbiased proteomics approach, we found that the morphology of the organoids predicts the EMT state, with solid organoids associated with a P-EMT signature. We also observed that exogenous TGFβ1 induces a solid organoid morphology that is associated with changes in the S100 family, C-EMT and the formation of high-grade tumors. S100A4 may represent a useful biomarker to predict EMT state, disease progression and outcome for pancreatic cancer patients.

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling., SOCS2 is a key regulator of growth hormone and cytokine signaling, which recognizes phosphotyrosine (pTyr)-modified targets via a central SH2 domain. Here, the authors discover and characterize an exosite on this SH2 domain that can bind a non-phosphorylated peptide to enhance SOCS2:pTyr affinity.

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease., LNK is a potent negative regulator of cytokine signaling implicated in blood cells proliferation. Here the authors present structures of the substrate recognition (SH2) domain of LNK in complex with phosphorylated motifs from JAK2 and EPOR; providing insight into its binding specificity and mode of action.

More than 50 cytokines signal via the JAK/STAT pathway to orchestrate hematopoiesis, induce inflammation and control the immune response. Cytokines are secreted glycoproteins that act as intercellular messengers, inducing proliferation, differentiation, growth, or apoptosis of their target cells. They act by binding to specific receptors on the surface of target cells and switching on a phosphotyrosine-based intracellular signaling cascade initiated by kinases then propagated and effected by SH2 domain-containing transcription factors. As cytokine signaling is proliferative and often inflammatory, it is tightly regulated in terms of both amplitude and duration. Here we review molecular details of the cytokine-induced signaling cascade and describe the architectures of the proteins involved, including the receptors, kinases, and transcription factors that initiate and propagate signaling and the regulatory proteins that control it.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied., Young et al. report on the diversity in regulation of nematode apoptosis. Results indicate that the apoptotic pathways of C. elegans are not fully representative of nematodes generally.

Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2V617F mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1V658F). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases., AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.

The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines., Cytokines are key molecules in controlling haematopoiesis that signal via the JAK/STAT pathway. Here the authors present the structures of SOCS1 bound to its JAK1 target as well as in complex with elonginB and elonginC, providing a molecular explanation for the potent JAK- inhibitory activity of SOCS1.

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.

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 α4β1 (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.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction.

Janus kinases (JAKs) initiate the intracellular signaling cascade triggered by exposure of cells to cytokines and interferons. In order to achieve this, JAKs are bound to the intracellular domain of specific cytokine receptors immediately adjacent to the cell membrane. In this issue of Structure, Ferrao et al. (2016) provide structural details of such an interaction and in doing so, identify for the first time the motif used by type II cytokine receptors to recruit JAK1.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

Crystal structures of human and prokaryotic ribosomal oxygenases reported here, with and without their ribosomal protein substrates, support their assignments as hydroxylases, and provide insights into the evolution of the JmjC-domain-containing hydroxylases and demethylases. Christopher Schofield and colleagues present a comprehensive structural study of a recently discovered family of 2-oxoglutarate and iron-dependent oxygenases termed ribosomal oxygenases (ROXs). They have solved 13 new structures of human and bacterial ROXs with or without substrate. While the analysis confirms their assignment as hydroxylases, comparison with JmjC-domain-containing hydroxylases and demethylases provides an understanding of the evolution of these related families of proteins, in part driven by flexibility in the coordination position. The general binding mode of the hydroxylated residues is conserved between prokaryotic and human ROXs. 2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Ne-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

SOCS3 (Suppressor of Cytokine Signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus Kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced poly-ubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalysed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

JAKs (Janus kinases) are essential mediators of almost all biological signalling events initiated by haemopoietic and immune cytokines. However, aberrant and/or prolonged JAK-induced signalling is detrimental and can give rise to a number of inflammatory and proliferative pathologies. For this reason, the tyrosine kinase activity of the JAKs is carefully regulated at a number of different levels. Primarily, this is achieved by: (i) ensuring that the catalytic domain is ‘switched off’ under basal conditions, and (ii) inhibiting the activity of JAK after it has been switched on. Whereas the first mode of inhibition is mediated by JAK's own pseudokinase domain as well as the action of phosphatases, the second is achieved by the action of the SOCS (suppressor of cytokine signalling) proteins, negative-feedback inhibitors of JAK-mediated signalling. The present review focuses on the mode of action of SOCS1 and SOCS3, the two most potent JAK inhibitors. Abbreviations: G-CSF, granulocyte colony-stimulating factor; gp130, glycoprotein 130; Grb14, growth factor receptor-bound protein 14; IL, interleukin; IRK, insulin receptor kinase; JAK, Janus kinase; JH1, JAK homology 1; KIR, kinase-inhibitory region; LIF, leukaemia-inhibitory factor; OSM, oncostatin M; SH2, Src homology 2; SOCS, suppressor of cytokine signalling; STAT, signal transducer and activator of transcription; TYK2, tyrosine kinase 2

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

SummaryJanus 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.

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses. DOI: http://dx.doi.org/10.7554/eLife.08698.001, eLife digest The immune system protects the body from invading microbes like bacteria and viruses. Upon recognizing the presence of these microbes, cells in the immune system are activated to destroy the foreign threat and clear it from the body. A type of immune cell called T follicular helper cells (or Tfh for short) are formed during an infection and are essential for coordinating other immune cells to produce high-quality antibody proteins that attack the microbes. Without Tfh cells, life-long production of these protective antibodies is severely crippled, which can cause common variable immune deficiency and other serious immunodeficiency diseases. On the other hand, the body must also avoid generating excessive numbers of Tfh cells, which can lead to the production of antibodies that attack healthy cells of the body. ROQUIN is a protein that inhibits the formation of Tfh cells and other types of active T cells. A region on the protein called the ROQ domain destabilizes particular molecules of ribonucleic acid (RNA) that are required for these specialist T cells to form and work properly. ROQUIN belongs to a large family of enzymes that have a so-called RING domain, which is a feature that enables these enzymes to attach tags onto specific target proteins to modify their activity or stability. However, it was not known whether the RING domain of ROQUIN was active. Ramiscal et al. now address this question in mice. Unexpectedly, the experiments show that the RING domain is required to promote the formation of Tfh cells, but not other types of active T cells. This domain allows ROQUIN to repress an enzyme called AMPK, which normally blocks cell growth by regulating cell metabolism. The findings suggest that the different roles of the ROQ and RING domains allow ROQUIN to fine-tune the numbers of Tfh cells so that they remain within a safe range. In the future, these findings may aid the development of vaccines that are more efficient at generating protective Tfh cells to prevent infectious diseases. DOI: http://dx.doi.org/10.7554/eLife.08698.002

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1-6, D1-D6) at 3.6 A resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4-D6) at 1.9 A. This represents the first atomic resolution structure of the complete ectodomain of any "tall" cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.

(3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A β-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P21 space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley-Liss, Inc.

The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ≈ 15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to > 95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate α and β subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an α2β2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the β subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the β subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major β subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate, but not the formation of N-acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N-acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the β subunit.

Introduction: Oral cavity squamous cell carcinomas (OSCCs) are the most prevalent head and neck malignancy and are a significant cause of mortality and morbidity, especially in the developing world. A better understanding of the molecular pathogenesis of OSCCs is essential for improved management of this disease. Mutations in the NOTCH1 gene have been identified in 9-18% patients with head and neck malignancies. A large proportion of these mutations lie in its extracellular domain and are thought to be inactivating, in contrast to the activating NOTCH1 mutations found in hematological malignancies such as T-ALL. This dichotomy raises interesting questions about the role of the Notch pathway in malignancies. In this study, we aim to characterize NOTCH1 mutations in a cohort of OSCC patients and use an in vitro model to investigate the role of NOTCH1 inactivation in OSCC tumorigenesis. Methods: Targeted next-generation sequencing of a custom 69 gene panel was carried out on fresh frozen and formalin fixed paraffin embedded (FFPE) tissue from prospectively and retrospectively recruited OSCC patients presenting to a tertiary cancer center (n = 94). Detailed clinical annotation was collected for all patients. The BWA/GATK pipeline was used for sequence alignment and mutation calling. Downstream sequencing pipelines were designed in the R statistical environment to optimize identification of somatic mutations in the FFPE samples. Notch pathway mutations were interrogated, and comparisons made against NOTCH1 mutations across all cancer subtypes in The Cancer Genome Atlas (TCGA) database. Five OSCC cell lines (H314, H357, H400, BICR16, BICR56) were sequenced and Notch1 expression characterized by Western blotting. A lentiviral CRISPR/Cas9 system using custom designed guides was used to knock out NOTCH1 in the cell lines expressing wild-type Notch1. Wild-typeNOTCH1-mCherry was expressed in the cell lines with mutated NOTCH1. Non-adherent clonal growth assays and RNAseq were used to assess the impact of these modifications. Results: In our patient cohort, a high frequency of mutations were identified in known tumor suppressor genes TP53 (57%) and CDKN2A (22%). Sixteen patients (17%) were NOTCH1 mutant. The majority of these mutations fell within the extracellular domain and 10 were predicted to be nonsense mutations. Ten patients (11%) had a NOTCH3 mutation. Two of the five OSCC cell lines were NOTCH1 mutant and expressed no detectable Notch1 protein. A wild-type Notch1-mCherry construct was introduced into the mutant cell lines and Notch1 was knocked out in the three cell lines in which functional Notch1 protein was detected. The results of the growth assays and RNA sequencing for all of the pre- and post- modification cell lines will be presented. Conclusions: We have built a bioinformatics pipeline to successfully analyze FFPE tissue and have identified potentially inactivating NOTCH1 mutations in our patients at similar frequencies to international cohorts. We have identified NOTCH3 mutations, which are of uncertain significance. Successful knockout of NOTCH1 in OSCC cell lines was achieved. Citation Format: Kendrick Koo, Christopher Angel, Nadia Kershaw, Dmitri Mouradov, Anderly Chueh, David Wiesenfeld, Tim A. Iseli, Michael McCullough, Oliver Sieber, Antony W. Burgess. Characterization of NOTCH1 mutations in oral cavity squamous cell carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A01.

Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.

The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B (EloB), and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison to other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and the rationale design of novel compounds that may modulate cullin–substrate receptor interactions.