Tofacitinib treatment modulates the levels of several inflammation-related plasma proteins in rheumatoid arthritis and baseline levels of soluble biomarkers associate with the treatment response.

The data on the effects of tofacitinib on soluble proteins in patients with rheumatoid arthritis (RA) is currently very limited. We analyzed how tofacitinib treatment and thus inhibition of the Janus kinase-signal transducer and activation of transcription pathway affects the in vivo levels of inflammation-related plasma proteins in RA patients. In this study, 16 patients with active RA [28-joint disease activity score (DAS28) >3.2] despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) started tofacitinib treatment 5 mg twice daily. Levels of 92 inflammation-related plasma proteins were determined by proximity extension assay at baseline and at 3 months. Tofacitinib treatment for 3 months, in csDMARD background, decreased the mean DAS28 from 4.4 to 2.6 (P < 0.001). Marked (>20%) and statistically significant (P < 0.05) changes were found in the levels of 21 proteins, 18 of which decreased and 3 increased. Of these proteins, 17 are directly involved in inflammatory responses or in the cellular response to cytokines. The highest (>50%) decrease was observed for interleukin-6 (IL-6), C-X-C motif chemokine ligand 1, matrix metalloproteinase-1, and AXIN1. Higher baseline levels of IL-6 and lower levels of C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptors were associated with DAS28 improvement. Our results indicate that tofacitinib downregulates several proinflammatory plasma proteins that may contribute to the clinical efficacy of tofacitinib. In addition, soluble biomarkers may predict the treatment response to tofacitinib.

Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations.

BACKGROUND: Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity. OBJECTIVE: We sought to characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors. METHODS: We performed a systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell-signaling assays, microscopy, and biochemical analysis. RESULTS: Distinct structural requirements were identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation, V617F, is the most sensitive to structural perturbations in multiple JH2 elements (C helix [αC], Src homology 2-JH2 linker, and ATP binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP binding site mutations have only a minor effect on signaling, whereas JH2 αC mutations reduce homomeric (JAK2-JAK2) erythropoietin signaling and almost completely abrogate heteromeric (JAK2-JAK1) IFN-γ signaling, potentially by disrupting a dimerization interface on JH2. CONCLUSIONS: These results suggest that therapeutic approaches targeting the JH2 ATP binding site and αC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-γ functions. Simultaneously, however, we identified the JH2 αC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.

Janus kinases to jakinibs: from basic insights to clinical practice.

Cytokines are critical mediators of diverse immune and inflammatory diseases. Targeting cytokines and cytokine receptors with biologics has revolutionized the treatment of many of these diseases, but targeting intracellular signalling with Janus kinase (JAK) inhibitors (jakinibs) now represents a major new therapeutic advance. We are still in the first decade since these drugs were approved and there is still much to be learned about the mechanisms of action of these drugs and the practical use of these agents. Herein we will review cytokines that do, and just as importantly, do not signal by JAKs, as well as explain how this relates to both efficacy and side effects in various diseases. We will review new, next-generation selective jakinibs, as well as the prospects and challenges ahead in targeting JAKs.

The regulation of JAKs in cytokine signaling and its breakdown in disease.

The JAK-STAT signal transduction pathway is responsible for mediating signals of over fifty cytokines, growth factors and hormones. Signaling through the JAK-STAT pathway is regulated on multiple levels, including intramolecular regulation by the JAK pseudokinase domain, and intermolecular regulation by a host of regulatory proteins. The advent of accessible genomic tools have provided a wealth of information on disease-associated mutations in the JAK-STAT pathway and its regulatory components. The vast number of these mutations in diseases ranging from immunodeficiencies and obesity to many cancers highlight the importance of correct regulation of JAK-STAT signaling for biological processes such as hematopoiesis, regulation of the immune system, metabolism, and growth. Simultaneously, JAK inhibitors are gaining traction in clinical use, both for treatment of diseases driven by JAK mutations, and for a host of inflammatory disorders, in which proinflammatory cytokine signaling through the JAK-STAT pathway is an integral part of pathogenesis. The elucidation of molecular mechanisms in the pathogenesis of complex diseases has also, however, brought the limitations of our current understanding on the regulation of cytokine signaling to the foreground. Indeed, deeper understanding of these regulatory mechanisms are a prerequisite for the development of the next generation of pharmacological modulators of the JAK-STAT pathway. In this review we discuss the current state of knowledge of the intra- and intermolecular regulation of the JAK-STAT pathway, with a focus on diseases arising from disruptions in the regulatory apparatus.

Proprotein convertase enzyme FURIN is upregulated in primary Sjögren's syndrome.

OBJECTIVES: The proprotein convertase enzyme FURIN is a critical regulator of the anti-inflammatory TGFß-1 cytokine and peripheral immune tolerance. In T cells, FURIN is co-regulated with IFN-γ and thus highly expressed in T helper 1 type cells. Previous studies have demonstrated that FURIN is upregulated in inflammatory conditions, including atherosclerosis, rheumatoid arthritis and systemic lupus erythematosus. Here, we evaluated the levels of FURIN in the plasma and peripheral blood mononuclear cells (PBMCs) of patients with primary Sjögren's syndrome (pSS) and in healthy controls. METHODS: FURIN plasma levels were determined by ELISA, and the mRNA expression in PBMCs was quantitated using qPCR. FURIN levels in the plasma were correlated with the clinical and demographic characteristics of the patients. RESULTS: FURIN was found to be significantly upregulated at both the protein and mRNA level in pSS patients compared to healthy controls. In pSS patients, high FURIN protein levels were significantly associated with elevated IFN-γ levels in the plasma as well as a longer duration of sicca symptoms in the eyes. pSS patients with high FURIN levels in their plasma showed a trend towards lower levels of serum beta-2 microglobulin, ESR and a lower systemic disease activity index ESSDAI. CONCLUSIONS: The proprotein convertase FURIN is significantly upregulated in pSS. Elevated FURIN levels associate with high levels of the Th1 type cytokine IFN-γ and long duration of dry eye symptoms. Patients with high FURIN levels show signs of lower disease activity suggesting that FURIN might have a protective role in pSS.

ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation.

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.

Molecular insights into regulation of JAK2 in myeloproliferative neoplasms.

The critical role of Janus kinase-2 (JAK2) in regulation of myelopoiesis was established 2 decades ago, but identification of mutations in the pseudokinase domain of JAK2 in myeloproliferative neoplasms (MPNs) and in other hematologic malignancies highlighted the role of JAK2 in human disease. These findings have revolutionized the diagnostics of MPNs and led to development of novel JAK2 therapeutics. However, the molecular mechanisms by which mutations in the pseudokinase domain lead to hyperactivation of JAK2 and clinical disease have been unclear. Here, we describe recent advances in the molecular characterization of the JAK2 pseudokinase domain and how pathogenic mutations lead to constitutive activation of JAK2.

Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications.

Tudor staphylococcal nuclease (Tudor-SN), a novel regulator facilitating G1/S phase transition, acting as a co-activator of E2F-1 in cell cycle regulation.

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein implicated in a variety of cellular processes. In the present study, we identified Tudor-SN as a novel regulator in cell cycle. Tudor-SN was abundant in proliferating cells whereas barely expressed in terminally differentiated cells. Functional analysis indicated that ectopic overexpression of Tudor-SN promoted the G1/S transition, whereas knockdown of Tudor-SN caused G1 arrest. Moreover, the live-cell time-lapse experiment demonstrated that the cell cycle of MEF(-/-) (knock-out of Tudor-SN in mouse embryonic fibroblasts) was prolonged compared with wild-type MEF(+/+). We noticed that Tudor-SN was constantly expressed in every cell cycle phase, but was highly phosphorylated in the G1/S border. Further study revealed that Tudor-SN was a potential substrate of Cdk2/4/6, supportively, we found the physical interaction of endogenous Tudor-SN with Cdk4/6 in G1 and the G1/S border, and with Cdk2 in the G1/S border and S phase. In addition, roscovitine (Cdk1/2/5 inhibitor) or CINK4 (Cdk4/6 inhibitor) could inhibit the phosphorylation of Tudor-SN, whereas ectopic overexpression of Cdk2/4/6 increased the Tudor-SN phosphorylation. The underlying molecular mechanisms indicated that Tudor-SN could physically interact with E2F-1 in vivo, and could enhance the physical association of E2F-1 with GCN5 (a cofactor of E2F-1, which possesses histone acetyltransferase activity), and promote the binding ability of E2F-1 to the promoter region of its target genes CYCLIN A and E2F-1, and as a result, facilitate the gene transcriptional activation. Taken together, Tudor-SN is identified as a novel co-activator of E2F-1, which could facilitate E2F-1-mediated gene transcriptional activation of target genes, which play essential roles in G1/S transition.

Cytokine-induced STAT1 activation is increased in patients with primary Sjögren's syndrome.

Limited data are available regarding the intracellular responses to different cytokines in primary Sjögren's syndrome (pSS). We studied the signal transducer and activator of transcription (STAT) activation profile in response to cytokine stimulations in peripheral blood mononuclear cells (PBMCs) from pSS patients by multicolor flow cytometry. The expression of the suppressors of cytokine signaling (SOCS), and interferon (IFN)-γ target genes in PBMCs was studied using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The induction of STAT1 phosphorylation in response to stimulation with IFN-α, IFN-γ or interleukin (IL)-6 was significantly increased in B cells and monocytes from pSS patients. Accordingly, the STAT1-mediated gene responses were significantly enhanced in PBMCs from pSS patients. Finally, the expression of SOCS1 and SOCS3 mRNA was increased in pSS patients. The results indicate increased sensitivity of immune cells from pSS patients to STAT1-activating signals, and may partly explain the IFN signature observed in pSS.

Tudor-SN, a novel coactivator of peroxisome proliferator-activated receptor γ protein, is essential for adipogenesis.

Adipogenesis, in which mesenchymal precursor cells differentiate into mature adipocytes, is a well orchestrated process. In the present study we identified Tudor-SN as a novel co-activator of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ). We provide the first evidence that Tudor-SN and PPARγ exist in the same complex. Both are up-regulated by the early factor C/EBPß during adipogenesis and significantly influence the regulation of PPARγ target genes in both 3T3-L1 pre-adipocyte and mouse embryonic fibroblasts (MEF) upon exposure to a mixture of hormonal mixture. Moreover, aP2-PPARγ response element (PPRE) interacts with both PPARγ and Tudor-SN, and the gene transcriptional activation of PPRE-luc is enhanced by ectopic expression of Tudor-SN. Deletion of Tudor-SN protein (MEF-KO) affects but does not completely abolish the association of PPARγ and aP2-PPRE. Loss-of-function studies further verified that Tudor-SN is required for adipogenesis, as deletion of Tudor-SN (MEF-KO) impairs dexamethasone, 3-isobutyl-1-methylxanthine, and insulin (DMI)-induced adipocyte differentiation and the expression of PPARγ target genes, such as aP2 and adipsin. Furthermore, H3 acetylation levels were lower in MEF-KO than MEF-WT. Both HDAC1 and HDAC3 are stably associated with PPARγ in MEF-KO, whereas only a small amount of association was observed in MEF-WT after 5 days of treatment during adipogenesis. PPARγ requires various co-activators or co-repressors, which may dynamically associate with and regulate the higher order chromatin remodeling of the promoter region of PPARγ-bound target genes; Tudor-SN is likely one of these co-activators.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP.

The activity of JAK-STAT pathways in rheumatoid arthritis: constitutive activation of STAT3 correlates with interleukin 6 levels.

OBJECTIVE: Many cytokines involved in RA activate the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Therapeutic drugs that inhibit these pathways are being developed for RA. To investigate disease-related alterations in the activity of JAK-STAT pathways in RA, we studied the expression and activation of STAT1 and STAT3 in unstimulated and cytokine-stimulated cells and determined the levels of circulating cytokines. METHODS: The expression of STAT1 and STAT3 mRNA in peripheral blood (PB) and SF T cells and monocytes was studied in RA patients and healthy volunteers by RT-PCR. Basal and cytokine (IFN-γ, IL-6, IL-10)-induced STAT phosphorylation was analysed in PB T cells and monocytes using multicolour flow cytometric analysis. RESULTS: STAT3 mRNA levels were up-regulated in both PB and SF T cells and monocytes from RA patients. STAT1 expression was elevated in SF monocytes. The levels of phospho-STAT3 in resting PB T cells and monocytes were significantly higher in patients with RA than in healthy volunteers. IL-6 levels were elevated in RA plasma and correlated with the level of STAT3 phosphorylation in CD4(+) T cells and monocytes. IL-6-mediated STAT3 activation was deregulated in T cells from RA patients. IL-6-induced phosphorylation of STAT3 was decreased in CD4(+) T cells from patients with high plasma IL-6 levels and constitutive STAT3 phosphorylation. CONCLUSION: The results suggest that IL-6 induces hyperactivation of STAT3 in circulating immune cells in active RA, and this subsequently desensitizes the IL-6 response in T cells.

Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses.

JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haemopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase domain (JH2) of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase domain (JH1) and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator SOCS3 (suppressor of cytokine signalling 3) have remained unclear. In the present study, using recombinant purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. SAXS (small-angle X-ray scattering) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Th17 cells in multiple sclerosis express higher levels of JAK2, which increases their surface expression of IFN-γR2.

IFN-ß inhibits the expansion of Th17 cells in active multiple sclerosis (AMS), and this might contribute to improve the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. In this study, we report that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2) and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells and clones from patients with AMS compared with those with inactive multiple sclerosis (IMS) or healthy subjects (HS). IFN-γ decreased the frequency of Th17 peripheral cells and proliferation of Th17 clones from AMS patients. Stimulation of PBMCs from HS in Th17-polarizing conditions resulted in the enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from AMS patients, making them sensitive to IFN-γ. These data may have clinical implications for a better use of IFNs in multiple sclerosis and possibly other inflammatory diseases.

Functional and Structural Characterization of Clinical-Stage Janus Kinase 2 Inhibitors Identifies Determinants for Drug Selectivity.

Janus kinase 2 (JAK2) plays a critical role in orchestrating hematopoiesis, and its deregulation leads to various blood disorders, most importantly myeloproliferative neoplasms (MPNs). Ruxolitinib, fedratinib, momelotinib, and pacritinib are FDA-/EMA-approved JAK inhibitors effective in relieving symptoms in MPN patients but show variable clinical profiles due to poor JAK selectivity. The development of next-generation JAK2 inhibitors is hampered by the lack of comparative functional analysis and knowledge of the molecular basis of their selectivity. Here, we provide mechanistic profiling of the four approved and six clinical-stage JAK2 inhibitors and connect selectivity data with high-resolution structural and thermodynamic analyses. All of the JAK inhibitors potently inhibited JAK2 activity. Inhibitors differed in their JAK isoform selectivity and potency for erythropoietin signaling, but their general cytokine inhibition signatures in blood cells were comparable. Structural data indicate that high potency and moderate JAK2 selectivity can be obtained by targeting the front pocket of the adenosine 5'-triphosphate-binding site.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Tudor staphylococcal nuclease (Tudor-SN) participates in small ribonucleoprotein (snRNP) assembly via interacting with symmetrically dimethylated Sm proteins.

Human Tudor staphylococcal nuclease (Tudor-SN) is composed of four tandem repeats of staphylococcal nuclease (SN)-like domains, followed by a tudor and SN-like domain (TSN) consisting of a central tudor flanked by two partial SN-like sequences. The crystal structure of the tudor domain displays a conserved aromatic cage, which is predicted to hook methyl groups. Here, we demonstrated that the TSN domain of Tudor-SN binds to symmetrically dimethylarginine (sDMA)-modified SmB/B' and SmD1/D3 core proteins of the spliceosome. We demonstrated that this interaction ability is reduced by the methyltransferase inhibitor 5-deoxy-5-(methylthio)adenosine. Mutagenesis experiments indicated that the conserved amino acids (Phe-715, Tyr-721, Tyr-738, and Tyr-741) in the methyl-binding cage of the TSN domain are required for Tudor-SN-SmB interaction. Furthermore, depletion of Tudor-SN affects the association of Sm protein with snRNAs and, as a result, inhibits the assembly of uridine-rich small ribonucleoprotein mediated by the Sm core complex in vivo. Our results reveal the molecular basis for the involvement of Tudor-SN in regulating small nuclear ribonucleoprotein biogenesis, which provides novel insight related to the biological activity of Tudor-SN.