Objective: Numerous studies have described the role of STAT3 (signal transducer and activator of transcription 3) in infections, but little is known on whether this transcription factor is linked to negative affectivity (NA) and social inhibition (SI), leading to social withdrawal as a typical symptom of various infections. Methods: In this study, we isolated peripheral blood mononuclear cells (PBMCs) from 63 consecutive depressed patients (mean age 41.4 +/- 16.1 years; 40 females) before and after psychotherapeutic intervention and measured STAT3 tyrosine phosphorylation (pSTAT3) with and without in vitro interleukin-6 (IL-6) stimulation of these cells using flow cytometry. In addition, all study participants were assessed for NA and SI using the German version of the Type D Scale-14 (DS-14) questionnaire with a cut-off level of >= 10 for each subscale. Results: While NA was unrelated to STAT3 activity, PBMCs from SI-positive patients had an increased baseline STAT3 activation level, which made the cells less sensitive to in vitro IL-6 stimulation (11.5% vs. 9.1%, p = 0.036). The stimulatory capacity, defined as the difference in pSTAT3 levels from IL-6-stimulated to unstimulated cells during hospitalization, was significantly lower in PBMCs from SI-positive than from SI-negative patients (-1.7% vs. 6.6%, p = 0.007). The sensitivity of PBMCs to IL-6 stimulation was negatively correlated with the SI score (r = -0.295, p = 0.019). Of note, the altered sensitivity to STAT3 phosphorylation remained stable, when adjusted for clinically relevant confounders in multivariate analysis (Exp((3) = 0.891, 95%-confidence interval = 0.804-0.988, p = 0.029). Conclusion: These findings point towards a possible relationship between STAT3 signaling and social inhibition in depressed patients.

The cytokine-driven transcription factor STAT1 (signal transducer and activator of transcription 1) executes anti-microbial and pro-apoptotic functions, and loss-of-function mutations are associated with increased susceptibility to various infections and the development of tumors. A targeted mutation in mice expressing an N-terminally truncated STAT1 protein (STAT1-ΔN) typically develops splenomegaly in animals older than 6 months due to the formation of splenic non-Hodgkin lymphomas. The expression of the STAT1-ΔN variant resulted in the disruption of normal spleen architecture by malignant CD3- and CD20-negative tumor cells, which stained positively for both tyrosine-phosphorylated STAT1 and STAT3. Immunoblotting of lysates from isolated tumor cells revealed the cytokine-independent hyperphosphorylation of both STAT proteins, whereas the expression level of NF-κB was significantly reduced. Gel-shift assays showed that the DNA-binding activity of STAT1-ΔN was increased compared to the wild-type protein. This elevated level of tyrosine-phosphorylated STAT1-ΔN did not further increase upon stimulation of isolated tumor cells with either interferon-γ (IFNγ), lipopolysaccharide (LPS), or the combination of both. Since the truncation mutant was unable to accumulate in the nucleus upon cytokine stimulation, real-time PCR data from tumor tissue as well as from isolated, IFNγ/LPS-treated lymphoma cells demonstrated significantly reduced STAT1-regulated target gene expression despite its observed hyperphosphorylation. The nuclear import defect of tyrosine-phosphorylated STAT1-ΔN was associated with an elevated tyrosine-phosphorylation level of its antagonistic homolog STAT3, which is a known oncogene. These data demonstrate that the lack of STAT1 nuclear accumulation interferes with the functional balance between the two STAT proteins and, thereby, promotes the formation of phospho-STAT3-expressing CD3-/- CD20-/- non-Hodgkin lymphomas in the spleens of the diseased animals.

Background STAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation. Results In this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner. Conclusion Based on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process.

Summary Background Despite conflicting data, some studies have suggested a pathophysiological relationship between inflammation and attention-deficit/hyperactivity disorder (ADHD). Methods Using data from the nationwide and representative German Health Interview and Examination Survey for Children and Adolescents (KiGGS; n = 6922 study participants aged 11–17 years), this post hoc analysis assessed the associations between ADHD and three common inflammatory diseases. Results Results showed univariate associations between ADHD and lifetime inflammatory diseases including atopic dermatitis ( p = 0.002), otitis media ( p = 0.001), and herpes simplex infection ( p = 0.032). In logistic regression models adjusted for clinically relevant confounders, we found that ADHD remained a significant predictor of all three inflammatory diseases (atopic dermatitis, Exp(β) = 1.672, 95% confidence interval [CI] 1.239–2.257, p = 0.001; otitis media, Exp(β) = 1.571, 95% CI 1.209–2.040, p = 0.001; herpes simplex, Exp(β) = 1.483, 95% CI 1.137–1.933, p = 0.004). Conclusion Our findings demonstrate a positive link between ADHD and peripheral inflammatory diseases, including atopic dermatitis, otitis media, and herpes simplex infection. Further studies are needed to understand the exact pathophysiological mechanisms underlying these associations.

In this randomized, sham-controlled study, we explored the effects of acute transcutaneous vagus nerve stimulation (tVNS) on serum aldosterone in 20 younger (21–26 years) and 19 older (40–70 years) healthy participants. Blood samples were collected on two different days before and after a 20-min application of active tVNS at the inner tragus or sham stimulation of the earlobe. Irrespective of the stimulation mode, aldosterone levels decreased from pre- to post-stimulation in both the young (active: β = − 1.610 (− 2.855, − 0.365), p = 0.022; sham: β = − 0.857 (− 2.102, 0.388), p = 0.257) and the old cohort (active: β = − 1.969 (− 3.234, − 0.703), p = 0.005; sham: β = − 1.334 (− 2.600, − 0.069), p = 0.063). Although this decline was significant during active tVNS, the difference in estimated β-coefficients between active and sham stimulation was not statistically significant in either cohort. Nevertheless, aldosterone concentrations showed a significant interaction effect between sex and age (p = 0.001). Among all study participants, younger women (23.3 ± 1.6 years) had the highest mineralocorticoid levels (pre active: 172.1 ± 102.0 pg/ml, pre sham: 214.3 ± 82.3 pg/ml), whereas the lowest were observed in older females (59.4 ± 9.4 years) (pre active: 104.9 ± 85.8 pg/ml, pre sham: 81.1 ± 53.8 pg/ml). This post hoc analysis did not suggest that active auricular tVNS reduces serum aldosterone levels compared to sham stimulation in healthy subjects. However, serum aldosterone levels differed among subjects depending on their age and sex, irrespective of tVNS.

Background Previous clinical studies have reported elevated levels of depressive symptoms in selected samples of patients with gastritis. The objective of this study was to examine the associations of specific biomarkers of inflammation expressed in mucosal tissue from the stomach with mood and anxiety symptoms in adult patients with upper gastrointestinal symptoms. Methods In this monocentric, observational study, a total of 32 study participants were included who were referred for a routine diagnostic upper endoscopic assessment based on the suspected clinical diagnosis of gastritis. All participants completed the Hospital Anxiety and Depression Scale (HADS) before undergoing gastroscopy. Immunohistochemical stainings from biopsy sections were performed to evaluate the expression level of nuclear factor kappa B (NF-κ B), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS). Results Our findings confirmed that nearly half of the study cohort ( n = 13; 41%) displayed positive HADS depression scores above the clinically relevant cut-off level of ≥ 8. Regression models demonstrated that depressive symptoms were significantly and positively associated with the expression level of NF-κ B in biopsies from the upper gastrointestinal tract. Conclusions In summary, our study showed a significant association between NF-κ B expression and clinically relevant depressive symptoms in patients with gastritis, as assessed by a self-rated psychometric questionnaire. Further investigations are needed to confirm this relationship and to identify the pathophysiological mechanisms involved.

In this study, we addressed the functional significance of co-operative DNA binding of the cytokine-driven transcription factor STAT1 (signal transducer and activator of transcription 1) in an experimental murine model of acute myocardial infarction (MI). STAT1 knock-in mice expressing a phenylalanine-to-alanine substitution at position 77 in the STAT1 amino-terminal domain were examined for the early clinical effects produced by ligation of the left anterior descending coronary artery (LAD), an established model for MI. The F77A mutation has been previously reported to disrupt amino-terminal interactions between adjacent STAT1 dimers resulting in impaired tetramerization and defective co-operative binding on DNA, while leaving other protein functions unaffected. Our results demonstrate that a loss of STAT1 tetramer stabilization improves survival of adult male mice and ameliorates left ventricular dysfunction in female mice, as determined echocardiographically by an increased ejection fraction and a reduced left intra-ventricular diameter. We found that the ratio of STAT3 to STAT1 protein level was higher in the infarcted tissue in knock-in mice as compared to wild-type (WT) mice, which was accompanied by an enhanced infiltration of immune cells in the infarcted area, as determined by histology. Additionally, RNA sequencing of the infarcted tissue 24 h after LAD ligation revealed an upregulation of inflammatory genes in the knock-in mice, as compared to their WT littermates. Concomitantly, genes involved in oxidative phosphorylation and other metabolic pathways showed a significantly more pronounced downregulation in the infarcted tissue from STAT1F77A/F77A mice than in WT animals. Based on these results, we propose that dysfunctional STAT1 signalling owing to a lack of oligomerisation results in a compensatory increase in STAT3 expression and promotes early infiltration of immune cells in the infarcted area, which has beneficial effects on left ventricular remodelling in early MI following LAD ligation.

Unphosphorylated signal transducer and activator of transcription 1 (U-STAT1) has been reported to elicit a distinct gene expression profile as compared to tyrosine-phosphorylated STAT1 (P-STAT1) homodimers. However, the impact of U-STAT1 on the IFNγ-induced immune response mediated by P-STAT1 is unknown. By generating a double mutant of STAT1 with mutation R602L in the Src-homology 2 (SH2) domain and Y701F in the carboxy-terminal transactivation domain mimicking U-STAT1, we investigated the effects of U-STAT1 on P-STAT1-mediated signal transduction. In this study, we discovered a novel activity of U-STAT1 that alters the nucleo-cytoplasmic distribution of cytokine-stimulated P-STAT1. While the dimerization-deficient mutant R602L/Y701F was not able to display cytokine-induced nuclear accumulation, it inhibited the nuclear accumulation of co-expressed IFNγ-stimulated wild-type P-STAT1. Disruption of the anti-parallel dimer interface in the R602L/Y701F mutant via additional R274W and T385A mutations did not rescue the impaired nuclear accumulation of co-expressed P-STAT1. The mutant U-STAT1 affected neither the binding of co-expressed P-STAT1 to gamma-activated sites in vitro, nor the transcription of reporter constructs and the activation of STAT1 target genes. However, the nuclear accumulation of P-STAT1 was diminished in the presence of mutant U-STAT1, which was not restored by mutations reducing the DNA affinity of mutant U-STAT1. Whereas single mutations in the amino-terminus of dimerization-deficient U-STAT1 similarly inhibited the nuclear accumulation of co-expressed P-STAT1, a complete deletion of the amino-terminus restored cytokine-stimulated nuclear accumulation of P-STAT1. Likewise, the disruption of a dimer-specific nuclear localization signal also rescued the U-STAT1-mediated inhibition of P-STAT1 nuclear accumulation. Our data demonstrate a novel role of U-STAT1 in affecting nuclear accumulation of P-STAT1, such that a high intracellular concentration of U-STAT1 inhibits the detection of nuclear P-STAT1 in immunofluorescence assays. These observations hint at a possible physiological function of U-STAT1 in buffering the nuclear import of P-STAT1, while preserving IFNγ-induced gene expression. Based on these results, we propose a model of a hypothetical import structure, the assembly of which is impaired under high concentrations of U-STAT1. This mechanism maintains high levels of cytoplasmic STAT1, while simultaneously retaining signal transduction by IFNγ.

OBJECTIVES:Patients undergoing coronary artery bypass graft (CABG) surgery are exposed to multiple treatment-related stressors, which can impact coping and health-related quality of life (HRQoL). The objective of this trial was to analyse the feasibility and preliminary efficacy of a multi-component intervention that combines psychological support and reduction of hospital-specific stressors on HRQoL, length of hospital and intensive care unit stay, self-efficacy, and plasma interleukin (IL)-6 and -8 levels in CABG patients.METHODS:This three-arm, randomized controlled, single-centre pilot trial assessed the Intervention for CABG to Optimize Patient Experience in 88 patients undergoing elective CABG. Standard medical care (SMC, n = 29) was compared with 2 intervention groups: (i) psychological interventions to optimize treatment expectations (IA group, n = 30) and (ii) multi-component intervention (IB group, n = 29) with psychological interventions plus an additional treatment package (light therapy, noise reduction, music, and if desired, 360° images delivered via virtual reality).RESULTS:The implementation of psychological interventions in routine medical treatment was feasible (91.5% of participants completed all intervention sessions). Both interventions were associated with significantly shorter hospital stay compared to SMC (IA/IB 9.8/9.3 days vs SMC 12.5 days). Self-efficacy expectations at post-surgery were significantly higher compared to SMC both in the IA group (P = 0.011) and marginally in the IB group (P = 0.051). However, there were no treatment effects of the interventions on HRQoL and plasma levels of IL-6 or IL-8 after CABG.CONCLUSIONS:A perioperative multi-component intervention may lead to shorter hospital stay and higher self-efficacy after CABG. Further studies are needed to determine its impact on HRQoL and inflammation.CLINICAL TRIAL REGISTRATION NUMBER:Ethical approval (# 21/2/18) for the study was obtained from the Research Ethics Committee of the University of Göttingen Medical Center, and the trial was registered in the German Clinical Trials Register (DRKS00015309, https://www.drks.de/drks_web/setLocale_EN.do).

Background Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that promotes cell proliferation and immunomodulation in untransformed cells and maintains stemness of transformed cells, facilitating invasion and metastasis. Numerous point mutations in the STAT3 protein have been identified that drive malignancy in various tumor entities. The missense mutation D427H localized in the STAT3 DNA-binding domain has been previously reported in patients with NK/T cell lymphomas. To assess the biological activity of this missense mutation, we compared the STAT3-D427H mutant to wild-type (WT) protein as well as the known hyper-active mutant F174A. Results Although previously reported as an activating mutation, the STAT3-D427H mutant neither showed elevated cytokine-induced tyrosine phosphorylation nor altered nuclear accumulation, as compared to the WT protein. However, the D427H mutant displayed enhanced binding to STAT-specific DNA-binding sites but a reduced sequence specificity and dissociation rate from DNA, which was demonstrated by electrophoretic mobility shift assays. This observation is consistent with the phenotype of the homologous E421K mutation in the STAT1 protein, which also displayed enhanced binding to DNA but lacked a corresponding increase in transcriptional activity. Conclusions Based on our data, it is unlikely that the D427H missense mutation in the STAT3 protein possesses an oncogenic potential beyond the WT molecule.

Objective: The vagus nerve constitutes the main component of the parasympathetic nervous system and plays an important role in the regulation of neuro-immune responses. Invasive stimulation of the vagus nerve produces anti-inflammatory effects; however, data on humoral immune responses of transcutaneous vagus nerve stimulation (tVNS) are rare. Therefore, the present study investigated changes in serum cytokine concentrations of interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor α (TNFα) following a short-term, non-invasive stimulation of the vagus nerve. Methods: Whole blood samples were collected before and after a short-lived application of active tVNS at the inner tragus as well as sham stimulation of the earlobe. Cytokine serum concentrations were determined in two healthy cohorts of younger (n = 20) and older participants (n = 19). Differences between active and sham conditions were analyzed using linear mixed models and post hoc F tests after applying Yeo-Johnson power transformations. This trial was part of a larger study registered on ClinicalTrials.gov (NCT05007743). Results: In the young cohort, IL-6 and IL-1β concentrations were significantly increased after active stimulation, whereas they were slightly decreased after sham stimulation (IL-6: p = 0.012; IL-1β: p = 0.012). Likewise, in the older cohort, IL-1β and IL-8 concentrations were significantly elevated after active stimulation and reduced after sham application (IL-8: p = 0.007; IL-1β: p = 0.001). In contrast, circulating TNFα concentrations did not change significantly in either group. Conclusion: Our results show that active tVNS led to an immediate increase in the serum concentrations of certain pro-inflammatory cytokines such as IL-1β, IL-6, and/or IL-8 in two independent cohorts of healthy study participants.

This paper presents data from a transcutaneous vagus nerve stimulation experiment that point towards a blunted cardiac baroreceptor sensitivity (cBRS) in young males compared to females during electrical stimulation of the forearm and a rhythmic breathing task. Continuous electrocardiography, impedance cardiography and continuous blood-pressure recordings were assessed in a sex-matched cohort of twenty young healthy subjects. Electrical stimulation of the median nerve was conducted by using a threshold-tracking method combined with two rhythmic breathing tasks (0.1 and 0.2 Hz) before, during and after active or sham transcutaneous vagus nerve stimulation. Autonomic and hemodynamic parameters were calculated, and differences were analyzed by using linear mixed models and post hoc F-tests. None of the autonomic and hemodynamic parameters differed between the sham and active conditions. However, compared to females, male participants had an overall lower total cBRS independent of stimulation condition during nerve stimulation (females: 14.96 ± 5.67 ms/mmHg, males: 11.89 ± 3.24 ms/mmHg, p = 0.031) and rhythmic breathing at 0.2 Hz (females: 21.49 ± 8.47 ms/mmHg, males: 15.12 ± 5.70 ms/mmHg, p = 0.004). Whereas vagus nerve stimulation at the left inner tragus did not affect the efferent vagal control of the heart, we found similar patterns of baroreceptor sensitivity activation over the stimulation period in both sexes, which, however, significantly differed in their magnitude, with females showing an overall higher cBRS.

Studies have reported controversial results on the relationship between headache and blood pressure. The aim of this post hoc study was twofold: first, to further investigate this relationship and, second, to assess the impact of psychosocial factors on this association in a population-based study of German children and adolescents. The analysis was conducted on study participants aged between 11 and 17 years (n = 5221, weighted from the total study cohort) from the nationwide German Health Interview and Examination Survey for Children and Adolescents (KiGGS). Health-related quality of life was assessed by self- and parent-rated German-language KINDL-R questionnaires (Children’s Quality of Life Questionnaire), while mental problems were analyzed using the Strengths and Difficulties Questionnaire (SDQ). Our findings confirmed that blood pressure was significantly lower in adolescents reporting episodes of headache than in those without headache (114.0 ± 10.2 mmHg vs. 115.5 ± 11.0 mmHg, p < 0.001). Logistic regression models adjusted to sex, age, body mass index, contraceptive use, and serum magnesium concentration demonstrated that headache was significantly associated with self-rated KINDL-R (Exp(B) = 0.96, 95% confidence interval (95% Cl) = 0.96–0.97, p < 0.001), parent-rated KINDL-R (Exp(B) = 0.97, 95% CI = 0.96–0.98, p < 0.001), as well as self-rated SDQ (Exp(B) = 1.08, 95% CI = 1.07–1.10, p < 0.001), and parent-rated SDQ (Exp(B) = 1.05, 95% CI = 1.04–1.06, p < 0.001). There was evidence that quality of life and mental problems mediated the effect of blood pressure on headache, as revealed by mediation models. Our results from the nationwide, representative KiGGS survey showed that low blood pressure is a significant predictor of headache, independent of quality of life and mental problems. However, these psychosocial factors may mediate the effect of blood pressure on headache in a still unknown manner.

Signal transducer and activator of transcription 3 (STAT3) gain-of-function mutations have been widely reported in patients with tumors and haematological malignancies. However, the molecular mechanisms of these pathogenic mutations remain largely uninvestigated. In this study, we have extensively characterized two STAT3 missense mutations, namely a valine-to-alanine exchange in the amino-terminal region (V77A) and a phenylalanine-to-alanine substitution (F174A) in the coiled-coil domain. The two mutants displayed elevated levels of tyrosine phosphorylation, premature nuclear accumulation, and differential transcriptional responses following stimulation of cells with interleukin-6 and interferon-ɣ. In line with their hyper-phosphorylated status, a greater fraction of V77A and F174A proteins was bound to DNA on high-affinity binding sites termed sis-inducible elements (SIE) as compared to the wild-type (WT) protein. Unexpectedly, these STAT3 variants displayed similar kinetics using in vitro kinase and dephosphorylation assays performed with recombinant Janus kinase 2 (JAK2) and Tc45 phosphatase, respectively. This indicates that the two mutations neither affected the susceptibility of STAT3 to the enzymatic activity of the inactivating tyrosine phosphatase nor to the activating kinase. However, experiments triggering intracellular dephosphorylation by the addition of the tyrosine-kinase inhibitor staurosporine to cytokine-pretreated cells showed that the two mutants partially resisted dephosphorylation. From these data, we propose that the F174A missense mutation hinders the exchange from a parallel to an anti-parallel dimer conformation, thereby increasing the ratio of tyrosine-phosphorylated molecules bound to DNA and enhancing gene-dependent transcription. Our data point to the physiological importance of the anti-parallel dimer conformation in the inactivation of the cytokine-induced STAT3 signalling pathway.

Genetic variations affecting the course of depressive symptoms in patients with coronary artery disease (CAD) have not yet been well studied. Therefore, we set out to investigate whether distinct haplotypes of the two insertion/deletion polymorphisms in the serotonin-transporter-linked polymorphic region (5-HTTLPR) and the angiotensin I-converting enzyme (ACE) gene located on chromosome 17 can be identified as risk factors for trajectories of depression. Clinical and genotyping data were derived from 507 depressed CAD patients participating in the randomized, controlled, multicenter Stepwise Psychotherapy Intervention for Reducing Risk in Coronary Artery Disease (SPIRR-CAD) trial, of whom the majority had an acute cardiac event before study inclusion. Depression scores on the Hospital Anxiety and Depression Scale (HADS) were assessed at baseline and at five follow-up time points up to 2 years after study entrance. At baseline, depression scores did not significantly differ between patients carrying the risk haplotype ACE D/D, 5-HTTLPR I/I (n = 46) and the non-risk haplotypes (n = 461, 10.9 ± 2.7 versus 10.4 ± 2.5, p = 0.254). HADS-depression scores declined from study inclusion during the first year irrespective of the genotype. At each follow-up time point, HADS-depression scores were significantly higher in ACE D/D, 5-HTTLPR I/I carriers than in their counterparts. Two years after study inclusion, the mean HADS depression score remained 1.8 points higher in patients with the risk haplotype as compared to subjects not carrying this haplotype (9.9 ± 4.2 versus 8.1 ± 4.0, p = 0.009). In summary, the presence of the ACE D/D, 5-HTTLPR I/I haplotype may be a vulnerability factor for comorbid depressive symptoms in CAD patients.

Heterozygous gain-of-function (GOF) mutations in the interferon-driven transcription factor STAT1 (signal transducer and activator of transcription 1) cause chronic mucocutaneous candidiasis (CMC). In this study, we characterized the molecular basis of a CMC-associated missense mutation by introducing a threonine-to-alanine exchange in the STAT1 DNA-binding domain at position 387. This substitution had previously been described in a CMC patient with suppurative eyelid infection and cutaneous abscesses, which are both unusual symptoms in this immunodeficiency. The STAT1-T387A mutant generated was compared to the wild-type protein and, in addition, to the missense mutant in the neighbouring position 386. Our results showed that the T387A mutant displayed distinct properties different from the wild-type molecule, namely elevated levels of tyrosine phosphorylation in conjunction with increased DNA-binding activity, hyperactive transcriptional regulation, and prolonged nuclear accumulation. The elevated tyrosine phosphorylation of the T387A mutant did not result in an increased mRNA production above the level of the wild-type molecule for all transcripts tested, indicating that the transcriptional activity of this mutant is largely gene-dependent. Nonetheless, these data demonstrate that the pathogenic T387A mutation associated with an atypical CMC symptomatology is biochemically similar to other well-characterized GOF mutants, while the H386A mutant was indistinguishable from the wild-type molecule. Our findings are in line with the assumption that the phenotype of this dominant STAT1 GOF mutation probably results from a disturbed shift in the equilibrium between the parallel and antiparallel dimer conformation, which is required for physiological gene activation.

Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.