Intermolecular disulfide bond influences unphosphorylated STAT3 dimerization and function

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. Recently, the roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in the maintenance of heterochromatin stability. It has been reported that U-STAT3 dimerizes, shuttles between the cytoplasm and nucleus, and binds to DNA, thereby driving genes transcription. Although many reports describe the active role of U-STAT3 in oncogenesis in addition to phosphorylated STAT3, the U-STAT3 functional pathway remains elusive.In this report, we describe the molecular mechanism of U-STAT3 dimerization, and we identify the presence of two intermolecular disulfide bridges between Cys367 and Cys542 and Cys418 and Cys426, respectively. Recently, we reported that the same cysteines contribute to the redox regulation of STAT3 signaling pathway both in vitro and in vivo The presence of these disulfides is here demonstrated to largely contribute to the structure and the stability of U-STAT3 dimer as the dimeric form rapidly dissociates upon reduction in the S-S bonds. In particular, the Cys367-Cys542 disulfide bridge is shown to be critical for U-STAT3 DNA-binding activity. Mutation of the two Cys residues completely abolishes the DNA-binding capability of U-STAT3. Spectroscopic investigations confirm that the noncovalent interactions are sufficient for proper folding and dimer formation, but that the interchain disulfide bonds are crucial to preserve the functional dimer. Finally, we propose a reaction scheme of U-STAT3 dimerization with a first common step followed by stabilization through the formation of interchain disulfide bond

Effects of pet exposure in the first year of life on respiratory and allergic symptoms in 7-yr-old children. The SIDRIA-2 study

The effects of pet exposure on the development of respiratory symptoms have recently been the matter of vivid discussion. Our objective was to determine the effects of exposure to cat or dog in the first year of life on subsequent respiratory/allergic symptoms in children in a large Italian multicentre study. As part of the SIDRIA-2 Study (Studi Italiani sui Disturbi Respiratori dell'Infanzia e l'Ambiente 2002), the parents of 20016 children (median age 7 yr) provided information on indoor exposures at different times in life and respiratory/allergic symptoms through questionnaires. Logistic regression analyses were performed taking into account cat or dog exposure at different times in life and adjusting for the presence of the other pet, mould exposure, gender, age, parental education, maternal smoking during the first year of life, current passive smoking, family history of asthma/rhinitis/eczema and other potential confounders. Neither significant effects of dog exposure in the first year of life nor in other periods were found on respiratory/allergic symptoms after adjusting for the other covariates. Cat exposure in the first year of life was significantly and independently associated with current wheezing [OR (95% CI) 1.88 (1.33-2.68), p < 0.001] and current asthma [1.74 (1.10-2.78), p < 0.05] and border-line associated with current rhinoconjunctivitis [1.43 (0.97-2.11), p = 0.07]. No other effects of cat exposure were found on respiratory/allergic symptoms. Cat, but not dog, exposure in the first year of life is an independent risk factor for current wheezing, current asthma and current rhinoconjunctivitis at the age of 7

Profile, Healthcare Resource Consumption and Related Costs in ANCA-Associated Vasculitis Patients: A Real-World Analysis in Italy

Introduction: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are rare autoimmune diseases triggering inflammation of small vessels. This real-world analysis was focused on the most common AAV forms, granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), to describe patients’ demographic and clinical characteristics, therapeutic management, disease progression, and the related economic burden. Methods: A retrospective analysis was conducted on administrative databases of a representative sample of Italian healthcare entities, covering approximately 12 million residents. Between January 2010 and December 2020, adult GPA patients were identified by payment waiver code or hospitalization discharge diagnosis, and MPA patients by payment waiver code with or without hospitalization discharge diagnosis. Clinical outcomes were evaluated through AAV-related hospitalizations, renal failure onset, and mortality. Economic analysis included healthcare resource utilization deriving from drugs, hospitalizations, and outpatient specialist services. The related mean direct costs year/patient were also calculated in patients stratified by presence/absence of glucocorticoid therapy and type of inclusion criterion (hospitalization/payment waiver code). Results: Overall, 859 AAV patients were divided into GPA (n = 713; 83%) and MPA (n = 146; 17%) cohorts. Outcome indicators highlighted a clinically worse phenotype associated with GPA compared to MPA. Cost analysis during follow-up showed tendentially increased expenditures in glucocorticoid-treated patients versus untreated (overall AAV: €8728 vs. €7911; GPA: €9292 vs. €9143; MPA: €5967 vs. €2390), mainly driven by drugs (AAV: €2404 vs. €874; GPA: €2510 vs. €878; MPA: €1881 vs. €854) and hospitalizations. Conclusion: Among AAV forms, GPA resulted in a worse clinical picture, higher mortality, and increased costs. This is the first real-world pharmaco-economic analysis on AAV patients stratified by glucocorticoid use on disease management expenditures. In both GPA and MPA patients, glucocorticoid treatment resulted in higher healthcare costs, mostly attributable to medications, and then hospitalizations, confirming the clinical complexity and economic burden for management of patients with autoimmune diseases under chronic immunosuppression

Predictors of children's secondhand smoke exposure at home: a systematic review and narrative synthesis of the evidence

BACKGROUND: Children's exposure to secondhand smoke (SHS) has been causally linked to a number of childhood morbidities and mortalities. Over 50% of UK children whose parents are smokers are regularly exposed to SHS at home. No previous review has identified the factors associated with children's SHS exposure in the home. AIM: To identify by systematic review, the factors which are associated with children's SHS exposure in the home, determined by parent or child reports and/or biochemically validated measures including cotinine, carbon monoxide or home air particulate matter. METHODS: Electronic searches of MEDLINE, EMBASE, PsychINFO, CINAHL and Web of Knowledge to July 2014, and hand searches of reference lists from publications included in the review were conducted. FINDINGS: Forty one studies were included in the review. Parental smoking, low socioeconomic status and being less educated were all frequently and consistently found to be independently associated with children's SHS exposure in the home. Children whose parents held more negative attitudes towards SHS were less likely to be exposed. Associations were strongest for parental cigarette smoking status; compared to children of non-smokers, those whose mothers or both parents smoked were between two and 13 times more likely to be exposed to SHS. CONCLUSION: Multiple factors are associated with child SHS exposure in the home; the best way to reduce child SHS exposure in the home is for smoking parents to quit. If parents are unable or unwilling to stop smoking, they should instigate smoke-free homes. Interventions targeted towards the socially disadvantaged parents aiming to change attitudes to smoking in the presence of children and providing practical support to help parents smoke outside the home may be beneficial

Biophysical and biochemical characterization of two novel GCAP1 mutants associated with cone-rod dystrophy

Purpose Guanylate Cyclase Activating Protein 1 (GCAP1) is a Ca2+-sensor protein involved in the regulation of Guanylate Cyclase (GC) during the phototransduction cascade, which initiates the visual process. An increasing number of GCAP1 mutants has been found to be associated with degenerative retinal diseases such as cone dystrophy (COD) and cone-rod dystrophy (CORD). This study is focused on the structural and functional characterization of two novel CORD-associated GCAP1 mutants, namely I107T and L84F. Methods Circular dichroism spectroscopy was employed to investigate changes in protein secondary and tertiary structure and in thermal stability both in the absence and in the presence of physiological 1 mM Magnesium (Mg2+) and saturating Ca2+ concentration. Variations in hydrodynamic radius of the mutants in the aforementioned conditions was monitored by dynamic light scattering. Ca2+-binding constants were estimated by a chromophoric chelator assay. The conformational transition range upon Mg2+ or Ca2+ binding was investigated by monitoring the tryptophan fluorescence in titration experiments. Results I107T-GCAP1 exhibited similarities with the wild type in terms of conformational and hydrodynamic radius changes upon Mg2+ or Ca2+ binding, while its Ca2+ affinity was severely impaired and its stability was increased independently on the presence of Ca2+ . Ca2+ fluorescence titrations showed a biphasic pattern similar to the COD-associated G159V mutant. L84F-GCAP1 showed structural features significantly different from the wild type, with small differences in secondary structure but major differences in tertiary structure upon Ca2+ binding. Moreover this mutant showed higher thermal stability than the wild type particularly in the presence of Ca2+ and appeared to be oligomeric both in the presence and in the absence of Ca2+ or Mg2+. Conclusion Our results suggest that these two novel CORD-associated GCAP1 mutants could affect GC regulation via different processes. Indeed I107T-GCAP1 might alter the Ca2+ regulation of GC by its impaired Ca2+-sensitivity, while L84F-GCAP1 may lead to different supramolecular assemblies as a consequence of its oligomeric state

Structural effects of Mg2+ on the regulatory states of Neuronal Calcium Sensors operating in vertebrate phototransduction

Several lines of evidence suggest that free Mg2+ plays an important role in phototransduction, as the Neuronal Calcium Sensors (NCS) Recoverin and Guanylate Cyclase Activating Proteins 1 and 2 (GCAP1 and GCAP2) are also capable of binding Mg2+ via their EF-hand motifs. Previous studies showed that a Mg2+ -bound state is required for GCAP1 in order to activate GC and that Recoverin binds Mg2+ without triggering its physiological conformational change. No structural studies were performed so far about GCAP2, for which the effects that Mg2+ could exert were only hypothesized. Here we compared the effects of physiological [Mg2+] (1 mM) on the switch states of these three NCS in their myristoylated (myr) and non myristoylated (nonmyr) form over the extreme conditions of high and low [Ca2+], mimicking respectively dark and light states of the photoreceptor cell. We performed Circular Dichroism spectroscopy measurements to assess the differences in thermal stability, secondary and tertiary structure of all NCS in the aforementioned conditions. Intrinsic fluorescence spectroscopy titrations and Isothermal Titration Calorimetry were performed for monitoring the binding of Mg2+ to GCAP2. Molecular dynamics simulations (200 ns, all-atom force field) were performed to assess structural properties of GCAP1 in putatively activator, inhibitor and transitory states. Our results confirm that Mg2+ is unable to trigger the physiological conformational change of Recoverin (myristoyl switch) and that it decreases its thermal stability. Mg2+ induces a conformational change in GCAP2 both at high and low [Ca2+], however these variations are more substantial for apo-myrGCAP2. Apo-GCAP1 is responsive to Mg2+, acquiring a different tertiary structure from both apo and Ca2+-bound states, though this difference is lost when Ca2+ is saturating. GCAP1 seems to be stabilized by the presence of Mg2+ in solution, more notably its Ca2+-bound form. Molecular dynamics simulations point out that myrGCAP1 has a highly flexible loop (125-135) when at least one divalent cation is bound to EF-3. In line with experimental data, this is sufficient to stabilize the entire structure. Moreover all simulated transitory states show very similar dynamic properties, which differ from both apo and Ca2+- or Mg2+- loaded forms

Conformational Changes in Calcium-Sensor Proteins under Molecular Crowding Conditions.

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca2+ sensors, which undergo conformational changes in response to oscillating intracellular Ca2+ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca2+ -binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca2+ -sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca2+ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca2+ -occupied functional state under crowding conditions

Nanodevice-induced conformational and functional changes in a prototypical calcium sensor protein.

Calcium (Ca2+) plays a major role in a variety of cellular processes. Fine changes in its concentration are detected by calcium sensor proteins, which adopt specific conformations to regulate their molecular targets. Here, two distinct nanodevices were probed as biocompatible carriers of Ca2+-sensors and the structural and functional effects of protein-nanodevice interactions were investigated. The prototypical Ca2+-sensor recoverin (Rec) was incubated with 20-25 nm CaF2 nanoparticles (NPs) and 70-80 nm liposomes with lipid composition similar to that found in photoreceptor cells. Circular dichroism and fluorescence spectroscopy were used to characterize changes in the protein secondary and tertiary structure and in thermal stability upon interaction with the nanodevice, both in the presence and in the absence of free Ca2+. Variations in the hydrodynamic diameter of the complex were measured by dynamic light scattering and the residual capability of the protein to act as a Ca2+-sensor in the presence of NPs was estimated spectroscopically. The conformation, thermal stability and Ca2+-sensing capability of Rec were all significantly affected by the presence of NPs, while liposomes did not significantly perturb Rec conformation and function, allowing reversible binding. NP-bound Rec maintained an all-helical fold but showed lower thermal stability and high cooperativity of unfolding. Our analysis can be proficiently used to validate the biocompatibility of other nanodevices intended for biomedical applications involving Ca2+-sensors

Impact of cone dystrophy-related mutations in GCAP1 on a kinetic model of phototransduction.

Cone dystrophy-related mutations in guanylate cyclase-activating protein 1 (GCAP1) are known to cause severe disturbance of their Ca2+-sensing properties affecting also their regulatory modes. However, crucial biochemical properties of mutant GCAP1 forms have not been fully elucidated and regulatory parameters of GCAP1 mutants have not been considered within the context of a comprehensive description of the phototransduction cascade kinetics. We investigated therefore the structure-function relationships of four dystrophy-relevant point mutations in GCAP1 harboring the following amino acid substitutions: E89K, D100E, L151F, and G159V. All mutations decrease the catalytic efficiency in regulating the target guanylate cyclase and decrease the affinity of Ca2+-binding in at least one, but in most cases two EF-hand Ca2+-binding sites. Although the wild type and mutants of GCAP1 displayed large differences in Ca2+-binding and regulation, circular dichroism (CD) spectroscopy revealed that all proteins preserved an intact secondary and tertiary structure with a significant rearrangement of the aromatic residues upon binding of Ca2+. To gain insight into the dynamic changes of cyclic GMP levels in a photoreceptor cell, we incorporated parameters describing the regulation of target guanylate cyclase by GCAP1 mutants into a comprehensive kinetic model of phototransduction. Modeling led us to conclude that the contribution of GCAP1 to the dynamic synthesis of cyclic GMP in rod cells would depend on the expression level of the wild-type form. Although the synthesis rate controlled by GCAP1 remains at a constant level, in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca2+-sensor GCAP2