Selenomethionine mis-incorporation and redox-dependent voltage-gated sodium channel gain of function.

Selenomethionine (SeMet) readily replaces methionine (Met) residues in proteins during translation. Long-term dietary SeMet intake results in the accumulation of the amino acid in tissue proteins. Despite the high rates of SeMet incorporation in proteins and its stronger susceptibility to oxidation compared to Met, little is known about the effect of SeMet mis-incorporation on electrical excitability and ion channels. Fast inactivation of voltage-gated sodium (NaV ) channels is essential for exact action potential shaping with even minute impairment of inactivation resulting in a plethora of adverse phenotypes. Met oxidation of the NaV channel inactivation motif (Ile-Phe-Met) and further Met residues causes a marked loss of inactivation. Here, we examined the impact of SeMet mis-incorporation on the function of NaV channels. While extensive SeMet incorporation into recombinant rat NaV 1.4 channels preserved their normal function, it greatly sensitized the channels to mild oxidative stress, resulting in loss of inactivation and diminished maximal current, both reversible by dithiothreitol-induced reduction. SeMet incorporation similarly affected human NaV 1.4, NaV 1.2, NaV 1.5, and NaV 1.7. In mouse dorsal root ganglia (DRG) neurons, 1 day of SeMet exposure exacerbated the oxidation-mediated broadening of action potentials. SeMet-treated DRGs also exhibited a stronger increase in the persistent NaV current in response to oxidation. SeMet incorporation in NaV proteins coinciding with oxidative insults may therefore result in hyperexcitability pathologies, such as cardiac arrhythmias and neuropathies, like congenital NaV channel gain-of-function mutations.

Serum matrix metalloproteinase-3 levels monitor the therapeutic efficacy in Syrian patients with rheumatoid arthritis.

Background: The plans for the successful treatment of rheumatoid arthritis (RA) seek to attain low disease activity or reach clinical remission. Objective: Our study aimed to compare the serum MMP-3 levels with predictors of response to therapy of rheumatoid arthritis in Syrian patients and explore its worth as a new valuable biomarker for RA therapy outcomes in daily practice. Methods: Serum samples were gathered from 43 RA patients at diagnosis and 12 weeks of therapy. Related clinical and laboratory tests were estimated, levels of serum MMP-3 were measured by ELISA method and the disease activity was assessed using disease activity scores in 28 joints with an erythrocyte sedimentation rate (DAS28-ESR) before and after therapy. Results: The mean of Serum MMP-3 levels significantly decreased (322.3 ± 43.83 ng/ml) after therapy (12 weeks) in RA patients compared to its mean at baseline (486.49 ± 34.5 ng/ml). There wasn't a statistically significant difference in the mean of MMP-3 levels before and after therapy (P = 0.137) in non-responder patients. Patients who showed a good response (N = 38) presented higher MMP-3 levels at first which subsequently decreased significantly at the 12-week follow-up (P < 0.05). Also, there was a statistically significant difference in MMP-3 levels between the two groups of patients after therapy (P = 0.002). To differentiate between RA patients who responded to therapy and who did not, our results found that the cut-off value of serum MMP-3 was 317.8 ng/ml (sensitivity was 80%, specificity was 73%, AUC was 0.818, 95% CI: 1.114-112.5; P = 0.045) and the best cut-off value of DAS28-ESR was 5.325 (sensitivity 100%, specificity 100%, AUC = 100%,95% CI: 15.2 to 47203.8). Conclusion: serum MMP-3 can be added as a novel and valuable biomarker in estimating the therapeutic response in RA patients, but it isn't better than DAS28-ESR.

The association of serum RANKL levels with disease activity and hematological parameters in Syrian patients with rheumatoid arthritis.

Our study aims to detect whether the serum RANKL could be a novel potential biomarker for activity and diagnosis of rheumatoid arthritis (RA). It included fifty-eight of RA patients and thirty of equal age and sex matched controls. Disease activity was determined by using DAS28-ESR. Serum Levels of RANKL were assayed by ELISA and compared with parameters such as ESR, CRP, Rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA). The serum RANKL levels were higher in RA patients compared to controls. There was an increase in its levels mean among post-menopausal patients compared to post-menopausal healthy group. RANKL levels were also higher in ACPA positive patients than ACPA negative. Our study found a correlation between RANKL levels and some parameters: DAS28, ACPA, CRP, and symptom duration. There was a moderate inverse correlation between RANKL levels and BMD. By ROC curve, our results displayed that the best cut-off value of RANKL was 178.99 pg/ml (sensitivity 79.31%; specificity 90%) to differentiate between RA patients and controls. In conclusion, elevated serum RANKL can be used as an indicator of disease activity and a diagnostic new biomarker in patients with early RA.

Selenomethionine incorporation in proteins of individual mammalian cells determined with a genetically encoded fluorescent sensor.

Selenomethionine (SeMet) randomly replaces methionine (Met) in protein translation. Because of strongly differing redox properties of SeMet and Met, SeMet mis-incorporation may have detrimental effects on protein function, possibly compromising the use of nutritional SeMet supplementation as an anti-oxidant. Studying the functional impact of SeMet in proteins on a cellular level is hampered by the lack of accurate and efficient methods for estimating the SeMet incorporation level in individual viable cells. Here we introduce and apply a method to measure the extent of SeMet incorporation in cellular proteins by utilizing a genetically encoded fluorescent methionine oxidation probe. Supplementation of SeMet in mammalian culture medium resulted in >84% incorporation of SeMet, and SeMet labeling as low as 5% was readily measured. Kinetics and extent of SeMet incorporation on the single-cell level under live-cell imaging conditions provided direct access to protein turn-over kinetics and SeMet redox properties in a cellular context. The method is furthermore suited for experiments utilizing high-throughput fluorescence microplate readers or fluorescence-activated cell sorting (FACS) analysis.

A GFP-based ratiometric sensor for cellular methionine oxidation.

Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with ∼400 and ∼470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.

Fine-Tuning of Neuronal Ion Channels-Mapping of Residues Involved in Glucose Sensitivity of Recombinant Human Glycine Receptors.

The inhibitory glycine receptor (GlyR) mediates synaptic inhibition in the spinal cord, brain stem, and other regions of the mammalian central nervous system. Glucose was shown to potentiate α1 GlyRs by interacting with K143. Here, additional amino acids involved in glucose modulation were identified using a structure-based approach of site-directed mutagenesis followed by whole-cell patch-clamp analysis. We identified two additional lysine residues in the α1 GlyR extracellular domain, K16 and K281, that were involved in glucose modulation. Mutation of either residue to alanine abolished glucose potentiation. Residue K281 is located in the same pocket as K143 and could thus contribute to glucose binding. The double mutant K143A-K281A showed a 6-fold increase of EC50, while EC50 of both single mutants K143A and K281A was only slightly increased (1.7- and 1.3-fold, respectively). K16 is located at an analgesic binding site that is distant from the agonist or glucose sites, and the K16A mutation may generate a receptor species that is not potentiated. GlyR position α1-S267 is close to the postulated glucose binding site and known for interactions with ethanol and anesthetics. In the presence of glucose, GlyR α1 mutants S267A, S267I, and S267R showed potentiation, no effect, and reduction of current responses, respectively. This pattern follows that of ethanol modulation and suggests that the interaction sites of glucose and ethanol are identical or located close to each other. Our results support the presence of a distinct binding site for glucose on the glycine receptor, overlapping with the ivermectin/ethanol binding pocket near the transmembrane region and the TM2-3 loop.

The Effects of Genetic and Epigenetic Alterations of BARD1 on the Development of Non-Breast and Non-Gynecological Cancers.

Breast Cancer 1 (BRCA1) gene is a well-characterized tumor suppressor gene, mutations of which are primarily found in women with breast and ovarian cancers. BRCA1-associated RING domain 1 (BARD1) gene has also been identified as an important tumor suppressor gene in breast, ovarian, and uterine cancers. Underscoring the functional significance of the BRCA1 and BARD1 interactions, prevalent mutations in the BRCA1 gene are found in its RING domain, through which it binds the RING domain of BARD1. BARD1-BRCA1 heterodimer plays a crucial role in a variety of DNA damage response (DDR) pathways, including DNA damage checkpoint and homologous recombination (HR). However, many mutations in both BARD1 and BRCA1 also exist in other domains that significantly affect their biological functions. Intriguingly, recent genome-wide studies have identified various single nucleotide polymorphisms (SNPs), genetic alterations, and epigenetic modifications in or near the BARD1 gene that manifested profound effects on tumorigenesis in a variety of non-breast and non-gynecological cancers. In this review, we will briefly discuss the molecular functions of BARD1, including its BRCA1-dependent as well as BRCA1-independent functions. We will then focus on evaluating the common BARD1 related SNPs as well as genetic and epigenetic changes that occur in the non-BRCA1-dominant cancers, including neuroblastoma, lung, and gastrointestinal cancers. Furthermore, the pro- and anti-tumorigenic functions of different SNPs and BARD1 variants will also be discussed.

Modulation of Glycine Receptor-Mediated Pain Signaling in vitro and in vivo by Glucose.

The inhibitory glycine receptor (GlyR) plays an important role in rapid synaptic inhibition in mammalian spinal cord, brainstem, higher brain centers, and is involved in transmission of nociceptive signals. Glucose and related mono- and disaccharides potentiate currents mediated by recombinant α1, α1-ß, and α3 GlyRs. Here, we confirmed the specific potentiation of α3 GlyR signaling by glucose through: (i) patch-clamp electrophysiology on recombinant receptors; and (ii) by verifying in vitro data in a mouse model in vivo. Mice were intraperitoneally (IP) injected with glucose (2 g/kg) or vehicle, and then challenged with sublethal doses of strychnine (0.2 mg/kg and 0.5 mg/kg). Pain-related behavior was assessed using two established models: (i) touch sensitivity tests using von Frey filaments; and (ii) hotplate assay. We observed a reduction of pain sensitivity in glucose-treated mice relative to vehicle-treated control mice. Injection of strychnine resulted in an increased sensitivity to tactile and heat stimuli, which was reversed in the presence of glucose. Analgesic effects of glucose were more pronounced in von Frey experiments, consistent with the established use of this model for neuropathic pain. Overall, glucose showed mild analgesic effects and was able to compensate for strychnine-induced allodynia in mice. Since the action of strychnine is specific for GlyR, these experiments show for the first time an in vivo potentiation of GlyR activity by glucose and suggest a molecular mechanism for glucose-mediated analgesia.

Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.