Transfected HEK293 Cells Research Articles

Role of sorting nexin adaptor proteins in GPCR regulation

Signaling by the G protein-coupled receptor CXCR4 is essential to a number of developmental and physiological processes, including stem cell mobilization, organ development, and immune responses. In addition, CXCR4 signaling has been linked to disease, including cancer and certain immune disorders. Despite this, the mechanisms governing CXCR4 signaling remain poorly defined. Typically, upon binding to its cognate ligand CXCL12, CXCR4 signaling occurs via G protein- and β-arrestin-dependent pathways. Although G protein-dependent CXCR4-mediated signaling is well understood, β-arrestin1-dependent signaling remains poorly understood. With this in mind, we performed proximity biotin labeling by ascorbic acid peroxidase (APEX2) fused to β-arrestin1 in order to identify proteins that are part of the β-arrestin1 interaction network downstream of CXCR4. Labeled proteins from cells treated with vehicle or CXCL12 for 5 or 60 minutes were selectively purified and identified by mass spectrometry. Several proteins known to be part of the b-arrestin network downstream of CXCR4 including STAM1 and FAK were identified. Several other proteins of biological relevance were identified that were enriched in cells treated with CXCL12 for 5 or 60 min. Orthogonal biochemical experiments verified that two of these proteins, WAVE2 and SNX9, interact with β-arrestin1. We selected SNX9 for further study of its functional role in CXCR4 signaling. SNX9 (sorting nexin 9), and related proteins SNX18 and SNX33, are multifunctional adaptor proteins best known for their role in clathrin-mediated endocytosis of multiple cell surface receptors. Whether they mediate endocytosis of GPCRs remains unknown. To address this, we transfected HEK293 cells stably expressing epitope-tagged CXCR4 with siRNA against SNX9, SNX18, and SNX33, and examined agonist-induced endocytosis of CXCR4 by whole cell ELISA. Simultaneous transfection with siRNA against SNX9 and SNX18 significantly reduced CXCR4 endocytosis when compared with control siRNA or when cells were transfected with individual siRNA against SNX9, SNX18 or SNX33. These data provide evidence for the first time for a role of members of the SNX9-family in GPCR endocytosis, likely by serving as adaptors for clathrin-mediated endocytosis. Additional work is needed to conclusively demonstrate that SNX9 functions as an adaptor protein for CXCR4 endocytosis and to determine the mechanism by which this occurs. To accomplish this we will use a combination of biochemical, molecular, imaging and biophysical approaches. In addition, we will investigate how broadly SNX9-family members impact CXCR4 endocytosis across multiple cell types and also address the impact of the SNX9-family on GPCR endocytosis in general.

Compensatory expression regulation of highly homologous proteins HNRNPA1 and HNRNPA2.

Heterogeneous nuclear ribonucleoprotein (HNRNP) A1 and A2 are the most abundant HNRNPs with nearly identical functions, and play important roles in regulating gene expression at multiple levels (i.e. transcription, posttranscription, and translation). However, the expression and regulation mechanism of HNRNPA1 and A2 themselves remain unclear. In this study, the amino acid sequences of HNRNPA1 and HNRNPA2 were compared and found to have 78% and 86% homology in key functional domains. Transfection of HEK293 cells with small interfering RNA and overexpression vectors of HNRNPA1 and HNRNPA2 demonstrated that HNRNPA1 and HNRNPA2 paralogs regulate each other’s expression in a compensatory manner at both the RNA and protein levels. Multiprimer reverse transcription-polymerase chain reaction showed that HNRNPA1 and HNRNPA2 did not affect splicing of the HNRNPA2 and HNRNPA1 gene. Using luciferase reporting system, we found that compensatory degradation was mediated by the 3′UTR of the two genes rather than by the promoter. Moreover, treatment with cycloheximide inhibited the compensatory regulation. Our results indicate a novel regulation mechanism of HNRNPA1 and A2 expression. Through compensatory regulation, the expression levels of HNRNPA1 and HNRNPA2 are strictly controlled within a certain range to maintain normal cellular activities under different physiological conditions.

8-Substituted Triazolobenzodiazepines: In Vitro and In Vivo Pharmacology in Relation to Structural Docking at the α1 Subunit-Containing GABAA Receptor.

In order to develop improved anxiolytic drugs, 8-substituted analogs of triazolam were synthesized in an effort to discover compounds with selectivity for α2/α3 subunit-containing GABAA subtypes. Two compounds in this series, XLi-JY-DMH (6-(2-chlorophenyl)-8-ethynyl-1-methyl-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine) and SH-TRI-108 [(E)-8-ethynyl-1-methyl-6-(pyridin-2-yl)-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine], were evaluated for in vitro and in vivo properties associated with GABAA subtype-selective ligands. In radioligand binding assays conducted in transfected HEK cells containing rat αXβ3γ2 subtypes (X = 1,2,3,5), no evidence of selectivity was obtained, although differences in potency relative to triazolam were observed overall (triazolam > XLi-JY-DMH > SH-TRI-108). In studies with rat αXβ3γ2 subtypes (X = 1,2,3,5) using patch-clamp electrophysiology, no differences in maximal potentiation of GABA-mediated Cl− current was obtained across subtypes for any compound. However, SH-TRI-108 demonstrated a 25-fold difference in functional potency between α1β3γ2 vs. α2β3γ2 subtypes. We evaluated the extent to which this potency difference translated into behavioral pharmacological differences in monkeys. In a rhesus monkey conflict model of anxiolytic-like effects, triazolam, XLi-JY-DMH, and SH-TR-108 increased rates of responding attenuated by shock (anti-conflict effect) but also attenuated non-suppressed responding. In a squirrel monkey observation procedure, both analogs engendered a profile of sedative-motor effects similar to that reported previously for triazolam. In molecular docking studies, we found that the interactions of the 8-ethynyl triazolobenzodiazepines with the C-loop of the α1GABAA site was stronger than that of imidazodiazepines XHe-II-053 and HZ-166, which may account for the non-sedating yet anxiolytic profile of these latter compounds when evaluated in previous studies.

Metabolism and transporter based drug–drug interaction of tacrolimus with nine co-medicated injections

BackgroundTacrolimus, also named FK506, is a well-known potent immunosuppressant agent, which is usually co-medicated with multiple drugs in solid organ transplant. The objective of this study was to evaluate DDI between tacrolimus and nine injections co-administered in patients receiving liver organ transplant. MethodsIn this study, the cytochrome P450 enzymes (CYPs) inhibition assay were conducted by using pooled human liver microsomes, for supporting the evaluation of metabolism-mediated DDI. The transport substrate assay used to determine the transporter-mediated DDI, was conducted in the transfected HEK293 cell line expressing specific human transport proteins. ResultsAll the tested drugs, especially alprostadil, methylprednisolone, and pantoprazole showed apparent inhibition on CYP3A4/5 with the half-maximal inhibitory concentration (IC50) from >0.5 μg/mL to 89.6 μg/mL, which also inhibited metabolism of tacrolimus in human liver microsomes, whose half time (T1/2) was prolonged from 5.04 to 35.3 min. However, tacrolimus was not the substrates of solute carrier (SLC) transporters including OATP1B1, OATP1B3, OAT1, OAT3, and OCT2, which indicated that the risk of transport-mediated DDI between tacrolimus and these nine drugs was likely to be low. ConclusionsThese findings indicate that the metabolism-based DDI between tacrolimus and co-administered nine drugs seems to be the major cause of the high individual variability of tacrolimus exposure in patients with solid organ transplant and should be further studied.

COR758, a negative allosteric modulator of GABAB receptors

Allosteric modulators of G protein coupled receptors (GPCRs), including GABABRs (GABABRs), are promising therapeutic candidates. While several positive allosteric modulators (PAM) of GABABRs have been characterized, only recently the first negative allosteric modulator (NAM) has been described. In the present study, we report the characterization of COR758, which acts as GABABR NAM in rat cortical membranes and CHO cells stably expressing GABABRs (CHO-GABAB). COR758 failed to displace the antagonist [3H]CGP54626 from the orthosteric binding site of GABABRs showing that it acts through an allosteric binding site. Docking studies revealed a possible new allosteric binding site for COR758 in the intrahelical pocket of the GABAB1 monomer. COR758 inhibited basal and GABABR-stimulated O-(3-[35Sthio)-triphosphate ([35S]GTPγS) binding in brain membranes and blocked the enhancement of GABABR-stimulated [35S]GTPγS binding by the PAM GS39783. Bioluminescent resonance energy transfer (BRET) measurements in CHO-GABAB cells showed that COR758 inhibited G protein activation by GABA and altered GABABR subunit rearrangements. Additionally, the compound altered GABABR-mediated signaling such as baclofen-induced inhibition of cAMP production in transfected HEK293 cells, agonist-induced Ca2+ mobilization as well as baclofen and the ago-PAM CGP7930 induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in CHO-GABAB cells. COR758 also prevented baclofen-induced outward currents recorded from rat dopamine neurons, substantiating its property as a NAM for GABABRs. Altogether, these data indicate that COR758 inhibits G protein signaling by GABABRs, likely by interacting with an allosteric binding-site. Therefore, COR758 might serve as a scaffold to develop additional NAMs for therapeutic intervention.

Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)—a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids—disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible β(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.

Response of TRPM2 Channel to Hypercapnic Acidosis and Role of Zn, Se, and GSH.

Hypercapnia can increase the production of reactive oxygen species (ROS) by inducing oxidative stress in cells. Transient receptor potential melastatin 2 (TRPM2) channel activation that is realized by ROS plays a critical role in the cellular mechanism. It was shown that antioxidants such as zinc (Zn), selenium (Se), and glutathione (GSH) can partake in the structures of enzymes and create a protective effect against oxidative stress. This study revealed the relationship between TRPM2 channel and hypercapnia, and the interaction of zinc, selenium, and glutathione. In our study, normoxia, hypercapnia, hypercapnia + Zn, hypercapnia + Se, and hypercapnia + GSH were created, in transfected HEK293 cells. The cells were exposed to normoxia or hypercapnia gasses in two different times (30 min and 60 min), while Zn, Se, and GSH were applied to the cells in the other groups before being exposed to the gas mixtures. The statistical evaluation showed a significant increase in lipid peroxidation (LPO) level and lactate dehydrogenase (LDH)% in the hypercapnia 30 min and 60 min groups, compared to the normoxia 30 min and 60 min groups, and an increase in LPO level and LDH% in the hypercapnia groups that Zn, Se, and GSH were applied. It was determined that in comparison with the normoxia 30 min and 60 min groups, the amount of inward Ca+2 current across TRPM2 channels and mean current density increased in the groups that were exposed to hypercapnia for 30 min and 60 min, while the same values significantly decreased in the hypercapnia groups that Zn, Se, and GSH were applied. Also, it was shown that oxidative stress rose as the duration of hypercapnia exposure increased. It was concluded that hypercapnia increased oxidative stress and caused cellular membrane damage, while the addition of Zn, Se, and GSH could protect the cell membrane from these damaging effects.

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

A SPRY1 domain cardiac ryanodine receptor variant associated with short-coupled torsade de pointes.

Idiopathic ventricular fibrillation (IVF) causes sudden death in young adult patients without structural or ischemic heart disease. Most IVF cases are sporadic and some patients present with short-coupled torsade de pointes, the genetics of which are poorly understood. A man who had a first syncope at the age of 35 presented with frequent short-coupled premature ventricular beats with bursts of polymorphic ventricular tachycardia and then died suddenly. By exome sequencing, we identified three rare variants: p.I784F in the SPRY1 of the ryanodine receptor 2 (RyR2), p.A96S in connexin 40 (Cx40), reported to affect electrical coupling and cardiac conduction, and a nonsense p.R244X in the cardiac-specific troponin I-interacting kinase (TNNI3K). We assessed intracellular Ca2+ handling in WT and mutant human RYR2 transfected HEK293 cells by fluorescent microscopy and an enhanced store overload-induced Ca2+ release in response to cytosolic Ca2+ was observed in RyR2-I784F cells. In addition, crystal structures and thermal melting temperatures revealed a conformational change in the I784F-SPRY1 domain compared to the WT-domain. The novel RyR2-I784F variant in SPRY1 domain causes a leaky channel under non-stress conditions. The presence of several variants affecting Ca2+ handling and cardiac conduction suggests a possible oligogenic origin for the ectopies originating from Purkinje fibres.

Identification and characterization of a novel 5-hydroxytryptamine receptor in the sea cucumber Apostichopus japonicus (Selenka).

Serotonin (5-hydroxytryptamine [5-HT]) receptors (5-HTRs) mediate neuroendocrine signaling via interactions with the ligand serotonin (5-HT). The 5-HT signaling system has been well studied in vertebrates, but rarely known in invertebrate animals, especially in the marine invertebrates. In this study, we identified and characterized a novel 5-HTR from the sea cucumber Apostichopus japonicus (Aj5-HT4/6 ). The cloned Aj5-HT4/6 open reading frame comprised 1290 bp and encoded 429 amino acids. Bioinformatic analysis of the receptor indicated that it was a member of the class A of the G protein-coupled receptor family. Further experiments using Aj5-HT4/6 -transfected HEK293 cells demonstrated that treatment with 5-HT could induce rapid internalization of Aj5-HT4/6 fused with enhanced green fluorescent protein from the cell surface into the cytoplasm and triggered a significant increase in levels of the second messenger cAMP as well as mitogen-activated protein kinase phosphorylation in a 5-HT dose-dependent manner. Quantitative real time-polymerase chain reaction demonstrated that Aj5-HT4/6 was predominantly expressed in the muscle and respiratory tree, and its expression was significantly decreased during estivation. Taken together, these results imply that Aj5-HT4/6 is potentially involved in the movement and metabolism of the sea cucumber.

Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter.

Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C4. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser612, Arg615, His622, Glu624) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg615 and Glu624 might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe619 and distal Lys406 in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.

Whole exome sequencing reveals a biallelic frameshift mutation in GRXCR2 in hearing impairment in Cameroon.

BackgroundHearing impairment (HI) genes are poorly studied in African populations.MethodsWe used whole exome sequencing (WES) to investigate pathogenic and likely pathogenic (PLP) variants in 10 individuals with HI, from four multiplex families from Cameroon, two of which were previously unresolved with a targeted gene enrichment (TGE) panel of 116 genes. In silico protein modelling, western blotting and live imaging of transfected HEK293 cells were performed to study protein structure and functions.ResultsAll PLP variants previously identified with TGE were replicated. In one previously unresolved family, we found a homozygous frameshift PLP variant in GRXCR2 (OMIM: 615762), NM_001080516.1(GRXCR2):c.251delC p.(Ile85SerfsTer33), in two affected siblings; and additionally, in 1/80 unrelated individuals affected with non‐syndromic hearing impairment (NSHI). The GRXCR2‐c.251delC variant introduced a premature stop codon, leading to truncation and loss of a zinc‐finger domain. Fluorescence confocal microscopy tracked the wild‐type GRXCR2 protein to the cellular membrane, unlike the mutated GRXCR2 protein.ConclusionThis study confirms GRXCR2 as a HI‐associated gene. GRXCR2 should be included to the currently available TGE panels for HI diagnosis.

Piezo1 Is Essential for Cell Remodeling on Micropatterns

Adherent cells are able to integrate the mechanical input from the substrate, such as patterns, features and stiffness, to modify their shape, movement, and cytoskeletal tension. We have previously shown that HEK293 cells grown on microprinted fibronectin stripes developed elongated shape. We speculated that Piezo1 could be the key element that detects the confinement at the substrate and facilitate cells’ remodeling. We tested Piezo1 knockout HEK cells (P1KO) on the same patterns, and found that P1KO cells were not able to stretch to the full extent on the stripes compared with wild-type. By following GFP-tagged Piezo1 in permanently transfected HEK cells during their stretching, we found that the substrate confinement promoted punctuate Piezo1 plaques to translocate to the extrusion edges in a cell during cell expansion. In comparison, Piezo1 were mostly located on the nuclear envelope in non-stretching cells. To access whether Piezo1 functions as Ca2+ permeable ion channels, we inhibited Piezo1 channels using specific and non-specific MSC inhibitors, GsMTx4 and Gd3+, and both inhibited cells’ expansion on the pattern, suggesting Piezo1 channels are activated during cell spreading. These results demonstrate that Piezo1 plays an essential role in cells’ response to the mechanical inputs from the local environments.

Functional analysis of the F337C mutation in the CLCN1 gene associated with dominant myotonia congenita reveals an alteration of the macroscopic conductance and voltage dependence.

ABSTRACTBackground Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one.MethodsA patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties.ResultsConfocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence.ConclusionTo our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.

Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ∼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.

Enhanced Immune Responses and Protective Immunity to Zika Virus Induced by a DNA Vaccine Encoding a Chimeric NS1 Fused With Type 1 Herpes Virus gD Protein.

Zika virus (ZIKV) is a globally-distributed flavivirus transmitted to humans by Aedes mosquitoes, usually causing mild symptoms that may evolve to severe conditions, including neurological alterations, such as neonatal microcephaly and Guillain-Barré syndrome. Due to the absence of specific and effective preventive methods, we designed a new subunit vaccine based on a DNA vector (pgDNS1-ZIKV) encoding the non-structural protein 1 (NS1) genetically fused to the Herpes Simplex Virus (HSV) glycoprotein D (gD) protein. Recombinant plasmids were replicated in Escherichia coli and the expression of the target protein was confirmed in transfected HEK293 cells. C57BL/6 and AB6 (IFNAR1–/–) mice were i.m. immunized by electroporation in order to evaluate pgDNS1-ZIKV immunogenicity. After two doses, high NS1-specific IgG antibody titers were measured in serum samples collected from pgDNS1-ZIKV-immunized mice. The NS1-specific antibodies were capable to bind the native protein expressed in infected mammalian cells. Immunization with pgDNS1-ZIKV increased both humoral and cellular immune responses regarding mice immunized with a ZIKV NS1 encoding vaccine. Immunization with pgDNS1-ZIKV reduced viremia and morbidity scores leading to enhanced survival of immunodeficient AB6 mice challenged with a lethal virus load. These results give support to the use of ZIKV NS1 as a target antigen and further demonstrate the relevant adjuvant effects of HSV-1 gD.

A polymorphism in the 3'-untranslated region of the matrix metallopeptidase 9 gene is associated with susceptibility to idiopathic calcium nephrolithiasis in the Chinese population.

ObjectiveTo investigate whether single nucleotide polymorphisms (SNPs) in the 3′ untranslated region (UTR) of the matrix metallopeptidase 9 gene (MMP9) are associated with susceptibility to calcium oxalate stones.MethodsA total of 428 patients with kidney stone disease (KSD) and 450 control individuals were enrolled. Three MMP9 SNPs (rs20544, rs9509, and rs1056628) were genotyped, and MMP9 mRNA and protein expression was determined in patients and controls. The dual luciferase reporter gene assay was conducted by transfecting HEK293 cells with miR-491-5p mimics and plasmids containing MMP9 with rs1056628 AA/CC genotypes.ResultsThe rs1056628 CC genotype was significantly increased in KSD patients compared with controls (CC vs AA: odds ratio [OR] = 2.279, 95% confidence interval [CI] = 1.048–4.956). The rs1056628 C allele frequency was higher in KSD patients than controls. The increased KSD risks associated with rs1056628 were more evident in individuals aged <30 years (OR = 3.504, 95% CI = 1.102–11.139) and men (OR = 2.522, 95% CI = 1.004–6.334). mRNA and protein levels of MMP9 were significantly higher in KSD patients with the CC genotype than in those with the AA genotype.ConclusionThis study demonstrates that MMP9 SNP rs1056628 is associated with a significant KSD risk in Chinese Han individuals.

Re-visiting autoimmunity to sodium-iodide symporter and pendrin in thyroid disease.

Iodide transport across thyrocytes constitutes a critical step for thyroid hormone biosynthesis, mediated mainly by the basolateral sodium-iodide-symporter (NIS (SLC5A5)) and the apical anion exchanger pendrin (PDS (SLC26A4)). Both transmembrane proteins have been described as autoantigens in thyroid disease, yet the reports on autoantibody (aAb) prevalence and diagnostic usefulness are conflicting. Reasons for the inconclusive findings may be small study groups and principle differences in the technologies used. We decided to re-evaluate this important issue by establishing novel non-radioactive tests using full-length antigens and comparable protocols, and analyzing a large cohort of thyroid patients (n = 323) and control samples (n = 400). NIS and PDS were recombinantly expressed as fusion protein with firefly luciferase (Luc). Stably transfected HEK293 cells were used as reproducible source of the autoantigens. Recombinant NIS-Luc showed iodide transport activity, indicating successful expression and correct processing. Commercial antibodies yielded dose-dependent responses in the newly established assays. Reproducibility of assay signals from patient sera was verified with respect to linearity, stability and absence of matrix effects. Prevalence of PDS-aAb was similar in thyroid patients and controls (7.7% vs 5.0%). NIS-aAb were more prevalent in patients than controls (7.7% vs 1.8%), especially in Graves' Disease (12.3%). Neither NIS-aAb nor PDS-aAb concentrations were related to TPO-aAb or TSH-receptor-aAb concentrations, or to serum zinc or selenium status. Our data highlight a potential relevance of autoimmunity against NIS for thyroid disease, whereas an assessment of PDS-aAb in thyroid patients seems not to be of diagnostic value (yet).

Voltage-dependent conformational changes of Kv1.3 channels activate cell proliferation.

The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+ channels caninfluence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independentof K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently ofK+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here,we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.

Kv11 (ether-à-go-go-related gene) voltage-dependent K+ channels promote resonance and oscillation of subthreshold membrane potentials.

Key points Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called ‘resonant properties’).Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor‐like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+) channels, encoded by ether‐á‐go‐go‐related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole‐cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3‐, Kv11.1(Merg1b)‐ and Kv11.1(Merg1a)‐expressing cells were 2–6 Hz, 2–4 Hz, and 0.6–0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor–inductor–capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11‐dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at –30 to –60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency‐dependent responses in a subtype‐dependent manner from resting to depolarized membrane potentials.