HEK293 Expression Research Articles

Elucidating the molecular basis of PECAM-1 and Tie2 interaction from binding dynamics and complex formation

BackgroundEndothelial hyperpermeability-induced vascular dysfunction is a prevalent and significant characteristic in critical illnesses such as sepsis and other conditions marked by acute systemic inflammation. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and Tie2 serve as transmembrane receptors within endothelial cells (ECs), playing pivotal roles not only in maintaining EC-EC junctions but also in influencing vasculogenesis, vessel homeostasis, and vascular remodeling. ObjectivesAt present, the molecular basis of the PECAM-1-Tie2 interaction remains inadequately elucidated. In the study, recombinant soluble PECAM-1 (sPECAM-1) and Tie2 (sTie2) were expressed by Drosophila S2 and HEK293 expression systems, respectively. The interactions between sPECAM-1 and sTie2 were investigated using the Surface Plasmon Resonance (SPR) and size-exclusion chromatography methods. An immunofluorescence assay was used to detect the binding of sPECAM-1 and sTie2 on endothelial cells. ResultsPECAM-1 was found to bind with sTie2 in a sodium and pH-dependent manner as confirmed by the ELISA, the D5-D6 domains of PECAM-1 might play a crucial role in binding with sTie2. Surface Plasmon Resonance (SPR) results showed that the full length of sPECAM-1 has the strongest binding affinity (KD = 48.4 nM) with sTie2, compared to sPECAM-1-D1-D4 and sPECAM-1-D1-D2. This result is consistent with that in the ELISA. In addition, size-exclusion chromatography demonstrated that sPECAM-1, sTie2, and Ang1 can form a ternary complex. ConclusionIn this study, we determined that sPECAM-1 binds to sTie2 in a pH and sodium-dependent manner. The full length of sPECAM-1 has the strongest binding affinity, and the D5-D6 domains in sPECAM-1 play a crucial role in the interaction between sPECAM-1 and sTie2.

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

BackgroundThe advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting.ResultsHere, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA.ConclusionsOur data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway.

The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

Stimuli-Responsive Polypeptide Nanoparticles for Enhanced DNA Delivery

The development of non-viral delivery systems for effective gene therapy is one of the current challenges in modern biomedicinal chemistry. In this paper, the synthesis of pH- and redox-responsive amphiphilic polypeptides for intracellular DNA delivery is reported and discussed. Two series of polypeptides consisting of L-lysine, L-phenylalanine, L-histidine, and L-cysteine as well as the same amino acids with L-glutamic acid were synthesized by a combination of copolymerization of N-carboxyanhydrides of α-amino acids and post-polymerization modification of the resulting copolymers. The presence of histidine provided pH-sensitive properties under weakly acidic conditions specific to endosomal pH. In turn, the presence of cysteine allowed for the formation of redox-responsive disulfide bonds, which stabilized the self-assembled nanoparticles in the extracellular environment but could degrade inside the cell. The formation of intraparticle disulfide bonds resulted in their compactization from 200-250 to 55-100 nm. Empty and pDNA-loaded cross-linked nanoparticles showed enhanced stability in various media compared to non-crosslinked nanoparticles. At the same time, the addition of glutathione promoted particle degradation and nucleic acid release. The delivery systems were able to retain their size and surface charge at polypeptide/pDNA ratios of 10 or higher. GFP expression in HEK 293 was induced by the delivery of pEGFP-N3 with the developed polypeptide nanoparticles. The maximal transfection efficacy (70%) was observed when the polypeptide/pDNA ratio was 100.

An in silico-in vitro pipeline for drug cardiotoxicity screening identifies ionic pro-arrhythmia mechanisms.

Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic. An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug. Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IKr (a source of arrhythmias) by all strong IKr blockers, including cisapride, quinidine and quinine. The protocol also detected block of ICaL by verapamil and Ito by quinidine. Further demonstrating the power of the approach, the VC data uncovered a previously unidentified If block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp. We developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening.

FGD5-AS1 Is a Hub lncRNA ceRNA in Hearts With Tetralogy of Fallot Which Regulates Congenital Heart Disease Genes Transcriptionally and Epigenetically.

Heart development requires robust gene regulation, and the related disruption could lead to congenital heart disease (CHD). To gain insights into the regulation of gene expression in CHD, we obtained the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in 22 heart tissue samples with tetralogy of Fallot (TOF) through strand-specific transcriptomic analysis. Using a causal inference framework based on the expression correlations and validated microRNA (miRNA)–lncRNA–mRNA evidences, we constructed the competing endogenous RNA (ceRNA)-mediated network driven by lncRNAs. Four lncRNAs (FGD5-AS1, lnc-GNB4-1, lnc-PDK3-1, and lnc-SAMD5-1) were identified as hub lncRNAs in the network. FGD5-AS1 was selected for further study since all its targets were CHD-related genes (NRAS, PTEN, and SMAD4). Both FGD5-AS1 and SMAD4 could bind with hsa-miR-421, which has been validated using dual-luciferase reporter assays. Knockdown of FGD5-AS1 not only significantly reduced PTEN and SMAD4 expression in HEK 293 and the fetal heart cell line (CCC-HEH-2) but also increased the transcription of its interacted miRNAs in a cell-specific way. Besides ceRNA mechanism, RNAseq and ATACseq results showed that FGD5-AS1 might play repression roles in heart development by transcriptionally regulating CHD-related genes. In conclusion, we identified a ceRNA network driven by lncRNAs in heart tissues of TOF patients. Furthermore, we proved that FGD5-AS1, one hub lncRNA in the TOF heart ceRNA network, regulates multiple genes transcriptionally and epigenetically.

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool

Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2" from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.

TMEM63C, a Potential Novel Target for Albuminuria Development, Is Regulated by MicroRNA-564 and Transforming Growth Factor beta in Human Renal Cells

Introduction: Transmembrane protein (TMEM) 63C is a member of the TMEM gene family and was recently linked to glomerular filtration barrier function and albuminuria. Its molecular function and expression regulation are largely unknown. Objective: In this study, we set out to characterize the regulating impact of microRNAs (miRNAs) such as miRNA-564 (miR-564) on TMEM63C expression in renal cells. Also, we examined the influence of transforming growth factor beta (TGF-ß) on TMEM63C expression and the potential impact of TMEM63C inhibition on epithelial-mesenchymal transition (EMT) in renal cells and on cell viability in human embryonic kidney 293 cells (HEK 293). Methods: Expression analyses were done using real-time PCR and Western blot. Dual luciferase assay was performed to determine the miRNA-mediated expression control. Cell viability was assessed via trypan blue exclusion staining. Results and Conclusions: MiR-564 reduced TMEM63C expression in HEK 293 and human podocytes (hPC). The treatment of renal cells with TGF-ß led to an increased expression of TMEM63C. Moreover, a reduced TMEM63C expression was associated with a changed ratio of EMT marker proteins such as α-smooth muscle actin versus E-cadherin in HEK 293 and decreased nephrin expression in hPC. In addition, cell viability was reduced upon inhibition of TMEM63C expression in HEK 293. This study demonstrates first mechanisms involved in TMEM63C expression regulation and a link to EMT in renal cells.

RyR2 mutation-linked arrhythmogenic diseases and its therapeutic strategies

The type 2 ryanodine receptor (RyR2) is a sarcoplasmic reticulum Ca2+ release channel that plays a central role in cardiac excitation-contraction coupling. Abnormal activity of the RyR2 is linked to abnormal Ca2+ signaling in cardiac cells, which often results in cardiac arrhythmias. For example, amino acid mutations in RyR2 have been reported to cause various types of arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation, and left ventricular non-compaction. At present, the total number of disease-associated RyR2 mutations exceeds 300. In addition, in chronic heart failure, modification of RyR2 by phosphorylation, oxidation or S-nitrosylation may cause abnormal channel activity. Arrhythmogenic mechanisms of these various disorders are not yet fully understood. We have recently established a method to quantitatively evaluate the effects of various arrhythmogenic mutations and modifications on RyR2 channels by using HEK293 expression system. We found that arrhythmogenic mutations in RyR2 are classified into two groups: gain-of-function and loss-of-function of the channel. Since they are indistinguishable in clinical diagnosis, our analysis is very useful for diagnosis and choice of treatment strategies for RyR2-linked arrhythmogenic diseases. This review describes the current advances and issues of research on RyR2 mutation-related arrhythmogenic disorders.

Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.

P2RX7B is a new theranostic marker for lung adenocarcinoma patients.

Rationale: The characterization of new theranostic biomarkers is crucial to improving the clinical outcome of patients with advanced lung cancer. Here, we aimed at characterizing the P2RX7 receptor, a positive modulator of the anti-tumor immune response, in patients with lung adenocarcinoma.Methods: The expression of P2RX7 and its splice variants was analyzed by RT-qPCR using areas of tumor and non-tumor lung adenocarcinoma (LUAD) tissues on both immune and non-immune cells. The biological activity of P2RX7 was studied by flow cytometry using fluorescent dyes. Bi-molecular fluorescence complementation and confocal microscopy were used to assess the oligomerization of P2RX7. Tumor immune infiltrates were characterized by immunohistochemistry.Results: Fifty-three patients with LUAD were evaluated. P2RX7A, and 3 alternative splice variants were expressed in LUAD tissues and expression was down regulated in tumor versus adjacent non-tumor tissues. The protein retained biological activity only in immune cells. The P2RX7B splice variant was differentially upregulated in immune cells (P < 0.001) of the tumor and strong evidence of oligomerization of P2RX7A and B was observed in the HEK expression model, which correlated with a default in the activity of P2RX7. Finally, LUAD patients with a high level of P2RX7B had non-inflamed tumors (P = 0.001).Conclusion: Our findings identified P2RX7B as a new theranostic tool to restore functional P2RX7 activity and open alternative therapeutic opportunities to improve LUAD patient outcome.

Expression of Enterocin-P in HEK Platform: Evaluation of Its Cytotoxic Effects on Cancer Cell Lines and Its Potency to Interact with Cell-Surface Glycosaminoglycan by Molecular Modeling

Cancer remains one of the leading causes of death worldwide. Introduction of natural compounds with anticancer properties can be an effective step in the prevention and treatment of cancer. Antimicrobial peptides (AMPs) are short peptides whose anticancer activities have been proved previously. In the present study, Enterocin-P (EntP) as a bacteriocin of E. faecium was expressed in HEK expression system by pcDNA3.1 + vector. The recombinant peptide was purified from culture medium using Ni2+ affinity chromatography with an average yield of 0.6 mg/ml. The cytotoxic activity of the recombinant peptide was determined toward some cancer cell lines including: SW1353, HUH7, Huh-7.5, C26, B16F0 and NIH3T3 as a normal cell line. Our results showed that EntP peptide had selective cytotoxicity activity only against C26 (IC50: 0.32 mg/ml) and SW1353 (IC50: 1.36 mg/ml) as cancer cell and did not show cytotoxic properties against normal cell. In the second phase of our study, to better understand the mechanisms of EntP peptide, we have tried to predict the possible interaction of this peptide to predominantly negative charge molecules in the cell membrane of cancer cells. Our in-silico analysis revealed that EntP peptide has strong tendency to chondroitin sulfate (− 189 ± 6.24 kJ/mol) and heparan sulfate (− 115 ± 5.12 kJ/mol) as two well-known anionic molecules on surface cancer cells.

Positions in the N-methyl-D-aspartate Receptor GluN2C Subunit M3 and M4 Domains Regulate Alcohol Sensitivity and Receptor Kinetics.

Alcohol alters synaptic transmission in the brain. The N-methyl-D-aspartate (NMDA) receptor (NMDAR), a subtype of glutamate-gated ion channel, is an important synaptic target of alcohol in the brain. We and others have previously identified 4 alcohol-sensitive positions in the third and fourth membrane-associated (M) domains, designated M31-2 and M41-2 , of the GluN1, GluN2A, and GluN2B NMDAR subunits. In the present study, we tested whether the corresponding positions in the GluN2C subunit also regulate alcohol sensitivity and ion channel gating. We performed alanine- and tryptophan-scanning mutagenesis in the GluN2C subunit followed by expression in HEK 293 cells and electrophysiological patch-clamp recording. Alanine substitution at the M31 (F634) and M41-2 (M821 and M823) positions did not alter ethanol (EtOH) sensitivity, whereas substitution of alanine at the M32 position (F635) yielded nonfunctional receptors. Tryptophan substitution at the M31-2 positions did not change EtOH sensitivity, whereas tryptophan substitution at the M41 position increased, and at the M42 position decreased, EtOH sensitivity. The increased EtOH sensitivity of the tryptophan mutant at M41 is in marked contrast to previous results observed in the GluN2A and GluN2B subunits. In addition, this mutant exhibited increased desensitization, but to a much lesser extent compared to the corresponding mutations in GluN2A and GluN2B. A series of mutations at M41 altered EtOH sensitivity, glutamate potency, and desensitization. Seven amino acid substitutions (of 15 tested) at this position yielded nonfunctional receptors. Among the remaining mutants at M41 , EtOH sensitivity was not significantly correlated with hydrophobicity, molecular volume, or polarity of the substituent, or with glutamate EC50 values, but was correlated with maximal steady-state-to-peak current ratio, a measure of desensitization. The identity and characteristics of alcohol-sensitive positions in the GluN2C subunit differ from those previously reported for GluN2A and GluN2B subunits, despite the high homology among these subunits.

The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis.

Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein. To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts. G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts. Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.

Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

BackgroundDysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa).MethodsSerum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis.ResultsMiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and β-catenin levels were down-regulated in miR-186-5p inhibited PCa cells.ConclusionsOur findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer.

Dual expression of constitutively active Gαs-protein-coupled receptors differentially establishes the resting activity of the cAMP-gated HCN2 channel in a single compartment

The hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channel is a major subtype of the HCN channel family expressed in the nervous system that sets the membrane potential, regulates cell excitability and senses changes in the extracellular environment. Neurons express various Gαs-protein-coupled receptors (GPCRs), many of which show ligand-independent constitutive activity. These membrane-bound proteins are expressed in various subcellular compartments of neurons. Therefore, some proportion of HCN2 channels opens in response to the basal cAMP pool size produced by constitutively active GPCRs. Here, we employed an exogenous HEK293 expression system and voltage-clamp patch-clamp recordings to investigate basal HCN2 channel activity in the presence of two GPCRs with diverse basal activities in a single compartment. We utilized the β2-adrenoceptor (β2AR) together with odorant receptors (ORs), as both GPCR families are known to show strong basal activity. Consequently, β2AR alone strongly enhanced the activity of HCN2 channels, and co-expression of ORs further diversified the HCN2 channel activity, which was totally abolished by an adenylate cyclase inhibitor.Thus, we conclude that the dual expression of constitutively active GPCRs establishes the diverse range of the basal cAMP pool size in resting cells through mutual additive or suppressive interactions, even in the absence of external stimulation.

NS1643 enhances ionic currents in a G604S-WT hERG co-expression system associated with long QT syndrome 2.

Loss of function mutations in the human ether-a-go-go-related gene (hERG) cause long QT syndrome type 2 (LQT2). Most LQT2 patients are heterozygous mutation carriers in which the mutant hERG exerts potent dominant-negative effects. 1, 3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) is known to enhance IKr in WT-hERG. We investigated its actions following lipofectamine-induced expression of both mutant G604S- and WT-hERG in the heterologous HEK293 expression system. Cells transfected with pcDNA3-G604S-hERG did not lead to any expression of detectable currents whether before or following NS1643 challenge. Cells transfected with both pcDNA3-WT-hERG and pcDNA3-G604S-hERG showed reduced hERG currents compared to those transfected with pcDNA3-G604S-hERG consistent with the reduced trafficking and formation of modified heteromeric WT-G604S channels reported on earlier occasions. Nevertheless, NS1643 then continued to produce concentration- and voltage-dependent increases in hERG current amplitude. It did not affect the voltage dependence of activation, recovery from inactivation and deactivation. However, NS1643 (30μmol/L) slowed steady state inactivation and shifted the steady state half maximal activation voltage (V1/2 ) of the inactivation curve by +10mV, and significantly increased the time constants of inactivation. Our present experimental results suggest that NS1643 significantly increases ion current and attenuates its inactivation in cells co-expressing G604S-hERG and WT-hERG. These findings raise the possibility that hERG channel activators offer potential treatment strategies for inherited LQT2.

Correlation between human ether-a-go-go-related gene channel inhibition and action potential prolongation.

Background and PurposeHuman ether‐a‐go‐go‐related gene (hERG; Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro‐arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs). The interference from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model.Experimental ApproachDrug‐induced changes in APD were assessed in hiPSC‐CMs using voltage‐sensitive dyes. The IC50 values for dofetilide and 13 derivatives on hERG current were estimated in an HEK293 expression system. The relative potency of each drug on APD was estimated by calculating the dose (D150) required to prolong the APD at 90% (APD90) repolarization by 50%.Key ResultsThe D150 in hiPSC‐CMs was linearly correlated with IC50 of hERG current. In silico simulations supported this finding. Three derivatives inhibited hERG without prolonging APD, and these compounds also inhibited Cav1.2 and/or Nav1.5 in a channel state‐dependent manner. Adding Cav1.2 and Nav1.2 block to the in silico model recapitulated the direction but not the extent of the APD change.Conclusions and ImplicationsPotency of hERG current inhibition correlates linearly with an index of APD in hiPSC‐CMs. The compounds that do not correlate have additional effects including concomitant block of Cav1.2 and/or Nav1.5 channels. In silico simulations of hiPSC‐CMs APs confirm the principle of the multiple ion channel effects.