Mechanism Of Allosteric Modulation Of The Cys-Loop Receptors

The cys-loop receptor family is a major family of neurotransmitter-operated ion channels. They play important roles in fast synaptic transmission, controlling neuronal excitability, and brain function. These receptors are allosteric proteins, in that binding of a neurotransmitter to its binding site remotely controls the channel function. The cys-loop receptors also are subject to allosteric modulation by many pharmaceutical agents and endogenous modulators. By binding to a site of the receptor distinct from the neurotransmitter binding site, allosteric modulators alter the response of the receptors to their agonists. The mechanism of allosteric modulation is traditionally believed to be that allosteric modulators directly change the binding affinity of receptors for their agonists. More recent studies support the notion that these allosteric modulators are very weak agonists or antagonists by themselves. They directly alter channel gating, and thus change the distribution of the receptor across multiple different affinity states, indirectly influencing receptors\u27 sensitivity to agonists. There are two major locations of allosteric modulator binding sites. One is in subunit interfaces of the amino-terminal domain. The other is in the transmembrane domain close to the channel gating machinery. In this review, we also give some examples of well characterized allosteric binding pockets. © 2010 by the authors

Fastcloning: A Highly Simplified Purification-Free Sequence- And Ligation-Independent Pcr Cloning Method

Background: Although a variety of methods and expensive kits are available, molecular cloning can be a timeconsuming and frustrating process. Results: Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion: Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA

Endothelial Cell-Specific Molecule 2 (Ecsm2) Localizes To Cell-Cell Junctions And Modulates Bfgf-Directed Cell Migration Via The Erk-Fak Pathway

Background: Despite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. Limited data suggest its involvement in cell migration and apoptosis. However, the underlying signaling mechanisms and novel functions of ECSM2 remain to be explored. Methodology/Principal Findings: A rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated and used to characterize the endogenous ECSM2 protein. Immunoblotting, immunoprecipitation, deglycosylation, immunostaining and confocal microscopy validated that endogenous ECSM2 is a plasma membrane glycoprotein preferentially expressed in vascular endothelial cells (ECs). Expression patterns of heterologously expressed and endogenous ECSM2 identified that ECSM2 was particularly concentrated at cell-cell contacts. Cell aggregation and transwell assays showed that ECSM2 promoted cell-cell adhesion and attenuated basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overexpression or knockdown of ECSM2 in ECs demonstrated that ECSM2 modulated bFGF-directed EC motility via the FGF receptor (FGFR)-extracellular regulated kinase (ERK)-focal adhesion kinase (FAK) pathway. The counterbalance between FAK tyrosine phosphorylation (activation) and ERK-dependent serine phosphorylation of FAK was critically involved. A model of how ECSM2 signals to impact bFGF/FGFR-driven EC migration was proposed. Conclusions/Significance: ECSM2 is likely a novel EC junctional protein. It can promote cell-cell adhesion and inhibit bFGF-mediated cell migration. Mechanistically, ECSM2 attenuates EC motility through the FGFR-ERK-FAK pathway. The findings suggest that ECSM2 could be a key player in coordinating receptor tyrosine kinase (RTK)-, integrin-, and EC junctional component-mediated signaling and may have important implications in disorders related to endothelial dysfunction and impaired EC junction signaling. © 2011 Shi et al

Swimming exercise ameliorates hypertension-induced kidney dysfunction via alleviating renal interstitial fibrosis and apoptosis

Background: Hypertensive nephropathy is one of the major causes of ESRD. Exercise has been considered a nonpathological therapy for hypertension and its complications, yet mechanisms remain unclear. We sought to investigate whether periodic swimming could ameliorate hypertension-induced kidney dysfunction and its underlying mechanisms. Methods: Four-week male spontaneously hypertensive rats (SHRs) were randomly divided into the hypertension group (SHR, n = 8) and exercise group (SE, n = 8, 60 min swimming/day, 6 days per week, for 8 weeks). Wistar-Kyoto rats (WKY, n = 8) were served as a sedentary normotensive group. Bodyweight and blood pressure (BP) were recorded weekly. After 8-week sedentary or swimming exercise, lipids profile, BUN, and Cr were measured. The renal interstitial fibrosis was examined by the histopathological analysis using Masson\u27s trichrome staining and hematoxylin and eosin staining. The kidney cell apoptosis was tested by TUNEL staining. The expressions of critical proteins responsible for the TGF-β1/Smad signaling of fibrosis, that is, TGF-β1, Smad2/3, and Smad7, as well as apoptosis related proteins, Bax and Bcl-2 in kidney cortex tissues were measured. Results: The 8-week swimming exercise reduced BP and bodyweight, lowered concentrations of BUN, and serum Cr, compared with SHR. Exercise remarkably inhibited hypertension-induced tubular degeneration, cellular cluster, and tubular cell swelling as well as glomerular degeneration in the kidney cortical tissues, attenuated renal interstitial fibrosis, and renal cell apoptosis. Moreover, expressions of TGF-β1, Smad2/3, and Bax were higher in the SHR than the WKY, which were significantly suppressed by the exercise. In contrast, hypertension-reduced expressions of Smad7 and Bcl-2 were enhanced by the swimming exercise. Strong correlations were found between kidney function indices, blood lipids, and key protein expressions. Conclusion: Our results demonstrate beneficial effects of the periodic swimming on ameliorating hypertension-induced kidney dysfunction highlighting the potential of swimming exercise as a nonpathological therapy for early prevention of hypertension-caused kidney diseases

Exosomal Dna Aptamer Targeting α-Synuclein Aggregates Reduced Neuropathological Deficits In A Mouse Parkinson\u27S Disease Model

The α-synuclein aggregates are the main component of Lewy bodies in Parkinson\u27s disease (PD) brain, and they showed immunotherapy could be employed to alleviate α-synuclein aggregate pathology in PD. Recently we have generated DNA aptamers that specifically recognize α-synuclein. In this study, we further investigated the in vivo effect of these aptamers on the neuropathological deficits associated with PD. For efficient delivery of the aptamers into the mouse brain, we employed modified exosomes with the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface. We demonstrated that the aptamers were efficiently packaged into the RVG-exosomes and delivered into neurons in vitro and in vivo. Functionally, the aptamer-loaded RVG-exosomes significantly reduced the α-synuclein preformed fibril (PFF)-induced pathological aggregates, and rescued synaptic protein loss and neuronal death. Moreover, intraperitoneal administration of these exosomes into the mice with intra-striatally injected α-synuclein PFF reduced the pathological α-synuclein aggregates and improved motor impairments. In conclusion, we demonstrated that the aptamers targeting α-synuclein aggregates could be effectively delivered into the mouse brain by the RVG-exosomes and reduce the neuropathological and behavioral deficits in the mouse PD model. This study highlights the therapeutic potential of the RVG-exosome delivery of aptamer to alleviate the brain α-synuclein pathology

Functional Impact Of 14 Single Nucleotide Polymorphisms Causing Missense Mutations Of Human α7 Nicotinic Receptor

The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders. Copyright

Desensitization Of α7 Nicotinic Receptor Is Governed By Coupling Strength Relative To Gate Tightness

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR)

Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR

Integrative transcriptomic profiling of mRNA, miRNA, circRNA, and lncRNA in alveolar macrophages isolated from PRRSV-infected porcine

IntroductionThe porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry, attributed largely to its immunosuppressive properties and the chronic nature of its infection. The absence of effective vaccines and therapeutics amplifies the urgency to deepen our comprehension of PRRSV’s intricate pathogenic mechanisms. Previous transcriptomic studies, although informative, are partially constrained by their predominant reliance on in vitro models or lack of long-term infections. Moreover, the role of circular RNAs (circRNAs) during PRRSV invasion is yet to be elucidated.MethodsIn this study, we employed an in vivo approach, exposing piglets to a PRRSV challenge over varied durations of 3, 7, or 21 days. Subsequently, porcine alveolar macrophages were isolated for a comprehensive transcriptomic investigation, examining the expression patterns of mRNAs, miRNAs, circRNAs, and long non-coding RNAs (lncRNAs).ResultsDifferentially expressed RNAs from all four categories were identified, underscoring the dynamic interplay among these RNA species during PRRSV infection. Functional enrichment analyses indicate that these differentially expressed RNAs, as well as their target genes, play a pivotal role in immune related pathways. For the first time, we integrated circRNAs into the lncRNA-miRNA-mRNA relationship, constructing a competitive endogenous RNA (ceRNA) network. Our findings highlight the immune-related genes, CTLA4 and SAMHD1, as well as their associated miRNAs, lncRNAs, and circRNAs, suggesting potential therapeutic targets for PRRS. Importantly, we corroborated the expression patterns of selected RNAs through RT-qPCR, ensuring consistency with our transcriptomic sequencing data.DiscussionThis study sheds lights on the intricate RNA interplay during PRRSV infection and provides a solid foundation for future therapeutic strategizing