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Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (Scl N93A ), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that Scl N93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of Scl N93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (Scl N93A Fc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance., (© 2021. The Author(s).)

The aims of this study was to investigate the influences of hsa_circ_0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has_circ_0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa_circ_0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ_0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa_circ_0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa_circ_0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa_circ_0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.

Background: Long intergenic non-protein coding RNA00665 (LINC00665) plays a crucial tumorigenic role in many cancers, such as gastric cancer and lung adenocarcinoma. However, its role and mechanism of action in the progression of breast cancer (BC) are unknown., Methods: LINC00665 expression levels were determined using quantitative polymerase chain reaction analysis with BC tissues and cell lines. BC cell proliferation was tested by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, whereas BC cell migration and invasion capabilities were analyzed by performing transwell migration assays. Percentages of apoptotic cells were measured by flow cytometry. Interactions between LINC00665 and miR-3169-5p were examined by performing luciferase reporter assays, and the expression levels of proteins, such as β-catenin, were examined by western blot analysis., Results: LINC00665 was expressed at high levels in BC tissues and cells. Upregulated LINC00665 expression correlated with tumor size and tumor, node, and metastasis stages, but not with the age of patients. LINC00665 knockdown inhibited BC cell proliferation, migration, and invasion, whereas it promoted apoptosis. Moreover, bioinformatics analysis and the luciferase reporter assay revealed that LINC00665 bound the microRNA (miR) miR-3619-5p. miR-3619-5p expression correlated negatively with LINC00665 expression in BC tissues. miR-3619-5p overexpression inhibited BC cell proliferation, migration, and invasion, but promoted apoptosis. Simultaneous knockdown of LINC00665 and miR-3619-5p led to increased cell proliferation, migration, and invasion, and inhibited apoptosis. Additionally, catenin beta 1, which encodes the β-catenin protein, was the target gene of miR-3619-5p. β-catenin expression clearly decreased after LINC00665 knockdown and miR-3619-5p overexpression, but increased after simultaneous knockdown of LINC00665 and miR-3619-5p., Conclusion: LINC00665 knockdown inhibited BC cell proliferation and invasion by binding miR-3619-5p and inhibiting β-catenin expression., Competing Interests: Competing interestsThe authors declare no conflict of interest., (© The Author(s) 2020.)

Circular RNAs are a class of endogenous noncoding RNAs that play an important role in gene regulation. These RNAs are involved in the development and progression of various cancers, but their roles in gastric cancer have not yet been thoroughly elucidated. This study showed that hsa&#95;circ&#95;0000467 expression was higher in gastric cancer tissues than in corresponding adjacent tissues ( P < 0.050) and that hsa&#95;circ&#95;0000467 expression levels were correlated with gastric cancer histological grade ( P < 0.050). In addition, hsa&#95;circ&#95;0000467 was remarkably upregulated in gastric cancer cell lines ( P < 0.001). Cell function experiments indicated that hsa&#95;circ&#95;0000467 downregulation decreased the proliferation and invasion ability of BGC-823 and SGC-7901 cells and the number of cells entering the G2/M phase. A direct binding interaction was detected between hsa&#95;circ&#95;0000467 and miR-326-3p by dual-luciferase reporter assays. In addition, the results showed that inhibition of miR-326-3p reversed the decreases in the proliferation and invasion of BGC-823 and SGC-7901 cells caused by hsa&#95;circ&#95;0000647 downregulation. Inhibition of miR-326-3p also decreased the number of cells entering the G2/M phase and the expression of cyclin D1. In conclusion, hsa&#95;circ&#95;0000467 plays a regulatory role in the development and progression of gastric cancer by regulating miR-326-3p, and this circRNA may be a potential diagnostic marker and therapeutic target of gastric cancer., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Wei Lie Mo et al.)

We aimed to assess the roles of small nucleolar RNA host gene 6 ( SNHG6 ) in hepatocellular carcinoma (HCC) progression, and establish the lncRNA-miRNA-mRNA regulation mechanism for HCC therapy. SNHG6 is one of the host genes in small nucleolar RNAs (snoRNAs), which make a difference in the development of human cancers. SERPINH1 is a gene encoding a member of the serpin superfamily of serine proteinase inhibitors with miRNA predicted by TargetScan and DIANA Tools. SNHG6 , serpin family H member 1 ( SERPINH1 ) and miR-139-5p expression levels in HCC tissues and cells were determined by quantitative real-time PCR (qRT-PCR). Migration and invasion of HCC cells were measured by transwell assay. Cell cycle analysis was determined by using flow cytometry. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay were performed for cell viability analysis. The expression of SERPINH1 was detected by qRT-PCR and western blot. Dual-luciferase reporter gene assay was conducted to identify the targeted relationship between miR-139-5p and SNHG6 , as well as SERPINH1 and miR-139-5p. The positive regulation between SNHG6 and SERPINH1 was demonstrated in this study. In contrast, miR-139-5p was significantly down-regulated in HCC cells, the inhibition of miR-139-5p promotes the proliferation of HCC cells, and accelerated the cell cycle of HCC cells. Our study demonstrated the co-expression of SNHG6 and SERPINH1 in HCC cells for the first time, which revealed that SNHG6 could serve as a novel oncogene for the HCC therapy by its regulation.

Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell-cell or cell-extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.

Crystal structures can identify ligand-receptor interactions and assist the development of novel therapeutics, but experimental challenges sometimes necessitate the use of homologous proteins. Tropisetron is an orthosteric ligand at both 5-HT 3 and α7 nACh receptors and its binding orientation has been determined in the structural homologue AChBP (pdbid: 2WNC). Co-crystallisation with a structurally-related ligand, granisetron, reveals an almost identical orientation (pdbid; 2YME). However, there is a >1000-fold difference in the affinity of tropisetron at 5-HT 3 versus α7 nACh receptors, and α7 nACh receptors do not bind granisetron. These striking pharmacological differences prompt questions about which receptor the crystal structures most closely represent and whether the ligand orientations are correct. Here we probe the binding orientation of tropisetron and granisetron at 5-HT 3 receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT 3 receptor mutants transiently expressed in HEK 293 cells, and synthetic modification of the ligands. For 15 of the 23 cysteine substitutions, the effects on tropisetron and granisetron were different. Structure-activity relationships on synthesised derivatives of both ligands were also consistent with different orientations, revealing that contrary to the crystallographic evidence from AChBP, the two ligands adopt different orientations in the 5-HT 3 receptor binding site. Our results show that even quite structurally similar molecules can adopt different orientations in the same binding site, and that caution may be needed when using homologous proteins to predict ligand binding., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.

Background: CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin., Methods: Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis., Results: Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose., Conclusions: Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs., General Significance: Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [³H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among five pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [³H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. The dopamine transporter (DAT) can exist as an oligomer but it is unknown whether the protomers function independently. The present results indicate that protomers that are superpotent or deficient in cocaine analog binding can confer enhanced or reduced potency to the oligomer, respectively. In this respect, positive or negative cooperativity is revealed in the DAT oligomer., (© 2015 International Society for Neurochemistry.)

Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.

Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process)., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.

It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/β1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.

Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni-ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12-8.75 μM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49-1.78 μM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis., (© 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

Imaging fibrillar amyloid-β deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-β pathology in Alzheimer's disease. The most widely used amyloid-β imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-β tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-β fibrils, offering the same ability to detect the regional amyloid-β burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Analysis of correlates of risk of infection in the RV144 HIV-1 vaccine efficacy trial demonstrated that plasma IgG against the HIV-1 envelope (Env) variable region 1 and 2 inversely correlated with risk, whereas HIV-1 Env-specific plasma IgA responses directly correlated with risk. In the secondary analysis, antibody-dependent cellular cytotoxicity (ADCC) was another inverse correlate of risk, but only in the presence of low plasma IgA Env-specific antibodies. Thus, we investigated the hypothesis that IgA could attenuate the protective effect of IgG responses through competition for the same Env binding sites. We report that Env-specific plasma IgA/IgG ratios are higher in infected than in uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env glycoprotein 120 (gp120). An Env-specific monomeric IgA mAb isolated from an RV144 vaccinee also inhibited the ability of natural killer cells to kill HIV-1-infected CD4(+) T cells coated with RV144-induced IgG antibodies. We show that monomeric Env-specific IgA, as part of postvaccination polyclonal antibody response, may modulate vaccine-induced immunity by diminishing ADCC effector function.

The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.

Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.

The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the α/β hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 × 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the 'M2-M3 segment' in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium channels (VGSC) contribute to the initiation and propagation of action potentials within the nervous system. These channels are important targets for inhibition by several classes of drugs, including antiarrhythmics and local anesthetics. Structural and pharmacological studies have localized the binding of these drugs to a common site near the channel's intracellular pore region. Point mutations within this region disrupt local anesthetic inhibition of cardiac, CNS, and skeletal muscle VGSC subtypes. This study was designed to test whether a similar structural requirement for drug binding exists on the peripheral neuronal VGSC subtype; Na(v)1.7. In support of this hypothesis, an alanine substitution for phenylalanine at position 1737 (F1737A) in the pore lining S6 segment of domain IV in human Na(v)1.7 reduced both use- and state- dependent inhibition of the local anesthetics, lidocaine and tetracaine, by 8-21-fold. We also saw a 2-3-fold reduction in tonic inhibition with the F1737A mutant. The voltage dependence of both activation and inactivation were unaffected by the F1737A mutation, however, fast inactivation kinetics were impaired, such that a significant portion of inward current remained at the end of a 20-ms depolarization. These data suggest that F1737 forms a part of the high affinity binding of local anesthetics as well as mediating inactivation processes of neuronal Na(v)1.7 channels.

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.

The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca(2+) from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.

Background: Gliomas frequently contain mutations in the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both., Methodology/principal Findings: We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein)., Conclusions: Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.

The expression of the histone genes is regulated during the cell cycle to provide histones for nucleosome assembly during DNA replication. In budding yeast, histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. Here, we show that the major activator of HTA1-HTB1 is Spt10, a sequence-specific DNA binding protein with a putative histone acetyltransferase (HAT) domain. Spt10 binds to two pairs of upstream activation sequence (UAS) elements in the HTA1-HTB1 promoter: UAS1 and UAS2 drive HTA1 expression, and UAS3 and UAS4 drive HTB1 expression. UAS3 and UAS4 also contain binding sites for the cell cycle regulator SBF (an Swi4-Swi6 heterodimer), which overlap the Spt10 binding sites. The binding of Spt10 and binding of SBF to UAS3 and UAS4 are mutually exclusive in vitro. Both SBF and Spt10 are bound in cells arrested with α-factor, apparently awaiting a signal to activate transcription. Soon after the removal of α-factor, SBF initiates a small, early peak of HTA1 and HTB1 transcription, which is followed by a much larger peak due to Spt10. Both activators dissociate from the HTA1-HTB1 promoter after expression has been activated. Thus, SBF and Spt10 cooperate to control the timing of HTA1-HTB1 expression.

Small RNAs (sRNAs) regulate diverse pathways, including stress responses, virulence, and metabolism in Escherichia coli. At the center of this large sRNA regulatory network is the Hfq protein. Hfq mediates the binding of sRNAs to their target mRNAs; without Hfq, most sRNAs cannot efficiently regulate target mRNA expression. Here, we show in vivo that Hfq can be a limiting factor for sRNA activity and that it can be easily depleted, causing disruption of the sRNA network. Depletion of the available Hfq can occur when sRNAs and target mRNAs are transcribed at high levels without their partners, resulting in the sequestration of Hfq into sRNA-Hfq and target mRNA-Hfq complexes. This can be avoided by coordinating the transcription of sRNAs with their target mRNAs so that they are turned on and off together to maximize duplex formation and minimize Hfq sequestration. Therefore, the limited availability of Hfq results in a highly interdependent sRNA network, wherein the activity of each sRNA depends on the activity of the other sRNAs and target mRNAs in the network.

Galanin is a neuropeptide found throughout the central and peripheral nervous systems of a wide range of species, ranging from human and mouse to frog and tuna. Galanin mediates its physiological roles through three receptors (GalR1-3), all members of the G-protein coupled receptor family. In mapping these roles, receptor subtype selective ligands are crucial tools. To facilitate the ligand design, data on receptor structure and interaction points are of great importance. The current study investigates the mechanism by which galanin interacts with GalR3. Mutated receptors were tested with competitive binding analysis in vitro. Our studies identify six mutagenic constructs that lost receptor affinity completely, despite being expressed at the cell surface. Mutations of the Tyr103(3.33) in transmembrane helix (TM) III, His251(6.51) in TM VI, Arg273(7.35) or His277(7.39) in TM VII, Phe263(6.63) or Tyr270(7.32) in the extracellular loop III all result in complete reduction of ligand binding. In addition, docking studies of an in silico model of GalR3 propose that four of the identified residues interact with pharmacophores situated within the galanin(2-6) sequence. This study provides novel insights into the interaction between ligands and GalR3 and highlights the requirement for correct design of targeting ligands., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 requires two viral proteins, A20 and D4, for processive DNA synthesis, yet the mechanism of how this tricomplex functions is unknown. This study confirms that these three proteins are necessary and sufficient for processivity, and it focuses on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. A series of D4 mutants was generated to discover which sites are important for processivity. Three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis, though they retained UDG catalytic activity, were identified. The mutants were able to compete with wild-type D4 in processivity assays and retained binding to both A20 and DNA. The crystal structure of R187V was resolved and revealed that the local charge distribution around the substituted residue is altered. However, the mutant protein was shown to have no major structural distortions. This suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. Consistent with this is the ability of the conserved mutant K126R to function in processivity. These mutants may help unlock the mechanism by which D4 contributes to processive DNA synthesis.

Kainate receptors (KARs) modulate synaptic transmission and plasticity, and their dysfunction has been linked to several disease states such as epilepsy and chronic pain. KARs are tetramers formed from five different subunits. GluK1-3 are low affinity kainate binding subunits, whereas GluK4/5 bind kainate with high affinity. A number of these subunits can be present in any given cell type, and different combinations of subunits confer different properties to KARs. Here we report the characterization of a new GluK1 subunit-selective radiolabeled antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione ([(3)H]UBP310) using human recombinant KARs. [(3)H]UBP310 binds to GluK1 with low nanomolar affinity (K(D) = 21 ± 7 nM) but shows no specific binding to GluK2. However, [(3)H]UBP310 also binds to GluK3 (K(D) = 0.65 ± 0.19 μM) but with ~30-fold lower affinity than that observed for GluK1. Competition [(3)H]UBP310 binding experiments on GluK1 revealed the same rank order of affinity of known GluK1-selective ligands as reported previously in functional assays. Nonconserved residues in GluK1-3 adjudged in modeling studies to be important in determining the GluK1 selectivity of UBP310 were point-mutated to switch residues between subunits. None of the mutations altered the expression or trafficking of KAR subunits. Whereas GluK1-T503A mutation diminished [(3)H]UBP310 binding, GluK2-A487T mutation rescued it. Likewise, whereas GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [(3)H]UBP310 binding activity. These data show that Ala487 in GluK2 and Asn691 in GluK3 are important determinants in reducing the affinity of UBP310 for these subunits. Insights from these modeling and point mutation studies will aid the development of new subunit-selective KAR antagonists.

Vesicular acetylcholine transporter (VAChT; TC 2.A.1.2.13) mediates storage of acetylcholine (ACh) by synaptic vesicles. A three-dimensional homology model of VAChT is available, but the binding sites for ACh and the allosteric inhibitor (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) are unknown. In previous work, mutations of invariant W331 in the lumenal beginning of transmembrane helix VIII (TM VIII) of rat VAChT led to as much as ninefold loss in equilibrium affinity for ACh and no loss in affinity for vesamicol. The current work investigates the effects of additional mutations in and around W331 and the nearby lumenal end of the substrate transport channel. Mutants of human VAChT were expressed in the PC12(A123.7) cell line and characterized using radiolabeled ligands and filtration assays for binding and transport. Properties of a new and a repeat mutation in W331 are consistent with the original observations. Of 16 additional mutations in 13 other residues (Y60 in the beginning of lumenal Loop I/II, F231 in the lumenal end of TM V, W315, M316, K317, in the lumenal end of TM VII, M320, A321, W325, A330 in lumenal Loop VII/VIII, A334 in the lumenal beginning of TM VIII, and C388, C391, F392 in the lumenal beginning of TM X), only A334F impairs binding. This mutation decreases ACh and vesamicol equilibrium binding affinities by 14- and 4-fold, respectively. The current results, combined with previous results, demonstrate existence of a spatial cluster of residues close to vesicular lumen that decreases affinity for ACh and/or vesamicol when the cluster is mutated. The cluster is composed of invariant W331, highly conserved A334, and invariant F335 in TM VIII and invariant C391 in TM X. Different models for the locations of the ACh and vesamicol binding sites relative to this cluster are discussed., (© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.)

Family B of G protein-coupled receptors (GPCRs) is composed of receptors that bind peptides, such as secretin, glucagon, parathyroid hormone, and corticotropin releasing factor (CRF), which play critical physiological roles. These receptors, like all GPCRs, share a common structural motif of seven membrane-spanning segments, which have been proposed to bind small ligands, such as antalarmin, a nonpeptide antagonist of the type 1 receptor for CRF (CRF(1)). This leads to the hypothesis that as for family A GPCRs, the binding sites of small ligands for family B GPCRs are on the surface of a water-accessible crevice, the binding-site crevice, which is formed by the membrane-spanning segments and extends from the extracellular surface of the receptor into the plane of the membrane. To test this hypothesis we have begun to obtain structural information about family B GPCRs, using as a prototype the CRF(1), by determining the ability of sulfhydryl-specific methanethiosulfonate derivatives, such as the methanethiosulfonate-ethylammonium (MTSEA), to react with CRF(1) and thus irreversibly inhibit (125)I-Tyr(0)-sauvagine binding. We found that MTSEA inhibited (125)I-Tyr(0)-sauvagine binding to CRF(1) and that antalarmin protected against this irreversible inhibition. To identify the susceptible cysteine(s), we mutated, one at a time, four endogenous cysteines to serine. Mutation to serine of Cys211, Cys233, or Cys364 decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. Thus, Cys211, Cys233, and Cys364 at the cytoplasmic ends of the third, fourth, and seventh membrane-spanning segments, respectively, are exposed in the binding site crevice of CRF(1).

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Abeta), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for beta-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPbeta, but not sAPPalpha. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that--in addition to its role in APP trafficking--LRP1 affects APP processing by competing for cleavage by BACE1., (Copyright 2010 Elsevier Inc. All rights reserved.)

The nucleotide substitution C797T in the Chrm2 gene causes substitution of leucine for proline at position 266 (P266L) of the CHRM2 protein. Because Chrm2 codes for the type 2 muscarinic receptor, this mutation could influence physiologic and behavioral phenotypes of mice. Chrm2 mRNA was not differentially expressed in 2 brain regions with high cholinergic innervation in a mouse strain that does (BALB/cByJ) or does not (C57BL/6J) have the mutation. In addition, strains of mice with and without the C797T point mutation in Chrm2 did not differ significantly in muscarinic binding properties. Variation across strains was detected in terms of acoustic startle, prepulse inhibition, and the physiologic effects of the muscarinic agonist oxotremorine. However, interstrain differences in these measures did not correlate with the presence of the mutation. Although we were unable to associate a measurable phenotype with the Chrm2 mutation, assessment of the mutation on other genetic backgrounds or in the context of other traits might reveal differential effects. Therefore, despite our negative findings, evaluation of characteristics that involve muscarinic function should be undertaken with caution when comparing mice with different alleles of the Chrm2 gene.

The constitutive interaction between the P1 domain (a 67-amino-acid functional domain within the proline-rich region) of SLP76 and the SH3 domain of phospholipase Cγ1 (PLCγ1) has been shown. To determine the significance of the interaction between SLP76 and PLCγ1 in resting T cells, we examined molecules associated with PLCγ1 in the absence of both SLP76 and, more specifically, the P1 domain of SLP76. Using a mutant Jurkat T-cell line, we showed that PLCγ1 associated with LAT when the constitutive association with SLP76 was blocked. We also found that the PLCγ1 association with LAT occurred in the membranes of resting T cells. Further experiments demonstrated that LAT competed with SLP76 for PLCγ1 binding and that the LAT interaction with PLCγ1 was mediated by the SH3 domain of PLCγ1. Collectively, these results suggest that the constitutive association of SLP76 with PLCγ1 is required to prevent the association with LAT as well as the premature recruitment of PLCγ1 to the cell membrane.

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhDelta97), a single carbohydrate at position 330 (C1inhDelta97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhDelta97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding K(D) values of 16.7 x 10(-8) M (C1 inhibitor), 2.3 x 10(-8) M (C1inhDelta97), and 3.6 x 10(-8) M (C1inhDelta97DM). C1s also bound to heparin, with lower affinity (K(D) = 108 x 10(-8) M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a "sandwich" mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

We used (11)C-raclopride PET, a marker of D(2) dopamine receptor binding, and statistical parametric mapping (SPM) to localise cortical D(2) receptor dysfunction in individual Huntington's disease (HD) gene carriers (16 symptomatic and 11 premanifest subjects) and assess its clinical significance. 62.5% of symptomatic HD patients and 54.5% of premanifest carriers showed cortical reductions in D(2) binding. The most frequent decreases in cortical binding in individual HD subjects were seen in temporal and frontal areas. Symptomatic HD subjects with decreased cortical D(2) binding had worse scores on neuropsychological tests assessing attention and executive functions than subjects without cortical dopamine dysfunction, notwithstanding comparable reduction in striatal D(2) binding and motor disability. Our results indicate that cortical dopaminergic dysfunction is common in both symptomatic and premanifest HD gene carriers. It is an early event in HD pathophysiology and could contribute to the impairment in neuropsychological performance in these patients.

The mechanism by which HLA-DM (DM) promotes exchange of peptides bound to HLA-DR (DR) is still unclear. We have shown that peptide interaction with DR1 can be considered a folding process as evidenced by cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative, which could be a function of the breaking of a critical interaction. The hydrogen bond (H-bond) between beta-chain His(81) and the peptide backbone at the -1 position is a candidate for such a target. In this study, we analyze the exchange of peptides bound to a DR1 mutant in which formation of this H-bond is impaired. We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond contributes to the overall energetics of the complex and its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable complex.

The A/M2 proton channel of influenza A virus is a target for the anti-influenza drugs amantadine and rimantadine, whose effectiveness was diminished by the appearance of naturally occurring point mutants in the A/M2 channel pore, among which the most common are S31N, V27A, and L26F. We have synthesized and characterized the properties of a series of compounds, originally derived from the A/M2 inhibitor BL-1743. A lead compound emerging from these investigations, spiro[5.5]undecan-3-amine, is an effective inhibitor of wild-type A/M2 channels and L26F and V27A mutant ion channels in vitro and also inhibits replication of recombinant mutant viruses bearing these mutations in plaque reduction assays. Differences in the inhibition kinetics between BL-1743, known to bind inside the A/M2 channel pore, and amantadine were exploited to demonstrate competition between these compounds, consistent with the conclusion that amantadine binds inside the channel pore. Inhibition by all of these compounds was shown to be voltage-independent, suggesting that their charged groups are within the N-terminal half of the pore, prior to the selectivity filter that defines the region over which the transmembrane potential occurs. These findings not only help to define the location and mechanism of binding of M2 channel-blocking drugs but also demonstrate the feasibility of discovering new inhibitors that target this binding site in a number of amantadine-resistant mutants.

CD32A, the major phagocytic FcgammaR in humans, exhibits a polymorphism in the ligand binding domain. Individuals homozygous for the R allelic form of CD32A (CD32A(R) allele) are more susceptible to bacterial infections and autoimmune diseases as compared with H allelic CD32A (CD32A(H)) homozygous and CD32A(R/H) heterozygous individuals. To understand the mechanisms behind this differential susceptibility, we have investigated the dynamics of the interaction of these allelic forms of CD32A when they are simultaneously exposed to immune complexes (IC). Binding studies using Ig fusion proteins of CD32A alleles showed that the R allele has significantly lower binding not only to human IgG2, but also to IgG1 and IgG3 subtypes. Competition assays using purified molecules demonstrated that CD32A(H)-Ig outcompetes CD32A(R)-Ig for IC binding when both alleles simultaneously compete for the same ligand. CD32A(H)-Ig blocked the IC binding mediated by both the allelic forms of cell surface CD32A, whereas CD32A(R)-Ig blocked only CD32A(R) and was unable to cross-block IC binding mediated by CD32A(H). Two-dimensional affinity measurements also demonstrated that CD32A(R) has significantly lower affinity toward all three subtypes as compared with CD32A(H). Our data suggest that the lower binding of CD32A(R) not only to IgG2 but also to IgG1 and IgG3 might be responsible for the lack of clearance of IC leading to increased susceptibility to bacterial infections and autoimmune diseases. Our data further suggests that in humans, inflammatory cells from CD32A(R/H) heterozygous individuals may predominantly use the H allele to mediate Ab-coated target cell binding during phagocytosis and Ab-dependent cellular cytotoxicity, resulting in a phenotype similar to CD32A(H) homozygous individuals.