Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.Fil: Aprile García, Fernando. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Metzger, Michael W.. Max Planck Institute of Psychiatry; AlemaniaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Stadler, Herbert. Affectis Pharmaceuticals; AlemaniaFil: Acuña, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hoijman, Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Refojo, Damian. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mitkovski, Mišo. Max Planck Institute of Experimental Medicine; AlemaniaFil: Panhuysen, Markus. Affectis Pharmaceuticals; AlemaniaFil: Stühmer, Walter. Max Planck Institute of Experimental Medicine; AlemaniaFil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania. HMNC Brain Health; AlemaniaFil: Deussing, Jan M.. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Proteomics-based monitoring of pathway activity reveals that blocking diacylglycerol biosynthesis rescues from alpha-synuclein toxicity

Proteinaceous inclusions containing alpha-synuclein (α-Syn) have been implicated in neuronal toxicity in Parkinson’s disease, but the pathways that modulate toxicity remain enigmatic. Here, we used a targeted proteomic assay to simultaneously measure 269 pathway activation markers and proteins deregulated by α-Syn expression across a panel of 33 Saccharomyces cerevisiae strains that genetically modulate α-Syn toxicity. Applying multidimensional linear regression analysis to these data predicted Pah1, a phosphatase that catalyzes conversion of phosphatidic acid to diacylglycerol at the endoplasmic reticulum membrane, as an effector of rescue. Follow-up studies demonstrated that inhibition of Pah1 activity ameliorates the toxic effects of α-Syn, indicate that the diacylglycerol branch of lipid metabolism could enhance α-Syn neuronal cytotoxicity, and suggest a link between α-Syn toxicity and the biology of lipid droplets

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-Cell NMR

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

<div><p>Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.</p> </div

RSV enters human bronchial epithelial cells by macropinocytosis.

<p>(A). Human bronchial epithelial cells (16HBE14o) were polarized for 9 days and then infected with rgRSV for 20 h. After fixation cells were stained with anti-ZO-1-AF594 antibody (red) and TO-PRO-3 nuclear dye (blue) to examine polarization of the cell monolayer. RSV infection was visualized by GFP expression (green). (B) Polarized 16HBE14o cells were pretreated with solvent (MOCK), low or high concentrations of dynasore (10, 20 µM), cytochalasin D (CytoD 1.5, 3 µM), jasplakinolide (Jas 0.5, 1 µM), IPA-3 (40, 80 µM), genistein (Gen 50, 100 µM), iressa (10, 20 µM), Ly294002, EIPA (40, 80 µM), and dec-RVKR-CMK (50, 100 µM). The cells were infected with RSV for 20 h in the presence of the inhibitors, fixed and counterstained with DAPI. RSV infection was quantified by an image-based approach and normalized to mock infected controls.</p

Rapid RSV endocytosis is followed by the intracellular fusion.

<p>(A). HeLa cells were incubated with RSV-DiOC (moi ∼3) for 30 min at 37°C. Single focal plane image of live cell samples was acquired with a confocal microscope before (green) and after TB addition (pseudocolored red/white). Arrowheads indicate virus spots that were not quenched after TB addition. (B). RSV-DiOC (moi ∼1–5) was bound to versene detached HeLa cells at 4°C for 1 h, unbound virus was washed away and cells were incubation at 37°C. (top) At the indicated times, cells were fixed and the mean fluorescence intensity (MFI) of DiOC measured by FACS in the presence of TB. (bottom) In parallel experiments at indicated times cells were trypsinized on ice for 10 min, washed, permeabilized, stained with anti-N-AF647 or anti-F-AF647 and the MFI of AF647 measured by FACS. (C). RSV-R18/DiOC (moi ∼5) was bound to HeLa cells at 4°C, unbound virus was removed and cells were incubated at 37°C. At indicated times, single focal planes of live cell samples were acquired with confocal microscope. (D). RSV-R18/DiOC (moi ∼5) was bound to versene detached HeLa cells at 4°C, unbound virus was washed away and cells were incubated at 37°C. At indicated times cells were fixed and the MFI of DiOC measured by FACS with or without TB. (E). RSV (moi ∼1 to 30) was bound to HeLa cells at 4°C for 1 h, unbound viruses were removed, and cells warmed to 37°C. At indicated times, samples were placed on ice and trypsinized for 10 min to remove virus remaining on the cell surface. Cells were washed and re-plated for up to 10 h before the percent of GFP expressing cells determined by FACS.</p

Purified RSV is efficient in cell binding and infection.

<p>(A). Gradient purifier RSV (∼10⁵ particles/50 µl) was resolved on the SDS-PAGE, followed by the blue silver gel staining. (B). After binding to a glass slide, purified RSV was stained with anti-F-AF488 (green), anti-N-AF594 (red), and phalloidin-AF647 (pseudocolored white). The particles (n = 457) were imaged with a confocal microscope and analyzed for colocalization by Imaris. Arrowheads show particles with all three stains. (C). Equal volumes of the virus input (moi 10), the cell bound virus lysates, and the unbound virus (sup) were resolved by a SDS-PAGE. Western blots were developed with anti-P or anti-N RSV specific antibody. (left) Representative western blots. (right) Densitometry quantification of the P- and N- protein bands intensities for the virus input and cell bound virus samples. (D). HeLa cells were infected with RSV moi (3–10) for 1 h at 37°C. Virus inoculum was replaced with medium and the infection was carried for indicated times. The percentage of infected cells expressing GFP was measured by FACS.</p

RSV induces bulk fluid phase uptake.

<p>(A). Serum starved HeLa cells were incubated with 20% FCS or purified RSV (moi ∼10, 30), at 4°C for 1 h. The inoculum was replaced with medium containing 10 kDa dextran-AF488 and transferred for 15 min to 37°C before fixation. The MFI of AF-488 measured by FACS. (B). Purified RSV (moi ∼10) was bound at 4°C to serum-starved HeLa cells. The input virus was replaced with medium containing 10 kDa dextran-AF488 (green) and transferred to 37°C. At indicated times cells were fixed, permeabilized and stained with anti-F-AF594 (red) and anti-P-AF647 (blue) antibody. Images represent a Z-stack projection acquired with the same confocal microscope settings. (C). HeLa cells were pretreated with solvent (MOCK) or EIPA at indicated concentration. (left) Cells were infected with RSV (moi ∼3) at 37°C for 6 hours before FACS analysis of GFP expressing cells. (right) RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS. (D). Serum starved HeLa cells were pretreated with EIPA at indicated concentration and incubated with purified RSV (moi ∼30) or no virus control at 4°C. The inoculum was replaced with medium containing 10 kDa dextran-AF488 and EIPA and transferred for 15 min to 37°C. Cells were fixed and the MFI of AF-488 measured by FACS.</p

RSV virions traffic through Rab5 positive vesicles.

<p>(A–B). HeLa cells were transiently transfected with a GFP expressing constructs of Rab5 WT, -Rab5 Q79L (C/A), -Rab5 S34N (D/N), -Rab7 WT, -Rab7 Q67L (C/A), -Rab7 T22N (D/N) for 12 h. (A). RSV (moi ∼3) was bound to HeLa cells at 4°C, unbound virus was washed away and cells warmed at 37°C. At indicated time cells were fixed and stained with anti-F-AF594 (red) and anti-P-AF647 (blue) antibody. Images represent a single 0.37 µm thick focal planes acquired with the same confocal microscope settings. Arrowheads point the discussed in text phenotype of RSV and Rab colocalization. (B). Cells were infected with RSV-A2 (moi ∼0.5) for 16 hours before fixation and staining with anti-N-AF647. The number of AF647 positive cells among the population of GFP expressing cells was measured FACS. (C). HeLa cells were pretreated with 30 µM PIKfyve inhibitor (CAS 371942-96-7) or a solvent control (MOCK) and infected with RSV (moi ∼3) or ZsGreen-SFV (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells.</p