R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

<p>Abstract</p> <p>Background</p> <p>Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain.</p> <p>Results</p> <p>We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain.</p> <p>Conclusion</p> <p>A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed Gβ5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the Gβ5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the Gβ5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex.</p

Critically Ill COVID-19 Patients Exhibit Anti-SARS-CoV-2 Serological Responses

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health care emergency. Anti-SARS-CoV-2 serological profiling of critically ill COVID-19 patients was performed to determine their humoral response. Blood was collected from critically ill ICU patients, either COVID-19 positive (+) or COVID-19 negative (−), to measure anti-SARS-CoV-2 immunoglobulins: IgM; IgA; IgG; and Total Ig (combined IgM/IgA/IgG). Cohorts were similar, with the exception that COVID-19+ patients had a greater body mass indexes, developed bilateral pneumonias more frequently and suffered increased hypoxia when compared to COVID-19-patients (p \u3c 0.05). The mortality rate for COVID-19+ patients was 50%. COVID-19 status could be determined by anti-SARS-CoV-2 serological responses with excellent classification accuracies on ICU day 1 (89%); ICU day 3 (96%); and ICU days 7 and 10 (100%). The importance of each Ig isotype for determining COVID-19 status on combined ICU days 1 and 3 was: Total Ig, 43%; IgM, 27%; IgA, 24% and IgG, 6%. Peak serological responses for each Ig isotype occurred on different ICU days (IgM day 13 \u3e IgA day 17 \u3e IgG persistently increased), with the Total Ig peaking at approximately ICU day 18. Those COVID-19+ patients who died had earlier or similar peaks in IgA and Total Ig in their ICU stay when compared to patients who survived (p \u3c 0.005). Critically ill COVID-19 patients exhibit anti-SARS-CoV-2 serological responses, including those COVID-19 patients who ultimately died, suggesting that blunted serological responses did not contribute to mortality. Serological profiling of critically ill COVID-19 patients may aid disease surveillance, patient cohorting and help guide antibody therapies such as convalescent plasma

Etude du comportement cinétique des lipases de différentes origines sur les esters vinyliques et les triacylglycérols en solution et en émulsion : comparaison entre lipases et estérases

On admet que les lipases, au contraire des estérases, agissent aux interfaces et montrent une forte augmentation d'activité en présence d'une émulsion de substrat (activation interfaciale). La structure de nombreuses lipases montre qu'il existe une boucle peptidique (volet) masquant l'accés au site actif. On a considéré ce domaine comme une caractéristique des lipases. L'étude du comportement cinétique des lipases de R.oryzae, M.miehei, P.cyclopium, P.camembertii, H.lanuginosa, C.rugosa, C.antarctica, des lipases de pancréas humain et de Cobaye, de l'estérase hépatique, de l'acétylcholinestérase et de la cutinase, à l'aide d'esters vinyliques et triglycérides, a montré que toutes ces enzymes, dont certaines ne possèdent pas de volet, sont actives sur des solutions d'esters sans montrer d'activation interfaciale. Les estérases ont une très grande affinité pour les esters en solution et se distinguent des lipases par leur incapacité à hydrolyser les émulsions trioléine, la trioctanoine et laurate de vinyle.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-3

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>ractionated into Triton X-100 soluble and insoluble fractions. Proteins in each fraction were probed for HA-R7BP, Gβ5 and RGS7 and α-tubulin by immunoblotting with specific antibodies as shown. The data shown are representative of three experiments performed with similar results. B. The percentage of total transfected HA-R7BP or endogenous Gβ5 or RGS7 protein in the Triton X-100 soluble (Sol) or insoluble (Insol) fractions was determined by densitometry for the different transfection conditions as shown, with error bars representing the S.E.M

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-0

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>AGE gels and subjected to immunoblotting using anti-R7BP (N-terminal), anti-RGS7, anti-Gβ5, and actin antibodies as described in Methods. The relative mobility of the major immunoreactive bands (in kDa) is indicated on the right. B. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in PC12 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation as detailed in Methods. Fractions were analyzed by immunoblotting for flotillin-1, LAT and PSD-95 as markers for DRM and the β2 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. C. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells. PC12 cells were lysed, and subjected to a sucrose density gradient. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5 and RGS7 proteins as indicated. D. Densitometric quantification of the distribution of endogenous Gβ5 and RGS7 proteins among DRM and non-DRM fractions in sucrose density gradients. Immunoblots of sucrose density gradients such as that shown in C were analyzed by densitometry and the distribution of the indicated immunoreactivity between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblots, with error bars representing the S.E.M. The results shown are representative of three experiments completed with similar results

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-5

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>d rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation. Fractions were analyzed by immunoblotting for Fyn as a marker for DRM and the α1 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. B. Localization of transfected AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were transfected with the indicated constructs, lysed and subjected to a sucrose density gradient centrifugation. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the transfected Gβ5 and RGS7 proteins as indicated. Immunoblots of the sucrose density gradients shown were analyzed by densitometry and the distribution of the indicated immunoreactive bands between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblot, with error bars representing the S.E.M. C. Localization of co-transfected wild type HA-R7BP, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicted and analyzed as indicated in the legend to panel B. D. Localization of co-transfected HA-R7BP-SS, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicated and analyzed as indicated in the legend to panel B. These results are representative of four total such experiments performed with similar findings

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-6

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>alyzed by immunoblotting for flotillin-1, as marker for DRM, and the transferrin receptor (TfR) as a non-DRM marker. B. Sucrose density gradient analysis of endogenous Gβ5, RGS7, and R7BP synaptosomal membrane proteins in mouse brain. A detergent extract of mouse brain synaptosomes was subjected to a sucrose density gradient fractionation. Equal aliquots from all eleven fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5, RGS7, and R7BP (using antibody TRS) proteins as indicated. Immunoblots of the sucrose density gradients shown were analyzed by densitometry and the distribution of the indicated immunoreactive bands between DRM and non-DRM fractions among (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblot, with error bars representing the S.E.M

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-7

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>AGE gels and subjected to immunoblotting using anti-R7BP (N-terminal), anti-RGS7, anti-Gβ5, and actin antibodies as described in Methods. The relative mobility of the major immunoreactive bands (in kDa) is indicated on the right. B. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in PC12 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation as detailed in Methods. Fractions were analyzed by immunoblotting for flotillin-1, LAT and PSD-95 as markers for DRM and the β2 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. C. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells. PC12 cells were lysed, and subjected to a sucrose density gradient. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5 and RGS7 proteins as indicated. D. Densitometric quantification of the distribution of endogenous Gβ5 and RGS7 proteins among DRM and non-DRM fractions in sucrose density gradients. Immunoblots of sucrose density gradients such as that shown in C were analyzed by densitometry and the distribution of the indicated immunoreactivity between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblots, with error bars representing the S.E.M. The results shown are representative of three experiments completed with similar results

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-2

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>d nuclear fractions were prepared from PC12 cells transfected with the cDNAs indicated on the left. Immunoblots to monitor HA-R7BP expression, α-tubulin expression as a marker for the cytosolic fraction, and p84/N5 expression as marker for the nuclear fraction are shown. B. Immunocytofluorescent localization of R7BP in PC12 cells. PC12 cells transfected with vector (panels in left column), wild type (wt) HA-R7BP (middle column) or HA-R7BP SS (right column), were subjected to the immunostaining using anti-HA primary antibody and fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG antibody (green signal) as a secondary antibody. The Hoechst 33342 nuclear dye (blue signal) was used to counterstain transfected PC12 cells. The single or combined (merge) fluorescent signal was recorded by laser confocal microscopy. The results shown are representative of three total experiments performed with similar findings