Journal: ACS Chemical Neuroscience

Article Title: Improving Proteostasis of Trafficking-Deficient GABA A Receptor Variants by Activating IRE1

doi: 10.1021/acschemneuro.5c00227

Figure Lengend Snippet: Mechanism of action of IRE1/XBP1s activators on enhancing GABA A R variant proteostasis. Certain GABA A R variants misfold in the ER and undergo excessive ER-associated degradation (ERAD), leading to their trafficking deficiency to the plasma membrane. IRE1/XBP1s activators enhance the ER folding capacity to promote the folding of a variant by increasing the interaction between the variant and BiP, a pro-folding chaperone, and further promote the assembly into pentameric receptors in the ER membrane. In addition, such IRE1/XBP1s activators inhibit the ERAD of the variant by decreasing the interaction between the variant and Grp94, Hrd1-Sel1L, and VCP, known ERAD factors for GABA A Rs. Consequently, IRE1/XBP1s activators enhance the anterograde trafficking of the variant to the plasma membrane by increasing the interaction between the variant and LMAN1. The receptor variant is also stabilized after reaching the plasma membrane.

Article Snippet: The rabbit polyclonal anti-Grp94 antibody (#14700-1-AP, 1:250) was obtained from Proteintech.

Techniques: Variant Assay, Clinical Proteomics, Membrane

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-GRP94 , Cell Signaling Technology , Cat# 2104: RRID: AB_823506.

Techniques: Virus, Recombinant, Protease Inhibitor, Amplified Luminescent Proximity Homogenous Assay, Lysis, TA Cloning, Sequencing, Software

Journal: PLoS ONE

Article Title: Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

doi: 10.1371/journal.pone.0086198

Figure Lengend Snippet: ( A ) A representative fraction of peak 2 from gel filtration was analyzed in SDS-PAGE (8.5% polyacrylamide gel), in both non-reducing and reducing conditions (*) of samples (3 µg proteins/lane). Mass analysis (MALDI-TOF-TOF) for amino-acid sequence determinations was applied to bands (resolved in SDS-PAGE in reducing conditions) after controlled tryptic digestion. The more frequent peptide sequences of each band are indicated on right of the lane, whereas Western blotting (monoclonal anti-Grp94 Abs) is performed on the same fraction following SDS-PAGE in non-reducing conditions. ( B ) Native Grp94 of peak 2 (0.1 mg/ml, 10 mM Tris, pH 7.0) was incubated at 37°C for 120 min in absence of ATP, both alone (control) and with IgG at the indicated concentrations. Three µg of Grp94 were analyzed in native PAGE (8% acrylamide, pH 8.0) and gels stained with Coomassie blue. Lane on right, control IgG at the highest concentration. a, b and c: Grp94 bands with increasing electrophoretic mobility. ( C ) Western blotting for Grp94 on samples as in (B) processed in native PAGE in both absence and presence of ATP (1 mM) preincubated with Grp94 for 15 min before the addition of IgG. ( D ) 2D-PAGE of Grp94 alone (control) and with IgG (at the Grp94∶IgG molar ratio of 1∶2) after incubation at 37°C for 120 min in the presence of ATP, following native PAGE of samples, as in (B) and (C). The lanes of Grp94 alone (3 µg) and with IgG were cut and submitted to the second dimension on 10% acrylamide gel (see Methods). Above and on the left of each gel are lanes of reference of the first dimension and of SDS-PAGE (in non-reducing conditions), respectively. Long arrows indicate the direction of the run (from the cathode to the anode) in both PAGEs, whereas short arrows in the gels mark Grp94 bands that disappear after co-incubation with IgG (gel on right). ( E ) Native PAGE of Grp94 incubated with 0.3 mg/ml IgG and processed as in B at the indicated incubation times, in both absence and presence of ATP (as in C). On right: histograms representing the densitometric analysis of bands b and c of each lane (Gel-Pro Analyzer software, version 3.1). Heights of histograms indicate the optical density (in arbitrary units).

Article Snippet: Primary Abs used in Western blot analyses were: anti-Grp94 rat monoclonal, anti-calnexin and anti-calreticulin rabbit polyclonal and anti-Grp78 goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Grp94 rabbit polyclonal (Enzo Life Sciences, Lausen, Switzerland).

Techniques: Filtration, SDS Page, Sequencing, Western Blot, Incubation, Clear Native PAGE, Staining, Concentration Assay, Acrylamide Gel Assay, Software

Journal: PLoS ONE

Article Title: Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

doi: 10.1371/journal.pone.0086198

Figure Lengend Snippet: ( A ) DLS of native Grp94, IgG and Grp94-IgG complex. Spectra represent the frequency distribution of sized species of Grp94 (empty area), IgG (dashed area), and Grp94-IgG complex (gray area). Each peak is the mean of three records, from 14 consecutive measurements over 3 min. Solutions of Grp94 and IgG, both alone and mixed together were analyzed in the cuvette located in the instrument at 37°C (see Methods). Peaks are numbered in decreasing order of intensity of the area. Spectra of a representative experiment of three others made on different occasions are shown. ( B ) Glycerol density gradient centrifugation. Solutions of native Grp94 (0.1 mg/ml) and IgG (0.3 mg/ml), both alone and mixed together after incubation for 120 min, were subjected to fractionation by glycerol density gradient centrifugation (10–40% from fractions 1 to 18) (see Methods). Each fraction (8 µg proteins) was analyzed in SDS-PAGE in reducing conditions and boiling of samples, followed by Western blotting for detecting Grp94 and IgG. Fractions are numbered below blotting. For IgG, only the most representative band at 50 kDa (among the bands that form after reducing treatment of integer IgG) are represented for sake of clarity and uniformity with Grp94 bands. The mass of standard proteins is above the blotting, whereas masses of Grp94 and IgG are on right. The graph below shows the pattern of the optical density of the bands in each fraction expressed as percentage of the maximal intensity (100%) that in each blotting is represented by the fraction with the highest optical density. ( C ) The complex formed with native Grp94 (0.1 mg/ml) and IgG (0.3 mg/ml) after co-incubation at 37°C for 120 min in the presence of ATP was submitted to papain digestion for 8 h at 37°C (see Methods). The extent of proteolysis was followed in SDS-PAGE (10% acrylamide gel) in non-reducing conditions of samples and compared with that of both IgG and Grp94 alone. Western blotting for Grp94 was performed on samples of Grp94 alone and in complex with IgG. 2.5 µg of Grp94 and 7.5 µg IgG were loaded in both control lanes and in the lane of the complex. Arrows indicate the bands of IgG after digestion in both the control and complex. Bands of Grp94 remaining in the bulk of IgG after digestion of the complex are marked by asterisks. On right, molecular masses of reference in kDa.

Article Snippet: Primary Abs used in Western blot analyses were: anti-Grp94 rat monoclonal, anti-calnexin and anti-calreticulin rabbit polyclonal and anti-Grp78 goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Grp94 rabbit polyclonal (Enzo Life Sciences, Lausen, Switzerland).

Techniques: Gradient Centrifugation, Incubation, Fractionation, SDS Page, Western Blot, Acrylamide Gel Assay

Journal: PLoS ONE

Article Title: Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

doi: 10.1371/journal.pone.0086198

Figure Lengend Snippet: ( A ) Representative electron microscopy images of negatively stained native Grp94, treated as specified in Methods, in both absence (control, left panel) and presence of ATP (1.0 mM) (right panel) at the same magnification with a 4.2 Å pixel size. Images are taken from several pictures made on Grp94 solutions (fractions of peak 2 from gel-filtration) in three separate occasions. Arrowheads indicate some of the typical structures of Grp94. ( B ) Representative images of Grp94-IgG complexes obtained by incubating native Grp94 (0.1 mg/ml) with IgG at the Grp94∶IgG molar ratio of 1∶2, in both absence and presence of ATP (1.0 mM) at different magnifications. Images of the complex are obtained from different fields. The larger and smaller boxes have, respectively, 25 Å and 5 Å pixel sizes. Calibration bars are reported below each panel.

Article Snippet: Primary Abs used in Western blot analyses were: anti-Grp94 rat monoclonal, anti-calnexin and anti-calreticulin rabbit polyclonal and anti-Grp78 goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Grp94 rabbit polyclonal (Enzo Life Sciences, Lausen, Switzerland).

Techniques: Electron Microscopy, Staining, Filtration

Journal: PLoS ONE

Article Title: Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

doi: 10.1371/journal.pone.0086198

Figure Lengend Snippet: ( A ) Both recombinant rabbit and Con-A native Grp94 (obtained as specified in Methods) were incubated at the concentration of 0.1 mg/ml in 10 mM Tris HCl (pH = 7.0) both alone and with IgG (Grp94∶IgG molar ratio of 1∶2) for 120 min at 37°C in both absence and presence of ATP (1.0 mM). Three µg of proteins were loaded in each lane and submitted to native PAGE (8% acrylamide, pH = 8.0) and gels stained with Coomassie blue. a, b, c, bands as in . ( B ) Both recombinant and Con-A Grp94 (2.5 µg) were incubated with human plasma, both whole and deprived of IgG, as specified in Methods, for 6 and 18 h at 37°C. Three µg of proteins were loaded in each lane and submitted to SDS-PAGE in non-reducing conditions of samples. Lanes of control Grp94 in absence of incubation are on the left of upper panels. Recombinant rabbit Grp94 appears prevalently in its monomeric form, whereas Con-A native Grp94 is visible as a dimer. Bands are shown that are positive for both Grp94 and IgG. Lanes of WB for both Grp94 and IgG in control whole plasma are on right of lower panels.

Article Snippet: Primary Abs used in Western blot analyses were: anti-Grp94 rat monoclonal, anti-calnexin and anti-calreticulin rabbit polyclonal and anti-Grp78 goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Grp94 rabbit polyclonal (Enzo Life Sciences, Lausen, Switzerland).

Techniques: Recombinant, Incubation, Concentration Assay, Clear Native PAGE, Staining, SDS Page

Journal: PLoS ONE

Article Title: Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

doi: 10.1371/journal.pone.0086198

Figure Lengend Snippet: ( A ) N-terminus and C-terminus of recombinant rabbit Grp94 were prepared as reported in Methods. Each Grp94 fragment (0.1 mg/ml in 10 mM Tris, pH = 7.0) was incubated in absence of ATP for 120 min at 37°C without (control) and with IgG at the indicated concentrations, corresponding to the molar ratios of N- and C-Grp94-to-IgG of 1∶1 and 1∶2 (for the lower and higher IgG concentration, respectively). 2.5 µg Grp94 were loaded and analyzed in SDS- (not shown) and native PAGE. Arrows mark bands of N-Grp94 and of C-Grp94. ( B ) 2D PAGE of N-Grp94 incubated both alone and with IgG at 0.5 mg/ml. Above the gel and on left, are lanes of reference from the first dimension PAGE (8% acrylamide, pH = 8.0) and from control SDS-PAGE (10% acrylamide, in non-reducing conditions). On right, the molecular mass of reference in kDa. In both gels, circled bands are those that disappear following incubation with IgG.

Article Snippet: Primary Abs used in Western blot analyses were: anti-Grp94 rat monoclonal, anti-calnexin and anti-calreticulin rabbit polyclonal and anti-Grp78 goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Grp94 rabbit polyclonal (Enzo Life Sciences, Lausen, Switzerland).

Techniques: Recombinant, Incubation, Concentration Assay, Clear Native PAGE, SDS Page