Review

rhbdl4 (Proteintech)

Proteintech is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Proteintech rhbdl4
Rhbdl4, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rhbdl4/product/Proteintech
Average 93 stars, based on 9 article reviews

rhbdl4 - by Bioz Stars, 2026-06

93/100 stars

Images

Image Search Results

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Schematic representation of the wild-type RHBDL4 and proteolytically inactive RHBDL4-S144A, highlighting their conserved rhomboid motifs (WR and Gx3G), VBM, and UIM domains. In the inactive RHBDL4, residue S144 is mutated to an alanine. ( B ) Schematic representation of RHBDL4 purification (His-tagged at its C terminus), solubilized in 3% DDM, and subjected to Ni 2+ affinity purification. ( C ) Size-exclusion chromatography profile of wild-type RHBDL4 and inactive RHBDL4-S144A, being eluted as a monomeric protein. (D) Coomassie staining and western blotting with α-RHBDL4 antibody (Sigma) of concentrated purified wild-type RHBDL4 and inactive RHBDL4-S144A. (E) Purified wild-type RHBDL4 and inactive RHBDL4-S144A incubated with TAMRA-FP probe. The reactions were quenched with 1×Laemmli buffer and analyzed by SDS-PAGE on a 12% gel. Post-transfer membranes were visualized using a fluorescence Cy3 filter and subjected to western blotting using an α-RHBDL4 antibody.

Article Snippet: pcDNA3.1-HisA plasmids expressing wild-type RHBDL4, RHBDL4-S144A, RHBDL4-W65A, and RHBDL4-G198A were transfected into Freestyle TM 293-F mammalian suspension cells (Thermo Fisher Scientific) in Gibco Freestyle TM 293-F media, according to the specified manufacturer’s protocol. pEGFP-N1-RHBDL4 was transfected as a 1:10 mix with empty pcDNA3.1 plasmid into Hek293T cells (ATTC) using 25-kDa linear polyethylenimine (Polysciences) as had been described ( ).

Techniques: Residue, Purification, Affinity Purification, Size-exclusion Chromatography, Staining, Western Blot, Incubation, SDS Page, Fluorescence

(A) Freestyle 293-F suspension cells were transfected with the RHBDL4 plasmid and cells were grown for the indicated number of days. 50 µg of lysate was subjected to immunoblotting for polyclonal α-RHBDL4 antibody and monoclonal α-GAPDH antibody. (B) Same as (A) except RHBDL4-S144A was transfected.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Freestyle 293-F suspension cells were transfected with the RHBDL4 plasmid and cells were grown for the indicated number of days. 50 µg of lysate was subjected to immunoblotting for polyclonal α-RHBDL4 antibody and monoclonal α-GAPDH antibody. (B) Same as (A) except RHBDL4-S144A was transfected.

Techniques: Suspension, Transfection, Plasmid Preparation, Western Blot

(A) Proteolytic rate of indicated IQ substrates cleaved by RHBDL4. (B) Catalytic parameters of IQ6, IQ40, and IQ41 substrate cleavage by RHBDL4.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Proteolytic rate of indicated IQ substrates cleaved by RHBDL4. (B) Catalytic parameters of IQ6, IQ40, and IQ41 substrate cleavage by RHBDL4.

Techniques:

( A ) Schematic representation of the FRET-based cleavage assay using internally quenched (IQ) substrates. ( B ) Saturation curves of RHBDL4 using the fluorescent IQ substrates, IQ6, IQ41, and IQ46. ( C ) pH dependence of the RHBDL4 protease activity using substrates IQ6, IQ41, and IQ46. ( D ) Salt concentration dependence of the RHBDL4 protease activity using substrates IQ6, IQ41, and IQ46. All the assays were performed in triplicate, and data points are represented by the average ± standard deviation

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: ( A ) Schematic representation of the FRET-based cleavage assay using internally quenched (IQ) substrates. ( B ) Saturation curves of RHBDL4 using the fluorescent IQ substrates, IQ6, IQ41, and IQ46. ( C ) pH dependence of the RHBDL4 protease activity using substrates IQ6, IQ41, and IQ46. ( D ) Salt concentration dependence of the RHBDL4 protease activity using substrates IQ6, IQ41, and IQ46. All the assays were performed in triplicate, and data points are represented by the average ± standard deviation

Techniques: Cleavage Assay, Activity Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) RHBDL4 representation highlighting TM1–6 ( in green ), the VBM and UIM binding motifs ( in blue ), the conserved rhomboid motifs (WR and Gx3G), and the serine-histidine catalytic dyad ( in pink ). The table indicates the RHBDL4 region, the conserved rhomboid motifs, and amino acid mutation. the RHBDL4 proteolytic activity. (B) Size-exclusion chromatography profile of RHBDL4-AR and RHBDL4-Ax3G, being eluted as a monomeric protein. (C) Coomassie staining and western blotting using an α-RHBDL4 antibody of concentrated purified RHBDL4-AR and RHBDL4-Ax3G. (D) Activity profiles of the wild-type RHBDL4 and mutant RHBDL4-S144A, RHBDL4-AR, and RHBDL4-Ax3G proteins, as measured by cleavage of the fluorescent substrate IQ6. Assay was performed in triplicate, and data points are represented by the average ± standard deviation.

Techniques: Binding Assay, Mutagenesis, Activity Assay, Size-exclusion Chromatography, Staining, Western Blot, Purification, Standard Deviation

(A) Conservation of RHBDL4. Alignment of RHBDL4 ( H. sapiens, M. musculus, E. norvegicus, D. rerio, P. troglodytes ) with GlpG ( E. coli ). Identical residues are highlighted in red. (B) Alphafold model of RHBDL4. Positions of WxR and Gx 3 G motif is highlighted in yellow. (C) Freestyle 293-F suspension cells were transfected with RHBDL4-AxR and RHBDL4-Ax3G and cells were grown for the indicated amount of days. 50 µg of lysate was subjected to immunoblotting for RHBDL4 with monoclonal α-His antibody and monoclonal α-GAPDH antibody.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Conservation of RHBDL4. Alignment of RHBDL4 ( H. sapiens, M. musculus, E. norvegicus, D. rerio, P. troglodytes ) with GlpG ( E. coli ). Identical residues are highlighted in red. (B) Alphafold model of RHBDL4. Positions of WxR and Gx 3 G motif is highlighted in yellow. (C) Freestyle 293-F suspension cells were transfected with RHBDL4-AxR and RHBDL4-Ax3G and cells were grown for the indicated amount of days. 50 µg of lysate was subjected to immunoblotting for RHBDL4 with monoclonal α-His antibody and monoclonal α-GAPDH antibody.

Techniques: Suspension, Transfection, Western Blot

(A) Wild-type RHBDL4 and inactive RHBDL4-S144A were incubated with 10 μM of the IQ6 substrate Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys (DNP)-OH for 4 h at room temperature, and the reaction was quenched with 8 M GuHCl, desalted, eluted, and analyzed via tandem mass spectrometry (MS/MS). (B) Depiction of IQ6 cleavage sites as determined from tandem mass spectrometry (MS/MS). (C) Peptide sequence of GlpG-derived and PARL-derived fluorescent substrates IQ4 and IQ15, respectively. (D) Activity profiles of the wild-type RHBDL4 and mutant RHBDL4-S144A, as measured by cleavage of the fluorescent substrates IQ4 and IQ15. Assays was performed in triplicate, and data points are represented by the average ± standard deviation

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Wild-type RHBDL4 and inactive RHBDL4-S144A were incubated with 10 μM of the IQ6 substrate Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys (DNP)-OH for 4 h at room temperature, and the reaction was quenched with 8 M GuHCl, desalted, eluted, and analyzed via tandem mass spectrometry (MS/MS). (B) Depiction of IQ6 cleavage sites as determined from tandem mass spectrometry (MS/MS). (C) Peptide sequence of GlpG-derived and PARL-derived fluorescent substrates IQ4 and IQ15, respectively. (D) Activity profiles of the wild-type RHBDL4 and mutant RHBDL4-S144A, as measured by cleavage of the fluorescent substrates IQ4 and IQ15. Assays was performed in triplicate, and data points are represented by the average ± standard deviation

Techniques: Incubation, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Derivative Assay, Activity Assay, Mutagenesis, Standard Deviation

(A) Proteolytic rates of the fluorescent substrate IQ6 by RHBDL4 in the presence of increasing concentrations of the general protease inhibitors, Dicyclocoumarin (DCI), tosyl-phenylalanine-chloromethyl ketone (TPCK) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). (B) Schematic representation of the peptidyl-α-ketoamide scaffold with peptide sequence P5-P1 at the non-prime side and hydrophobic substituent (R) at the prime side. (C) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 1a/1b. (D) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 1a. (E) Same as (D), except inhibition curve was derived from using increasing concentrations of compound 1b. (F) Wild-type RHBDL4 and inactive RHBDL4-S144A were preincubated with TAMRA-FP probe followed by incubation with peptidyl-α-ketoamide inhibitor 1a and 1b. Reactions were quenched with 1×Laemmli buffer and loaded on 12% SDS-PAGE gel. Post-transfer membranes were visualized using a fluorescence Cy3 filter and by western blotting using an α-RHBDL4 antibody. For (A), (D), and (E), all inhibitors were pre-incubated with RHBDL4 for 1hr and allowed to react with the IQ6 substrate for 2 h. Assays were performed in triplicate, and data points represent the average ± standard deviation.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Proteolytic rates of the fluorescent substrate IQ6 by RHBDL4 in the presence of increasing concentrations of the general protease inhibitors, Dicyclocoumarin (DCI), tosyl-phenylalanine-chloromethyl ketone (TPCK) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). (B) Schematic representation of the peptidyl-α-ketoamide scaffold with peptide sequence P5-P1 at the non-prime side and hydrophobic substituent (R) at the prime side. (C) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 1a/1b. (D) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 1a. (E) Same as (D), except inhibition curve was derived from using increasing concentrations of compound 1b. (F) Wild-type RHBDL4 and inactive RHBDL4-S144A were preincubated with TAMRA-FP probe followed by incubation with peptidyl-α-ketoamide inhibitor 1a and 1b. Reactions were quenched with 1×Laemmli buffer and loaded on 12% SDS-PAGE gel. Post-transfer membranes were visualized using a fluorescence Cy3 filter and by western blotting using an α-RHBDL4 antibody. For (A), (D), and (E), all inhibitors were pre-incubated with RHBDL4 for 1hr and allowed to react with the IQ6 substrate for 2 h. Assays were performed in triplicate, and data points represent the average ± standard deviation.

Techniques: Sequencing, Inhibition, Derivative Assay, Incubation, SDS Page, Fluorescence, Western Blot, Standard Deviation

(A) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 2. (B) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 2 . (C) Catalytic parameters of KSp207 substrate cleavage by RHBDL4.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 2. (B) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 2 . (C) Catalytic parameters of KSp207 substrate cleavage by RHBDL4.

Techniques: Inhibition, Derivative Assay

(A) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 3 and 4. (B) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 3 . (C) Same as (B), except inhibition curve was derived from using increasing concentrations of compound 4 . (D) Wild-type RHBDL4 and inactive RHBDL4-S144A were preincubated with TAMRA-FP probe followed by incubation with peptidyl-α-ketoamide inhibitor 3 and 4 . Reactions were quenched with 1×Laemmli buffer and loaded on 12% SDS-PAGE gel. Post-transfer membranes were visualized using a fluorescence Cy3 filter and by western blotting using an α-RHBDL4 antibody. (E) Saturation curve of RHBDL4 using fluorescent substrate, KSp207. For (B) and (C), all inhibitors were pre-incubated with RHBDL4 for 1hr and allowed to react with the IQ6 substrate for 2 h. All assays were performed in triplicate, and data points represent the average ± standard deviation.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: (A) Schematic representation of the peptidyl-α-ketoamide inhibitor, compound 3 and 4. (B) Representative inhibition curve derived from measuring rates of RHBDL4 proteolysis of the fluorescent substrate IQ6 with increasing concentrations of compound 3 . (C) Same as (B), except inhibition curve was derived from using increasing concentrations of compound 4 . (D) Wild-type RHBDL4 and inactive RHBDL4-S144A were preincubated with TAMRA-FP probe followed by incubation with peptidyl-α-ketoamide inhibitor 3 and 4 . Reactions were quenched with 1×Laemmli buffer and loaded on 12% SDS-PAGE gel. Post-transfer membranes were visualized using a fluorescence Cy3 filter and by western blotting using an α-RHBDL4 antibody. (E) Saturation curve of RHBDL4 using fluorescent substrate, KSp207. For (B) and (C), all inhibitors were pre-incubated with RHBDL4 for 1hr and allowed to react with the IQ6 substrate for 2 h. All assays were performed in triplicate, and data points represent the average ± standard deviation.

Techniques: Inhibition, Derivative Assay, Incubation, SDS Page, Fluorescence, Western Blot, Standard Deviation

ER-enriched microsomes were preincubated with 100 μM of compounds or DMSO vehicle control followed by addition of 500 nM of TAMRA-FP serine hydrolase probe. RHBDL4-GFP was immunoprecipitated with a α-GFP antibody and loaded on SDS-PAGE. TAMRA-FP labeling was analyzed via Cy3 filter followed by western blotting using α-GFP antibody.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: ER-enriched microsomes were preincubated with 100 μM of compounds or DMSO vehicle control followed by addition of 500 nM of TAMRA-FP serine hydrolase probe. RHBDL4-GFP was immunoprecipitated with a α-GFP antibody and loaded on SDS-PAGE. TAMRA-FP labeling was analyzed via Cy3 filter followed by western blotting using α-GFP antibody.

Techniques: Control, Immunoprecipitation, SDS Page, Labeling, Western Blot

RHBDL4 structure modeling and peptide ensemble docking. (A) Workflow of modeling RHBDL4 structural dynamics and docking of peptide substrate. Human RHBDL4 model was obtained from AlphaFold 2 and was equilibrated with all-atom MD simulations. Ensemble docking was done in AutoDock Vina with peptide (n=4) and RHBDL4 clustered structures from MD simulations. (B) Membrane composition and box size dimension. (C) Peptide structures, binding site, and calculated ensemble average binding energy. RHBDL4 open structure was used for the ensemble docking with the crevice adjacent to the catalytic dyad S144 and H195 exposed. (D) P2 residue and active site distance evolution.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: RHBDL4 structure modeling and peptide ensemble docking. (A) Workflow of modeling RHBDL4 structural dynamics and docking of peptide substrate. Human RHBDL4 model was obtained from AlphaFold 2 and was equilibrated with all-atom MD simulations. Ensemble docking was done in AutoDock Vina with peptide (n=4) and RHBDL4 clustered structures from MD simulations. (B) Membrane composition and box size dimension. (C) Peptide structures, binding site, and calculated ensemble average binding energy. RHBDL4 open structure was used for the ensemble docking with the crevice adjacent to the catalytic dyad S144 and H195 exposed. (D) P2 residue and active site distance evolution.

Techniques: Membrane, Binding Assay, Residue

RHBDL4 (apo) most populated clusters from all-atom MD simulations. Structures were clustered based from the solvent-accessibility-surface-areas (SASA) values of residues within 10 Angstrom of HIS144 and SER195. Each cluster was represented with the frames that has max SASA values.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: RHBDL4 (apo) most populated clusters from all-atom MD simulations. Structures were clustered based from the solvent-accessibility-surface-areas (SASA) values of residues within 10 Angstrom of HIS144 and SER195. Each cluster was represented with the frames that has max SASA values.

Techniques: Solvent

RHBDL4 and peptide Ca Root-mean-square deviation from the initial frame of molecular dynamics simulations. Trajectories were aligned. Four replicates for each system were analyzed.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: RHBDL4 and peptide Ca Root-mean-square deviation from the initial frame of molecular dynamics simulations. Trajectories were aligned. Four replicates for each system were analyzed.

Techniques:

2D representation of RHBDL4 close interacting residues with the peptide side chains. All residues that were shared by the three substrate-derived peptides were marked with yellow stars. Ligplot+ was used to render this figure.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: 2D representation of RHBDL4 close interacting residues with the peptide side chains. All residues that were shared by the three substrate-derived peptides were marked with yellow stars. Ligplot+ was used to render this figure.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet:

Techniques:

Time evolution snapshot of MESA interaction with the RHBDL4 active site.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: Time evolution snapshot of MESA interaction with the RHBDL4 active site.

Techniques:

Time evolution snapshot of PGFSA interaction with the RHBDL4 active site.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: Time evolution snapshot of PGFSA interaction with the RHBDL4 active site.

Techniques:

Time evolution snapshot of QMESA interaction with the RHBDL4 active site.

Journal: bioRxiv

Article Title: An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

doi: 10.1101/2024.10.13.618094

Figure Lengend Snippet: Time evolution snapshot of QMESA interaction with the RHBDL4 active site.

Techniques:

RHBDL4 generates Aη-like peptides in vitro . A , investigation of RHBDL4-mediated APP-CTFs at the cell surface using cell surface biotinylation. Co-transfection of APP and RHBDL2 (R2), active RHBDL4 (R4), or inactive RHBDL4 (R4in). The input consists of lysates without neutravidin to serve as loading controls ( left panels ). Biotinylated cell surface proteins were pulled down using neutravidin ( right panels ). Integrin-β1 is a positive control for successful pulldown of plasma membrane proteins. Detection of APP fl. with 6E10, CTFs with 6E10 and C1/6.1; RHBDL4 with rabbit anti-RHBDL4 antibody; Integrin-β1 with rabbit anti-integrin-β1 antibody. Representative Western blot of three individual experiments is shown. B and C , immunoprecipitation of Aη species from cell culture supernatant and lysate. Total cell culture lysates or supernatant are used as input ( left panels ) while immunoprecipitation (IP) was performed using the 6E10 antibody ( right panels ). Detection of APP full length (fl.), sAPP⍺ and Aη with 2E9, CTFs with Y188; RHBDL4 with rabbit-anti-RHBDL4 antibody; RHBDL2 with mouse-anti-flag antibody and β-actin as a loading control. A representative Western blot of three individual experiments is shown. D–F , RHBDL4-mediated Aη generation is independent of canonical processing by ⍺-, β- or γ-secretases. Cells were treated with either ⍺-secretase inhibitor (⍺-Sec. Inh.), BACE-1 inhibitor (BACE1 Inh.) or γ-secretase inhibitor (γ-sec. Inh). Total cell culture supernatant is used as input ( left panels ) while immunoprecipitation (IP) was performed using the 6E10 antibody ( right panels ). Detection of sAPP⍺ and Aη with 2E9 and 6E10. Representative western blots of each inhibitor experiment are shown, n = 3 per inhibitor. Asterix indicates signals derived from the antibody used in the immunoprecipitation.

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Figure Lengend Snippet: RHBDL4 generates Aη-like peptides in vitro . A , investigation of RHBDL4-mediated APP-CTFs at the cell surface using cell surface biotinylation. Co-transfection of APP and RHBDL2 (R2), active RHBDL4 (R4), or inactive RHBDL4 (R4in). The input consists of lysates without neutravidin to serve as loading controls ( left panels ). Biotinylated cell surface proteins were pulled down using neutravidin ( right panels ). Integrin-β1 is a positive control for successful pulldown of plasma membrane proteins. Detection of APP fl. with 6E10, CTFs with 6E10 and C1/6.1; RHBDL4 with rabbit anti-RHBDL4 antibody; Integrin-β1 with rabbit anti-integrin-β1 antibody. Representative Western blot of three individual experiments is shown. B and C , immunoprecipitation of Aη species from cell culture supernatant and lysate. Total cell culture lysates or supernatant are used as input ( left panels ) while immunoprecipitation (IP) was performed using the 6E10 antibody ( right panels ). Detection of APP full length (fl.), sAPP⍺ and Aη with 2E9, CTFs with Y188; RHBDL4 with rabbit-anti-RHBDL4 antibody; RHBDL2 with mouse-anti-flag antibody and β-actin as a loading control. A representative Western blot of three individual experiments is shown. D–F , RHBDL4-mediated Aη generation is independent of canonical processing by ⍺-, β- or γ-secretases. Cells were treated with either ⍺-secretase inhibitor (⍺-Sec. Inh.), BACE-1 inhibitor (BACE1 Inh.) or γ-secretase inhibitor (γ-sec. Inh). Total cell culture supernatant is used as input ( left panels ) while immunoprecipitation (IP) was performed using the 6E10 antibody ( right panels ). Detection of sAPP⍺ and Aη with 2E9 and 6E10. Representative western blots of each inhibitor experiment are shown, n = 3 per inhibitor. Asterix indicates signals derived from the antibody used in the immunoprecipitation.

Article Snippet: Plasmid pCMV6 encoding cDNA for human RHBDL1, RHBDL2, and RHBDL4 with a C-terminal myc-FLAG tag was obtained from OriGene, USA. cDNA encoding APP695 untagged (in pcDNA3.1, Invitrogen) was a kind gift of Dr Claus Pietrzik, Johannes Gutenberg University, Mainz, Germany.

Techniques: In Vitro, Cotransfection, Positive Control, Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation, Cell Culture, Control, Derivative Assay

Identification of RHBDL4 cleavage sites in APP . A , identification of RHBDL4 cleavage sites by mass spectrometry. Immunoprecipitation of N-terminally myc-tagged APP fragments after co-transfection with either active or inactive (inac.) RHBDL4 (R4) in HEK293T cells. Samples were digested with LysC and analyzed by electrospray ionization mass spectrometry (ESI-MS). Representative extracted ion chromatograms showing retention times for different identified peptides. The table lists the identified retention time per peak, the peptide mass per charge (m/z), and peptide sequences along with APP695 amino acid numbering for fragments or cleavage sites. Due to subtle differences in the automated injections, the retention time of the complete LysC peptide differs between the samples containing inactive RHBDL4 (55.7 min) and active RHBDL4 (56.4 min). B , schematic representation of the identified RHBDL4 cleavage sites in APP created with BioRender.com . The previously identified η-secretase cleavage site, as well as conventional APP processing enzymes, are indicated, scheme is not to scale. Antibody binding sites for 6E10, M3.2, 2E9, 22C11, Y188 and C1/6.1 antibodies are indicated. C–E , analysis of RHBDL4-mediated (R4-med.) cleavage of the APP deletion (APPΔ) mutant. Amino acid stretches comprising two amino acids N- and C-terminal of both identified cleavage sites were deleted (as shown in C ). Comparison of RHBDL4 cleavage pattern for APP WT and APPΔ upon co-transfection. Different gel systems were used to optimally analyse the fragments, 4 to 12% bis-tris ( D ), 8% tris-glycine ( upper panel E ) and 10 to 20% tris-tricine ( lower panel E ). Blue arrows indicate novel bands in the APPΔ samples. Detection of APP full length (APP fl.) and APP ectodomain (APP ecto.) with 22C11, CTFs with 6E10 and Y188; RHBDL4 with anti-myc antibody. β-actin or tubulin as loading controls. A representative Western blot of three individual experiments is shown. F , schematic representation of the luciferase constructs used for the RHBDL4 activity assay. All constructs are N-terminally tagged with a Flag sequence. GLuc-APP-KDEL and GLuc-KDEL contain the ER-retention motif KDEL at their C terminus. GLuc-APP-KDEL contains the APP sequence with the RHBDL4 cleavage sites. G and H , luciferase activity measured in the cell culture supernatant (luciferase released from ER, ( G ) or in cell lysates ( H ). GLuc-APP-KDEL ( green bars ) only yields extracellular luciferase activity when co-expressed with RHBDL4 (R4), but not with RHBDL1 (R1) or inactive RHBDL4. GLuc-KDEL ( dark grey ) is not cleaved by RHBDL4 and yields only luciferase activity in the lysate (ER retained). GLuc ( light grey ) is constitutively secreted and serves as a positive control. R1 + GLuc luminescence signal ( G ) or R4 + GLuc-KDEL ( H ) was set to one for normalization to plot other conditions as a fold change between biological replicates. Mean ± SEM is displayed, n = 4 to 5, one-way ANOVA ( p < 0.0001) with Tukey’s multiple comparison test. Selected statistical differences are indicated. I , detection of GLuc constructs with mouse anti-flag antibody by Western blot; RHBDL4 and RHBDL1 with direct antibodies and β-actin as a loading control. A representative Western blot of three individual experiments is shown.

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Figure Lengend Snippet: Identification of RHBDL4 cleavage sites in APP . A , identification of RHBDL4 cleavage sites by mass spectrometry. Immunoprecipitation of N-terminally myc-tagged APP fragments after co-transfection with either active or inactive (inac.) RHBDL4 (R4) in HEK293T cells. Samples were digested with LysC and analyzed by electrospray ionization mass spectrometry (ESI-MS). Representative extracted ion chromatograms showing retention times for different identified peptides. The table lists the identified retention time per peak, the peptide mass per charge (m/z), and peptide sequences along with APP695 amino acid numbering for fragments or cleavage sites. Due to subtle differences in the automated injections, the retention time of the complete LysC peptide differs between the samples containing inactive RHBDL4 (55.7 min) and active RHBDL4 (56.4 min). B , schematic representation of the identified RHBDL4 cleavage sites in APP created with BioRender.com . The previously identified η-secretase cleavage site, as well as conventional APP processing enzymes, are indicated, scheme is not to scale. Antibody binding sites for 6E10, M3.2, 2E9, 22C11, Y188 and C1/6.1 antibodies are indicated. C–E , analysis of RHBDL4-mediated (R4-med.) cleavage of the APP deletion (APPΔ) mutant. Amino acid stretches comprising two amino acids N- and C-terminal of both identified cleavage sites were deleted (as shown in C ). Comparison of RHBDL4 cleavage pattern for APP WT and APPΔ upon co-transfection. Different gel systems were used to optimally analyse the fragments, 4 to 12% bis-tris ( D ), 8% tris-glycine ( upper panel E ) and 10 to 20% tris-tricine ( lower panel E ). Blue arrows indicate novel bands in the APPΔ samples. Detection of APP full length (APP fl.) and APP ectodomain (APP ecto.) with 22C11, CTFs with 6E10 and Y188; RHBDL4 with anti-myc antibody. β-actin or tubulin as loading controls. A representative Western blot of three individual experiments is shown. F , schematic representation of the luciferase constructs used for the RHBDL4 activity assay. All constructs are N-terminally tagged with a Flag sequence. GLuc-APP-KDEL and GLuc-KDEL contain the ER-retention motif KDEL at their C terminus. GLuc-APP-KDEL contains the APP sequence with the RHBDL4 cleavage sites. G and H , luciferase activity measured in the cell culture supernatant (luciferase released from ER, ( G ) or in cell lysates ( H ). GLuc-APP-KDEL ( green bars ) only yields extracellular luciferase activity when co-expressed with RHBDL4 (R4), but not with RHBDL1 (R1) or inactive RHBDL4. GLuc-KDEL ( dark grey ) is not cleaved by RHBDL4 and yields only luciferase activity in the lysate (ER retained). GLuc ( light grey ) is constitutively secreted and serves as a positive control. R1 + GLuc luminescence signal ( G ) or R4 + GLuc-KDEL ( H ) was set to one for normalization to plot other conditions as a fold change between biological replicates. Mean ± SEM is displayed, n = 4 to 5, one-way ANOVA ( p < 0.0001) with Tukey’s multiple comparison test. Selected statistical differences are indicated. I , detection of GLuc constructs with mouse anti-flag antibody by Western blot; RHBDL4 and RHBDL1 with direct antibodies and β-actin as a loading control. A representative Western blot of three individual experiments is shown.

Article Snippet: The following primary antibodies were used: 22C11 (Millipore), 6E10 (human Aβ region-specific, Biolegend), 2E9 (epitope as determined by Willem et al. : PWHSFGADSVP, N-terminal of β-cleavage and C-terminal of η-cleavage site; Millipore), M3.2 (mouse Aβ region-specific, Biolegend), Y188 (APP C-terminus, ab32136, Abcam), C1/6.1 (APP C-terminus, Biolegend) mouse-anti-myc (9B11, Cell Signaling), mouse-anti-β-actin (8H10D10, Cell Signaling), rabbit-anti-β-tubulin (2128, Cell Signaling), rabbit-anti-flag (D6W5B, Cell Signaling), mouse-anti-flag M2 (F3165, Sigma), rabbit-anti-RHBDL4 (HPA013972 Sigma), goat-anti-RHBDL1 (sc139041, Santa Cruz Biotechnology) rabbit-anti-integrin-β1 (D2E5, Cell signaling).

Techniques: Mass Spectrometry, Immunoprecipitation, Cotransfection, Binding Assay, Mutagenesis, Comparison, Western Blot, Luciferase, Construct, Activity Assay, Sequencing, Cell Culture, Positive Control, Control

Familial APP mutations do not affect the RHBDL4-mediated processing of APP. A and B , RHBDL4-mediated (R4-med.) processing of familial AD mutants of APP. Co-transfection of various familial APP mutants with active RHBDL4. Detection of APP full length (APP fl.) and APP ectodomain (APP ecto.) with 22C11, CTFs with 6E10 and Y188; RHBDL4 with anti-myc antibody. β-actin as loading controls. APP-CTFs were quantified and normalized first to β-actin and then the fold change compared to WT was calculated. WT was always set to one in each individual experiment ( green dashed line ). Mean ± SEM is displayed, n = 3 to 6, p values for Bonferroni-corrected one sample t-tests are reported.

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Figure Lengend Snippet: Familial APP mutations do not affect the RHBDL4-mediated processing of APP. A and B , RHBDL4-mediated (R4-med.) processing of familial AD mutants of APP. Co-transfection of various familial APP mutants with active RHBDL4. Detection of APP full length (APP fl.) and APP ectodomain (APP ecto.) with 22C11, CTFs with 6E10 and Y188; RHBDL4 with anti-myc antibody. β-actin as loading controls. APP-CTFs were quantified and normalized first to β-actin and then the fold change compared to WT was calculated. WT was always set to one in each individual experiment ( green dashed line ). Mean ± SEM is displayed, n = 3 to 6, p values for Bonferroni-corrected one sample t-tests are reported.

Techniques: Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Techniques: In Vitro, Cotransfection, Positive Control, Membrane, Western Blot, Immunoprecipitation, Cell Culture, Control, Derivative Assay

Scheme of APP processing and Aη formation in different compartments. In the absence of RHBDL4, full-length APP traffics to the cell surface where MT5-MMP as well as α- or β-secretases will process APP to generate Aη at the cell surface ( left panels ). In the presence of RHBDL4, APP will be cleaved by RHBDL4 in the ER, and RHBDL4-derived large APP C-terminal fragments do not reach the cell surface. RHBDL4-derived Aη-like peptides are directly generated in the ER ( right panels ).

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Figure Lengend Snippet: Scheme of APP processing and Aη formation in different compartments. In the absence of RHBDL4, full-length APP traffics to the cell surface where MT5-MMP as well as α- or β-secretases will process APP to generate Aη at the cell surface ( left panels ). In the presence of RHBDL4, APP will be cleaved by RHBDL4 in the ER, and RHBDL4-derived large APP C-terminal fragments do not reach the cell surface. RHBDL4-derived Aη-like peptides are directly generated in the ER ( right panels ).

Techniques: Derivative Assay, Generated

RHBDL4 knockout affects APP levels and Aη production in vivo . A , Aη extraction was performed according to , extraction of soluble proteins upon DEA extraction from brain tissue homogenates of WT and RHBDL4 KO mice at 10 to 11 months of age. sAPPα and Aη were detected with M3.2 antibody (specific for mouse Aβ), β-actin as loading control. Representative Western blot for n = 8 brain samples (WT and KO, each). B and C , full-length APP levels in brain tissue lysates of 10 to 11 months old RHBDL4 knockout (R4 KO) mice as compared to age-matched wild-type (WT) mice. Equal amounts of protein were loaded per lane. Detection of APP full length (fl) with 22C11, endogenous RHBDL4 with rabbit anti-RHBDL4 antibody, and β-actin as a loading control. Quantification with ImageJ, normalized to β-actin, mean ± SEM, n = 14 to 19, p -value for unpaired two-tailed t test is reported. D , schematic representation of cortical dissociation and primary cell culture procedure followed by Aη immunoprecipitation from cell medium and downstream analysis via Western blot. Created using BioRender. E , full-length APP expression from primary cortical cell culture lysates prepared from WT or RHBDL4 knockout brains. Representative Western blot of three individual experiments is shown; APP quantification of untreated condition normalized to ponceau S with ImageJ, mean ± SEM, unpaired two-tailed t test performed. Detection of APP full length (fl.) with M3.2, endogenous RHBDL4 with rabbit-anti-RHBDL4 antibody, β-tubulin, and ponceau S as loading controls. F , Immunoprecipitation of Aη species from primary cortical cell culture supernatant prepared from WT or RHBDL4 knockout mouse brains. Input consists of total cell culture supernatant ( left panels ) while immunoprecipitation (IP) was performed using the M3.2 antibody ( right panels ). sAPP and Aη detection using M3.2 antibody. Representative Western blot of three individual experiments is shown; quantification of untreated condition with ImageJ normalized to full-length APP from lysates, mean ± SEM, the p -value for unpaired two-tailed t test is reported. Treatment with metalloprotease inhibitor (MP Inh.) TIMP2.

Journal: The Journal of Biological Chemistry

Article Title: Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

doi: 10.1016/j.jbc.2024.107541

Figure Lengend Snippet: RHBDL4 knockout affects APP levels and Aη production in vivo . A , Aη extraction was performed according to , extraction of soluble proteins upon DEA extraction from brain tissue homogenates of WT and RHBDL4 KO mice at 10 to 11 months of age. sAPPα and Aη were detected with M3.2 antibody (specific for mouse Aβ), β-actin as loading control. Representative Western blot for n = 8 brain samples (WT and KO, each). B and C , full-length APP levels in brain tissue lysates of 10 to 11 months old RHBDL4 knockout (R4 KO) mice as compared to age-matched wild-type (WT) mice. Equal amounts of protein were loaded per lane. Detection of APP full length (fl) with 22C11, endogenous RHBDL4 with rabbit anti-RHBDL4 antibody, and β-actin as a loading control. Quantification with ImageJ, normalized to β-actin, mean ± SEM, n = 14 to 19, p -value for unpaired two-tailed t test is reported. D , schematic representation of cortical dissociation and primary cell culture procedure followed by Aη immunoprecipitation from cell medium and downstream analysis via Western blot. Created using BioRender. E , full-length APP expression from primary cortical cell culture lysates prepared from WT or RHBDL4 knockout brains. Representative Western blot of three individual experiments is shown; APP quantification of untreated condition normalized to ponceau S with ImageJ, mean ± SEM, unpaired two-tailed t test performed. Detection of APP full length (fl.) with M3.2, endogenous RHBDL4 with rabbit-anti-RHBDL4 antibody, β-tubulin, and ponceau S as loading controls. F , Immunoprecipitation of Aη species from primary cortical cell culture supernatant prepared from WT or RHBDL4 knockout mouse brains. Input consists of total cell culture supernatant ( left panels ) while immunoprecipitation (IP) was performed using the M3.2 antibody ( right panels ). sAPP and Aη detection using M3.2 antibody. Representative Western blot of three individual experiments is shown; quantification of untreated condition with ImageJ normalized to full-length APP from lysates, mean ± SEM, the p -value for unpaired two-tailed t test is reported. Treatment with metalloprotease inhibitor (MP Inh.) TIMP2.

Techniques: Knock-Out, In Vivo, Extraction, Control, Western Blot, Two Tailed Test, Cell Culture, Immunoprecipitation, Expressing

Review

Structured Review

Images

Similar Products

Image Search Results