dysferlin cdna  (Addgene inc)

Journal: Nature Communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: a Domain organization of dysferlin (237 kDa). Individual domains are color-coded. b PageBlue-stained SDS-PAGE gel of purified full-length dysferlin. Three independent experiments were performed. c Representative electron micrograph shows negatively stained dysferlin protein. More than 50 images were collected in two independent sessions. d Cryo-EM micrograph (left) and 2D class average (right) of the dysferlin monomer. e Cryo-EM reconstruction of dysferlin. The front and back views of the cryo-EM maps are shown. Individual domains are color-coded according to panel a . f The front and back view of the molecular model of the dysferlin monomer in cartoon representation. Individual domains are color-coded according to panel a . g Electrostatic potential distribution in the dysferlin monomer. The electrostatic potential of the monomer is calculated using default electrostatic surface potential parameters in ChimeraX, where the acidic surface is shown in red, and the basic surface is in blue. Source data are provided as a Source Data file 2.

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch) was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Staining, SDS Page, Purification, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: a Structures of the classical C2 domains (C2B-C2G) of dysferlin. Inserts of variable length extend from the consensus β-sandwich of two antiparallel four-stranded β-sheets. b Structure of the composite C2 domain (C2-FerA-FerB-C2). The topology diagram shows the position and orientation of secondary structures. c Structure of the nested DysF region composed of the DysFN, DysFI, and DysFC. d Residue-level interactions between the C2C insert and the FerB. e Residue-level interactions between the C2C and the composite C2 domain. f Residue-level interactions between the DysF region and the C2-FerA domain.

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch) was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Residue

Journal: Nature Communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: a Native PAGE of dysferlin shows the presence of monomers, dimers, and higher-order oligomers. M denotes marker. Two independent experiments were performed. b SEC-MALS shows the presence of dysferlin monomers, dimers, and high-order oligomers. The molecular weight of GDN micelles is in the range of ~ 140 to 160 kDa. c Side-view of the asymmetric parallel dysferlin homodimer. The homodimer shows DysF regions on the proximal side and the C2G domains on the distal side of the homodimer. d Close-up view of the dimer interface shows how the two protomers are docked to each other. The homodimer is rotated 180° relative to the overview representation. Individual domains are color coded according to panel c . e Clinically significant dysferlinopathy mutations in the dimer interface. Individual domains are color coded according to panel c . The dimer interface is indicated by a dashed line. f Close-up view of the dimer interface shows the location of select disease-causing mutations. * indicates a terminal codon. Source data are provided as a Source Data file 2.

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch) was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Clear Native PAGE, Marker, Molecular Weight

Journal: Nature Communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: Upon membrane damage and Ca 2+ influx, dysferlin containing vesicles are transported to the sites of membrane repair. These vesicles dock to the membrane. Dysferlin then accumulates in the form of dimers and larger oligomers at the sites of membrane damage, where it, together with binding partners, facilitates repair. The precise structural and molecular mechanisms underlying the interaction between dysferlin, and its binding partners are largely unknown. Schematic not drawn to scale. MG53: Mitsugumin 53, also known as TRIM72; A1: Annexin A1; A2: Annexin A2.

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch) was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Membrane, Binding Assay

Journal: Nature communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin.

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: Fig. 3 | Key interactions in the dysferlin monomer. a Two views (front, back) of the pentameric ring formed by the C2B, C2E, C2D, C2C, and composite C2 domain and stabilized by the C2D-C2E linker. Individual domains are color-coded. b Schematic representation of the pentameric ring. Color code according to panel a. c Residue-level interactions at interdomain interfaces in the pentameric ring. d Residue-level interactions of the FerI with the C2B, C2C, and C2E. Color code according to panel a. The electrostatic potential distribution is shown for the C2B, C2C, and C2E (left). e The C2E-C2F (grey) and C2F-C2G (grey) linkers connect the respective domains. The C2E insert interacts with the C2E (yellow), C2F (blue), and

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch)60 was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Structure based primary sequence alignment of the C2A domains of the ferlin family. Secondary structure assignments are based on the known and predicted structures of dysferlin as calculated by DSSP. The η symbol refers to a 3 10 -helix. α -helices, 3 10 -helices and π -helices are displayed as medium, small and large squiggles, respectively. β -strands are rendered as arrows, strict β -turns as TT letters and strict α -turns as TTT. Residues that are absolutely conserved between all C2A domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the dysferlin sequence. The mean evolutionary relatedness between the C2A domains of dysferlin, otoferlin, myoferlin, and Fer1L5 is 26.5% identity and 56.7% similarity.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2B domains of the ferlin family. Secondary structure assignments are based on the predicted structures of dysferlin C2B as calculated by DSSP. The η symbol refers to a 3 10 -helix. α -helices, 3 10 -helices and π -helices are displayed as medium, small and large squiggles, respectively. β -strands are rendered as arrows, strict β -turns as TT letters and strict α -turns as TTT. Residues that are absolutely conserved between all C2B domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The mean evolutionary relatedness between the C2B domains of dysferlin, otoferlin, myoferlin, Fer1L4, Fer1L5, and Fer1L6 is 44.4% mean identity and 63.8% mean sequence similarity across all six C2B domains.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2C domains of the ferlin family. Secondary structure assignments are based on the predicted structures of dysferlin C2C as calculated by DSSP. The η symbol refers to a 3 10 -helix. α -helices, 3 10 -helices and π -helices are displayed as medium, small and large squiggles, respectively. β -strands are rendered as arrows, strict β -turns as TT letters and strict α -turns as TTT. Residues that are absolutely conserved between all C2C domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin C2C sequence. The mean evolutionary relatedness between the C2C domains of dysferlin, otoferlin, myoferlin, Fer1L4, Fer1L5, and Fer1L6 is 38.2% mean identity and 60.7% mean sequence similarity.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2-FerA domains of the ferlin family. Secondary structure assignments are based on the predicted structures of dysferlin C2-FerA as calculated by DSSP. The η symbol refers to a 3 10 -helix. α -helices, 3 10 -helices and π -helices are displayed as medium, small and large squiggles, respectively. β -strands are rendered as arrows, strict β -turns as TT letters and strict α -turns as TTT. Residues that are absolutely conserved between all C2-FerA domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The blue helical cartoons demarcate the FerA helices between β strand-4 and β strand-5. The mean evolutionary relatedness between the C2-FerA domains of dysferlin, otoferlin, myoferlin, Fer1L4, Fer1L5, and Fer1L6 shows 34.9% mean identity and 55.3% mean sequence similarity for all six C2-FerA domains.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the DysF domains of the ferlin family. Secondary structure assignments are based on the known and predicted structures of dysferlin DysF as calculated by DSSP. The blue secondary structure is predicted to constitute the “outer” DysF domain, while the green secondary structure demarcates the “inner” DysF domain. The η symbol refers to a 3 10 -helix. α -helices, 3 10 -helices and π -helices are displayed as medium, small and large squiggles, respectively. β -strands are rendered as arrows, strict β -turns as TT letters and strict α -turns as TTT. Residues that are absolutely conserved between all DysF domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The fold of the predicted dysferlin outer DysF domain superimposes with the known structure of the dysferlin inner DysF structure with an RMSD of 3.5 Å across all C- α residues ( . The mean evolutionary relatedness between the three DysF regions of the Type-I ferlins is 45.0% mean identity and 68.4% mean sequence similarity.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2D domains of the ferlin family. Secondary structure assignments are based on the predicted structures of dysferlin C2D as calculated by DSSP. α -helices are displayed as medium squiggles. β -strands are rendered as arrows, strict β -turns as TT letters. Residues that are absolutely conserved between all C2D domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The mean evolutionary relatedness between all six C2D domains shows 63.4% mean sequence similarity and 40.9% mean identity.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2E domains of the ferlin family. Secondary structure assignments are based on the known and predicted structures of dysferlin C2E as calculated by DSSP. α -helices are displayed as medium squiggles. β -strands are rendered as arrows, strict β -turns as TT letters. Residues that are absolutely conserved between all C2D domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The mean evolutionary relatedness between the C2E domains of all ferlins is 54.1% mean sequence similarity and 34.7% mean identity.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2F domains of the ferlin family. Secondary structure assignments are based on the predicted structures of dysferlin C2F as calculated by DSSP. α -helices are displayed as medium squiggles. β -strands are rendered as arrows, strict β -turns as TT letters. Residues that are absolutely conserved between all C2F domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. beta strands are labeled according to the main body of the C2 domain. The accessory domain between β -6 and β -7 are colored in blue. The mean evolutionary relatedness between the six C2F domains shows 66.7% mean sequence similarity and 48.2% mean identity for all six C2F domains.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue, Labeling

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: Model-based primary sequence alignment of the C2G domains of the ferlin family. Secondary structure assignments are based on the known and predicted structures of dysferlin C2G as calculated by DSSP. α -helices are displayed as medium squiggles. β -strands are rendered as arrows, strict β -turns as TT letters. Residues that are absolutely conserved between all C2G domains are highlighted in red. Conserved residues are boxed in blue. Numbers along the top of the alignment correspond to residue numbers in the human dysferlin sequence. The mean evolutionary relatedness between the six C2G domains shows 69.5% mean sequence similarity and 51.2% mean identity for all six C2G domains.

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: A. SDS-PAGE showing the purified dysferlin C2-FerA domain versus molecular weight size markers. B. Far-UV CD spectrum of purified dysferlin C2-FerA (red curve) and predicted C2-FerA spectrum derived from the model (blue curve). The inset table reports the secondary structure summary of the purified dysferlin C2-FerA domain (Experimental), and the dysferlin C2-FerA model (Predicted).

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: SDS Page, Purification, Molecular Weight, Derivative Assay

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: The gray spheres are the divalent cations from the crystal structures of dysferlin C2A (4IHB, chain E) and myoferlin C2A (6EEL). The amino acid boundaries from each respective C2 domain are listed with the domain assignment. The superposition of all four C2A domains are labeled as ‘Overlay’. The ferlins without a C2A domain are listed as Not Applicable (N/A).

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: Labeling

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet: A. Superimposed β 6-7 subdomain of Dysferlin C2F (green) and RNA binding domain of Staufen (1STU) in cyan; RMSD = 2.7 Å . B. Superimposed β 6-7 subdomain of Dysferlin C2F (green) and dsRNA binding domain of TARBP2 (4WYQ) in magenta; RMSD = 0.86 Å .

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques: RNA Binding Assay, Binding Assay

Journal: bioRxiv

Article Title: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

doi: 10.1101/2022.01.18.476802

Figure Lengend Snippet:

Article Snippet: The gene with codon-optimized human dysferlin cDNA was obtained from Addgene (Plasmid 67878) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: The full-length model indicated super-tertiary domain interactions in the dysferlin model. The RoseTTAFold models that were used in this study were flexibly aligned using FATCAT . Inconsistencies in the 3D models that were generated as a result of the elastic alignment process were repaired using PyMod . Figures were rendered with PyMol and displayed as 180° views of the model. The various domains of dysferlin are shown as colored surfaces and similarly colored labels. The other ferlin full-length models can be found in the supplemental information (S1-S17 Figs and S1-S7 Tables in ).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Generated

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Dysferlin C2A (green, 4IHB), otoferlin C2A (blue, 3L9B), myoferlin C2A (cyan, 6EEL), and Fer1L5 C2A (yellow). The gray spheres are the divalent cations from the crystal structures of dysferlin C2A (4IHB, chain E) and myoferlin C2A (6EEL). The amino acid boundaries from each respective C2 domain are listed with the domain assignment. The superposition of all four C2A domains is labeled as ‘Overlay’. The ferlins without a C2A domain are listed as Not Applicable (N/A).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Labeling

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Primary sequence alignment of the FerI region of the six human ferlin proteins.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A. C2-FerA domain schematic showings the secondary structure connectivity and the large insertion of the β 4–5 FerA subdomain. B. SDS-PAGE showing the purified dysferlin C2-FerA domain versus molecular weight size markers. C. Far-UV CD spectrum of purified dysferlin C2-FerA (red curve) and predicted C2-FerA spectrum derived from the model (blue curve). The inset table reports the secondary structure summary of the purified dysferlin C2-FerA domain (Experimental), and the dysferlin C2-FerA model (Predicted).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: SDS Page, Purification, Molecular Weight, Derivative Assay

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A. Superimposed β 6–7 subdomain of Dysferlin C2F (green) and RNA binding domain of Staufen (1STU) in cyan; RMSD = 2.7 Å. B. Superimposed β 6–7 subdomain of Dysferlin C2F (green) and dsRNA binding domain of TARBP2 (4WYQ) in magenta; RMSD = 0.86 Å.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: RNA Binding Assay, Binding Assay

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Predicted ferlin transmembrane span boundaries and extracellular residues.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A: Dysferlin C2A structure (4IHB) colored to highlighted the various insertions of subdomains. B: The schematic highlights the loops with embedded subdomains. The Type-2 C2 domain β -strand topology is shown as grey arrows. The colored loops: β 1–2 (green, C2C), β 2–3 (orange, C2-FerA), β 4–5 (blue, C2-FerA, C2G), β 6–7 (red, C2C, C2E, C2G, C2F), and β 7–8 (purple, C2C) show where conserved subdomains are present.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques:

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Myoferlin cleavage products are detected in breast tumours, breast cancer cell lines and transfected cells. (A) Western blot analysis of five mouse xenograft tumour samples derived from MDA-MB-231 cells transplanted into nude mice, showing an N-terminal myoferlin cleavage product. (B) Western blot analysis of endogenous myoferlin in four different human breast cancer cell lines (BT-474, EVSA-T, MCF-7 and MDA-MB-231) with (+) or without (−) scrape-injury in +Ca2+-PBS. A ~75kDa C-terminal cleavage product is detected with the K-16 antibody recognizing a C-terminal myoferlin epitope, and a ~180 kDa counter fragment detected with 7D2 that recognizes an N-terminal myoferlin epitope. K16 works less effectively than 7D2 with a higher background, thus 30 μg total protein is loaded on K16 gel and 10 μg total protein loaded on the 7D2 gel. (C) ~75 kDa C-terminal myoferlin fragments (doublet bands) and an ~180 N-terminal counter fragment are also detected in triple negative human breast cancer samples (#88 and #89). H&E staining of fresh frozen tumour sections of the same samples run on the western blot. The purple stain represents tumour tissue and the pink stain normal breast tissue (H&E staining provided by the ABCTB). Scale bar 500 μm. (D) Western blot analysis of HEK293 and COS-7 cells transfected with full length myoferlin (MFL) or dysferlin containing the calpain cleavage site in exon 40a (D40a) with (+) or without (−) scrape-injury in +Ca2+-PBS.

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: Transfection, Western Blot, Derivative Assay, Staining

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Myoferlin bears two predicted calpain cleavage sites. (A) Calpain cleavage prediction (GPS-CCD, ccd.biocuckoo.org; [29]) of myoferlin resulted in two highly predicted cleavage sites consistent with production of a 75–80 kDa C-terminal cleavage fragment. (B) Schematic of the myoferlin protein structure showing the location of the two predicted calpain cleavage sites. The first predicted cleavage site (SLLS|APPC) is encoded by constitutive in exon 38, at the end of the C2DE domain and would result in a ~74 kDa mini-myoferlin fragment. The second predicted cleavage site (SKMA|SPAT) is encoded by alternatively spliced exon 38a in the linker directly adjacent to C2DE, which would result in a slightly smaller mini-myoferlin (~69 kDa). (C) Phylogenetic amino acid sequence alignment showing that exon 38a is evolutionary conserved down to the fish class, and that myoferlin exon 38a aligns to the same position as exon 40a in dysferlin. (D) PCR analysis of human tissue cDNA showing myoferlin RNA is expressed throughout the different tissues tested and reveals that exon 38a is expressed as a minor species in human tissues (~10% of total transcripts). PCR amplification was performed for 35 and 40 cycles to control for saturation. +38a and -38a are plasmid controls with and without exon 38a.

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: Sequencing, Amplification, Control, Plasmid Preparation

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Molecular modelling of predicted calpain cleavage sites in myoferlin. (A) Homology modelling of the C2DE domain of myoferlin based on the closest solved crystal structure of myoferlin C2A (2DMH, see methods) indicates the first cleavage site (encoded by exon 38, red arrow) is located in a loop between the last two beta strands (β7 and β8) of myoferlin C2DE (red loop). The cleavage site in exon 38a (yellow arrow) is located in the linker region (dashed line) that cannot be modelled and was drawn freely onto the model (yellow dashed line). (B) Alignment of the C2DE sequence of dysferlin and myoferlin in relation to the β-strand (purple) shows that the region of the first cleavage site of myoferlin has the least homology between dysferlin and myoferlin.

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: Sequencing

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Chimeric constructs of myoferlin and dysferlin. (A) Graphical illustration of the design of chimeric expression constructs. Myoferlin sequence is displayed in purple and dysferlin sequence in grey scale. The first predicted calpain cleavage site (SLLS|APPC) site in myoferlin is coloured in red and the second cleavage site in exon 38a is highlighted in yellow (SKMA|SPAT). The cleavage site in exon 40a in dysferlin is coloured in orange (TNTA|SPPS). (B) Amino acid sequences of the chimeric constructs. To confirm the cleavage site in myoferlin resided within the non-conserved loop between the 7th and 8th β-strand of the C2DE domain (see schematic in Fig. 3), we reciprocally substituted residues comprising the 7th and 8th β-strand and intervening linker C2DE between dysferlin and myoferlin (DM38 and MD40).

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: Construct, Expressing, Sequencing

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Evaluation of predicted calpain-cleavage sites in myoferlin. A) In vitro cleavage of dysferlin and myoferlin constructs immunopurified from transfected HEK293 cells and incubated with recombinant calpain-1. Protein-bound Sepharose beads were incubated in buffer containing 2 mM CaCl2 in the presence of purified 0.2 A.U. of recombinant calpain-1 at 30 °C for 10 or 120 s, or in the absence of calpain (−). Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94 °C. Digested samples were analyzed by SDS–PAGE and western blot. B) In cell cleavage of dysferlin and myoferlin expression constructs is induced via scrape-harvesting of transfected HEK293 cells. (+) with scrape injury in +Ca2+- PBS; (−) without scrape injury, harvested directly into ice-cold RIPA buffer with EDTA. Dysferlin without a calpain cleavage site (DFL) is not cleaved. Dysferlin with exon 40a (D40a) is cleaved by recombinant calpain (A) and during scrape-harvesting (B). Both predicted myoferlin cleavage sites in exon 38 and exon 38a can functionally substitute for exon 40a in in vitro and in cell cleavage assays (DM38, DM38a). Myoferlin (MFL) is cleaved by calpain-1 in vitro (A) and is cleaved independently of scrape injury in transfected HEK293 (B). Substitution of myoferlin exon 38 with the corresponding residues from dysferlin (encoded by exon 40; MD40) prevents cleavage, confirming myoferlin exon 38 sequences contain a cleavage motif. Paradoxically, myoferlin constructs with exon 38a (M38a) are not cleaved in vitro or in cells. Inclusion of exon 38a sequences regulates (precludes) cleavage both within exon 38a as well as at the exon 38 site.

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: In Vitro, Construct, Transfection, Incubation, Recombinant, Purification, Lysis, SDS Page, Western Blot, Expressing

Journal: Cellular signalling

Article Title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

doi: 10.1016/j.cellsig.2017.02.009

Figure Lengend Snippet: Myoferlin cleavage is independent of calpain-1 and -2. The dysferlin C-terminal domain confers calpain- and Ca2+-dependent cleavage on myoferlin. (A) Illustration of expression constructs; Full lengthMyoferlin(MFL),Dysferlinwith exon40a (D40a),Myoferlin with mini-dysferlinC72 C-terminal domains (MmDf). Antibody epitopes and epitope tags are annotated for each construct. (B)Western blot analysis of HEK293 cells(WT and CAPNS1−/−) transfected with MFL,MmDf, and D40a, or an untransfected control. In contrast to D40a, proteolytic cleavage of MFL is insensitive to targeted knock-out of CAPNS1, and does not require scrape injury. Substitution of the myoferlin C-terminus for the dysferlin C-terminal domains confers calpain-dependent, injury-dependent cleavage on MmDf. (C) WT and CAPNS1−/− mouse embryonic fibroblasts (MEFs) showing cleavage of endogenous myoferlin detectable with the N-terminal antibody (7D2). (D) In vitro cathepsin L cleavage of MFL transfected HEK293 cells shows the release of the same mini-myoferlin seen with the in vitro calpain-1 cleavage.

Article Snippet: Constructs The dysferlin cDNA construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1 ) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK), and was subcloned into pIRES2-EGFP (OriGene).

Techniques: Expressing, Construct, Western Blot, Transfection, Control, Knock-Out, In Vitro