Journal: Frontiers in Cell and Developmental Biology

Article Title: Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress

doi: 10.3389/fcell.2022.1008542

Figure Lengend Snippet: Vimentin cleavage is blocked by inhibitors of the proteasome and calpains, which are elevated in GAN. (A) Immunoblotting for vimentin (top) and actin (bottom; loading control) in Triton X insoluble pellet fractions from untreated (lanes 1–3, 7–9) or water-exposed (lanes 4–6, 10–12) control 56.3 and GAN 56.1 fibroblasts in the absence or presence of the DMSO vehicle control, calpain inhibitor (MDL-28170), or calpain/proteasome inhibitor (MG-132). Note partial cleavage inhibition by MDL and near-complete inhibition by MG-132. (B) Immunoblotting for vimentin (top) and actin (bottom; loading control) in reduced Triton X insoluble fractions and for calpain-1 (middle top), calpain-2 (middle), and calpastatin (middle bottom) in reduced Triton X soluble fractions from GAN and control fibroblasts exposed to 8 min of hypotonic stress and treated with the DMSO vehicle control or MG-132. Shown are technical replicates from an individual experiment. The experiment was independently repeated at least 3 times. (C) Quantification of the relative fragment 1 (FR1) and fragment 2 (FR2) band intensities normalized to the full length vimentin band for each cell line (GAN 56.1 fibroblasts; control 56.3 fibroblasts) and treatment group (from panel B). **** p < 0.0001; two-way ANOVA. (D) Quantification of calpain-1, calpain-2, and calpastatin levels (from panel B). ** p < 0.01; **** p < 0.0001; two-way ANOVA.

Article Snippet: The following primary antibodies and concentrations were utilized: rabbit anti-Vimentin (Cell Signaling Technology, D21H3, WB 1:1,000, IF 1:200), mouse anti-Vimentin (Invitrogen, V9, WB 1:1,000–1:2,500, IF 1:100), mouse anti-Actin (Thermo Fisher Scientific, ACTN05, WB 1:1,000), mouse anti-Actin (Santa Cruz, SPM161, WB 1:1,000), rabbit anti-Calpastatin (Cell Signaling Technology, WB 1:1,000), rabbit anti-Calpain 1 (Cell Signaling Technology, large subunit Mu-type, WB 1:1,000), rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, WB 1:1,000), and rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, E3M6E, IF 1:200).

Techniques: Western Blot, Inhibition

Journal: Frontiers in Genetics

Article Title: A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4)

doi: 10.3389/fgene.2021.801253

Figure Lengend Snippet: SMA4 causative p. G492R/p. F610C compound het mutation in CAPN1 . (A) Pedigree of SMA4 family. Squares indicate male, circles indicate females. Solid symbols denote affected individuals and open symbols unaffected individuals. Carriers are represented with internal dots. Asterisk indicates individuals in which whole exomes sequencing was performed and the black arrow shows the proband. (B) Sanger sequencing confirms the nucleotide changes c.1474G > A (p.G492R) and c.1829T > G (p.F610C) in the two affected siblings (II:2 and II:3). (C) The substituted amino acid residues (p.G492 and p. F610) are located within highly conserved functional domains of the CAPN1 protein. (D) CAPN1 secondary structure displaying the protease domain (yellow) and the C2 domain-like (C2L, orange) and penta-EF-hand (PEF, red) Ca 2+ binding domains bearing the p. G492R and p. F610C mutations respectively. Affected residues located in the 3D ribbon structure of CAPN1 using PyMOL and the Rattus norvegicus CAPN1 crystalized structure (PDB:1QXP). (E) The impact of the p. G492R and p. F610C missense mutations in the protein stability (∆∆G) of CAPN1 predicted using the PremPS online tool. For clarity, the ribbon was hidden and only the non-covalent interactions affected by the substitutions are displayed. Dotted lines represent hydrophobic (blue), polar (light blue), hydrogen bonds (violet) and Van der Waals (green) interactions in the wild-type and mutant structures. Positive ∆∆G predicts a reduction in the stability of the resulted protein.

Article Snippet: Membranes were probed with the following primary antibodies: rabbit α- CAPN1 (CST), 1:1,500; mouse α-SMN (BD Transduction Laboratories), 1:2000; rabbit α- p -Akt (CST), 1:1,000; mouse α-Pan-Akt (CST), 1:2000; rabbit α-LC3B-II (CST) 1:1,000; rabbit α-β-actin (CST) 1:2000; rabbit α-caspase-3 (Abcam) 1:4,000 and rabbit α-β-actin (CST) 1:2000.

Techniques: Mutagenesis, Sequencing, Functional Assay, Binding Assay

Journal: Frontiers in Genetics

Article Title: A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4)

doi: 10.3389/fgene.2021.801253

Figure Lengend Snippet: Consequences of p. G492R/p. F610C compound heterozygous mutation on CAPN1 protein levels and protease activity. (A) Levels of CAPN1 protein determined by western blot analysis in lysates from a SMA4 patient (proband II:2) compared to controls fibroblasts ( n = 3). β-actin was used as a loading control in these experiments. (B) Data in bar graphs are represented as mean ± SEM from 3 independent experiments. (C) Protease activity determined from 100 μg protein lysates in the presence of 0.5 mM EGTA and 3 mM CaCl 2 (Ca 2+ ) , with and without the specific calpain-1 inhibitor (C1I) at saturating (20 µM) and at half-maximal inhibitory concentration (1 µM). Data in bar graphs represents the mean ± SEM of the hydrolysis rate of 100 μM Succ-LLVY-AMC substrate determined from the linear portion of the curve measured every 30 s for 45 min. (D) Calpain-1 activity from 5 independent experiments. Data is represented as % of Succ-LLVY-AMC hydrolysis in the assay that can be attributed to CAPN1 , calculated by dividing the hydrolysis rate in the presence of 3 mM free Ca 2+ by the value obtained in the presence of 1 µM C1I. p values were obtained from a 2-way ANOVA test (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Article Snippet: Membranes were probed with the following primary antibodies: rabbit α- CAPN1 (CST), 1:1,500; mouse α-SMN (BD Transduction Laboratories), 1:2000; rabbit α- p -Akt (CST), 1:1,000; mouse α-Pan-Akt (CST), 1:2000; rabbit α-LC3B-II (CST) 1:1,000; rabbit α-β-actin (CST) 1:2000; rabbit α-caspase-3 (Abcam) 1:4,000 and rabbit α-β-actin (CST) 1:2000.

Techniques: Mutagenesis, Activity Assay, Western Blot, Concentration Assay

Journal: Frontiers in Genetics

Article Title: A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4)

doi: 10.3389/fgene.2021.801253

Figure Lengend Snippet: Effect of the CAPN1 compound heterozygous mutation on intracellular pathways affected in spinal muscular atrophy. (A) Protein levels of the autophagy marker LC3B (LC3B-II, 14 KDa and LC3B-I, 16 KDa) determined by western blot analysis in lysates from SMA4 patient (proband II:2) compared to controls fibroblasts. β-actin was used as a loading control in these experiments. (A) The expression of LC3B type I (LC3B-I) and LC3B type II (LC3B-II) was determined and shown in bar graphs as the mean ± SEM from 3 independent experiments. (B) Autophagy occurrence visualised by confocal microscopy using an anti-LC3B antibody (green) in cells stained with phalloidin (violet). (B) . Quantification of the LC3B cytoplasmic expression within each cell. Violin plot shows the full distribution of all data points acquired ( n > 250 cells). (C) ATP production was measured using the ATPlite assay kit. Arbitrary luminescence units (ALU) are shown for each experimental group from data obtained for 3 independent experiments. (D) Mitochondria membrane potential was assessed in live cells using 1 μM TMRE (green). 1 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) was used as a positive control to uncouple mitochondrial oxidative phosphorylation. (D) . Quantification of the TMRE stanning (integrated intensity) for untreated (solid bars) and FCCP treated (lined pattern) cells was calculated for 10 random images and represented using bar graphs as the mean ± SEM from 3 separate experiments for each cell line. (E) Protein levels of the PI3K-Akt neuronal survival pathway protein p -Akt (relative to total pan Akt) and the proapoptotic caspase-3 determined by western blot analysis. β-actin was used as a loading control in these experiments. (E) The expression of p -Akt and caspase-3 was determined and shown in bar graphs as the mean ± SEM from 3 independent experiments.

Article Snippet: Membranes were probed with the following primary antibodies: rabbit α- CAPN1 (CST), 1:1,500; mouse α-SMN (BD Transduction Laboratories), 1:2000; rabbit α- p -Akt (CST), 1:1,000; mouse α-Pan-Akt (CST), 1:2000; rabbit α-LC3B-II (CST) 1:1,000; rabbit α-β-actin (CST) 1:2000; rabbit α-caspase-3 (Abcam) 1:4,000 and rabbit α-β-actin (CST) 1:2000.

Techniques: Mutagenesis, Marker, Western Blot, Expressing, Confocal Microscopy, Staining, Positive Control

Journal: Frontiers in Genetics

Article Title: A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4)

doi: 10.3389/fgene.2021.801253

Figure Lengend Snippet: β-catenin is not altered in SMA4 patient fibroblasts. (A) β-catenin is the only differentially expressed protein in SMA synapses that is a CAPN1 known protease target. (B) Functional protein association network of all reported SMA genes and CTNNB1 using STRING. The thickness of the network edges indicates the strength of data support. Network clusters are differentiated by colour and their connection represented by a dotted line. (C) Protein levels of β-catenin determined by western blot analysis in lysates from a SMA4 patient compared to controls fibroblasts. β-actin was used as a loading control in these experiments. The expression of β-catenin is shown in bar graphs as the mean ± SEM from 3 independent experiments. (D) Immunofluorescence analysis in control and SMA4-derived fibroblasts detects β-catenin (green) distribution in cytoplasmic and within the nuclei (D) . Quantification of the β-catenin cytoplasmic expression and nuclear (integrated intensity) within each cell. Violin plot shows the full distribution of all data points acquired ( n > 250 cells).

Article Snippet: Membranes were probed with the following primary antibodies: rabbit α- CAPN1 (CST), 1:1,500; mouse α-SMN (BD Transduction Laboratories), 1:2000; rabbit α- p -Akt (CST), 1:1,000; mouse α-Pan-Akt (CST), 1:2000; rabbit α-LC3B-II (CST) 1:1,000; rabbit α-β-actin (CST) 1:2000; rabbit α-caspase-3 (Abcam) 1:4,000 and rabbit α-β-actin (CST) 1:2000.

Techniques: Functional Assay, Western Blot, Expressing, Immunofluorescence, Derivative Assay

Journal: medRxiv

Article Title: Deceased donor kidney degradomics indicates cytoskeletal proteolytic alterations impacting post-transplant function

doi: 10.1101/2021.06.10.21254305

Figure Lengend Snippet: A. Table showing the demographic and clinical characteristics of patients who have undergone nephrectomies from where PCKS were prepared, n=8 patients; B. PCKS stimulation with TGF-β (10ng) up to 24h caused the kidney slices to develop profibrotic morphological changes detected by serius red (top) and trichrome mason staining (bottom). 20x magnification and 20µm scale bar; C. Sirius red and trichrome mason staining revealed that the PCKSs developed profibrotic histological characteristics of cortex, podocyte and tubular structures (****P<0.0001,***P<0.001; paired t-test; dot plot shows biological and technical replicates) following treatment with TGF-β up to 24h, (evidence of profibrotic characteristics are derived from Collagen1A1 mRNA comparison as shown in the ; measurement of ATP levels showed that the integrity of the PCKS was maintained throughout ; D. Calpain-1 activity significantly (**P<0.01; paired t-test) increased in PCKSs treated with TGF-β up to 24 hours.

Article Snippet: Antibodies used were; rabbit polyclonal anti-calpain-1 (2556S, Cell Signalling), mouse monoclonal anti α-actinin-4 (SC390205, Santa Cruz Biotechnology), goat polyclonal anti Talin-1 (AF5456, R&D Systems) and rabbit anti GAPDH (5174s, Cell Signalling).

Techniques: Staining, Derivative Assay, Activity Assay

Journal: medRxiv

Article Title: Deceased donor kidney degradomics indicates cytoskeletal proteolytic alterations impacting post-transplant function

doi: 10.1101/2021.06.10.21254305

Figure Lengend Snippet: A. Representative contrast microscopy images showing changes to podocyte cells morphology when treated by TGF-β (10ng) for up to 24h; 20x magnification, scale bar is 100µm; B. Calpain activity of podocyte cells was significantly increased following stimulation with TGF-β (10ng) up to 24h when compared to control (****P<0.0001; t-test). Treatment of cells with Calpeptin (1µM) prior to and during TGF-β stimulation significantly reduced Calpain activation (**P<0.01; t-test); C. The degradation pattern of Talin-1 of suboptimal DBD kidneys compared to LD kidney with good outcome showed the generation of ∼125KDa fragment (cropped western blot presenting donor kidneys probed with anti-Talin-1 was derived from data presented in ). In vitro human kidney podocyte cells stimulated with TGF-β (0-20ng, up to 24h) showed a dose responsive loss of the Talin full length protein. Talin-1 proteolytic fragments were detected as ∼190 and ∼125kDa bands with a further lower intensity band at ∼100 kDa. The generation of these fragments was reduced when podocytes were treated with Calpain inhibitor Calpeptin (1µM) prior to and during TGF-β treatment. The two histograms show the alterations (fold changes) in the band intensities of the Talin-1 main band and the 125KDa fragment following treatment of podocytes with TGF-β in the absence (red bars) and presence of Calpain-1 inhibitor Calpeptin (grey bars); D. Phalloidin staining of actin stress fibres in cultured conditionally immortalised podocytes in response to TGF-β (10ng) and Calpeptin (1µM) treatments (three technical replicates); E. TGF-β treatment (10ng) decreased the length of actin fibres (**P<0.01; t-test) indicating dysregulation of the actin cytoskeleton. This loss of stress fibre area was recovered by treating cells with 1µM Calpeptin alongside the TGF-β treatment (*P<0.05; t-test). 10x magnification and 20µm scale bar; F. Schematic representation of the podocyte cytoskeletal matrix of healthy and after brain death kidney; brain stem death increases TGF-β triggering the activation of Calpain-1 and subsequent degradation of cytoskeletal Talin-1 affecting the integrity of actin cytoskeleton in the kidneys with suboptimal post-transplant function.

Article Snippet: Antibodies used were; rabbit polyclonal anti-calpain-1 (2556S, Cell Signalling), mouse monoclonal anti α-actinin-4 (SC390205, Santa Cruz Biotechnology), goat polyclonal anti Talin-1 (AF5456, R&D Systems) and rabbit anti GAPDH (5174s, Cell Signalling).

Techniques: Microscopy, Activity Assay, Activation Assay, Western Blot, Derivative Assay, In Vitro, Staining, Cell Culture

Journal: Science translational medicine

Article Title: Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis

doi: 10.1126/scitranslmed.aau2814

Figure Lengend Snippet: (A) Representative immunoblots (left, bracket indicates identical gel) and quantification (right, normalized to GAPDH) for indicated proteins and time points after TGFβ stimulation with or without CAST-IRES-GFP overexpression (n = 3). Representative αSMA immunoblots (left) for siRNA knockdown of dimeric calpain subunits. (B) CAPN1 (n = 4), (C) CAPN2 (n = 3), (D) CAPNS1 (n = 3), (E) CAPN9 (n = 5), and (F) CAPNS2 (n = 3) and quantification (right, normalized to GAPDH) with or without TGFβ stimulation. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, †P < 0.005, ‡P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Antibodies used were: mouse anti-αSMA (R&D clone 1A4, MAB 1420) at 1:1000, goat anti-GAPDH (Santa Cruz Biotechnology, sc-20457) at 1:5000, mouse anti-phosphoSMAD2 (Millipore clone A5S, 04–953) at 1:500, rabbit anti-filamin A-C-terminal fragment (Epitomics, 2242–1) at 1:15000, mouse anti-AcGFP (Clonetech clone JL-8, 632380) at 1:1000, rabbit anti-CAPN1 (CST, 2556) at 1:1000, rabbit anti-CAPN2 (CST, 2539) at 1:1000, mouse anti-CAPN9 (Abnova, H00010753-M02) at 1:1000, rabbit anti-CAPN9 (Sigma, HPA020398) at 1:1000, rabbit anti-CAPNS2 (Lifespan Biosciences, LS-C133503) at 1:1000, rabbit anti-phospho-β-catenin (Ser552) (CST, 9566).

Techniques: Western Blot, Over Expression

Journal: Science translational medicine

Article Title: Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis

doi: 10.1126/scitranslmed.aau2814

Figure Lengend Snippet: (A) Representative low power Masson trichrome stained lung from wildtype mice treated with saline or bleomycin and Capn9−/− mice treated with bleomycin. Scale bar: 500 µm. (B) Fibrosis scores of mouse lung stained with Masson trichrome (n = 3 to 8). (C) Collagen content of lungs from Capn9−/− or wildtype control mice treated with saline or bleomycin (Bleo) (n = 4 to 6). (D) Survival curves of Capn9−/− and wildtype control mice with saline or bleomycin treatment. Results are combined from three independent experiments. (E) Respiratory resistance (n = 3 to 6) (F) total lung capacity (n = 3 to 4) and (G) lung compliance (n = 3 to 6) of saline (Sal) and bleomycin treated wildtype and Capn9−/− mice. (H) Representative IF of mouse lung stained for αSMA (red) or DAPI (blue). Scale bar: 100 µm. (I) Calpain mRNA normalized to Gapdh in wildtype mouse lung with saline (S) or bleomycin (B) treatment (C1/2/9/s1/s1 are Capn1/2/9/s1/s2, respectively, n = 3 to 5), and (J) and representative Capn9 (top, scale bar: 20 µm, insets are 1.5x magnifications) and Acta2 and Capn9 (bottom, scale bar: 10 µm) RNA-ISH of mouse lungs. Dot plot data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, †P < 0.005, ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test (B, C, E-G), log-rank test (D), and Student’s t-test (I).

Article Snippet: Antibodies used were: mouse anti-αSMA (R&D clone 1A4, MAB 1420) at 1:1000, goat anti-GAPDH (Santa Cruz Biotechnology, sc-20457) at 1:5000, mouse anti-phosphoSMAD2 (Millipore clone A5S, 04–953) at 1:500, rabbit anti-filamin A-C-terminal fragment (Epitomics, 2242–1) at 1:15000, mouse anti-AcGFP (Clonetech clone JL-8, 632380) at 1:1000, rabbit anti-CAPN1 (CST, 2556) at 1:1000, rabbit anti-CAPN2 (CST, 2539) at 1:1000, mouse anti-CAPN9 (Abnova, H00010753-M02) at 1:1000, rabbit anti-CAPN9 (Sigma, HPA020398) at 1:1000, rabbit anti-CAPNS2 (Lifespan Biosciences, LS-C133503) at 1:1000, rabbit anti-phospho-β-catenin (Ser552) (CST, 9566).

Techniques: Staining

Journal: Cell metabolism

Article Title: Mitofusin 2 regulates axonal transport of calpastatin to prevent neuromuscular synaptic elimination in skeletal muscles

doi: 10.1016/j.cmet.2018.06.011

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Calpain1 Large Subunit (Mu-type) , Cell Signaling Technology , Cat# 2556S, RRID: AB_10692800.

Techniques: Histone Deacetylase Assay, Plasmid Preparation, Recombinant, Lysis, Western Blot, Transfection, Protease Inhibitor, Sample Prep, Transgenic Assay, Software