Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay

Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (β1c, β2c, β5c), the three homologous activities, β1i, β2i and β5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of β1i, β2i and β5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present

Quantitative proteomics data set

The folder 'Mannitol' contains data from the two mixtures (M1, M2) in the mannitol background. The folder 'Fructose' contains data from the two mixtures (M1, M2) in the fructose background. Mixtures were prepared as shown in Fig. 1a of the main paper: | Label: Strain, Carbon source (X = fructose or mannitol) | M1: | Light (L): M145, G+X | Medium (M): M145, X | Heavy (H): dGlkA, G+X | M2: | Light (L): dGlkA, X | Medium (M): M145, X | Heavy (H): dGlkA, G+X | Normalized ratios as found in proteinGroups.txt were used to create the data tables in the main paper. Descriptions of all tables and column headers can be found in 'tables.pdf'. All tables were created using MaxQuant 1.2.2.5

Proteomic Identification of in Vivo Interactors Reveals Novel Function of Skin Cornification Proteins

Protection against injurious external insults and loss of vital fluids is essential for life and is in all organisms, from bacteria to plants and humans, provided by some form of barrier. Members of the small proline-rich (SPRR) protein family are major components of the cornified cell envelope (CE), a structure responsible for the barrier properties of our skin. These proteins are efficient reactive oxygen species (ROS) quenchers involved not only in the establishment of the skin’s barrier function but also in cell migration and wound healing. Here, a proteomic analysis of <i>in vivo</i> SPRR-interacting proteins confirmed their function in CE-formation and ROS-quenching and also revealed a novel unexpected role in DNA-binding. Direct <i>in vitro</i> and <i>in vivo</i> evidence proved that the DNA-binding capacity of SPRRs is regulated by the oxidation state of the proteins. At low ROS levels, nuclear SPRR is able to bind DNA and prevent ROS-induced DNA damage. When ROS levels increase, SPRR proteins multimerize and form an effective antioxidant barrier at the cell periphery, possibly to prevent the production or infiltration of ROS. At even higher ROS exposure, DNA-binding is restituted. A molecular model explaining how the intracellular oxidation state of SPRRs likely influences their selective protective function is provided

PABPN1-Dependent mRNA Processing Induces Muscle Wasting

<div><p>Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse <i>tibialis anterior</i> (<i>TA</i>) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.</p></div

Pabpn1-DR causes a decrease in distal PAS utilization.

<p><b>A.</b> Paired dot plot shows the change in PAS utilization between Scram and shPab muscles for 6 candidate genes; Arih2 PAS utilization is unchanged. <b>B.</b> Paired dot plot shows the mRNA fold change between Scram and shPab muscles. Murf1 and Arih1 levels are unchanged. <b>A-B.</b> Paired muscles are connected with a line. Statistical significance (P-value <0.05) was assessed by paired Student’s t-tests. <b>C-D.</b> PAS utilization and mRNA fold-changes in shPab cell culture. Mean and SD are from three biological replicates. Statistical significance (P-value <0.05) was assessed by unpaired Student’s t-tests. Fold changes were determined after normalization to <i>Hprt</i> housekeeping gene and to Scram muscles/cultures. PAS utilization was measured by ratio distal to total expression values. P-value <0.05 is depicted with an asterisk.</p

Proteasome activity in Pabpn1-DR cell culture and muscle tissue.

<p><b>A-C.</b> Analyses of β-subunits catalytic activity in muscle cell culture using activity gel. Specific binding of LWA300 to β-subunits is demonstrated by pre-incubation with 0.5μM Epox for 1 hour (A). Proteasomal activity in Scram vs. shPab muscle cell cultures (B-C), activity gel shows a representative experiment (B) Equal loading was assessed by Coomassie staining (CS). Bar chart (C) shows LWA300 MFI in after normalization to CS. Averages are from triplicates. <b>D.</b> A representative western blot shows protein levels of proteasome-encoded genes in Scram and shPab cell cultures. <b>E.</b> Bar chart shows quantification of protein fold change after normalization to Scram cultures. Averages and standard variations are from three independent experiments. Statistical significance (p<0.05; *) was made with unpaired Student’s t-test. <b>F.</b> Images show subcellular localization of LWA300 in Scram or shPab cell cultures. Cultures were incubated with 62.5 nM LWA300, pre-incubation with 0.5μM Epox was carried out as control. Segmented nuclei are depicted with red circles. Scale bar is 5 μm. <b>G.</b> Plot shows cumulative distribution of nuclear LWA300 MFI in Scram or shPab C2C12 cells. Statistical significance (p<0.05) was assessed with the Kruskal-Wallis test. <b>H.</b> Bar chart shows flow cytometer analysis of LWA300 MFI in Scram or shPab cell cultures treated with or without Epox. Averages are from three experiments. Representative histogram of flow cytometer output is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006031#pgen.1006031.s002" target="_blank">S2 Fig</a>. <b>I-J.</b> LWA300 signal in muscle cryosections. <b>I.</b> Images show GFP or LWA300 fluorescence in successive cryosections from Scram or shPab. Examples of the four same myofibers in GFP and LWA300 are depicted with numbers. Nuclei were counterstained with DAPI. Scale bar is 50 μm. <b>J.</b> Plot shows cumulative distribution of nuclear LWA300 MFI in cryosections from three mice. Over 400 myofibers are included per condition per mouse. Statistical significance (p<0.05) was assessed with Kruskal-Wallis test.</p

Histopathological characteristics of Pabpn1-DR muscles.

<p><b>A-B.</b> Haematoxylin and eosin staining of muscle tissue. Myofibers with central nucleation are indicated with black arrows. Scale bar is 50 μm. B. Quantification of myofiber cross-sectional area. <b>C-D.</b> Quantitation of thickening of ECM in Pabpn1-DR muscles. (C) Shown are representative images of muscle sections stained for Collagen 1 and counterstained with DAPI. Scale bar is 75 μm. (D) Percentage of collagen positive area of muscle sections. Bars show SD from three mice. <b>E-F.</b> Western blot (E) shows Atrogin-1 and Murf1 protein levels in Scram and shPab muscles. Bar charts (F) show means of Atrogin-1 and Murf1 levels normalized to the average of three proteins (45, 47 and 51 kDa). Averages and SD are from three mice. <b>G-H.</b> Atrogin1 and Murf1 protein levels in Scram and shPab cell cultures. Gapdh is used as loading control. Averages and SD are from three biological replicates. Statistical significance was assessed by paired student’s t-test. P-value <0.05 was considered statistically significant and indicated by an asterisk. <b>I.</b> Dot plots show protein accumulation of Atrogin-1, Murf1 in Scram (in black) or shPab (in red) cell cultures after Chx treatment for 0, 1, 3, 5 hours. Protein levels were normalized to untreated in Scram cultures (time 0). Averages and standard deviations are from three independent experiments. Equal loading was assessed by Tubulin and Gapdh (representative blots are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006031#pgen.1006031.s005" target="_blank">S5 Fig</a>). <b>J.</b> Western blot shows protein accumulation in mock or Epox treated myoblasts cultures. Blots were incubated with antibodies to Pabpn1, Murf1, Atrogin-1, or Gapdh and Tubulin as loading controls.</p

Pabpn1-DR in mouse TA muscles.

<p><b>A.</b> A representative fluorescence image at four weeks post-injection of AAV containing shRNA construct to Pabpn1 or Scramble RNA. The GFP signal is localized into the injected TA muscles. <b>B.</b> GFP expression is stabilized three weeks post injection. <b>C.</b> Box plots show fold-change of eGFP mRNA expression in five mice and averages were normalized to PBS injected mice. <b>D-F.</b> Pabpn1 levels. <b>D.</b> Dot plot shows Pabpn1 mRNA Hprt and Gapdh levels in all injected mice. Hprt and Gapdh levels were normalized to mean Ct value in PBS injected mice. Pabpn1 levels were normalization to the mean of Gapdh and Hprt and PBS injected. Faded color circles mark mice with lower Pabpn1 levels. Means and SD are depicted with lines. <b>E.</b> A representative Western blot shows Pabpn1 and Tubulin proteins. Loading controls are denoted by Tubulin or Coomassie blue (CB) stained gel. <b>F.</b> Bar chart shows mean Pabpn1 accumulation is Scram and shPab muscles. Pabpn1 levels were normalized to the average of three proteins (45, 47 and 51 kDa) from the CB gel. Averages and SD are from three mice with lowest Pabpn1 levels. Statistical significance was assessed by paired Student’s t-tests. P-values <0.05 was considered statistically significant and indicated by an asterisk.</p

Papbn1-DR causes muscle fiber-type transition.

<p><b>A.</b> Merged images of muscle cross sections stained with antibodies to four MyHC (type -2b in green; -2x in yellow; -2a in red; and type-1 in blue) isotypes and to laminin. Scale bar is 100 μm. <b>B.</b> MFI distribution plots show the distribution of each MyHC isotype MFI per myofiber in Scram or shPab muscles. Distribution plots were made from N >3000 myofibers. Myofibers were sorted by MyHC-2b (high to low) and MyHC-2x (low to high). <b>C.</b> Plots show cumulative distribution of MFIs of MyHC isotypes per myofiber between Scram and shPab muscles. <b>D.</b> Changes in Spearman correlation indices between two MyHC isotypes in shPab muscles as compared with the Scram muscles. Statistical significance was assessed by a paired Student’s t-test from N = 3 mice and paired muscles per mouse are connected with a line. The differences in MFI distribution between scram and shPab muscle were statistically evaluated using Kruskal-Wallis test. P-value <0.05 was considered statistically significant and indicated by an asterisk. <b>E-F.</b> Epox treatment affects MyHC expression in shPAB cultures. 7304.1 human cultures were fused for three days and subsequently were incubated with Epox. MyHC-2b expression was determined with immunofluorescence, and stained cultures were imaged and MFI was quantified in segmented cytoplasmic regions (E). Bar chart (<b>F</b>) shows MFI quantification, means and standard errors of the means are from 3760 ± 1870 myotubes. The fold change between mock and Epox treatments is indicated above the bars.</p

A model for the effect of reduced PABPN1 levels on muscle atrophy.

<p>Reduced PABPN1 levels induced a alternative polyadenylation site (APA) utilization in the 3’-UTR of a subset of transcripts whose gene product regulates muscle waste, including Atrogin-1/Fbxo32 and proteasomal genes. In these muscles, Atrogin-1 is overexpressed, whilst proteasome activity is reduced. This leads to pathological hallmarks of muscle atrophy including reduction in myofiber cross-sectional area, increase in extracellular matrix (ECM) and a switch in myosin heavy chain (MyHC) expression pattern in myofibers.</p