Journal: bioRxiv

Article Title: Molecular aging is the main driver of Parkinson’s Disease

doi: 10.1101/2025.08.27.672359

Figure Lengend Snippet: (A-C) Determination of proteasomal activity of Caspase-like enzymes of full-brain lysates from S1/S2 animals by proteasome activity assay. Comparison of short-, long- and non-expressing animals with (A) 6 M, (B) 16 M and (C) 24 M of age. Luminescence signal (counts per second, CPS) is proportional to proteasomal activity and was normalized to total amount of 20S proteasome determined by Western blot analysis (student’s t-test, ***<0.001, **<0.01; data: mean ± SEM). (D) Top 20 PD Signature genes and 20 randomly sampled genes were used to calculate a Module Score on the human midbrain snRNA-seq dataset Smajic et al., 2022. P values were calculated by Wilcoxon test on sample wise pseudo-bulked samples. Reflecting our finding in mice GABA-Rgs9 Neurons (GABAergic BGNs), the PD Signature is found to be most affecting the GABAergic Neurons in the human midbrain. This effect is not observed in the random control. (E) Heatmap of scaled log2 Fold Changes of genes from the PD Signature that were concordantly changed across mice GABA-Rgs9 synON24M vs synOFF24M, Smajic et al., human PD vs Control in GABAergic neurons, and Martirosyan et al. PD vs Control in GABAergic neurons. We obtained a total of 53 concordant genes, including genes we deemed as central to our PD Signature, such as Ubb, Dynll1, or Calm1.

Article Snippet: For Western blots with the antibody against the 20S proteasome α1/α2/α3/α5/α6/α7 subunits (Santa Cruz), only 12 % polyacrylamide gels and 40 μg protein per sample was used.

Techniques: Activity Assay, Comparison, Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation

doi: 10.1074/jbc.RA119.010796

Figure Lengend Snippet: Roseltide rT7 is a human 20S proteasome inhibitor. A, representative Western blotting analysis on the ubiquitinated protein expressions in A549 cells for 2 h with 10 μm roseltide rT7; 1 μm MG132 was used as positive control. B, dose-response effect of roseltide rT7 and MG132 on human 20S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 20S chymotrypsin-like proteasome activities are 3.3 ± 0.04 and 0.13 ± 0.03 μm, respectively. C, in-gel fluorescence image of human 20S proteasome treated with roseltide rT7 or MG132 for 1 h followed by 30 min with Me4BodipyFL-Ahx3Leu3VS probe. D, MALDI-TOF MS profiles of roseltide rT7 (10 μm) without (upper panel) or with human 20S proteasome (5 nm) (lower panel) for 24 h at 37 °C. E, dose-response effect of roseltide rT7 and MG132 on human 26S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 26S chymotrypsin-like proteasome activities are 14.93 ± 0.04 and 0.05 ± 0.02 μm, respectively. F, effects of linear pentapeptides, IIMLP, IIMLA, IIMLK, IIMPA, and IIMPK, on human 20S chymotrypsin-like proteasome activities using a Proteasome-GloTM chymotrypsin-using assay kit. The proteasome cleavage of P1′-P1 site was determined using RP-HPLC and LC-MS analysis. P(X) refers to N-terminal to the substrate cleavage site. P(X)′ refers to C-terminal to the substrate cleavage site.

Article Snippet: Briefly, purified human 20S proteasome (100 p m ; BostonBiochem) or human 26S proteasome (500 p m ; BostonBiochem) was incubated with MG132 (Santa Cruz Biotechnology), roseltide rT7, or pentapeptide analogs in 10 m m HEPES buffer (pH 7.6).

Techniques: Western Blot, Positive Control, Fluorescence, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a , cryo-TEM low magnification lamellae map showing four human sperm heads. Arrows indicate regions with sperm lacunae targeted for tilt-series acquisition. Scale bar = 1 μ m. b, z-slices from reconstructed tomograms showing barrel-shaped protein complexes inside membrane-less nuclear compartments. Scale bar = 100nm. c, zoom-in from tomograms shown in b , showing barrel-shaped particles inside lacunae . Scale bar = 100 nm. d, subtomogram averaging classes showing three populations: 20S complexes (43,128 particles, 94.4% of total particles); 20S-PA200 complexes (2,430 particles, 5.3% of total particles); and PA200-20S-PA200 (245 particles, 0.3% of total particles).

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Membrane

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, cryo-tomographic slices from deconvolved tomograms of six nuclear lacunae filled with barrel-shaped proteins. Scale bar = 100 nm. b, Details of the subtomogram averaging processing pipeline of 20S, 20S-PA200 and PA200-20S-PA200 proteasomes. Subtomogram averages with fitted PDBs are shown. c , Gold standard Fourier Shell Correlation (FSC) curves for the 20S (which reached Nyquist), 20S-PA200 and PA200-20S-PA200 proteasome reconstructions (STA stands for subtomogram averaging). The resolution values at FSC = 0.143 (half-map FSC) are indicated.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques:

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a , z-slice of 3D reconstructed cryo-scanning electron microscopy volumetric data of human sperm cells. Sperm lacunae are visible in the nucleus. Scale = 1 μ m. b, detail of three human sperm heads from volumetric data shown in a . Segmented lacunae are shown in colour (purple, yellow and blue) at three different z heights. c, Relative frequency plot of Feret diameter of segmented human sperm lacunae (blue) and comparison with values from mouse data (red, ). Feret diameter was defined as the longest xy distance of a segmented lacuna. The histogram depicts the values for 2,147 segmented lacunae segmented from 9 human sperm cells, and 866 lacunae from 5 mouse sperm cells. Human lacunae mean Feret diameter ± standard error of the mean (s.e.m.) = 326 ± 6 nm. Mouse lacunae mean Feret diameter ± s.e.m = 173.3 ± 2.1 nm. d, Violin plots for data shown in d . Each data point represents the average lacunae diameter of an individual cell. Mean ± s.e.m. diameter is plotted for human (blue, 335.3 ± 30.5 nm, n = 9 cells) and mouse cells (red, 169.4 ± 13.6 nm, n = 5 cells). Statistical analysis: t-test, P = 0.0022. e, Stochastic Optical Reconstruction Microscopy (STORM) of sperm-specific proteasome subunit α 4s (PSMA8) in human sperm cells. White dashed line shows sperm nuclear region. Panels on the right show α 4s cluster identification following DBSCAN analysis of α 4s within nuclear region. Each cluster is shown in a different colour. Scale bars = 1 μ m. f, Histogram of α 4s clusters identified using STORM in human (blue) and mouse cells (red, ). Histogram represents values from 415 α 4s clusters obtained from 11 human sperm cells and 37 α 4s clusters from 3 mouse sperm cells. Human proteasome clusters mean Feret diameter ± standard error of the mean (s.e.m.) = 199.0 ± 7.2 nm. Mouse proteasome clusters mean Feret diameter ± s.e.m = 72.0 ± 5.9 nm. g, Violin plots of data shown in f . Data points represent average Feret diameter of α 4s clusters in each cell. Mean ± s.e.m. diameter is shown for human (blue, 157.3 ± 18.4 nm, n = 11 cells) and mouse cells (red, 59.7 ± 25.7 nm, n = 3 cells). For mouse data > 10 cells were measured by STORM, but clusters were only identified in 3 cells. Clusters were identified in all measured human cells. h, STORM imaging of human sperm cell DNA using SiR-DNA and immunofluorescence staining against α 4s. Regions of α 4s density segregated from DNA are shown within dashed yellow lines. Scale bar = 1 μ m. i, Structured Illumination Microscopy (SIM) showing proteasome subunit PSMB7 ( β 2) forming foci in the nucleus of sperm cells (magenta) in regions of no DNA staining (Hoechst). Scale bar = 5 μ m. j, SIM imaging of human sperm cells showing nuclear foci of α 4/ α 4s (PSMA7/8) proteasome subunit (green) and partial co-localisation with PA200 (magenta). Small α 4/ α 4s foci are also present in the tail. Scale bar = 5 μ m.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Electron Microscopy, Comparison, Microscopy, Imaging, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, STORM of sperm-specific proteasome subunit PSMA8 ( α 4s) in a mouse sperm cell. The white dashed line shows the sperm nuclear region. The right panel shows α 4s cluster identification in red, superimposed to non-clustered localisations (cyan/grey) following DBSCAN analysis. Scale bar = 1 μ m. b, SIM image of mouse sperm cell showing foci of α 4s (green) and partial co-localisation with less abundant PA200 (magenta). Scale bar = 5 μ m. c, SIM image of mouse sperm cell showing localisation of α 4s and 19S subunit PSMC1. Scale bar = 5 μ m. c, SIM image showing localisation of α 4s in the nucleus of human sperm cells, segregated from DNA signal (Hoechst, cyan). PA200 signal can be observed partially localising to the same region of the nucleus. Scale bar = 5 μ m. d, SIM image showing localisation of 19S subunit, PSMC1 (magenta), in the tail of human sperm cells. Scale bar = 10 μ m.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques:

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, size exclusion chromatography (SEC) of the sperm cell extract. b , negative-stain transmission electron microscopy micrographs corresponding to SEC B1 and C2 fractions from a . c, silver-stained SDS-PAGE of B1 and C2 fractions. d, immunoblotting confirms the proteasome presence in B1 and C2 fractions. e, cryo-EM micrographs of the C2 (left) and B1 (right) fractions. Protein particles are marked with a black box. In b and e , the scale bars correspond to 50 nm. f, single-particle cryo-EM data processing pipeline. “p.” - particle number at corresponding processing step. 2D class averages of the initial particle selection, as well as maps from key processing steps, are shown.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Size-exclusion Chromatography, Staining, Transmission Assay, Electron Microscopy, SDS Page, Western Blot, Cryo-EM Sample Prep, Single Particle, Selection

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, segmented map of a sperm 20S CP at 1.85 Å resolution. Key subunits are indicated: α4s* (orange), β5 (blue), β2 (cyan). b , constricted pore of the sperm 20S CP. The segmented map is shown as a transparent surface, and the corresponding structure is shown in a cartoon representation. The map and structure are coloured according to a . c , the canonical β2 and β5 subunits are present in the sperm proteasome, and an alternatively spliced α4s variant, referred to as α4s*, replaces the α4 subunit. Residues 119-127 β2/β2i , 141-149 β5/β5i , 217-224 α4s* /215-222 α4 , as well as 63-77 α4s* /63-83 α4s are shown as sticks and coloured as in a . The densities corresponding to these residue stretches in the sperm 20S core particle are shown as a transparent surface. β2, β5, and α4s coordinates from sperm 20S CP are shown. β2i and β5i immunoproteasome coordinates (PDB ID: 7b12) ( Klein et al ., 2021 ) were rigid-body fitted into the sperm 20S map for comparison. The canonical α4s (UniProt ID: Q8TAA3-1) is referred to as α4s FL. The alternatively spliced variant lacking the α4s-specific loop 77-82 α4s FL (UniProt ID: Q8TAA3-5) is referred to as α4s*. α4s FL coordinates were modelled as described in Materials and Methods. d, most substitutions in α4s occur at the solvent-accessible surface. α4s* (left) and α4 (right, PDB ID: 5LE5) coordinates are shown as an electrostatic surface, while other proteasomal subunits are shown as cartoons. The calculated mean coulombic electrostatic potential is of −0.09 kcal/mol· e for α4 and 0.14 kcal/mol· e for α4s *. The substitutions Asp39 α4 → Asn41 α4s , Thr141 α4 → Ile143 α4s , Glu182 α4 → Ala184 α4s , Asp185 α4 → Ser187 α4s , and Lys227 α4 → Leu229 α4s create an extensive, uncharged patch on the α4s* surface, shown as a dashed circle. Both side view (top) and top view (bottom) are shown. The shown regions of the structure are indicated in the insets by black rectangles. e, the segmented map of sperm 20S-PA200 proteasome at 2.9 Å resolution. Key subunits are indicated: PA200 (dark pink), α4s* (dark orange), β5 (dark blue), β2 (dark cyan). f, conformational changes in the α-subunits occurring upon PA200 engagement. The structures are shown as cartoons with α-helices as tubes. The 20S-PA200 structure is opaque in darker tones, while the 20S is transparent in brighter tones. α4s* is coloured in orange tones. g, PA200 binding opens the proteasome pore. The segmented map is shown as a transparent surface, and the corresponding structure is shown as cartoons. For PA200, only structural features interacting with the α-subunits are shown. h, conformational changes in α4s* upon binding of PA200. α4s* from the sperm 20S core particle is shown in orange, while the one from 20S-PA200 is in dark orange. The disordered terminus of 20S-PA200 and the 49-63 α4s* loop are marked with rectangles. i, Upon docking, α4s* interacts with PA200 via mostly conserved interface formed by loops 51-55 α4s* , and 198-205 α4s* , as well as with the 164-178 α4s* helix. α4 (grey) and α4s* (dark orange) structural features interacting with PA200 are indicated and shown as cartoons and sticks. PA200 of sperm 20S-PA200 is shown as an electrostatic surface. The mutation S167 α4 → T169 α4s is indicated, with the T169 α4s side chain shown in ball-and-stick representation.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Variant Assay, Residue, Comparison, Solvent, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: Local resolution maps (left), Fourier Shell Correlation (FSC) plots (middle), and angular distribution maps (right) are shown for the a) 20S, b) 20S-PA200, and c) 26S maps. In a and b , both the gold-standard half-map FSC (green) and the model-map FSC (black) are shown. The resolution values at FSC = 0.143 (half-map FSC) and FSC = 0.5 (model-map FSC) are indicated. The maps are locally coloured and filtered according to the resolutions of the colour key under each map.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques:

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, alignment of Homo sapiens α4 sequence (UniProt ID: O14818) with α4s sequences from five different species: Homo sapiens (α4s*, UniProt ID: Q8TAA3-5), Macaca mulatta (UniProt ID: A0A1D5Q503), Bos taurus (UniProt ID: A0AAA9S8Q9), Rattus norvegicus (UniProt ID: F1M6I7), Mus musculus (UniProt ID: Q9CWH6). The sequences are coloured by conservation. The underlines indicate solvent-exposed residues and are coloured by side-chain group conservation between α4 with α4s: black – conserved, orange – different side-chain group across all species, red – different side-chain group in some species. b, side-chain densities in the cryo-EM 20S proteasome map with modelled α4 (grey, PDB ID: 5LE5) and α4s* (yellow) side chains. The α4s* residue numbers are indicated. c, e, Sequence alignments of β2 (Uniprot ID: Q99436) and β2i (UniProt ID: P40306) (c) as well as β5 (UniProt ID: P28074) and β5i (UniProt ID: P28062) ( e) , coloured by conservation. The propeptide sequences are omitted. d, f, side-chain densities in the cryo-EM 20S proteasome map with modelled side chains of β2 (cyan) and β2i (grey, PDB ID: 7b12) (d) , as well as β5 (blue) and β5i (grey, PDB ID: 7b12) (f) . The residue numbers are indicated.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Sequencing, Solvent, Cryo-EM Sample Prep, Residue

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, α4s* structure (yellow) is highly conserved with α4 (grey, PDB ID: 5LE5). The models are shown in cartoon representation, and all non-conserved residues are shown as sticks. The solvent-exposed, non-conserved residues with a different side-chain group are indicated, including the substitutions creating the uncharged patch on the surface pf α4s*: Asp39 α4 → Asn41 α4s , Thr141 α4 → Ile143 α4s , Glu182 α4 → Ala184 α4s , Asp185 α4 → Ser187 α4s , and Lys227 α4 → Leu229 α4s b, interfaces between α4s* (cartoon, yellow) or α4 (cartoon, grey) and α3 (left, shown as electrostatic surface) or α5 (right, shown as electrostatic surface) subunits. All interfacial residues of α4s*/α4 are shown as sticks, and the Ile82 α4 → Val84 α4S mutation is indicated. c, the pores of somatic (PDB ID: 5LE5) and sperm 20S proteasome, shown as electrostatic surfaces and viewed from the catalytic chamber side, as indicated in the inset. R4 α4S* is indicated. d, e, f sperm 20S proteasome active sites in β1 (red, d ), β2 (cyan, e ), and β5 (blue, f ) subunits, shown as cartoons. Catalytic residues are shown as sticks, along with their corresponding densities. The conserved catalytic water molecule is in green. The corresponding residues in the somatic proteasome (PDB ID:5LE5) are shown as grey sticks. In all panels, amino acids are marked using single-letter amino acid code.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Solvent, Mutagenesis

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a-d, binding PA200 to sperm 20S CP opens the proteasomal gate. To achieve this, the 561-576 PA200 loop extends into the α1-α2 interface while the C-terminal 1838-1843 PA200 (C-term PA200 ) stretch displaces the α5-α6 subunits away from each other and shifts the 49-63 α6 loop downwards. Channel opening is further facilitated by disordering the gate-forming, N-terminal loops of α1-α4s (α1-7 N-term), the flexibility of the first α3 helix (residues 19-30 α3 ), and incorporating the α5, α6, and α7 subunit N-termini into the pockets on the PA200 surface. a, all the interactions between the α-subunits and PA200 activator in sperm 20S-PA200 structure. The α-subunit residues interacting with PA200 are shown as sticks. The secondary structural features harbouring these residues are shown as cartoons. The subunit names, as well as the only PA200-interacting residue in α4s* different to α4, Thr169 α4s replacing Ser167 α4 , are indicated. The rectangles mark structural features detailed in d . The main PA200 features interacting with the CP, i.e. the PA200 C-terminus and the 561-576 loop are shown as purple cartoons and sticks. b, conformational changes in the α-subunits occurring upon PA200 engagement lead to a 4 Å horizontal shift and a 5° downwards tilt of the α4s*. The structures are shown as cartoons with α-helices as tubes. The 20S-PA200 structure is opaque in darker tones, while the 20S is transparent and in brighter tones. The α4s is coloured in orange tones. The key features undergoing conformational changes, as well as the shift and tilt of α4s*, are indicated. c, comparison of somatic (PDB ID:6KWY, darker tones) and sperm (brighter tones) 20S-PA200 structures at the α-ring. α4 (grey) and α4s* (dark orange) subunits are indicated. RMSD between Cαs of all α-subunits is 0.599 ( Guan et al ., 2020 ). d, the main features stabilising the 20S-PA200 interface. The panels depict regions marked by rectangles in ( a ). In α5, α6, and α7 panels, PA200 is shown as an electrostatic surface, while the 20S is shown as cartoons and sticks. In C-term PA200 and 561-576 PA200 panels, the 20S subunits are shown as electrostatic surfaces, with names indicated, while PA200 is shown as purple cartoons and sticks. e, cryo-EM map of the 26S proteasome isolated from human sperm. The 19S activator, α4/α4s*, β2 and β5 subunits are annotated. f, the interface between 19S and α4/α4s*. The 26S proteasome structure (PSB ID:5L4G) was rigid-body fitted in the map and shown as cartoons. g, side-chain densities in the sperm cryo-EM 26S proteasome map with modelled α4 (grey, PDB ID: 5L4G) and α4s* (orange, 20S-PA200 coordinates) side chains. In all panels, amino acids are marked using single-letter amino acid code.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Binding Assay, Residue, Comparison, Cryo-EM Sample Prep, Isolation

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, the substrate tripeptide completes an antiparallel β-sheet in β2 subunit of sperm 20S core particle by forming hydrogen bonds with Thr21 β2 , Gly47 β2 , and Ala49 β2 . The residues coordinating the substrate polypeptide backbone (cyan), as well as the substrate itself (pink), are shown as sticks with the corresponding densities shown as transparent surfaces. The S1, S2, and S3 positions are indicated. The dashed lines indicate hydrogen bonding (distances shown). The water molecule, previously identified as a catalytic nucleophile for deacylation, is shown in green. b, density protruding from the Thr1 β2 β-hydroxyl group is identified as a tripeptide acyl-enzyme intermediate. It contains a clear Arg density (R3 P ) with its putative carbonyl carbon ∼1.3 Å away from the nucleophilic oxygen, consistent with a single C-O covalent linkage (Allen, 2006). The tripeptide is shown as pink sticks, covalently linked to the catalytic Thr1 β2 β-hydroxyl. The corresponding density is shown as a transparent surface. The catalytic residues Thr1 β2 , Asp17 β2 , and Lys33 β2 are indicated and shown as cyan sticks. The β2 subunit is shown as cyan cartoons. The inset shows the Cys31 β2 rearrangement required to incorporate the arginine side chain. In grey, the somatic 20S proteasome structure in the APO state is shown. The corresponding region in the sperm 20S CP is shown in cyan (the enzyme) and pink (the tripeptide). Cys31 β2 and the tripeptide are shown as sticks, with their corresponding densities shown as transparent surfaces. The distance between the thiol group and N δ of Arg is shown for both the sperm 20S CP (black dashed line) and the putative interaction in the APO somatic enzyme that would lead to a steric clash (grey dashed line). c, the coordination of substrate arginine side chain in the β2 active site of sperm 20S CP. The arginine, as well as its coordinating Asp53 β2 , Ser32 β2 , and water molecules, are shown as sticks with corresponding densities shown as transparent surfaces. Black/grey dashed lines indicate hydrogen bonds and salt bridges with distances shown. The distances between N ω of arginine and His35 β2 (4.0 Å), N ω of arginine and Gly45 β2 carbonyl (5.8 Å), N δ of arginine and Cys31 β2 (4.2 Å) preclude interactions and are indicated as red stars and dashed lines. d, the structure of β2 subunit distal from the PA200 activator (β2-distal) is conserved with the sperm 20S CP β2. The main difference is the apparent rearrangement of the D53 β2 side chain away from the Arg side chain. The β2 subunits from the sperm 20S structure (cyan), β2-distal from 20S-PA200 structure (dark cyan), as well as the tripeptide from the 20S structure (pink), are shown as sticks. The corresponding 20S-PA200 density is shown as a transparent surface. The inset shows the position of the β2-distal in the 20S-PA200 structure. In the left panel, the same view as in b is used. The distance between the N ω of arginine and the Asp53 β2 side chain in the β2-distal is marked with a dashed line and indicated. The right panel shows conformational conservation of the substrate-coordinating S1, S2, and S3 sites between the sperm 20S β2 subunits and the β2-distal of the sperm 20S-PA200. The distances between the substrate backbone and S1 residues are marked with grey dashed lines and indicated. e, engaging PA200 causes conformational changes in the β2 subunit proximal to the activator (β2-proximal), expanding the substrate binding site to facilitate product release. The same features as in d are shown for the β2-proximal subunit of sperm 20S-PA200. The tripeptide from the 20S structure is shown for reference, as only residual substrate density is present in the β2-proximal site. The distances between the hypothetical substrate and the key coordinating residues are marked with dashed lines and indicated. In d and e , the map is shown at a 0.291 density threshold level. In all panels, amino acids are marked using single-letter amino acid code.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, diagram showing the organisation of seminiferous tubules in human testes. Cells within seminiferous tubules are surrounded by a basal lamina (BL). Spermatogenesis progresses from the outer region of the tubule towards the central lumen. Spermatogonia (SG), localised close to the BL, undergo two rounds of meiosis. Spermatocytes (SC) – meiotic cells - occupy the median region of the tubules, and post-meiotic haploid spermatids (SD) lie close to the lumen, where they differentiate into mature spermatozoa (SZ). b, confocal microscopy image showing an overview of a seminiferous tubule from a healthy human donor. Immunohistochemistry staining shows distribution of proteasome subunit PSMA7/8 ( α 4/ α 4s) in green, proteasome adaptor PA200 in magenta and DNA (Hoechst in cyan). Individual channels for this image are shown in Extended Data Fig. 9a. White dashed lines represent boundaries between different cell types. Scale bar = 50 μ m c, Distribution of PSMA7/8 ( α 4/ α 4s) and PA200 at different differentiation stages in human testes. Panels represent zoomed-in regions of the overview shown in b. Each cell type (SG, SC, SD and SZ) is represented within white dashed line boundaries and/or by white stars. Top right insets in the SD and top left insets in the SZ panels represent zoomed-in regions of the areas inside the white rectangle in the respective panel. Scale bar = 5 μ m.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Confocal Microscopy, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, single-channel confocal image for merge shown in , showing an overview of a seminiferous tubule from a healthy human donor. Immunohistochemistry staining shows distribution of proteasome subunits PSMA7/8 ( α 4/ α 4s) in green, proteasome adaptor PA200 in magenta and DNA (Hoechst, in cyan). White dashed lines represent boundaries between different cell types, as shown in . Scale bar = 50 μ m. b , Quantification of nuclear/cytoplasmic proteasome ratios in differentiating germ cells. Quantification was performed from immunohistochemistry confocal microscopy data. Mean ± s.e.m. values are as follows: SG = 0.91 ± 0.01 (n= 83 cells, tissue from 3 donors); SC = 1.03 ± 0.01 (n= 136 cells, tissue from 3 donors); SD = 1.19 ± 0.02 (n=70 cells, tissue from 3 donors). Statistical analysis: One-way ANOVA Bonferroni-corrected ( ****p < 0.0001). p = 2.6×10 -11 (SG-SC); p = 1.9×10 -35 (SG-SD); p = 2.9×10 -17 (SC-SD). c, Distribution of PSMB7 (β2) subunit in human testis (magenta). DNA (Hoechst) is shown in cyan. Dashed lines show regions of different spermatogenic cell types. Scale bar = 50 μ m. d, Distribution of PSMA3 ( α 7) subunit in human testis (magenta). DNA (Hoechst) shown in cyan. Dashed lines show regions of different spermatogenic cell types. Scale bar = 50 μ m. e, Distribution of PSMC1, a subunit of the regulatory cap 19S in human testis (green). DNA (Hoechst) shown in cyan. Scale bar = 50 μ m. f, localisation of 19S subunit PSMC1 (green) to the mature tail of SZ, growing tail of SD and nucleus of Sertoli cells (*Srt). Scale bar = 10 μ m. g, merge showing co-localisation of PSMC1 (19S subunit, in green) with acetylated tubulin (magenta). Putative SC cilia are highlighted with orange stars. Other regions of co-localisation, including SZ and SD tails, are highlighted with white stars. DNA is shown in cyan (Hoechst). Scale bar = 50 μ m. h, individual channels for merge shown in g . i, additional example of PSMC1 (19S, green) localisation relative to acetylated tubulin (magenta) in a human seminiferous tubule. Scale bar = 50 μ m. j, merge of images shown in h. Regions of co-localisation are highlighted with white stars. Scale bar = 50 μ m. k, confocal image merge showing clustering of proteasomes (PSMA7/8, magenta) in the nucleus of differentiating cells in human testes, relative to protamine signal (PRM1, green). Spermatids and spermatozoa are shown within the white-dashed line. Scale bar = 50 μ m. l, single-channel images for merge shown in k. Scale bar = 50 μ m.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Immunohistochemistry, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

doi: 10.64898/2025.12.16.694293

Figure Lengend Snippet: a, confocal microscopy image showing mouse seminiferous tubule and distribution of proteasomal PSMA7/8 ( α 4/ α 4s) subunits during sperm cell differentiation. White-dashed lines show tissue sub-compartments enriched for different germ cells (SG, SC, SD and SZ). Scale bar = 50 μ m. b, zoom-in region of mouse seminiferous tubules showing proteasome subunit PSMA7/8 ( α 4/ α 4s) distribution during differentiation. White-dashed lines show regions with the different cell types: SG, SC, SD and SZ. Scale bar = 10 μ m. c, quantification, from confocal immunohistochemistry mouse data, of nuclear/cytoplasmic proteasome ratios in germ cells. Mean ± s.e.m. values are as follows: SG = 0.49 ± 0.01 (n= 80 cells); SC = 0.58 ± 0.01 (n= 88 cells); SD = 0.88 ± 0.02 (n= 112 cells). Statistical analysis: One-way ANOVA Bonferroni-corrected ( ***p <0.001; ****p < 0.0001). p = 2.7 x10 -4 (SG-SC); p = 2.3 x10 -48 (SG-SD); p = 1.7 x10 -34 (SC-SD). d, distribution of PSMC1 subunit of regulatory cap 19S (green) in mouse sperm testis, relative to acetylated tubulin (magenta). Scale bar = 50 μ m.

Article Snippet: The primary antibody, 20S Proteasome β7 (H-3) (sc-365725, Santa Cruz Biotechnology), was used at 1:1000 dilution.

Techniques: Confocal Microscopy, Cell Differentiation, Immunohistochemistry

Journal: Journal of Biomedical Science

Article Title: Activation of mitophagy and proteasomal degradation confers resistance to developmental defects in postnatal skeletal muscle

doi: 10.1186/s12929-025-01153-7

Figure Lengend Snippet: RNA sequencing analysis of gastrocnemius-plantaris muscle during development. A Representative visualization of genes analyzed from the publicly available Gene Expression Omnibus database (Accession ID: 1025). B Volcano plot displaying differential gene expression in skeletal muscle. The horizontal line delineates a significance threshold of p < 0.05. Vertical lines mark a z-score change greater than Log2. Green labels indicate upregulated differentially expressed genes (DEGs), while red labels indicate downregulated DEGs. C List of top 50 DEGs identified by DESeq2 analysis. D Gene ontology analyses and donut graph outputs summarizing changes in genes associated with five major remodeling processes: muscle remodeling, metabolism, apoptosis, mitochondria, autophagy, and the ubiquitin proteasome system (UPS). A total of 7402 genes related to these processes are upregulated while 5992 genes related to these processes are downregulated. E, F Gene ontology analyses output of the number of genes linked to the top five upregulated (green) and downregulated (red) biological processes. P7-P112 refers to animals collected at their respective postnatal timepoints. N = 3 mice per group

Article Snippet: Equal protein was loaded and separated using 8–14% SDS-PAGE, transferred on to PVDF membranes, and blocked with 2–5% non-fat milk powder in TBS-T at room temperature for 1 h. Membranes were briefly rinsed and incubated overnight in primary antibodies against: BNIP3 (#3769, Cell Signaling), BNIP3L (#12396, Cell Signaling), ATG7 (#8558, Cell Signaling), MAP1LC3B/LC3B (#2775, Cell Signaling), GAPDH (#2118, Cell Signaling), OPA1 (#80471, Cell Signaling), MFN2 (#9482, Cell Signaling), DNM1L (#8570, Cell Signaling), FOXO3A (#9467, Cell Signaling), p-FOXO3A (#9465, Cell Signaling), AMPK (#2532, Cell Signaling), p-AMPK (#2535, Cell Signaling), P70S6K (#2708, Cell Signaling), p-P70S6K (#9204, Cell Signaling), AKT1 (#9272, Cell Signaling), p-AKT1 (#9275, Cell Signaling), SQSTM1 (PM045, MBL), PINK1 (sc-33796, Santa Cruz), PRKN (sc-32282, Santa Cruz), VDAC1 (sc-390996, Santa Cruz), ANT1 (sc-9299, Santa Cruz), CYCS (sc-13256, Santa Cruz), TFAM (sc-166965, Santa Cruz), PPARGC1A (ST1202, Sigma), SOD1 (SOD-101, Stressgen), SOD2 (SOD-110, Stressgen), 4HNE (ab46545, Abcam), ubiquitin (sc-8017, Santa Cruz), 20S proteasome (sc-166073, Santa Cruz), and USP14 (sc-398009, Santa Cruz).

Techniques: RNA Sequencing, Gene Expression, Ubiquitin Proteomics

Journal: Journal of Biomedical Science

Article Title: Activation of mitophagy and proteasomal degradation confers resistance to developmental defects in postnatal skeletal muscle

doi: 10.1186/s12929-025-01153-7

Figure Lengend Snippet: Postnatal development-associated changes in mitophagy and mitochondrial function in autophagy-deficient skeletal muscle. A Representative confocal images of mitophagic flux from longitutudinal TA muscle sections of Atg7 f/f and Atg7 f/f: Acta1 −Cre mice crossed with mt-Keima reporter mice (mt-Keima: Atg7 f/f and mt-Keima: Atg7 f/f: Acta1 −Cre ). Green fluorescence indicates healthy mitochondria. Red fluorescence indicates degrading mitochondria. Scale bar indicates 50 μm. B Quantification of maximal oxygen consumption rate (i.e., respiration) of permeabilized bundles from TA muscle. Simultaneous quantification of C succinate-stimulated hydrogen peroxide (H 2 O 2 ) production, and D succinate + pyruvate/malate-stimulated H 2 O 2 production in permeabilized TA muscle bundles. E Quantification of mitochondrial fraction of electron leak. F Representative immunoblots of gastrocnemius mitochondrial fractions. SOD1 and SOD2 shown as subcellular fraction controls. G Quantification of oxidative phosphorylation (OXPHOS) coupling. Quantification of H LC3B-II, I BNIP3, J BNIP3L, K PINK1, and L PRKN from mitochondrial-enriched fractions. Quantification of M CASP9, N CASP3, O CAPN, and P 20S proteasome activity. * p < 0.05 compared to Atg7 f/f group. N = 4–8 mice per group

Article Snippet: Equal protein was loaded and separated using 8–14% SDS-PAGE, transferred on to PVDF membranes, and blocked with 2–5% non-fat milk powder in TBS-T at room temperature for 1 h. Membranes were briefly rinsed and incubated overnight in primary antibodies against: BNIP3 (#3769, Cell Signaling), BNIP3L (#12396, Cell Signaling), ATG7 (#8558, Cell Signaling), MAP1LC3B/LC3B (#2775, Cell Signaling), GAPDH (#2118, Cell Signaling), OPA1 (#80471, Cell Signaling), MFN2 (#9482, Cell Signaling), DNM1L (#8570, Cell Signaling), FOXO3A (#9467, Cell Signaling), p-FOXO3A (#9465, Cell Signaling), AMPK (#2532, Cell Signaling), p-AMPK (#2535, Cell Signaling), P70S6K (#2708, Cell Signaling), p-P70S6K (#9204, Cell Signaling), AKT1 (#9272, Cell Signaling), p-AKT1 (#9275, Cell Signaling), SQSTM1 (PM045, MBL), PINK1 (sc-33796, Santa Cruz), PRKN (sc-32282, Santa Cruz), VDAC1 (sc-390996, Santa Cruz), ANT1 (sc-9299, Santa Cruz), CYCS (sc-13256, Santa Cruz), TFAM (sc-166965, Santa Cruz), PPARGC1A (ST1202, Sigma), SOD1 (SOD-101, Stressgen), SOD2 (SOD-110, Stressgen), 4HNE (ab46545, Abcam), ubiquitin (sc-8017, Santa Cruz), 20S proteasome (sc-166073, Santa Cruz), and USP14 (sc-398009, Santa Cruz).

Techniques: Fluorescence, Western Blot, Phospho-proteomics, Activity Assay

Journal: Journal of Biomedical Science

Article Title: Activation of mitophagy and proteasomal degradation confers resistance to developmental defects in postnatal skeletal muscle

doi: 10.1186/s12929-025-01153-7

Figure Lengend Snippet: Developmental changes in skeletal muscle of Atg7 f/f and Atg7 f/f: Acta1 −Cre mice treated with either vehicle (VEH) or proteasome inhibitor MG132. A Representative visualization of Atg7 f/f: Acta1 −Cre mice treated with VEH or MG132 and collected at P42. B Quantification of 20S proteasome activity. Quantification of C Body mass, skeletal muscle mass of D tibialis anterior (TA), E gastrocnemius, F quadriceps, and G triceps, and H peak forelimb grip strength. I Representative immunofluorescent images of TA muscle cross sections from P42 Atg7 f/f and Atg7 f/f: Acta1 −Cre mice. Samples were stained with antibodies specific for individual MYH isoforms: type IIA (green), type IIX (red), type IIB (unstained), and dystrophin (DMD; red). Scale bars indicate 50 μm. J , K Quantification of mean cross-sectional area (CSA) and distribution of type IIA, IIX, and IIB fibers. L Quantification of maximal oxygen consumption rate (i.e., respiration) of permeabilized bundles from TA muscle. Simultaneous quantification of M succinate-stimulated hydrogen peroxide (H 2 O 2 ) production, and N succinate + pyruvate/malate-stimulated H 2 O 2 production in permeabilized TA muscle bundles. Quantification of O mitochondrial fraction of electron leak and P oxidative phosphorylation (OXPHOS) coupling. Q Representative immunoblots of whole gastrocnemius lysate of Atg7 f/f (P42) or Atg7 f/f: Acta1 −Cre (P42) mice treated with VEH or MG132. R Quantitative analysis of ubiquitin, 20S proteasome, USP14, VDAC1, ANT1, and CYCS. S Representative immunoblots of mitochondrial-enriched fraction from the gastrocnemius of Atg7 f/f (P42) or Atg7 f/f: Acta1 −Cre (P42) mice treated with VEH or MG132. T Quantitative analysis of mitochondrial localized BAX, BCL2, and BAX:BCL2 ratio. U Quantification of CASP9, CASP3, and CAPN activity. * p < 0.05 compared to VEH group. N = 4–8 mice per group. Atg7 f/f group data shown for visualization purposes only

Article Snippet: Equal protein was loaded and separated using 8–14% SDS-PAGE, transferred on to PVDF membranes, and blocked with 2–5% non-fat milk powder in TBS-T at room temperature for 1 h. Membranes were briefly rinsed and incubated overnight in primary antibodies against: BNIP3 (#3769, Cell Signaling), BNIP3L (#12396, Cell Signaling), ATG7 (#8558, Cell Signaling), MAP1LC3B/LC3B (#2775, Cell Signaling), GAPDH (#2118, Cell Signaling), OPA1 (#80471, Cell Signaling), MFN2 (#9482, Cell Signaling), DNM1L (#8570, Cell Signaling), FOXO3A (#9467, Cell Signaling), p-FOXO3A (#9465, Cell Signaling), AMPK (#2532, Cell Signaling), p-AMPK (#2535, Cell Signaling), P70S6K (#2708, Cell Signaling), p-P70S6K (#9204, Cell Signaling), AKT1 (#9272, Cell Signaling), p-AKT1 (#9275, Cell Signaling), SQSTM1 (PM045, MBL), PINK1 (sc-33796, Santa Cruz), PRKN (sc-32282, Santa Cruz), VDAC1 (sc-390996, Santa Cruz), ANT1 (sc-9299, Santa Cruz), CYCS (sc-13256, Santa Cruz), TFAM (sc-166965, Santa Cruz), PPARGC1A (ST1202, Sigma), SOD1 (SOD-101, Stressgen), SOD2 (SOD-110, Stressgen), 4HNE (ab46545, Abcam), ubiquitin (sc-8017, Santa Cruz), 20S proteasome (sc-166073, Santa Cruz), and USP14 (sc-398009, Santa Cruz).

Techniques: Activity Assay, Staining, Phospho-proteomics, Western Blot, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Microtubule architecture connects AMOT stability to YAP/TAZ mechanotransduction and Hippo signaling

doi: 10.1101/2025.08.08.669326

Figure Lengend Snippet: a) Representative immunofluorescence images of HEK293 cells seeded in mechano-ON (Stiff, 40 kPa hydrogels) versus mechano-OFF (Soft, 0.7 kPa hydrogels or dense culture) conditions. The proteasome was labelled by 20S/PSMA5 staining (proteasome, yellow) and nuclei were counterstained with Hoechst (cyan). Scale bar, 10μm. See also Extended Data Fig. 5a for quantifications of proteasome localization in cells seeded in the same conditions. b) Representative immunofluorescence images of HEK293 cells seeded in Mech.ON conditions and treated with the indicated siRNAs. The proteasome was labelled by 20S/PSMA5 staining (proteasome, yellow) and nuclei were counterstained with Hoechst (cyan). Scale bar, 10μm. See also Extended Data Fig. 5b for quantifications of proteasome localization in cells treated in the same way. c) Representative AMOT immunoblot of MCF10A cells seeded on hydrogels of the indicated stiffness. GAPDH serves as loading control. See also for AMOT immunoblots from cells experiencing independent mechano-OFF conditions by treatment with F-actin inhibitors, and Supplementary Fig. 1a for quantifications. d) Representative AMOT immunoblot of HEK293 cells treated with the indicated siRNAs. GAPDH serves as loading control. See also Supplementary Fig. 1a for quantifications. e) Left: representative images of proximity ligation assays (PLA) showing exclusively cytoplasmic interaction (magenta) between endogenous AMOT and YAP/TAZ in HEK293 cells seeded in Mech.OFF conditions. Mech.ON conditions (in absence of endogenous AMOT protein) serve as negative control. Nuclei were counterstained with Hoechst (cyan). Scale bar, 10μm. Right: quantifications of the mean number of PLA dots per cell (>50 cells were quantified for each independent experiment, indicated as dot, n=4). f) Representative stills from live fluorescence images of RFP-AMOT expressing MCF10A-YAP-EGFP KI cells showing that AMOT overexpression is sufficient to cause YAP cytoplasmic retention in mechano- ON conditions. Cells without AMOT overexpression (*), showing nuclear YAP accumulation, serve as negative control. Cell and nuclear borders are outlined in yellow and blue dashes, respectively. Scale bar, 20μm. g) Left: representative immunofluorescence images of YAP/TAZ in control (wt) versus AMOT- 130/AMOT-L1/AMOT-L2 triple KO (AMOT tKO) HEK293 cells in Mech.ON vs Mech.OFF conditions. Nuclei were counterstained with Hoechst (cyan). Scale bar, 20μm. Right: quantifications (n≥50) of YAP/TAZ nuclear-to-cytoplasmic subcellular localization (N/C) in cells seeded as in top panels. h) AMOT is epistatic to LINC proteins and MTs. Left: representative immunofluorescence images of YAP/TAZ in control versus AMOT tKO HEK293 cells treated with the indicated siRNAs. Scale bar, 10μm. Right: quantifications (n≥45) of YAP/TAZ nuclear-to-cytoplasmic subcellular localization (N/C) in cells treated as in top panels. P values were determined by unpaired Student’s t -test with Welch’s correction ( e ) or one-way ANOVA with Welch’s correction ( g,h ).

Article Snippet: Primary antibodies were YAP (Santa Cruz Biotechnology, sc-101199) previously validated as YAP/TAZ antibody , , , , LaminA/C (Santa Cruz Biotechnology, sc-376248), Paxillin (Abcam no. ab32084), GFP (Abcam ab13970), Proteasome subunit alpha 5 (PSMA5) (Origene, TA332887), AMOT (Santa Cruz Biotechnology, sc-166924), AMOTL1 (Sigma HPA001196), alpha Tubulin (Abcam, ab18251), γ- Tubulin (Santa Cruz Biotechnology, sc-17787), acetylated-Tubulin (ab24610), Pericentrin 1 (Santa Cruz Biotechnology, sc-376111), LaminC/C (Abcam, ab8984), RNF146 (Thermofisher, H00081847-B01P).

Techniques: Immunofluorescence, Staining, Western Blot, Control, Ligation, Negative Control, Fluorescence, Expressing, Over Expression