Journal: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t -test. * = p<0.001; n.s. not statistically significant.

Article Snippet: For co-localization studies, WT or mutant TULP1 plasmids were co-electroporated with the mCherry-tagged ER-marker Sec61b plasmid DNA (mCherry-Sec61b, a gift from Dr. Gia Voeltz (Addgene #49155)).

Techniques: Transfection, Mutagenesis, Activation Assay, Transgenic Assay, Expressing, Quantitative RT-PCR, Injection, Marker, Two Tailed Test

Journal: Communications Biology

Article Title: Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay

doi: 10.1038/s42003-022-03090-9

Figure Lengend Snippet: a , b Constructs designed for the ICP assay containing mEmerald with nuclear localization signal (NLS), cleavage site for Mpro or PLpro, Zika virus NS2B followed by Mpro ( a ) or PLpro (amino acids 1541–1855 of nsp3) ( b ). c – f Localization of mEmerald-NLS (green) and ER marker mCherry-Sec61 β (red) at 6 h post-transfection and stained with nuclear stain Hoechst 33342 dye (blue), in ICP assay. c Cells transfected with ICP construct A, d cells transfected with ICP construct A with inactive Mpro mutant (C145A). e Cells transfected with ICP construct B, f cells transfected with ICP construct B with inactive PLpro mutant (C1651A).

Article Snippet: In-cell protease assay plasmids were co-transfected with the ER marker mCherry Sec61 β C1 (Addgene Plasmid # 90994) to determine the colocalization of uncleaved protein with the ER .

Techniques: Construct, Virus, Marker, Transfection, Staining, Mutagenesis

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: The four types of Sec61 substrates are shown, with characteristic topogenic determinants and mode of insertion in Sec61: Signal peptide (SP, blue segment), final orientation (FO) of the first N-terminal transmembrane domain (TMD, pink). The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. (6), is indicated (Myco).

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Diagram illustrating the differential effects of mycolactone on Sec61 client translocation (in vitro) and production in living cells.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Translocation Assay, In Vitro

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Biological process analysis of MutuDC proteins upregulated by mycolactone The biological processes that were most significantly enriched in “mycolactone-upregulated” proteins, compared to “all quantified” proteins, are listed. Proteins significantly upregulated by mycolactone in each category are shown with Uniprot accession number, FDR, variation extent of mycolactone/control, gene name and an indication of whether the protein is a Sec61 substrate inferred from www.uniprot.org . Upregulated (FDR ≤ 0.1; log2(Variation) > 0.5).

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Ubiquitin Proteomics, Membrane, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Mycolactone-mediated Sec61 blockade prevents the production of viral Type I/II but not Type III TMPs. Surface expression of viral envelope proteins HA (A), NA (B) and M2 (C) as well as HLA components B2M (D) and HLA-A2b (E), in HEK293 cells infected with IAV for 1h prior to incubation with Mycolactone, or vehicle as control. MFI: mean fluorescence intensity; hpi: hours post infection. Data shown are MFI ± S.E. (n = 3) from one of two independent experiments, which gave similar results. MFIs of mycolactone-treated cells were compared with vehicle controls using a t test with Holm-Sidak correction for multiple testing. *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.001; ns: no significant difference.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Expressing, Infection, Incubation, Control, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Conserved and variable features of mycolactone-induced proteomic alterations. A, The proportion of Sec61 substrates in “all quantified” proteins is compared with that in “mycolactone downregulated” or “mycolactone-upregulated” proteins, in each cell type studied. Jurkat T cells (left), MutuDCs (middle) and MED17.11 cells (right) were treated with mycolactone or vehicle as control in the conditions outlined in Table II. Number of identified proteins in each subset are indicated on the top of each bar, **** p value < 0.0001, ns: not significant, Fisher exact test. B, Venn diagrams representing the overlap between mycolactone downregulated (left) or mycolactone-upregulated (right) proteins across cell types. Human proteins (Jurkat T cells) were matched to their mouse orthologues (MutuDCs and MED17.11 neurons). Proteins that were found downregulated or upregulated by mycolactone in 2 cell types or more are listed.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Primary determinants of Sec61 substrate susceptibility or resistance to mycolactone. A, Bar plot depicting the number of proteins that were upregulated, downregulated or not significantly modulated by mycolactone in MutuDCs. Sec: secretory proteins; C-tail: C-terminal tail-anchored proteins, depending on the Guided Entry of Tail-anchor (GET) pathway for insertion into the ER membrane; Mitoch: mitochondrial membrane proteins, depending on the TIM/TOM complexes for mitochondrial membrane insertion. B, The proportion of Sec61 substrates with a SP (secretory + Type I TMP) or without a SP (Type II/III TMPs) in ≪ all detected ≫ proteins (All) or downregulated proteins (Down) is shown for each cell type studied. Fisher exact tests comparing the proportions of Sec61 substrates with SP (orange) or without SP (gray) to all other proteins. *p < 0.05; *** p < 0.001; **** p < 0.0001. C, The proportions of mycolactone-downregulated proteins in each class of single-pass TMPs is shown compared with those in multi-pass TMPs. D, The proportion of mycolactone-downregulated proteins in single-pass TMPs with or without an SP is shown compared with those in multi-pass TMPs. Fisher exact test comparing the proportion of mycolactone-downregulated proteins in each subset. ns: not significant. E, F, Scatter dot plot representing the length (in amino acid residues) of the N-terminal domain before the first TMD in Type I (E) and Type II (F) TMPs of MutuDCs. A Mann-Whitney test was used to compare mean lengths in mycolactone-downregulated proteins and proteins not modulated by mycolactone. **p < 0.01; ns: no significant difference.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Membrane, MANN-WHITNEY

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Low doses of mycolactone-upregulate the transcription of selected Sec61 substrates. A, Surface expression of Slc3a2 and MHC class II (I-A/I-E) in MutuDCs treated with the indicated doses of mycolactone for 24 h. B, Assay of Slc3a2 insertion in SRM, in the presence of increasing amounts of mycolactone (Myco). Membrane integration was assessed by analyzing the change in SDS-PAGE mobility and autoradiography. Correctly integrated, glycosylated SLC3A2 species are indicated with arrowheads and non-translocated, unglycosylated protein species with an asterisk. Endoglycosidase H (EndoH) treatment demonstrates that the change in SDS-PAGE migration is because of glycosylation. C, qRT-PCR comparing the expression slc3a2 and b2m in MutuDCs treated with 25 or 100 nm mycolactone for 24 h. D, qRT-PCR comparing the expression of slc3a2 in wt or R66G Sec61-expressing lymphoma B cells following a 24h treatment with 25 or 100 nm mycolactone. E, Effect of mycolactone on CD98 surface expression by lymphoma B cells overexpressing wild-type (wt) Sec61 or the mycolactone-resistant R66G Sec61 mutant. Cells were treated with the indicated doses of mycolactone for 24h, prior to flow cytometric analysis. F, Kinetic effects of mycolactone on transcript levels of slc3a2, hmox1, vimp and herpud1, as measured by qPCR in MutuDCs treated with 25 nm mycolactone for the indicated times. Data are mean ± S.E. (n = 3) from one of two independent experiments, which gave similar results.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Expressing, Membrane, SDS Page, Autoradiography, Migration, Glycoproteomics, Quantitative RT-PCR, Mutagenesis

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Mycolactone-upregulated proteins outline an atypical stress response. A, Proportion of mycolactone-downregulated or -upregulated Sec61 substrates across cell compartments in MutuDCs. Compartements primarily composed of Sec61 substrates are indicated. The numbers above the bars indicate the number of proteins in each category. B, Kinetics of mycolactone effects (25 nm) on MutuDC expression of total, spliced and unspliced Xbp-1. C–E, Differential effects of Tunicamycin (1 μm), Thapsigargin (1 μm), MG132 (1 μm) and mycolactone (25 nm) on expression of Chop, Slc3a2 and Bip in MutuDCs. Data are mean ± S.E. from biological triplicates.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature *

doi: 10.1074/mcp.RA118.000824

Figure Lengend Snippet: Mycolactone upregulated proteins are enriched in targets of the ATF4 and CHOP transcription factor targets Mycolactone upregulated proteins (FDR ≤ 0.1; log2(Variation) > 0.5) of MutuDCs and MED17.11 neurons that are known targets of the ATF4 and CHOP transcription factors (37) are listed with Uniprot accession number, FDR, variation extent of mycolactone/control, gene name, presence of an ATF4 and/or CHOP binding site on their promoter region and type of Sec61 substrate type.

Article Snippet: The differential susceptibility of each type of Sec61 sustrates to mycolactone-mediated Sec61 blockade, as proposed by McKenna et al. ( 6 ), is indicated (Myco).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Self-supervised learning for a gene program-centric view of cell states

doi: 10.64898/2026.03.24.713961

Figure Lengend Snippet: a) Overview of experimental data generation for previously published cord blood in vitro differentiation dataset . ( b/c ) Heatmap showing min-max normalized GP importance score by erythroid cell type in vivo b ) and in vitro c ). Each row represents a GP, the top/bottom 5 correspond to GPs whose importance scores decrease/increase the most as a function of differentiation. Each value reflects the importance score for an individual cell, grouped by cell types. Importance scores are obtained by averaging results across 3 Tripso model training runs initialized with 3 random seeds. d ) UMAP of GATA1 GP latent space for in vivo and in vitro erythroid cells. Small dots represent individual cells, colored by cell type. Large labelled dots represent the real cell nearest to the cluster centroid for that cell type. Dashed lines represent pairings computed by unbalanced optimal transport. e ) Overview of experimental data generation for previously published stem cell maintenance dataset . f ) Barplot showing the Sinkhorn divergence between cell populations described on the x-axis and LT-HSCs from adult bone marrow of patients 29-50 years old. Error bars indicate the standard deviation across 50 bootstrap iterations (750 cells sampled per iteration). g ) Differential gene importance for PI3K GP genes in stem and progenitor cells compared to LT-HSC compared in vivo. h ) Differential gene importance for PI3K GP genes in HSPCs cultured in SR-1 media compared to 3a media. i ) Differential gene importance for PI3K GP genes in HSPCs cultured in UM171 media compared to 3a media. Gene cosine similarity scores represent averages across 3 model runs initialized with 3 random seeds. Gene names in bold correspond to genes which were significantly differently expressed (adjusted p-value <0.05) in pseudobulked differential gene expression. j ) Proportion of immunophenotypic HSCs (CD34+ CD45RA− CD90+ EPCR+, as measured by flow cytometry) detected under different concentrations of SEC61 inhibitor or control (no inhibitors and vehicle). Each cord blood (CB) represents a biological replicate, each dot represents a technical replicate.

Article Snippet: Cells were cultured at 37°C with 5% CO 2 and 20% O 2 , in their corresponding media with varying concentrations, 1, 10 and 100 ng/mL, of SEC61 inhibitor (Cambridge bioscience, T40146-5mg) or DMSO (vehicle control).

Techniques: In Vitro, In Vivo, Standard Deviation, Cell Culture, Gene Expression, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) SEC61B -knockout (KO) single cell clones were generated from HEK293T cells using CRISPR/Cas9. Sec61β expression in lysates of the indicated SEC61B -KO single cell clones was analyzed by immunoblot using polyclonal anti-Sec61β antibody. Expression of β-actin was analyzed as loading control. Results were confirmed in two additional experiments. ( B ) The indicated SEC61B -KO cell lines were stained with CFSE and analyzed via flow cytometry at day 0, 1, 2 and 3. The results of a single experiment are shown and were confirmed in two separate experiments. ( C ) Phase contrast microscopy of Sec61β single cell clones. Similar results were obtained in one additional experiment.

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Expressing, Western Blot, Control, Staining, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: Proteolytic processing of the Marburg virus glycoprotein depends on Sec61β and is required for cell entry

doi: 10.1101/2025.06.26.660697

Figure Lengend Snippet: ( A ) WT and SEC61B -KO cells were transfected with plasmids expressing C-terminal V5-tagged GPs of VSV, EBOV (Makona) and MARV (Musoke) or empty vector (EV) as control. Glycoprotein expression was detected by immunoblot using a mouse monoclonal anti-V5 antibody. Detection of β-actin served as a loading control. Results were confirmed by one to three additional experiments. ( B ) The experiment was conducted as for panel A but the effect of coexpression of Sec61β on MARV-GP expression and cleavage was examined. The results of a representative immunoblot are shown and were confirmed in two separate experiments. ( C-D ) The expression of MARV-GP and EBOV-GP with the indicated mutations in the furin cleavage sites was analyzed in cell lysates (C) and pseudotyped VSV particles (D). The expression of β-actin (cell lysates) and VSV-M (particles) served as loading control. Results were confirmed in two (EBOV-GP) or three (MARV-GP) additional experiments. ( E ) HEK293T cells were inoculated with VSV particles pseudotyped with the indicated glycoproteins. Luciferase activity in cell lysates were quantified at 18-20 h post inoculation. Luminescence signals were normalized to background (empty vector control). Shown is the mean ± SEM of four separate experiments each conducted with four technical replicates. Statistical significances were determined using unpaired, two-tailed t-test with Welch‘s correction (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **)

Article Snippet: Here, we report that the Sec61 subunit Sec61β, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Two Tailed Test