Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: A variety of ER stressors alter DR5 disulfide bonding. A, upper panel. ERp44-deficient HepG2 cells into which vector, wild type or catalytically null ERp44 were reintroduced were treated as indicated for 24 h and analyzed by non-reducing immunoblot. A, lower panel. Expanded region of the DR5 immunoblot showing altered DR5 disulfide bonding in the Thapsigargin/PERKi combination treatment. B. Non-reducing immunoblot of MDA-MB-468 cells treated as indicated for 24 h. C. MDA-MB-468 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. D. Protein synthesis assays of cells pre-treated for 24 h as indicated before protein synthesis was measured by 3 H-Leucine incorporation over a 2 h pulse. Data are plotted as the average (N = 6), with error bars representing standard deviation. E. HepG2 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. F. Control and PERK knockout HepG2 cells were treated for 24 h as indicated and subjected to non-reducing immunoblot analysis. G. Non-reducing immunoblot analysis of the indicated cell lines treated as specified for 24 h. Note the DR5 oligomerization in A431 cells when Thapsigargin, Tunicamycin, or dFtcyDTDO are combined with PERK inhibition. H. Non-reducing immunoblot analysis of WM793 cells treated as specified for 24 h. Unless otherwise specified, the following concentrations of compounds were used in A-H above: dFtcyDTDO (2.5 μM), Thapsigargin (400 nM), Tunicamycin (500 ng/ml), Cyclosporine A (10 μM), Dithiothreitol (DTT; (2.5 mM)), ISRIB (200 nM), or PERKi (1μM). O and M represent Oligomeric and Monomeric protein isoforms in panels A, C, E, G, and H.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Plasmid Preparation, Western Blot, Standard Deviation, Knock-Out, Inhibition

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Genetic disruption of multiple DR5 disulfide bonds induces its stabilization and pro-apoptotic signaling. A. Structural model of DR5 showing its disulfide bonds, and the positive patch autoinhibitory domain described in the literature. B. Non-reducing immunoblot analysis of MDA-MB-468 cells engineered with doxycycline-inducible expression of wild type (WT) DR5 or the indicated Cys to Ser disulfide bond mutants. Cells were treated as indicated for 24 h with 1 μg/ml doxycycline and 2.5 μM dMtcyDTDO. The red arrow denotes DR4 oligomers that coincide with DR5 oligomerization. C. Reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 10 μM Q-VD-OPH. The catalog numbers of DR5 and DR4 antibodies are shown. D. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type DR4 and DR5, and DR4 and DR5 C-terminal deletion constructs defective in apoptotic signaling. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. E. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. F. Non-reducing immunoblot analysis of the indicated MDA-MB-468 doxycycline-inducible stable cell lines. Cells were treated for 24 h as indicated. G. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. O and M represent Oligomeric and Monomeric protein isoforms in panels B and D-G.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Disruption, Western Blot, Expressing, Stable Transfection, Construct

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: DDAs upregulate TRAIL Decoy Receptor 2, an effect overridden by Cyclosporine A. A. Sequence alignment of the putative autoinhibitory motifs of DR4, DR5, DCR1, and DCR2. B. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle, 5 μM Cyclosporine A or 100 nM FK506. C. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. D. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. Data are plotted as the average (N = 3), with error bars representing standard error. Asterisks denote p < 0.05 compared to control using Student’s unpaired t -test. E. Densitometry analysis of the relative levels of total, monomeric, and oligomeric forms of DR5 (left panel) or total levels of Cyclophilin B or DCR2 (right panel) from panels 7B-D. O and M represent Oligomeric and Monomeric protein isoforms in panels B-D.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Sequencing, Western Blot

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Effects of altered disulfide bonding on DR5 cell surface localization and antibody recognition. A. Flow cytometry analysis of the indicated doxycycline-inducible MDA-MB-468 stable cell lines with an antibody to DR5 (Clone DJR2-4 (7-8)) (top panel) or DR4 (bottom panel). Prior to analysis, cells were treated for 24 h as indicated with 10 μM Q-VD-OPH, 1 μg/ml doxycycline, or 2.5 μM dFtcyDTDO. Dots represent the average values from three independent biological replicates performed in triplicate. * Represents p < 0.05, **** represents p < 0.0001, and ns represents not significant (p > 0.05). B. Non-reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines treated for 24 h as specified. Note the alternate staining patterns observed with different DR5 antibodies. O and M represent Oligomeric and Monomeric protein isoforms. C. The indicated MDA-MB-468 stable cell lines were treated for 24 h as indicated with 1 μg/ml doxycycline and 2.5 μM dFtcyDTDO and subjected to cell surface protein biotin labeling. Cell surface proteins (External; Ext.) were affinity purified with Streptavidin-agarose, and the unlabeled flow-through (Internal; Int.) proteins were also collected. Both fractions were analyzed by non-reducing immunoblot using the indicated antibodies. D. Cell surface protein labeling experiment as in panel C except that cell lines were treated with the indicated combinations of 1 μg/ml doxycycline, 2.5 μM dFtcyDTDO, and 10 μM Cyclosporine A.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Flow Cytometry, Stable Transfection, Western Blot, Staining, Labeling, Affinity Purification