Journal: Nature Cell Biology

Article Title: Caspase-2 is a condensate-mediated deubiquitinase in protein quality control

doi: 10.1038/s41556-024-01522-8

Figure Lengend Snippet: a , Representative images of live-cell fluorescence imaging during HS or BTZ treatment of HEK293T cells stably expressing mGFP–CASP2-CA. b , Representative fluorescence images of CASP2 and Ubc9ts in HEK293T cells stably expressing mGFP–CASP2-CA and transfected with mCherry-tagged Ubc9ts. The arrowheads indicate co-localization of CASP2 and Ubc9ts in a large punctum and the arrow points to diffusely distributed CASP2 without Ubc9ts overexpression. c , Fluorescence recovery after photobleaching (red outline) of an mGFP–CASP2-CA punctum in HEK293T cells. Time-lapse images of bleached cells. d , Representative fluorescence images of poly-ubiquitin (FK2) in HEK293T cells stably expressing mGFP–CASP2-CA after HS (42 °C, 2 h) and BTZ (1 μM, 8 h) treatment (left). The fluorescence intensities along the dashed line were plotted (right). e , Representative images of immunofluorescence staining of endogenous CASP2 and poly-ubiquitin (FK2) in SH-SY5Y cells after HS (42 °C, 2 h) and BTZ (1 μM, 8 h) treatment (left). Arrows indicate the ubiquitin-positive puncta. The fluorescence intensities along the dashed line were plotted (right). f , Representative images of phase separation by mixing Cy3-labelled maltose-binding protein (MBP)–CASP2-FL-CA protein (20 μM) with GFP–8Ub or GFP–monoUb (20 μM). g , Liquid-droplet fusion of Cy3–MBP–CASP2-FL-CA and GFP–8Ub proteins. h , Liquid droplets of Cy3–MBP–CASP2-FL-CA and GFP–8Ub proteins were treated with 1 M NaCl, 1,6-hexanediol (1,6-HD), propylene glycol (1,2-PG) or 2,5-hexanediol (2,5-HD). Scale bars, 10 µm ( a ), 5 µm ( b , d – f , h ), 2 µm ( c , g ). Data are representative of three independent experiments.

Article Snippet: Recombinant MBP, MBP–CASP2-FL, MBP–CASP2-CARD and MBP–CASP2-ΔCARD proteins were labelled with fluorescent dye Cy3 (Yeasen, 40777ES03).

Techniques: Fluorescence, Imaging, Stable Transfection, Expressing, Transfection, Over Expression, Ubiquitin Proteomics, Immunofluorescence, Staining, Binding Assay

Journal: Nature Cell Biology

Article Title: Caspase-2 is a condensate-mediated deubiquitinase in protein quality control

doi: 10.1038/s41556-024-01522-8

Figure Lengend Snippet: a , Representative images of phase separation by mixing GFP-8Ub proteins (20 μM) with MBP–CASP2-FL-CA, CARD and ΔCARD-CA proteins labelled Cy3 (20 μM). Scale bars, 5 µm. b , Alignment of UIML region sequences of CASP2 across species by Jalview. c , The structure model of CASP2 CARD domain predicted by AlphaFold2. d , e , MBP pull-down assay followed by immunoblotting and CBB staining to monitor the ubiquitin-binding capacity of MBP–CASP2-CARD mutants ( d ) and truncations ( e ). The ubiquitin chains were assembled in vitro . f – h , MBP pull-down assay followed by immunoblotting and CBB staining for monitoring the binding between HS-treated MBP–CASP2-CARD and the in vitro assembly ubiquitin chains. The ubiquitin chains were assembled using different E2 or E2/E3. The ubiquitin proteins used in ( c – g ) are a mixture of no-tagged ubiquitin and HA-tagged ubiquitin at a ratio of 9:1. i , Alignment of CARD domain sequences of CASP2, CED-3, caspase-1/4/5/9. MBP pull-down assay followed by immunoblotting and CBB staining for monitoring the binding between the HS-treated CARD domain and the cell lysate. Data are representative of three independent experiments.

Article Snippet: Recombinant MBP, MBP–CASP2-FL, MBP–CASP2-CARD and MBP–CASP2-ΔCARD proteins were labelled with fluorescent dye Cy3 (Yeasen, 40777ES03).

Techniques: Pull Down Assay, Western Blot, Staining, Ubiquitin Proteomics, Binding Assay, In Vitro