usp2 258 605  (Addgene inc)

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a . Schematic representation of human USP36 domain architecture with active site residues shown as stars. The protein boundaries used for crystallography studies are indicated. b . Schematic representation of human USP16 domain architecture. The protein boundaries used for in-vitro studies are indicated. The USP domain of USP16 contains various large insertions . c.-f . Quantification of Ubiquitin and Fubi C-terminal hydrolase activity using Ub-RhoG and Fubi-RhoG substrates, respectively, for both USP2 ( c , d ) and USP30 ( e , f ) as described in Fig. .

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: In Vitro, Ubiquitin Proteomics, Activity Assay

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a , Semisynthesis of the Fubi-PA probe from Fubi-MesNa (top) and characterization of these species by intact protein MS (bottom). b , Reactivity assessment of recombinant USP16 incubated with the indicated probes for 1 h at 37 °C. Chloroacetamide (CAA) pretreatment was performed where indicated. Protein samples were analyzed by SDS–PAGE and Coomassie staining; ABP, activity-based probe. c , Reactivity assessment of recombinant USP36 as described in b . d , Results of a DUB panel inhibition assay shown as a heat map. Recombinant DUBs were incubated with Fubi-PA (4 µM) for 15 min at room temperature, and their activities were subsequently assessed. Residual activities for USP16 and USP36 are given as numbers. Data show results from technical duplicates normalized for each DUB to its respective activity in the absence of probe; UCHs, ubiquitin carboxy-terminal hydrolases; OTUs, ovarian tumor family of DUBs. e , Reactivity assessment of recombinant USP2 as described in b and c . f – h , Thermal stability assessment of USP36 ( f ), USP16 ( g ) and USP2 ( h ) complexed with Fubi or ubiquitin probes. Means and individual results of three samples are plotted; T m , protein melting temperature.

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: Recombinant, Incubation, SDS Page, Staining, Activity Assay, Inhibition, Ubiquitin Proteomics

Journal: Nature Chemical Biology

Article Title: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

doi: 10.1038/s41589-023-01388-1

Figure Lengend Snippet: a , Cartoon and transparent surface representation of Fubi in two orientations colored according to sequence conservation, which was calculated from sequences of 156 species. Selected residues are shown as sticks and are labeled. b – f , Reactivity assessment of recombinant proteins USP36 ( b ), USP16 ( c ), USP2 ( d ), USP42 ( e ) and USP30 ( f ) with wild-type ubiquitin-PA, wild-type Fubi-PA or Fubi-PA probes carrying the indicated mutations (which substitute the ubiquitin-equivalent residues into Fubi). Cross-reactivity to mutated Fubi probes is shown with black arrowheads for USP42 and USP30. Data are representative of three independent experiments. g , Model summarizing important motifs on Fubi underlying the Fubi/ubiquitin cross-reactivity in USP16/USP36 and the restriction from other DUBs, thereby enabling spatially controlled maturation of Fubi-S30. h , Schematic representation of the proposed two-tier processing of Fubi-S30 and the Fubi system.

Article Snippet: Human Fubi 1–73 , HA–Fubi 1–73 , GST–3C-GS-HA–Fubi 1–73 , ubiquitin 1–75 , HA–ubiquitin 1–75 , HA–NEDD8 1-75 , HA–ISG15 1–156 , HA–UFM1 1–82 , HA–URM1 1–100 and HA–SUMO1 1–96 were cloned from Escherichia coli optimized DNA gene strings into pTXB1 using restriction enzymes. cDNA encoding catalytic domains of human USP16 191–823 (Medical Research Council Protein Phosphorylation and Ubiquitylation (MRC PPU), DU25374), USP42 70–446 (MRC PPU, DU15140), USP36 81–461 and USP36 81–424 (MRC PPU, DU49030) were cloned into pOPINK, and USP2 258–605 (Addgene, 22577), USP8 734–1110 (Addgene, 22608) and USP18 16–372 (MRC PPU, DU14320) were cloned into pOPINB using an In-Fusion HD cloning kit (Takara Clonetech).

Techniques: Sequencing, Labeling, Recombinant, Ubiquitin Proteomics