Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet: MCF7 cell lysate either undigested or digested with RNAse I was separated by sucrose density gradient centrifugation (UV traces, left panel). Trypsin‐like proteasome activity was assessed in sucrose density gradient fractions using an in vitro assay (middle panel). Results were plotted in a line graph relative to the values obtained to the untreated control ( n = 2, means ± SD, right panel). MCF7 and AD293 cells were exposed to the conditions indicated after which an equal amount of cell lysate was subjected to sucrose density gradient centrifugation. The abundance of proteasome subunit ADRM1 was determined by immunoblotting. Source data are available online for this figure.

Article Snippet: Membranes were probed with primary and secondary antibodies as follows: RPL7 (Abcam, ab72550) 1:2,000, RPS19 (Bethyl, A304‐002A) 1:2,000, Proteasome 20S/PSMA (Enzo, BML‐PW8195) 1:1,000, ADRM1/RPN13 (Enzo, ENZ‐ABS296) 1:500, PSMB1 (Proteintech, 11749‐1‐AP) 1:1,000, PSMC1/Rpt2/S4 (Enzo, BML‐PW0530) 1:1,000, DNAJC2/MPP11 (Proteintech,11971‐1‐AP) 1:1,000, Anti‐Puromycin (Millipore, MABE343) 1:3,000, Hsp70/HSPA1 (Proteintech, 10995‐1‐AP) 1:3,000, HSPH1(Millipore‐ABC264) 1:1,000, K48‐Linkage‐Specific Polyubiquitin (Cell Signaling, 8081) 1:3,000, Anti‐β‐Actin (SIGMA‐ALDRICH, A5441) 1:2,000, Goat Anti‐Mouse IgG (Thermo, 31430) 1:5,000, Goat Anti‐Rabbit IgG (Thermo,31460) 1:5,000.

Techniques: Gradient Centrifugation, Activity Assay, In Vitro, Western Blot

Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet:

Article Snippet: Membranes were probed with primary and secondary antibodies as follows: RPL7 (Abcam, ab72550) 1:2,000, RPS19 (Bethyl, A304‐002A) 1:2,000, Proteasome 20S/PSMA (Enzo, BML‐PW8195) 1:1,000, ADRM1/RPN13 (Enzo, ENZ‐ABS296) 1:500, PSMB1 (Proteintech, 11749‐1‐AP) 1:1,000, PSMC1/Rpt2/S4 (Enzo, BML‐PW0530) 1:1,000, DNAJC2/MPP11 (Proteintech,11971‐1‐AP) 1:1,000, Anti‐Puromycin (Millipore, MABE343) 1:3,000, Hsp70/HSPA1 (Proteintech, 10995‐1‐AP) 1:3,000, HSPH1(Millipore‐ABC264) 1:1,000, K48‐Linkage‐Specific Polyubiquitin (Cell Signaling, 8081) 1:3,000, Anti‐β‐Actin (SIGMA‐ALDRICH, A5441) 1:2,000, Goat Anti‐Mouse IgG (Thermo, 31430) 1:5,000, Goat Anti‐Rabbit IgG (Thermo,31460) 1:5,000.

Techniques: Recombinant, Sequencing, Magnetic Beads, Staining, Protease Inhibitor, DNA Purification, Bicinchoninic Acid Protein Assay, Modification, Software, Imaging

Journal: bioRxiv

Article Title: Proteasome-Bound UCH37 Debranches Ubiquitin Chains to Promote Degradation

doi: 10.1101/2020.02.21.960088

Figure Lengend Snippet: (A) Characterization of the UCH37•RPN13 co-purified complex: Coomassie gel (left), α-RPN13 immunoblot (middle), and α-UCH37 immunoblot (right). 1 = recombinant UCH37, 2 = recombinant RPN13, 3 = co-purified complex. (B) Ubiquitin-AMC hydrolysis of UCH37, UCH37•RPN13 DEUBAD , UCH37•RPN13, and UCH37•RPN13 L56A/F76R/D79N (20 nM). All curves are representative traces and fits are derived from averaging two independent experiments to pseudo first-order kinetics: Y = Ymax (1-e( k cat/ K m)•E°•t). (C) Michaelis-Menten plot for the hydrolysis of native K6/K48 branched tri-Ub by either UCH37•RPN13 DEUBAD or UCH37•RPN13 L56A/F76R/D79N (0.5 μM, left). Table of kinetic parameters measured for all experiments following the initial rates of di-Ub formation (right). All kinetic curves are representative traces and constants are derived from averaging fits of independent experiments with SD (n = 3). (D) Ub MiD MS analysis of HMW K11/K48 chains (top) and HMW K48/K63 chains (bottom) treated with UCH37•RPN13 (1 μM). Percentages correspond to the relative quantification values of the 11+ charge state for each Ub species: Ub 1-74 , 1xdiGly-Ub 1-74 , and 2xdiGly-Ub 1-74 . (E) Ub-AQUA analysis of HMW K6/K48, K11/K48, and K48/K63 chains before and after UCH37•RPN13 (1 μM) treatment. For all points, * P <0.025, ** P <0.01 (Student’s T-test). Quantification values are derived from averaging fits of 2 independent experiments shown with SEM. (F) Steady-state parameters for the hydrolysis of HMW K6/K48, K11/K48, and K48/K63 chains by UCH37•RPN13 (0.5 μM, left), Catalytic efficiencies ( k cat / K m ) are calculated from the 11+ charge state of the 2xdiGly-Ub 1-74 species. Table of kinetic parameters (right) measured for all experiments following the first-order decay rates of the 2xdiGly-Ub 1-74 species. All kinetic curves are representative traces and constants are derived from averaging fits of independent experiments with SD (n = 2).

Article Snippet: Anti UCH37 (Abcam, Cat. # ab124931) Anti RPN11 (Abcam, Cat. # ab109130) Anti RPN13 (Cell Signaling Technology, Cat. # D9Z1U) Anti RPT2 (Abcam, Cat. # ab3317) Anti PSMB7 (R&D Systems, Cat. #MAB7590) Anti USP14 (Abcam, Cat. # ab56210) Anti Ub (P4D1, Enzo Lifesciences, Cat. # BML-PW0930) Anti K48-linkage Specific (Cell Signaling Technology, Cat. # D9D5) Goat Anti Mouse IR Dye 800CW (LI-COR Biosciences, Cat. # 926-32210) Goat Anti Rabbit IR Dye 680RD (LI-COR Biosciences, Cat. # 926-68071) Goat Anti Rabbit IR Dye 800CW (LI-COR Biosciences, Cat. # 926-32211)

Techniques: Purification, Western Blot, Recombinant, Ubiquitin Proteomics, Derivative Assay, Quantitative Proteomics

Journal: bioRxiv

Article Title: Proteasome-Bound UCH37 Debranches Ubiquitin Chains to Promote Degradation

doi: 10.1101/2020.02.21.960088

Figure Lengend Snippet: (A) Western blot analysis showing the loss of USP14 during purification. For KO cell lines the loss of UCH37 or RPN13 are also observed. For replenish experiments, addition of recombinant UCH37 and RPN13 are found in the final elution. (B) Ubiquitin-AMC (top) and Suc-LLVY-AMC (bottom) hydrolysis of each indicated proteasome (1 μg). All curves are representative traces and fits are derived from averaging two independent experiments. (C) Western blot analysis of HMW K11/K48 (left) and K48/K63 (right) chain debranching with increasing concentration of proteasomes using the α-Ub P4D1 antibody. (D-E) Ub MiD MS analysis of HMW K11/K48 (D) and K48/K63 (E) chains subjected to each indicated Ptsm complex (10 μg). Percentages correspond to the relative quantification values of the 11+ charge state for each Ub species: Ub 1-74 , 1xdiGly-Ub 1-74 , and 2xdiGly-Ub 1-74 . (F) Quantitative western blot analysis to determine concentration of UCH37 in WT and UCH37•RPN13-replenished proteasomes for kinetic analysis of cleavage reactions. (G-H) Steady-state parameters for the hydrolysis of HMW K11/K48 (G) and K48/K63 (H) chains by WT proteasome (10 μg, black) and UCH37•RPN13-replenished proteasome (10 μg, blue). Catalytic efficiencies ( k cat / K m ) are calculated from the 11+ charge state of the 2xdiGly-Ub 1-74 species. All curves are averaged representative traces from averaging fits of independent experiments with SD (n = 2).

Article Snippet: Anti UCH37 (Abcam, Cat. # ab124931) Anti RPN11 (Abcam, Cat. # ab109130) Anti RPN13 (Cell Signaling Technology, Cat. # D9Z1U) Anti RPT2 (Abcam, Cat. # ab3317) Anti PSMB7 (R&D Systems, Cat. #MAB7590) Anti USP14 (Abcam, Cat. # ab56210) Anti Ub (P4D1, Enzo Lifesciences, Cat. # BML-PW0930) Anti K48-linkage Specific (Cell Signaling Technology, Cat. # D9D5) Goat Anti Mouse IR Dye 800CW (LI-COR Biosciences, Cat. # 926-32210) Goat Anti Rabbit IR Dye 680RD (LI-COR Biosciences, Cat. # 926-68071) Goat Anti Rabbit IR Dye 800CW (LI-COR Biosciences, Cat. # 926-32211)

Techniques: Western Blot, Purification, Recombinant, Ubiquitin Proteomics, Derivative Assay, Concentration Assay, Quantitative Proteomics