Complete Materials and Methods
Cell culture, lysis, electrophoresis and immunoblot: Cell lines were cultured in FCS-supplemented RPMI-1640 with penicillin/streptomycin. Medium for adapted cells additionally contained 20 nM bortezomib (Ortho Biotech, Neuss, Germany). Incubation time of cells with other inhibitors was 16 hours. Cells were lysed by 0.5% Triton X-100 in 0.1 M phosphate pH 7.4 / 0.1 mM EDTA, or, for cathepsin activity assays, in 0.1 M citrate pH 5.0. Equal amounts of protein were used for gel electrophoresis or in activity assays. Antibodies against proteasome subunits a4, b1, b2, b5, b1i, b5i, and PA28a were from Biomol (Hamburg, Germany); anti-b2i was from Santa Cruz (Santa Cruz, CA, USA), and anti-Rpn1 from Calbiochem (Merck, Darmstadt, Germany). Anti-BiP (Grp78) was purchased from Becton Dickinson (Franklin Lakes, NJ, USA), anti-Caspase 4 and anti-poly-Ub from Biomol, and anti-poly(ADP-ribose)-polymerase (PARP) 1 p85 from Promega (Mannheim, Germany). Anti-TPPII from chicken was bought at Immunsystem AB (Uppsala, Sweden); anti-GAPDH and anti-b-actin were from Sigma (Taufkirchen, Germany). Antisera against USP were generated by immunizing rabbits with synthetic peptides.
Activity assays: Active-site directed probes were employed for (semi-)quantitative assessment of protease activities: Labeling of active proteasome subunits was performed in live cells with the proteasome-specific affinity probes MV151 or dansyl-sulfonamidohexanoyl-(6-aminohexanoyl)2-(Leu)3 vinyl sulfone (DALVS), as described [18,19]. Cell lysates were separated by SDS-PAGE. The fluorescent probe MV151 allows for visualization of proteasome activities by scanning the gel for fluorescence of its bodipy moiety (FLA-5100 fluorescence image scanner, Fuji, Düsseldorf, Germany). Quantification of fluorescent bands was performed using the program AIDA (Raytest, Straubenhardt, Germany), and fluorescence signal intensities were normalized to protein loading control bands measured by densitometry. DALVS-labeled species were visualized after western blot by a Molecular Probes biotinylated anti-dansyl rabbit pAb (Invitrogen, Paisley, UK) and a streptavidin-peroxidase reagent (Vector Laboratories, Burlingame, CA, USA). Similarly, activities of cathepsins were profiled with biotinylated DCG-0N, and DUB activities with HA-tagged Ubiquitin-vinyl methyl ester (HAUbVME) from cell lysates, as described [10,27,28]. In vitro cleavage of fluorogenic substrate Ala-Ala-Phe-7-amino-4-methyl-coumarin (AAF-AMC; Bachem, Weil am Rhein, Germany) by equal amounts of protein from cell lysates was performed at 37°C in 0.1 M phosphate pH 7.4 / 0.1 mM EDTA and detected during the linear phase (Tecan multi plate reader, Crailsheim, Germany).
Immunoprecipitation: For immunoprecipitation of proteasome, cell lysates were incubated with anti-a2-agarose beads (Biomol) in IP buffer (25 mM Tris/HCl pH 7.3, 10 mM KCl, 0.5 mM EDTA, 2 mM MgCl2, 1 mM DTT, 1 mM ATP, 5% glycerol). Beads were washed with 0.1 M NaCl in IP buffer and eluted with 2 M NaCl in IP buffer (without DTT, ATP, glycerol). Eluates were concentrated by ultrafiltration and analyzed by SDS-PAGE and immunoblot.
To identify deubiquitinating enzymes, cell lysates were incubated with HAUbVME and active site-labeled species were bound to anti-HA-agarose (Sigma), followed by intensive washing with NET buffer (50 mM Tris/HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40). Proteins were eluted with 0.1 M Glycine pH 3 and separated by SDS-PAGE. Matrix-assisted laser desorption/ionization mass spectrometry-based identification of the separated proteins was performed as described [29].
Cytotoxicity assay: Cytotoxicity of proteasome inhibitors bortezomib, NLVS, ZL3VS, epoxomicin (Biomol) and lactacystin (Sigma), and of daunorubicin (Calbiochem), was determined by MTS-assay (Promega) and absorbance measured at 492 nm. Results represent mean values from triplicates.
Gene expression analysis: RNA was extracted from HL-60 and HL-60a cultured without bortezomib for 3 days. This set of samples was taken in duplicates from individual cultures. After amplification and labeling with Cy3 (HL-60) and Cy5 (HL-60a), cDNA was hybridized to a PIQOR Ubiquitin PS microarray (Miltenyi Biotec) containing quadruplicate spots of probes from ubiquitin-proteasome system (UPS) genes. A mean ratio > 1.7 of Cy5 and Cy3 fluorescence in both sets of samples served as indicator of differential gene expression.
Metabolic labeling: Metabolic labeling was essentially performed as published [30]. After treatment with inhibitors for 16 h, equal cell numbers were starved in methionine/cysteine-free medium for 15 minutes at 37°C and subsequently pulsed with 35S-methionine/cysteine cell labeling mix (GE Healthcare, Freiburg, Germany) for 30 minutes. After intensive washing, cells were lysed and protein from equal volumes of lysate was precipitated with 5% trichloro acetic acid on filter paper. Washing with ethanol and acetone served to remove free 35S-methionine/cysteine. Protein-bound radioactivity was then measured in a scintillation counter.
Supplemental references
[27] Reich M, van Swieten PF, Sommandas V, Kraus M, Fischer R, Weber E et al. Endocytosis targets exogenous material selectively to cathepsin S in live human dendritic cells, and cell-penetrating peptides mediate nonselective transport to cysteine cathepsins. J Leukoc Biol 2007; 81: 990-1001.
[28] van Swieten PF, Maehr R, van den Nieuwendijk AMCH, Kessler BM, Reich M, Wong C-S et al. Development of an isotope-coded activity-based probe for the quantitative profiling of cysteine proteases. Bioorg Med Chem Lett 2004; 14: 3131-3134.
[29] Skopeliti M, Kratzer U, Altenberend F, Panayotou G, Kalbacher H, Stevanovic S et al. Proteomic exploitation on prothymosin a-induced mononuclear cell activation. Proteomics 2007; 7: 1814-1824.
[30] Driessen C, Bryant RA, Lennon-Dumenil AM, Villadangos JA, Bryant PW, Shi GP et al. Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells. J Cell Biol 1999; 147: 775-790.
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