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Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits
利用活性探针pull-down天然蛋白酶体亚基及免疫蛋白酶体亚基   

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Abstract

Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques.

Keywords: Activity-based probe(基于酶活性的探针), Activity-based pull-down(基于活性的pull-down), Biotin(生物素), Streptavidin(链霉亲和素), Proteasome Activity(蛋白酶体活性), Immunoproteasome(免疫蛋白酶体), Macrophage(巨噬细胞)

Background

The proteasome is a barrel-shaped, multicatalytic enzyme complex that is present in the nucleus and cytoplasm of eukaryotic cells. It is essential for protein degradation, including processing of antigenic peptides for MHC I presentation, and regulates many cellular processes (Kammerl and Meiners, 2016). In cells of hematopoietic origin, the standard (constitutive) proteasome is often replaced by the immunoproteasome (Meiners et al., 2014), which differs in the incorporation of the three distinct catalytically active β-subunits (Figure 1).

To study the molecular function of single catalytic subunits and to modulate physiological processes, the development of subunit-specific proteasome inhibitors is indispensable. Large progress has recently been made in this area by de Bruin et al. (2014). Specific immunoproteasome inhibitors have proven as potential drug candidates for the treatment of inflammatory and autoimmune disease. The inhibition of the immunoproteasome subunit β5i may alter cytokine production by activated monocytes and lymphocytes. In a mouse model of rheumatoid arthritis, this reversed signs of the disease (Muchamuel et al., 2009).

The protocol was developed in the context of a study that investigated the effects of selective inhibition of β5i on the polarization of alveolar macrophages (Chen et al., 2016). The activity-based pull-down of catalytic standard and immunoproteasome subunits using the pan-reactive ABP Bio-EP allowed us to confirm the specific inhibition of β5i by the inhibitor ONX-0914, previously developed by ONYX Pharmaceuticals.

The use of ABPs with alternative specificity may allow for activity-based pull-down of other enzyme families in a similar experimental approach.


Figure 1. Structure of the 20S standard and immunoproteasome. The 20S core particle of the proteasome consists of two stacked inner rings containing the β-subunits 1-7 of which β1, β2 and β5 exhibit proteolytic activity. The β-rings are flanked by two α-rings containing the α-subunits 1-7. In the immunoproteasome β1, β2 and β5 are replaced by β1i, β2i and β5i, respectively, which leads to altered proteolytic activity.

Materials and Reagents

  1. 15 cm cell culture dishes
  2. Protein LoBind® tubes (1.7 ml tubes; OMNILAB-LABORZENTRUM, catalog number: 5409327 )
  3. 50 ml Falcon tube (Corning, Falcon®, catalog number: 352070 )
  4. Pipette tips  
  5. Fine Dosage Syringe Omnifix-F (1 ml) (OMNILAB-LABORZENTRUM, catalog number: 5421736 )
  6. 15 ml Falcon tube (Corning, Falcon®, catalog number: 352096
  7. Immun-Blot® PVDF membrane (Bio-Rad Laboratories, catalog number: 162-0177
  8. PD MidiTrap G-25 (store at RT) (GE Healthcare, catalog number: 28-9180-08 )
  9. Filter 0.2 µm (Sartorius, catalog number 16534-K )
  10. Fuji X-ray films RX, 18 x 24 cm (Kisker Biotech, model: RX1824 )
  11. Whatman blotting paper (Laborbedarf Lammel, catalog number: 3030690 )
  12. Murine alveolar macrophage cell line MH-S (ATCC, catalog number: CRL-2019 )
  13. Liquid nitrogen
  14. BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  15. Biotin-Epoxomicin (Bio-EP; dissolved in DMSO, stable for at least 12 months at -20 °C; Hermen Overkleeft Lab, synthesis described in Florea et al., 2010)
  16. Dimethyl sulfoxide (DMSO) (Carl Roth, catalog number: A994.2 )
  17. HEPES (Molecular biology grade) (AppliChem, catalog number: A3724,1000
  18. Ultrapure water (e.g., MilliQ)
  19. Streptavidin beads (Strep-Tactin® Superflow® 50% suspension; store at 4 °C) (IBA, catalog number: 2-1206-010 )
  20. Methanol (AppliChem, catalog number: A3493.1000 )
  21. Roti®-Block blocking solution (store at RT) (Carl Roth, catalog number: A151.3
  22. Anti-β5 antibody (store at -20 °C) (Abcam, catalog number: 90867 )
  23. Anti-β5i antibody (store at -20 °C) (Abcam, catalog number: 3329 )
  24. Anti-Rabbit IgG, HRP-linked antibody (store at -20 °C) (New England Biolabs, catalog number: 7074S )
  25. Amersham ECL prime Western blotting detection reagent (store at 4 °C) (GE Healthcare, catalog number: RPN2232 )
  26. RPMI-1640 cell culture medium (Thermo Fisher Scientific, GibcoTM, catalog number 11875093 )
  27. Fetal bovine serum (FBS) (Biochrom, catalog number S 0615 )
  28. β-mercaptoethanol (Molecular biology grade) (AppliChem, catalog number A1108-100 )
  29. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number 15140122 )
  30. Tris (buffer grade) (AppliChem , catalog number: A1379,1000 )
  31. Magnesium chloride 6-hydrate (MgCl2.6H2O) (AppliChem, catalog number: A1036,0500 )
  32. Sodium chloride (NaCl) (AppliChem, catalog number: A2942,1000 )
  33. Ethylenediaminetetraacetic acid (EDTA) (AppliChem, catalog number: A2937,1000 )
  34. Sodium azide (NaN3) (AppliChem, catalog number: A1430,0100 )
  35. Dithiothreitol (DTT) (molecular biology grade) (AppliChem, catalog number: A2948,0025 )
  36. Adenosine triphosphate (ATP), disodium salt (10 g) (Roche Diagnostics, catalog number: 10127531001 )
  37. Glycerol (87%) (molecular biology grade) (AppliChem, catalog number: A3739,1000 )
  38. cOmpleteTM protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001 )
  39. PhosSTOPTM (phosphatase inhibitor) (Roche Diagnostics, catalog number: 4906845001 )
  40. Sodium dodecyl sulfate (SDS) (pure) (AppliChem, catalog number: A1502,1000 )
  41. Bromophenol blue (AppliChem, catalog number: A2331,0025 )
  42. Hydrochloric acid (fuming) (37%) (Sigma-Aldrich, catalog number: 258148-2.5L )
  43. Rotiphorese® Gel 30 (37.5:1) (Carl Roth, catalog number: 3029.2 )
  44. Ammonium peroxodisulfate (APS) (AppliChem, catalog number: A0834,0250 )
  45. Tetramethylethylenediamine (TEMED) (AppliChem, catalog number: A1148,0100 )
  46. Tween 20 (Moleculare biology grade) (AppliChem, catalog number: A4974,1000 )
  47. Dulbecco’s phosphate-buffered saline (1x DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  48. Complete growth medium for MH-S cells (see Recipes)
  49. TSDG buffer (see Recipes)
  50. 500 µM Bio-EP in DMSO (see Recipes)
  51. 50 mM HEPES (pH 7.4) (see Recipes)
  52. 10% SDS (see Recipes)
  53. 6x Laemmli buffer (see Recipes)
  54. 4x SDS-PAGE resolving buffer (pH 8.8) (see Recipes)
  55. 4x SDS-PAGE stacking buffer (pH 6.8) (see Recipes)
  56. Stacking gel (see Recipes)
  57. Resolving gel (15% acrylamide) (see Recipes)
  58. PBST (0.1% Tween) (see Recipes)

Equipment

  1. Falcon tube centrifuge (Hettich Instruments, model: Rotina 420R )
  2. Table centrifuge ( Hettich Instruments, model: MIKRO 200R )
  3. Water bath (LAUDA , model: Aqualine AL12 LCB 0725 )
  4. Thermomixer ( Eppendorf, model: Thermomixer comfort )
  5. Forceps
  6. Pipettes  
  7. Scissors
  8. Intelli-mixer rotator (ELMI, model: RM 2M )
  9. Western blot chambers ( Bio-Rad Laboratories, model: Mini PROTEAN Tetra Cell )
  10. Power supply (PowerPacTM Basic Power supply) (Bio-Rad Laboratories, model: 1645050 )
  11. Western blot developer ( Agfa -Gevaert, model: Curix60 )
  12. Fume hood

Procedure

  1. Preparation of cell material
    1. Culture MH-S cells in complete growth medium.
      Note: This protocol was tested in the murine alveolar macrophage cell line MH-S purchased from ATCC (No. CRL-2019). Cells were passaged at 70-90% cell density in a 1:6 up to 1:12 ratio (splitting frequency approx. 3-5 days). 12 x 106 cells were seeded in 15 cm cell culture dishes the day before treatment, but numbers may vary if other cell lines are used. Further information about cell culture and treatment are stated in Chen et al. (2016).
    2. Harvest cells and pellet at 800 x g for 5 min at RT, remove supernatant.
    3. Wash cells with 5 ml of PBS and pellet at 800 x g for 5 min at RT.
    4. Re-suspend cells gently in 400 µl of TSDG buffer.
      Note: Lysis in TSDG buffer helps to sustain standard and immunoproteasome activity by stabilizing proteasome complexes.
    5. Lyse cells with seven repeated cycles of freezing tubes in liquid nitrogen and thawing them in a water bath with water at room temperature until crystals disappear.
      Note: The use of a floater facilitates the handling of several tubes in parallel.
    6. Centrifuge at 14,000 x g for 20 min to remove cell debris and transfer the supernatant to a fresh tube.
    7. Determine protein concentration (for example with BCA protein assay, Bio-Rad).
    8. Store samples at -80 °C.

  2. ABP labeling of the standard and immunoproteasome
    1. Treatment with Biotin-Epoxomicin (Bio-EP)
      Note: Bio-EP is a biotinylated ABP that irreversibly binds to active catalytic subunits of both standard and the immunoproteasome.
      1. Thaw cell lysate on ice and transfer volume equivalent to 500 µg of protein into a 1.7 ml tube.
        Note: For each cell lysate, prepare two tubes. One tube serves as vehicle control and will be incubated with DMSO instead of Bio-EP.
      2. Fill up to 500 µl with 50 mM HEPES (pH 7.4). The final protein concentration is 1 mg/ml.
      3. Add Bio-EP at a final concentration of 5 µM (for example 5 µl of a 500 µM stock), respectively, add the same amount of DMSO to the control tube.
      4. Incubate tubes for 120 min at 37 °C on a Thermomixer at 600 rpm.
        Note: Meanwhile, equilibrate the columns for size exclusion chromatography (steps B2a-B2e) and prepare the beads for pull-down (steps C1a-C1d).
      5. Fill up to 1 ml with 50 mM HEPES (pH 7.4). The final protein concentration is 0.5 mg/ml.
        Note: A volume of 1 ml for SEC conforms manufacturer’s recommendation.
    2. Size exclusion chromatography (SEC)
      Note: SEC is performed to remove unbound ABP from the sample. Free ABP is retained in the column resin whereas large proteins like the standard and immunoproteasome pass through. Use a fresh PD MidiTrap G-25 column for each sample. Perform SEC also on vehicle control samples.
      1. Remove the top cap from the column and pour off the storage solution.
      2. Use forceps to remove the filter on top of the column resin.
      3. Remove the bottom cap and insert the column into a 50 ml collection tube using the blue adapter delivered by the manufacturer.
      4. Equilibrate column 2 x with 5 ml of ultrapure water and discard the flow-through.
      5. Fill the column a third time with ultrapure water and spin down at 1,000 x g for 2 min in a centrifuge at RT.
        Note: Don’t let the column run dry for a longer period. Cap the wet column with the bottom cap before centrifugation to pause equilibration until all samples are ready for application. Remove the cap and centrifuge the remaining liquid to resume with the protocol as soon as all samples are ready.
      6. Discard the flow through and transfer the column with the adapter to a fresh 50 ml Falcon tube labeled with the samples name.
      7. Slowly pipette the sample dropwise in the middle of the packed bed and centrifuge at 1,000 x g for 2 min.
      8. Discard the column and store the 50 ml Falcon tube containing the eluate on ice.

  3. Pull-down of ABP-labeled subunits
    1. Preparation of streptavidin beads
      Note: Can be done during ABP incubation (step B1d).
      1. Transfer 40 µl of the 50% streptavidin bead slurry (= 20 µl of beads) to a 1.7 ml tube.
        Note: Vortex slurry before transfer. Use a 200 µl pipette and cut the end of the pipette tip with scissors, otherwise beads will plug the pipette tip.
      2. Wash beads 3 x with 500 µl of PBS. Centrifuge between washing steps at 2,000 x g for 2 min and discard the supernatant.
        Note: Use a narrow needled syringe and carefully aspirate the supernatant without removing beads. The diameter of the needle tip must be smaller than the bead diameter.
      3. Re-suspend beads in 1 ml of 50 mM HEPES (pH 7.4) and transfer them into a 15 ml Falcon tube.
        Note: Re-suspend the beads in 500 µl of HEPES buffer and transfer to a 15 ml Falcon tube. Repeat this to transfer the residual beads and hence prevent bead loss. Use a 1,000 µl pipette and cut the end of the pipette tip with scissors, otherwise beads will plug the pipette tip.
      4. Store the beads on ice until needed.
    2. Solubilization of the standard and immunoproteasome
      Note: Solubilization is necessary to disrupt the complex structure and to allow separation of ABP-labeled from non-labeled subunits.
      1. Transfer 200 µl (equals max. 100 µg protein) per eluate (from SEC) into a fresh 1.7 ml tube. Store the rest of the eluate at -20 °C.
      2. Add 20 µl of 10% SDS and boil at 95 °C for 8 min in a Thermomixer. Cool down to RT.
      3. Centrifuge tubes 1 min at 10,000 x g at RT to collect evaporated liquid from the lid of the tube.
    3. Pull-down
      Note: Bio-EP labeled subunits are purified in this step using streptavidin beads.
      1. Transfer sample to a 15 ml Falcon tube with pre-washed beads (from C1) and fill up to 10 ml with 50 mM HEPES (pH 7.4).
        Note: SDS may interfere with the pull-down. Therefore, SDS is diluted to 0.02% in this step.
      2. Incubate tubes for 2 h at 4 °C. Rotate tubes in a rotator during incubation (app. 20 rpm).
      3. Centrifuge tubes at 2,000 x g for 2 min (4 °C). Discard the supernatant.
      4. Re-suspend beads in 2 x 500 µl of PBS and transfer slurry into a 1.7 ml tube.
      5. Centrifuge tubes at 2,000 x g for 2 min (4 °C). Discard the supernatant.
      6. Wash beads two more times with 500 µl of PBS. Centrifuge at 2,000 x g for 2 min (4 °C).
      7. Re-suspend beads in 60 µl of 1x Laemmli buffer and boil for 15 min at 90 °C in a Thermomixer.
        Note: Bio-EP labeled subunits bound to the beads are eluted in this step.
      8. Centrifuge tubes at 2,000 x g for 2 min (4 °C). Transfer the supernatant into a fresh 1.7 ml tube using a syringe.
      9. Store samples at -20 °C.

  4. Western blot detection
    Note: Alternative modes of detection or further processing steps may be performed. The protocol exemplifies the detection of immunosubunit β5i and standard proteasome subunit β5.
    1. Separate proteins of the pull-down eluate in a 15% polyacrylamide gel. Load gel pocket with 25 µl of eluate and run for approximately 90 min at 120 V.
    2. Blot proteins onto a polyvinylidene difluoride membrane for 90 min at 250 mA.
      Note: Soak membrane in MeOH before performing the electroblot.
    3. Block the membrane for 60 min at RT in 1x Roti®-Block blocking solution.
    4. Incubate membrane with either:
      a. 1:2,000 anti-β5i (in Roti®-Block ) overnight/over the weekend at 4 °C;
      b. 1:1,000 anti-β5 (in Roti®-Block ) overnight/over the weekend at 4 °C.
    5. Wash membrane three times with PBST for 10 min in a plastic box.
    6. Incubate with 1:40,000 anti-rabbit HRP-coupled secondary antibody (New England Biolabs, diluted in PBST) for 60 min at RT.
    7. Wash membrane 3 x with PBST for 10 min at RT.
    8. Cover the membrane with freshly prepared ECL substrate.
    9. Place membrane in a plastic foil and cover it with an X-ray film in a dark chamber. Develop film after several seconds to minutes.


      Figure 2. Workflow activity-based pull-down. Active catalytic standard (brown) and immunoproteasome (yellow) subunits are covalently labeled with the ABP Bio-EP. After removal of unbound ABP via SEC the complex structure of the proteasome is solubilized in 1% SDS. A pull-down with streptavidin-coated agarose beads is performed to capture ABP-labeled subunits. Non-labeled proteins are removed in three consecutive steps of PBS washing, bound subunits are eluted via heating in 1x Laemmli.

Data analysis


Figure 3. Western blot of activity-based pull-down with Bio-EP. Western blots show the amount of active β5i and β5 in ONX-0914 treated MH-S cell lysates and are representative for three independent experiments. With increasing concentration of the β5i-specific inhibitor, the amount of detected β5i in the pull-down eluate decreases whereas β5 remains unaffected by ONX-0914 treatment.

Recipes

  1. Complete growth medium for MH-S cells
    500 ml RPMI-1640
    10% fetal bovine serum (FBS)
    50 μM β-mercaptoethanol
    100 U/ml penicillin-streptomycin
  2. TSDG buffer (10 ml)
    1 ml 100 mM Tris stock (pH 7.5)
    1.1 ml 10 mM MgCl2 stock
    100 µl 1 M NaCl stock
    20 µl 50 mM EDTA stock
    100 µl 100 mM NaN3 stock
    10 µl 1 M DTT stock
    100 µl 200 mM ATP stock
    1.15 ml 87% glycerol
    Fill up to 10 ml with ultrapure water
    Final concentrations:10 mM Tris/HCl, 1.1 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 1 mM NaN3, 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol
    Adjust pH to 7.0
    Aliquot and store at -20 °C
    Add protease and phosphatase inhibitor (Roche) immediately before use (1 tablet for 50 ml lysis buffer; alternatively, a 25x stock can be prepared in ultrapure water and freshly added as needed, store stock at -20 °C)
  3. 500 µM Bio-EP in DMSO
    1 mg Bio-EP (MW = 724.95)
    2.759 ml DMSO
    Aliquot (30-50 µl suggested) and store at -20 °C
  4. 50 mM HEPES (pH 7.4; 1 L )
    11.915 g HEPES
    Fill up to 1 L with ultrapure water
    Adjust pH to 7.4 with HCl or NaOH, as necessary
  5. 10% SDS (10 ml)
    1 g SDS
    Fill up to 10 ml with ultrapure water
    Prepare under a fume hood, wear appropriate breath protection.
  6. 6x Laemmli buffer (10 ml)
    3 ml 1 M Tris HCl (pH 6.8)
    1.5 ml glycerol
    0.6 g SDS
    0.5 g DTT
    1 mg bromophenol blue
    Fill up to 10 ml with ultrapure water
    Filter (0.2 µm)
    Aliquot and store at -20 °C
  7. 4x SDS-PAGE resolving buffer (pH 8.8 )
    36.4 g Tris
    Fill up to 150 ml with ultrapure water
    Adjust pH to 8.8 with 37% HCl
    Fill up to 200 ml with ultrapure water
    Filter (0.2 µm)
    0.48 g SDS
    Storage: RT
  8. 4x SDS-PAGE stacking buffer (pH 6.8)
    6.05 g Tris
    Fill up to 40 ml with ultrapure water
    Adjust pH to 6.8 with 37% HCl
    Fill up to 100 ml with ultrapure water
    Filter (0.2 µm)
    0.4 g SDS
    Storage: RT
  9. Stacking gel (4 ml)
    1 ml 4x stacking buffer
    480 µl 30% acrylamide
    2.52 ml ultrapure H2O
    50 µl 10% APS
    12 µl TEMED
  10. Resolving gel (15% acrylamide) (8 ml )
    2 ml 4x resolving buffer
    4 ml 30% acrylamide
    2 ml ultrapure H2O
    100 µl 10% APS
    12 µl TEMED
  11. PBST (0.1% Tween) (1 L)
    999 ml 1x PBS
    1 ml Tween 20

Acknowledgments

This protocol was used for the work previously published in Cell Death and Differentiation (Chen et al., 2016). The work was supported by intramural funding of the Helmholtz Zentrum München. The authors declare no conflict of interest.

References

  1. Chen, S., Kammerl, I. E., Vosyka, O., Baumann, T., Yu, Y., Wu, Y., Irmler, M., Overkleeft, H. S., Beckers, J., Eickelberg, O., Meiners, S. and Stoeger, T. (2016). Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages. Cell Death Differ 23(6): 1026-1037.
  2. de Bruin, G., Huber, E. M., Xin, B. T., van Rooden, E. J., Al-Ayed, K., Kim, K. B., Kisselev, A. F., Driessen, C., van der Stelt, M., van der Marel, G. A., Groll, M. and Overkleeft, H. S. (2014). Structure-based design of β1i or β5i specific inhibitors of human immunoproteasomes. J Med Chem 57(14): 6197-6209.
  3. Florea, B. I., Verdoes, M., Li, N., van der Linden, W. A., Geurink, P. P., van den Elst, H., Hofmann, T., de Ru, A., van Veelen, P. A., Tanaka, K., Sasaki, K., Murata, S., den Dulk, H., Brouwer, J., Ossendorp, F. A., Kisselev, A. F. and Overkleeft, H. S. (2010). Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t. Chem Biol 17(8): 795–801.
  4. Kammerl, I. E. and Meiners, S. (2016). Proteasome function shapes innate and adaptive immune responses. Am J Physiol Lung Cell Mol Physiol 311(2): L328-336.
  5. Li, N., Kuo, C. L., Paniagua, G., van den Elst, H., Verdoes, M., Willems, L. I., van der Linden, W. A., Ruben, M., van Genderen, E., Gubbens, J., van Wezel, G. P., Overkleeft, H. S. and Florea, B. I. (2013). Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS. Nat Protoc 8(6): 1155-1168.
  6. Meiners, S., Keller, I. E., Semren, N. and Caniard, A. (2014). Regulation of the proteasome: evaluating the lung proteasome as a new therapeutic target. Antioxid Redox Signal 21(17): 2364-2382.
  7. Muchamuel, T., Basler, M., Aujay, M. A., Suzuki, E., Kalim, K. W., Lauer, C., Sylvain, C., Ring, E. R., Shields, J., Jiang, J., Shwonek, P., Parlati, F., Demo, S. D., Bennett, M. K., Kirk, C. J. and Groettrup, M. (2009). A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat Med 15(7): 781-787. 

简介

基于活性的探针(ABP)是不可逆地结合特定酶家族的活性中心并且可以偶联至荧光团或亲和标签的小有机分子(Li等人,2013)。在这里,我们描述了使用生物素化的蛋白酶体特异性ABP生物素 - 环氧丙素(Bio-EP)在细胞裂解物中下拉活性催化标准和免疫蛋白酶体亚基的方法。用Bio-EP共价标记活性催化亚单位,然后使用链霉抗生物素蛋白包被的珠子进行下拉。从珠中洗脱后,可以通过Western印迹,串联质谱法(Li et al。,2013)或其他技术检测富集的亚单位。

背景 蛋白酶体是存在于真核细胞的细胞核和细胞质中的桶形多分子酶复合物。蛋白质降解是重要的,包括加工MHC I呈递的抗原肽,并调节许多细胞过程(Kammerl和Meiners,2016)。在造血起源的细胞中,标准(组成型)蛋白酶体通常被免疫蛋白酶体(Meiners等人,2014)所替代,其在三种不同的催化活性β-亚基的掺入中不同(图1)。
为了研究单个催化亚基的分子功能并调节生理过程,亚基特异性蛋白酶体抑制剂的发展是必不可少的。 de Bruin等人(2014)最近在这方面取得了巨大的进步。特异性免疫蛋白酶体抑制剂被证明是用于治疗炎症和自身免疫疾病的潜在药物候选物。免疫蛋白酶体亚单位β5i的抑制可以改变活化的单核细胞和淋巴细胞的细胞因子产生。在类风湿性关节炎的小鼠模型中,这种逆转疾病的迹象(Muchamuel等人,2009)。
该方案是在一项研究的背景下制定的,该研究调查了选择性抑制β5i对肺泡巨噬细胞极化的影响(Chen et al。,2016)。使用泛反应性ABP Bio-EP的催化标准和免疫蛋白酶体亚基的基于活性的下拉使我们能够通过ONYX Pharmaceuticals先前开发的抑制剂ONX-0914来证实β5i的特异性抑制。
使用具有替代特异性的ABP可以通过类似的实验方法来实现其他酶家族的基于活性的下拉。


图1. 20S标准和免疫蛋白酶体的结构。 蛋白酶体的20S核心颗粒由两个堆叠的含有β-亚基1-7的内环组成,其中β1,β2和β5具有蛋白水解活性。 β环位于两个包含α亚基1-7的α环的两侧。在免疫蛋白酶体β1中,β2和β5分别被β1i,β2i和β5i代替,导致蛋白水解活性的改变。

关键字:基于酶活性的探针, 基于活性的pull-down, 生物素, 链霉亲和素, 蛋白酶体活性, 免疫蛋白酶体, 巨噬细胞

材料和试剂

  1. 15厘米细胞培养皿
  2. 蛋白质LoBind ®管(1.7ml管; OMNILAB-LABORZENTRUM,目录号:5409327)
  3. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  4. 移液器提示
  5. 精细剂量注射器Omnifix-F(1 ml)(OMNILAB-LABORZENTRUM,目录号:5421736)
  6. 15 ml Falcon管(Corning,Falcon ®,目录号:352096) 
  7. 免疫印迹 PVDF膜(Bio-Rad Laboratories,目录号:162-0177) 
  8. PD MidiTrap G-25(存储在RT)(GE Healthcare,目录号:28-9180-08)
  9. 过滤0.2μm(Sartorius,目录号16534-K)
  10. 富士X射线胶片RX,18 x 24厘米(Kisker Biotech,型号:RX1824)
  11. Whatman印迹纸(Laborbedarf Lammel,目录号:3030690)
  12. 鼠肺泡巨噬细胞细胞系MH-S(ATCC,目录号:CRL-2019)
  13. 液氮
  14. BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23225)
  15. 生物素 - 环氧丙素(Bio-EP;溶于DMSO,-20℃下稳定至少12个月; Hermen Overkleeft Lab,Florea等人,2010)描述的合成)
  16. 二甲基亚砜(DMSO)(Carl Roth,目录号:A994.2)
  17. HEPES(分子生物学级)(AppliChem,目录号:A3724,1000) 
  18. 超纯水(例如,MilliQ)
  19. 链霉抗生物素蛋白珠(Strep-TactinSuperflow50%悬浮液;储存于4℃)(IBA,目录号:2-1206-010)
  20. 甲醇(AppliChem,目录号:A3493.1000)
  21. Roti ® - 封锁解决方案(在RT存储)(Carl Roth,目录号:A151.3) 
  22. 抗β5抗体(-20℃储存)(Abcam,目录号:90867)
  23. 抗β5i抗体(-20℃储存)(Abcam,目录号:3329)
  24. 抗兔IgG,HRP连接抗体(-20℃储存)(New England Biolabs,目录号:7074S)
  25. Amersham ECL主要蛋白质印迹检测试剂(存储在4°C)(GE Healthcare,目录号:RPN2232)
  26. RPMI-1640细胞培养基(Thermo Fisher Scientific,Gibco TM,目录号11875093)
  27. 胎牛血清(FBS)(Biochrom,目录号S 0615)
  28. β-巯基乙醇(分子生物学级)(AppliChem,目录号A1108-100)
  29. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号15140122)
  30. Tris(缓冲液级)(AppliChem,目录号:A1379,1000)
  31. 氯化镁六水合物(MgCl 2·6H 2 O)(AppliChem,目录号:A1036,0500)
  32. 氯化钠(NaCl)(AppliChem,目录号:A2942,1000)
  33. 乙二胺四乙酸(EDTA)(AppliChem,目录号:A2937,1000)
  34. 叠氮化钠(NaN 3 3)(AppliChem,目录号:A1430,0100)
  35. 二硫苏糖醇(DTT)(分子生物学级)(AppliChem,目录号:A2948,0025)
  36. 三磷酸腺苷(ATP),二钠盐(10g)(Roche Diagnostics,目录号:10127531001)
  37. 甘油(87%)(分子生物学级)(AppliChem,目录号:A3739,1000)
  38. cOmplete TM蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11697498001)
  39. PhosSTOP TM(磷酸酶抑制剂)(Roche Diagnostics,目录号:4906845001)
  40. 十二烷基硫酸钠(SDS)(纯)(AppliChem,目录号:A1502,1000)
  41. 溴酚蓝(AppliChem,目录号:A2331,0025)
  42. 盐酸(发烟)(37%)(Sigma-Aldrich,目录号:258148-2.5L)
  43. Rotiphorese ®凝胶30(37.5:1)(Carl Roth,目录号:3029.2)
  44. 过硫酸铵(APS)(AppliChem,目录号:A0834,0250)
  45. 四甲基乙二胺(TEMED)(AppliChem,目录号:A1148,0100)
  46. 吐温20(Moleculare biology grade)(AppliChem,目录号:A4974,1000)
  47. Dulbecco的磷酸缓冲盐水(1x DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190-144)
  48. MH-S细胞的完整生长培养基(见食谱)
  49. TSDG缓冲区(见配方)
  50. DMSO中的500μMBio-EP(参见食谱)
  51. 50 mM HEPES(pH 7.4)(见配方)
  52. 10%SDS(参见食谱)
  53. 6x Laemmli缓冲液(见配方)
  54. 4x SDS-PAGE分辨缓冲液(pH 8.8)(参见食谱)
  55. 4x SDS-PAGE堆叠缓冲液(pH 6.8)(见配方)
  56. 堆叠凝胶(参见食谱)
  57. 分解凝胶(15%丙烯酰胺)(见配方)
  58. PBST(0.1%吐温)(见配方)

设备

  1. Falcon管式离心机(Hettich Instruments,型号:Rotina 420R)
  2. 台式离心机(Hettich Instruments,型号:MIKRO 200R)
  3. 水浴(LAUDA,型号:Aqualine AL12 LCB 0725)
  4. Thermomixer(Eppendorf,型号:Thermomixer comfort)
  5. 镊子
  6. 移液器
  7. 剪刀
  8. Intelli-mixer旋转器(ELMI,型号:RM 2M)
  9. Western印迹室(Bio-Rad Laboratories,型号:Mini PROTEAN Tetra Cell)
  10. 电源(PowerPac TM 基本电源)(Bio-Rad Laboratories,型号:1645050)
  11. 蛋白质印迹开发者(Agfa-Gevaert,型号:Curix60)
  12. 通风柜

程序

  1. 细胞材料的制备
    1. 培养完整生长培养基中的MH-S细胞 注意:该方案在从ATCC购买的鼠肺泡巨噬细胞细胞系MH-S(No.CRL-2019)中测试。以1:6至1:12的比例(分裂频率约3-5天)以70-90%细胞密度传代细胞。在处理前一天将12×10 6个细胞接种在15cm细胞培养皿中,但如果使用其它细胞系,则数量可能变化。关于细胞培养和治疗的进一步信息在Chen et al。 (2016)。
    2. 在室温下将收获的细胞和沉淀物在800×g下培养5分钟,除去上清液
    3. 用PBS洗涤细胞,并在室温下以800 x g沉淀5分钟
    4. 在400μlTSDG缓冲液中轻轻悬挂细胞。
      注意:TSDG缓冲液中的溶解有助于通过稳定蛋白酶体复合物来维持标准和免疫蛋白酶体活性。
    5. Lyse细胞用液氮中的七个重复循环冷冻试管,并在室温下在水浴中解冻,直到晶体消失。
      注意:使用浮子有助于并行处理多根管。
    6. 以14,000 x g离心20分钟,以除去细胞碎片,并将上清液转移到新鲜管中。
    7. 确定蛋白质浓度(例如用BCA蛋白测定法,Bio-Rad)
    8. 将样品储存在-80°C。

  2. 标准和免疫蛋白酶的ABP标记
    1. 用生物素 - 环氧丙素(Bio-EP)治疗
      注意:Bio-EP是生物素化的ABP,其不可逆地结合标准和免疫蛋白酶体的活性催化亚基。
      1. 在冰上解冻细胞裂解物并将相当于500μg蛋白质的体积转移到1.7ml管中。
        注意:对于每个细胞裂解液,准备两个管。一个管用作媒介物对照,并与DMSO代替Bio-EP孵育。
      2. 用50mM HEPES(pH 7.4)填充高达500μl。最终的蛋白质浓度为1毫克/毫升
      3. 添加终浓度为5μM的Bio-EP(例如5μl500μM储备液),向对照管中加入相同量的DMSO。
      4. 孵育管在37℃下在Thermomixer上以600转/分进行120分钟。
        注意:同时,平衡排列色谱柱(步骤B2a-B2e)并准备下拉珠(步骤C1a-C1d)
      5. 用50mM HEPES(pH 7.4)加满1ml。最终的蛋白质浓度为0.5mg/ml 注意:SEC的体积为1毫升符合制造商的建议。
    2. 大小排阻色谱(SEC)
      注意:执行SEC从样品中去除未绑定的ABP。自由ABP保留在柱树脂中,而大蛋白质如标准品和免疫蛋白酶体通过。对每个样品使用新鲜的PD MidiTrap G-25色谱柱。在车辆控制样品上执行SEC。
      1. 从列中取出顶盖,倒出储存溶液。
      2. 使用镊子去除树脂顶部的过滤器。
      3. 取下底盖,并使用制造商交付的蓝色适配器将色谱柱插入50ml收集管
      4. 用5毫升超纯水平衡柱2×,并丢弃流经。
      5. 用超纯水将色谱柱第三次填充,然后在离心机中以1000 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 注意:不要让色谱柱长时间干燥。在离心前盖上带有底盖的湿柱,以暂停平衡,直到所有样品准备应用。一旦所有样品准备就绪,取下盖子并离心剩余的液体以便使用方案恢复。
      6. 丢弃流经并将适配器的柱转移到标有样品名称的新鲜的50ml Falcon管上
      7. 在填充床的中间缓缓移液,并以1,000×g离心2分钟。
      8. 丢弃色谱柱并将含有洗脱液的50ml Falcon管储存在冰上。

  3. ABP标记亚基的下拉
    1. 链霉亲和素珠的制备
      注意:可以在ABP孵化期间完成(步骤B1d)。
      1. 将40μl50%链霉亲和素珠浆(=20μl珠)转移至1.7ml管中 注意:转移前的涡流浆。使用200微升移液器,用剪刀切割移液器末端,否则珠子会堵塞吸头。
      2. 用500微升PBS清洗珠3×。在2,000 x g的洗涤步骤之间离心2分钟,弃去上清液 注意:使用窄针头注射器,小心吸取上清液而不除去珠粒。针尖的直径必须小于珠径。
      3. 将小珠重新悬浮在1ml 50mM HEPES(pH 7.4)中,并将其转移到15ml Falcon管中。
        注意:将珠粒重新悬浮在500μlHEPES缓冲液中,并转移到15 ml Falcon管中。重复此操作以转移残余珠粒,从而防止珠粒损失。使用1000μl移液器,用剪刀切割移液器末端,否则珠子会堵塞吸头。
      4. 将珠存放在冰上,直至需要。
    2. 标准溶蛋白和免疫蛋白酶
      注意:溶解是破坏复杂结构和允许分离ABP标记的非标记亚基所必需的。
      1. 每个洗脱液(从SEC)将200μl(等于最大100μg蛋白质)转移到新鲜的1.7 ml管中。将其余洗脱液储存在-20°C。
      2. 加入20μl10%SDS,并在Thermomixer中在95℃下煮沸8分钟。冷静到RT。
      3. 离心管在室温下以10,000 x g进行1分钟,从管的盖子收集蒸发的液体。
    3. 下拉
      注意:Bio-EP标记的亚基在本步骤中使用链霉亲和素珠。
      1. 将样品转移到带有预洗珠(来自C1)的15ml Falcon管中,并用50mM HEPES(pH 7.4)填充至10ml。
        注意:SDS可能会干扰下拉。因此,SDS在此步骤中稀释至0.02%。
      2. 在4℃孵育2小时。在孵育期间旋转管子(应用20 rpm)。
      3. 离心管以2,000 x g离心2分钟(4℃)。丢弃上清液。
      4. 将小珠重新悬浮在2×500μlPBS中并将浆液转移到1.7ml管中
      5. 离心管以2,000 x g离心2分钟(4℃)。丢弃上清液。
      6. 用500μlPBS洗涤珠子两次。以2,000 x g离心2分钟(4°C)。
      7. 重新悬浮在60μl1x Laemmli缓冲液中,并在90℃下在Thermomixer中煮沸15分钟。
        注意:结合珠子的Bio-EP标记亚基在该步骤中被洗脱。
      8. 离心管以2,000 x g离心2分钟(4℃)。使用注射器将上清液转移到新鲜的1.7ml管中
      9. 将样品储存在-20°C。

  4. 蛋白质印迹检测
    注意:可以执行替代的检测模式或进一步的处理步骤。该方案体现了免疫球蛋白β5i和标准蛋白酶体亚单位β5的检测。
    1. 在15%聚丙烯酰胺凝胶中分离下拉洗脱液的蛋白质。用25μl洗脱液加载凝胶袋并在120V下运行约90分钟
    2. 将蛋白质在250mA下将蛋白质印迹在聚偏二氟乙烯膜上90分钟 注意:在进行电泳前,将膜浸入MeOH中。
    3. 在1×Roti ®阻塞溶液中,在室温下封闭膜60分钟。
    4. 孵育膜与:
      一个。 1:2,000抗-β5i(Roti -Block)过夜/周末在4℃;
      b。 1:1,000抗β5(Roti -Block)过夜/周末4℃。
    5. 在塑料盒中用PBST洗涤膜三次,持续10分钟。
    6. 用1:40,000抗兔HRP偶联二抗(新英格兰Biolabs,PBST稀释)孵育60分钟。
    7. 在室温下用PBST将膜3×洗涤10分钟。
    8. 用新鲜制备的ECL底物覆盖膜。
    9. 将膜放在塑料箔中,并在暗室中用X射线胶片覆盖。几分钟后开发电影。


      图2.基于工作流程活动的下拉。活性催化标准(棕色)和免疫蛋白酶体(黄色)亚单位用ABP Bio-EP共价标记。通过SEC去除未结合的ABP后,将蛋白酶体的复合结构溶解在1%SDS中。进行用链霉抗生物素蛋白包被的琼脂糖珠的下拉以捕获ABP标记的亚单位。在PBS洗涤的三个连续步骤中除去未标记的蛋白质,结合的亚基通过1x Laemmli中的加热洗脱。

数据分析

图3.使用Bio-EP的基于活性的下拉蛋白的Western印迹。 Western印迹显示ONX-0914处理的MH-S细胞裂解物中活性β5i和β5的量,并代表三次独立实验。随着β5i特异性抑制剂浓度的增加,下拉洗脱液中检测到的β5i的量减少,而β5仍然不受ONX-0914处理的影响。

食谱

  1. MH-S细胞的完整生长培养基
    500 ml RPMI-1640
    10%胎牛血清(FBS)
    50μMβ-巯基乙醇
    100 U/ml青霉素 - 链霉素
  2. TSDG缓冲液(10ml)
    1ml 100mM Tris储备液(pH7.5)
    1.1ml 10mM MgCl 2 库存
    100μl1 M NaCl储备液 20μl50 mM EDTA储备液 100μl100mM NaN 3 库存
    10μl1 M DTT库存
    100μl200 mM ATP储备液 1.15毫升87%甘油
    用超纯水加满10毫升 最终浓度:10mM Tris/HCl,1.1mM MgCl 2,10mM NaCl,0.1mM EDTA,1mM NaN 3,1mM DTT,2mM ATP,10 %(v/v)甘油
    调节pH至7.0
    等分并储存于-20°C
    在使用前立即加入蛋白酶和磷酸酶抑制剂(Roche)(1 ml用于50ml裂解缓冲液;或者,25x原料可以在超纯水中制备,根据需要新鲜加入,储存于-20°C)
  3. 500μMBio-EP在DMSO中
    1mg Bio-EP(MW = 724.95)
    2.759ml DMSO
    等分试样(建议30-50μl),并储存于-20°C
  4. 50mM HEPES(pH 7.4; 1L)
    11.915 g HEPES
    用超纯水加满1升 根据需要用HCl或NaOH调节pH至7.4
  5. 10%SDS(10ml)
    1克SDS
    用超纯水加满10毫升 在通风橱下准备,佩戴适当的呼吸保护
  6. 6x Laemmli缓冲液(10ml)
    3ml 1M Tris HCl(pH6.8)
    1.5毫升甘油
    0.6克SDS
    0.5 g DTT
    1毫克溴酚蓝
    用超纯水加满10毫升 过滤器(0.2μm)
    等分并储存于-20°C
  7. 4x SDS-PAGE拆分缓冲液(pH8.8)
    36.4克Tris
    用超纯水加满150毫升 用37%HCl调节pH至8.8
    用超纯水加满200毫升 过滤器(0.2μm)
    0.48克SDS
    存储:RT
  8. 4x SDS-PAGE堆叠缓冲液(pH 6.8)
    6.05克Tris
    用超纯水加满40毫升 用37%的HCl将pH调节至6.8
    用超纯水加满100毫升 过滤器(0.2μm)
    0.4克SDS
    存储:RT
  9. 堆叠凝胶(4 ml)
    1 ml 4x堆叠缓冲液
    480μl30%丙烯酰胺
    2.52ml超纯H 2 O
    50μl10%APS
    12μlTEMED
  10. 溶解凝胶(15%丙烯酰胺)(8 ml)
    2 ml 4x分辨率缓冲液
    4ml 30%丙烯酰胺
    2ml超纯H 2 O
    100μl10%APS
    12μlTEMED
  11. PBST(0.1%吐温)(1升)
    999 ml 1x PBS
    1ml吐温20

致谢

该方案用于以前在细胞死亡和分化中发表的工作(Chen等人,2016)。这项工作得到了亥姆霍兹中兴慕尼黑的校内资金的支持。作者宣称没有利益冲突。

参考文献

  1. Chen,S.,Kammerl,IE,Vosyka,O.,Baumann,T.,Yu,Y.,Wu,Y.,Irmler,M.,Overkleeft,HS,Beckers,J.,Eickelberg,O.,Meiners, S.和Stoeger,T。(2016)。 免疫蛋白酶体功能障碍增强肺泡巨噬细胞的替代极化。 ;细胞死亡差异 23(6):1026-1037。
  2. de Bruin,G.,Huber,EM,Xin,BT,van Rooden,EJ,Al-Ayed,K.,Kim,KB,Kisselev,AF,Driessen,C.,van der Stelt,M.,van der Marel, GA,Groll,M.和Overkleeft,HS(2014)。 人类免疫蛋白酶体β1i或β5i特异性抑制剂的基于结构的设计。   J Med Chem 57(14):6197-6209。
  3. Florea,BI,Verdoes,M.,Li,N.,van der Linden,WA,Geurink,PP,van den Elst,H.,Hofmann,T.,de Ru,A.,van Veelen,PA,Tanaka,K ,Sasaki,K.,Murata,S.,den Dulk,H.,Brouwer,J.,Ossendorp,FA,Kisselev,AF和Overkleeft,HS(2010)。 基于活动的分析显示鼠类胸腺蛋白酶体特异性亚基β5t的反应性。  Chem Biol 17(8):795-801。
  4. Kammerl,I.E.和Meiners,S。(2016)。 蛋白酶体功能形成天生和适应性免疫反应。 ; Am J Physiol Lung Cell Mol Physiol 311(2):L328-336。
  5. Li,N.,Kuo,CL,Paniagua,G.,van den Elst,H.,Verdoes,M.,Willems,LI,van der Linden,WA,Ruben,M.,van Genderen,E.,Gubbens,J ,van Wezel,GP,Overkleeft,HS和Florea,BI(2013)。 通过基于活性的蛋白质分析和LC- MS/MS。  Nat Protoc 8(6):1155-1168。
  6. Meiners,S.,Keller,I.E.,Semren,N。和Caniard,A.(2014)。 调节蛋白酶体:评估肺部蛋白酶体作为新的治疗靶点。  Antioxid氧化还原信号 21(17):2364-2382。
  7. Muchamuel,T.,Basler,M.,Aujay,MA,Suzuki,E.,Kalim,KW,Lauer,C.,Sylvain,C.,Ring,ER,Shields,J.,Jiang,J.,Shwonek,P ,Parlati,F.,Demo,SD,Bennett,MK,Kirk,CJ和Groettrup,M。(2009)。 免疫蛋白酶体亚单位LMP7的选择性抑制剂阻断细胞因子产生并减弱进展的实验性关节炎。  Nat Med 15(7):781-787。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Baumann, T., Vosyka, O., Florea, B. I., Overkleeft, H. S., Meiners, S. and Kammerl, I. E. (2016). Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits. Bio-protocol 6(24): e2073. DOI: 10.21769/BioProtoc.2073.
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