搜索

1 user has reported that he/she has successfully carried out the experiment using this protocol.
In vitro AMPylation Assays Using Purified, Recombinant Proteins
使用纯化的重组蛋白进行体外AMP化实验   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Proceedings of the National Academy of Sciences of the United States of America
Jan 2017

Abstract

Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.

Keywords: AMPylation (AMP化), Adenylylation (腺苷酰化), Grp78/BiP (Grp78/BiP), HSP70 (HSP70), GTPase (GTP酶), Proteostasis (蛋白内稳态)

Background

Metazoan cell signaling is complex and requires tight control. Aberrations in this well-balanced system threaten cellular homeostasis and induce several maintenance systems aimed at restoring the balance (Kim et al., 2013). Protein AMPylation is directly linked to cellular stress: AMPylation of Rho GTPases by bacterial toxins rewires GTPase-dependent signaling, eventually leading to a collapse of the actin cytoskeleton and cell death (Yarbrough et al., 2009; Mattoo et al., 2011). In contrast, AMPylation of Grp78/BiP in the ER keeps this chaperone in a primed, yet silent conformation to be awoken and set in motion once the burden of unfolded protein in the ER surpasses a certain threshold (Preissler et al., 2015; Sanyal et al., 2015). We and others have extensively used in vitro AMPylation assays to study general properties, target selectivity as well as reaction kinetics of Fic domain-containing AMPylases. We used a combination of distinct in vitro AMPylation assays to i) identify novel targets in complex cell lysates, ii) validate suspected targets and iii) approach the role of AMPylase dimerization and auto-modification as prerequisites for enzymatic activity (Truttmann et al., 2015; 2016 and 2017). Our efforts aim at understanding the scope and impact of the AMPylome on cellular signaling. The in vitro AMPylation assays described herein present methods to achieve this goal.

Materials and Reagents

  1. 1.5 ml tubes (1.5 ml Snaplock Microcentrifuge Tube) (Corning, Axygen®, catalog number: MCT-150-C-S )
  2. Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 9400327 , 9401255 , 9401410 )
  3. Microcentrifuge Tube Locks (Sorenson BioScience, catalog number: 11870 )
  4. Autoradiography film (Carestream Health X-OmatTM LS Film) (Eastman Kodak, catalog number: 05-728-45 )
  5. Saran wrap (generic)
  6. Whatman 3MM filter paper (GE Healthcare, catalog number: 3030-6185 )
  7. Sterile, deionized water (generic)
  8. Ice in isolated containment (generic)
  9. Ethanol (Sigma-Aldrich, catalog number: 362808 )
  10. Purified recombinant AMPylase at 1.0 μg/μl or higher (HIS6-HYPEaa187-437; homemade; see Truttmann et al., 2015)
  11. Purified recombinant target proteins at 1.0 μg/μl or higher (i.e., HIS6-Histone H3; homemade; see Truttmann et al., 2015)
  12. Appropriate TRIS-glycine gels (CriterionTM TGXTM Precast Midi Protein Gel) (Bio-Rad Laboratories, catalog number: 5671023 )
  13. Molecular weight marker (Precision PlusTM Protein Dual Color Standard) (Bio-Rad Laboratories, catalog number: 1610374 )
  14. 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris-base) (Sigma-Aldrich, catalog number: 252859 )
  15. 11.8 M hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  16. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  17. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  18. [Alpha-33P]ATP, 10 mCi/ml; 3,000 Ci/mmol (Hartman Analytic, catalog number: SRF-207 )
    Note: It is of uttermost importance to use [Alpha-33P]ATP and not [Gamma-33P]ATP, which is used to study kinase-dependent phosphorylation events.
  19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  20. Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405 )
  21. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  22. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255 )
  23. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  24. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  25. Poly-Phenyl-Oxazole (PPO) (Sigma-Aldrich, catalog number: 216984 )
  26. Dimethyl sulfoxide (DMSO) Sigma-Aldrich, catalog number: 276855 )
  27. 1 M Tris-HCl (pH 7.5) (see Recipes)
  28. 1 M DTT (see Recipes)
  29. 1 M MgCl2 (see Recipes)
  30. 100 mM ATP (see Recipes)
  31. 5 M NaCl (see Recipes)
  32. Protein storage buffer (see Recipes)
  33. Reaction buffer (see Recipes)
  34. SDS-PAGE 6x sample buffer (see Recipes)
  35. DMSO/PPO solution (see Recipes)

Equipment

  1. Pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 4600170 , 4600240 and 4600250 )
  2. -20 °C freezer (generic)
  3. 4 °C refrigerator (generic)
  4. Geiger-counter (generic)
  5. Refrigerated tabletop centrifuge for 1.5 ml Eppendorf tubes (Eppendorf, model: 5810 R )
  6. 10 μl Hamilton syringe (Hamilton, catalog number: 80075 )
  7. Radiation safety gear and personal protection equipment (generic)
  8. Vacuum gel dryer (Bio-Rad Laboratories, model: Model 583 )
  9. Glass tray (generic; 5 x 10 inches at least)
  10. Timer (Alarm Timer) (Grainger, catalog number: 8RLR2 )
  11. pH meter (Thermo Fisher Scientific, Thermo ScientificTM, model: Orion StarTM A111 )
  12. Balance (Sartorius, model: Cubis® Precision Balance )
  13. Vortex (Vortex-Genie 2 Vortexer) (VWR, catalog number: VWR-VG3 )
  14. SDS-PAGE system (Bio-Rad Laboratories, model: CriterionTM Cell and PowerPacTM Basic Power Supply, catalog number: 1656019 )
  15. Autoradiography cassettes (FisherBiotech Electrophoresis Systems Autoradiography Cassette, 8 x 10 in) (Fisher Scientific, model: FBAC 810)
    Note: This product is not available anymore.

Software

  1. Fiji/ImageJ image analysis software (https://fiji.sc/)

Procedure

Note: 33P-ATP is radioactive. Please counsel with your radio safety officer regarding your institute’s radio waste disposal and radio protection routine and ensure to obtained all required protective equipment–that should include but may not be limited to a lab coat, double-gloves and safety goggles–before starting the experiment. Continuously monitor work surfaces using a Geiger-counter and decontaminate if required following your institute’s decontamination protocol.

  1. In vitro AMPylation reaction
    1. Mix 5 μg of recombinant AMPylase enzyme in 10 μl protein storage buffer (see Recipes) with 20 μl reaction buffer (see Recipes) supplemented with 0.5 μl/sample 33P-ATP.
      Notes:
      1. The amount of AMPylase to use in an in vitro reaction depends on the relative activity of the individual enzymes; very potent enzymes (i.e., VopS, IbpA) will in vitro modify a molar excess of target protein (i.e., Rac1, Cdc42); thus, 1 μg/reaction is sufficient. In contrast, wild-type versions of metazoan AMPylases in general perform poorly in vitro; thus, 5-10 μg/reaction is preferred.
      2. Wear proper protective equipment and monitor work surfaces during this step with a Geiger-counter.
    2. Incubate reaction for 60 min at 20 °C.
      Notes:
      1. This step pre-loads or primes the enzyme, which will maximize target AMPylation.
      2. Throughout the protocol, suggested 20 °C incubation steps can also be performed at room temperature (approximately 20 °C).
    3. Centrifuge sample for 30 sec at 7,000 x g.
    4. Add 2 μg of target protein in 10 μl protein storage buffer to the primed enzyme (final reaction volume: 40 μl).
    5. Incubate reaction for 60 min at appropriate temperature (VopS, IbpA: 20 °C; FIC-1: 30 °C; HYPE: 37 °C).
      Note: Reactions can be performed at 20 °C; however, incubation at higher temperatures (37 °C) enhances target AMPylation.
    6. Quench the reaction by adding 8 μl of 6x SDS-PAGE loading buffer (see Recipes).
    7. Properly close 1.5 ml tubes and put on tube locks to prevent lids from opening inadvertently during step A9.
    8. Boil samples for 10 min at 95 °C.
      Note: Tube locks are essential, as they will minimize the chance for tubes to pop open while boiling (see next step); should accidental opening of a tube have occurred, identify likely [33P]-contamination using a Geiger-counter and decontaminate according to your radiation safety office’s guidelines.
    9. Proceed with gel electrophoresis or store samples at -80 °C.

  2. Sample analysis by gel electrophoresis and autoradiography
    1. Equilibrate samples on ice for 10 min.
    2. In the meantime, choose appropriate gel: if available, a TRIS-glycine gradient gel (i.e., 4-16%) should be used; alternatively, select the polyacrylamide percentage according to your target protein’s mass: i.e., 12% for HSP70 (approximately 70 kDa), 15% for Histones (approximately 15 kDa) (acrylamide: Bis-acrylamide = 30:0.8); prepare gel running buffer according to gel manufacturer’s instructions.
    3. Centrifuge samples for 30 sec at 7,000 x g.
    4. Load gel: add molecular weight marker and individual samples to each well of the SDS-PAGE gel.
      Note: The use of gel loading tips is recommended to minimize cross-well spilling. Alternatively, use a 10 μl Hamilton syringe for loading. Load only ~50% of your total sample; store remaining half in case you need to re-run the gel.
    5. Run the gel at 60-100 V during the stacking stage, then adjust to120 V (constant voltage) for ~2 h.
      Note: The running time depends on the gel system used.

      Note: Steps B6-B17 are referred to as fluorography, a method developed by Bonner and Laskey (Bonner and Laskey, 1974) to be able to detect 3H in autoradiograms of SDS-PAGE gels. The method also improves sensitivity for other soft emitters, less so for higher energy isotopes such as 33P. An added advantage is that the procedure confers mechanical stability to low-percentage gels, owing to the precipitation of PPO. Gels with acrylamide percentages lower than 5% should not be subjected to fluorography, as the mechanical properties of these gels can yield an irregular final product. Commercially available solutions for impregnation of gels with fluorophores are available, often at higher cost than home-made DMSO-PPO solutions. Wear gloves at all times, since the DMSO-PPO solution can penetrate the skin and cause local precipitation of PPO in tissues.

    6. Carefully transfer the gel into a metal or glass tray (a baking dish will serve the purpose).
    7. Add 500 ml of DMSO for a large gel of 100 ml volume, smaller gels can be handled with less.
      Notes:
      1. Add ~5 times the volume of the gel of DMSO to cover the gel completely.
      2. Incubate at room temperature for 60 min with agitation.
    8. Discard DMSO
      Note: This DMSO must be considered as potentially radioactive; dispose accordingly.
    9. Add a fresh volume (see step B7) of DMSO and incubate for 60 min at room temperature.
    10. Discard DMSO.
      Note: This DMSO must be considered as potentially radioactive; dispose accordingly.
    11. Cover the gel in DMSO-PPO solution (22.2 g of PPO and 80 ml of DMSO; scale volume according to need) (see Recipes).
    12. Incubate for 2 h at room temperature with agitation.
    13. Remove DMSO-PPO as completely as possible.
      Note: The DMSO-PPO solution can be recycled and reused for up to 3 gels/experiments. Replenish PPO in the DMSO-PPO solution after each cycle with an amount of PPO approximately equal to that now precipitated in the gel.
    14. Cover the gel in the glass tray with 2 cm of ddH2O and incubate for 60 min at room temperature (Figure 1A and Video 1).
      Notes:
      1. Adding ddH2O to the dehydrated, PPO-soaked gel will result in immediate precipitation of PPO and will rapidly turn the transparent gel into a milky-white gel.
      2. Use a paper towel to remove any precipitated PPO from the tray.


        Figure 1. PPO treatment and final autoradiography plot. A. Typical appearance of SDS-PAGE gel after successful dehydration and PPO loading (see Video 1 for more details); B. Representative autoradiography plot depicting auto-AMPylation of the conferring enzyme (FIC-1(E274G), a constitutive-active FIC-1 version), as well as target modification (HSP40).

        Video 1. PPO precipitation in dehydrated SDS-PAGE gel. Dehydrated and PPO-loaded SDS-PAGE gel is kept in a glass tray and distilled water is added. Upon contact with water, PPO precipitates within the gel. Excess PPO still present in the glass tray precipitates, too. To achieve best results, incubate SDS-PAGE gel for 60 min at room temperature (step B14) before proceeding with washing (step B15).

    15. Discard ddH2O.
    16. Add fresh ddH2O.
    17. Incubate gel in ddH2O for 10 min.
    18. Discard ddH2O.
    19. Repeat previous step 5 times.
    20. Pre-soak filter paper (Whatman 3MM) with ddH2O.
    21. Transfer gel face-up onto soaked filter paper.
    22. Transfer filter-gel stack to gel drying unit.
    23. Cover the filter-gel stack with Saran wrap.
      Note: Adding a single layer of Saran wrap or similar prevents the gel from sticking the gel dryer’s rubber cover and facilitates the removal of the gel once dried onto the filter.
    24. Dry gel for 90 min at 65 °C. It is essential that a strong vacuum be maintained to avoid cracking of the gel. If necessary, use a lyophilizer-style set-up with a cold trap.
    25. Transfer gel into autoradiography cassette.
      Note: At this stage, it is sometimes worthwhile to use a Geiger-counter to get a feeling for the signal intensity attained; if audible with a sensitive Geiger-counter, an overnight exposure of the film will usually yield an interpretable signal.
    26. In a dark room, expose to film.
    27. Expose autoradiogram for 10 min to 10 days, depending on the expected signal intensity.
      Note: This is a rather arbitrary factor and often must be evaluated empirically; 33P has a half-life time of ~30 days.
    28. If the film is exposed for longer than 12 h, store the autoradiography cassette at -80 °C to enhance sensitivity of detection.
    29. Develop film according to manufacturer’s instructions (Figure 1B).

Data analysis

  1. In vitro AMPylation assays as presented in this protocol will primarily result in a qualitative assessment of target AMPylation. It is strongly recommended to repeat experiments and to verify target AMPylation in three independent replica.
  2. If required, protein AMPylation can be quantified as follows:
    1. Scan develop autoradiography film.
    2. Open scan image in Fiji/ImageJ image analysis software (https://fiji.sc/).
    3. Use the integrated analyze ==> gels plugin and redeem the intensities of individual bands depicting AMPylated proteins.
    4. Determine average and standard deviation combining data from at least 3 independent replica.
    5. Use appropriate statistical methods to evaluate significance of observed AMPylation levels (e.g., Student’s t-test).
      Notes:
      1. The appropriate statistical test will depend on the study design
      2. Users are strongly encouraged to consult available online tutorials prior to using this tool for the first time (https://www.youtube.com/watch?v=JlR5v-DsTds).

Notes

  1. If performed under identical experimental conditions, in vitro AMPylation assays are highly reproducible and should provide a similar qualitative assessment of target modification.
  2. [33P] has a half-life time of 25.3 days; thus, signal intensity of particles emitted from AMPylated targets will decrease with daily increments. If quantification of signal intensities is desired, it might be necessary to use relative (intensity as compared to an internal control) rather than absolute quantification strategies.

Recipes

  1. 1 M Tris-HCl (pH 7.5)
    For 100 ml:
    12.1 g Tris-base
    90 ml ddH2O
    Adjust to pH 7.5 with HCl
    Adjust volume with ddH2O to 100 ml
    Store at room temperature
  2. 1 M DTT
    For 100 ml:
    15 g DTT in 100 ml ddH2O
    Store in aliquots at -20 °C
  3. 1 M MgCl2
    For 100 ml:
    47.6 g MgCl2
    Adjust volume with ddH2O to 100 ml
    Store in aliquots at -20 °C
  4. 100 mM ATP
    For 100 ml:
    5.51 g ATP
    Adjust volume with ddH2O to 100 ml
    Store in aliquots at -20 °C
  5. 5 M NaCl
    For 100 ml:
    29.22g NaCl in 100 ml ddH2O
  6. Protein storage buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 10% glycerol [v/v])
    For 10 ml:
    500 μl 1 M Tris-HCl pH 7.5
    300 μl 5 M NaCl
    1 ml glycerol
    Adjust volume with ddH2O to 10 ml
  7. Reaction buffer (50 mM TRIS-HCl pH 7.5, 10 mM MgCl2, 150 mM NaCl, 2 mM DTT)
    For 10 ml:
    500 μl 1 M Tris-HCl pH 7.5
    100 μl 1 M MgCl2
    300 μl 5 M NaCl
    20 μl 1 M DTT
    9.08 ml ddH2O
  8. SDS-PAGE 6x sample buffer (375 mM Tris-HCl pH 6.8, 6% SDS [w/v], 48% glycerol [v/v], 9% 2-mercaptoethanol [v/v], 0.03% bromophenol blue [w/v])
    For 100 ml:
    5.91 g Tris-HCl
    6 g SDS
    48 ml 100% glycerol
    9 ml 14.7 M 2-mercaptoethanol
    30 mg bromophenol blue
  9. DMSO/PPO solution
    30 g PPO in 120 ml DMSO

Acknowledgments

The authors like to thank the members of the Ploegh lab for helpful discussions and input. This work was supported by awards from the National Institute of Health to H.L.P. M.C.T. is supported by a Young Investigator Award from Emerald Foundation, Inc. This protocol was modified from previous work as described in Truttmann et al., 2016.

References

  1. Bonner, W. M. and Laskey, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46(1): 83-88.
  2. Engel, P., Goepfert, A., Stanger, F. V., Harms, A., Schmidt, A., Schirmer, T. and Dehio, C. (2012). Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. Nature 482(7383): 107-110.
  3. Ham, H., Woolery, A. R., Tracy, C., Stenesen, D., Kramer, H. and Orth, K. (2014). Unfolded protein response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP chaperone during endoplasmic reticulum homeostasis. J Biol Chem 289(52): 36059-36069.
  4. Hedberg, C. and Itzen, A. (2015). Molecular perspectives on protein adenylylation. ACS Chem Biol 10(1): 12-21.
  5. Itzen, A., Blankenfeldt, W. and Goody, R. S. (2011). Adenylylation: renaissance of a forgotten post-translational modification. Trends Biochem Sci 36(4): 221-228.
  6. Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M. and Hartl, F. U. (2013). Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82: 323-355.
  7. Kinch, L. N., Yarbrough, M. L., Orth, K. and Grishin, N. V. (2009). Fido, a novel AMPylation domain common to fic, doc, and AvrB. PLoS One 4(6): e5818.
  8. Mattoo, S., Durrant, E., Chen, M. J., Xiao, J., Lazar, C. S., Manning, G., Dixon, J. E. and Worby, C. A. (2011). Comparative analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other fic domain-containing enzymes reveals differences in substrate and nucleotide specificities. J Biol Chem 286(37): 32834-32842.
  9. Preissler, S., Rato, C., Chen, R., Antrobus, R., Ding, S., Fearnley, I. M. and Ron, D. (2015). AMPylation matches BiP activity to client protein load in the endoplasmic reticulum. Elife 4: e12621.
  10. Sanyal, A., Chen, A. J., Nakayasu, E. S., Lazar, C. S., Zbornik, E. A., Worby, C. A., Koller, A. and Mattoo, S. (2015). A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response. J Biol Chem 290(13): 8482-8499.
  11. Truttmann, M. C., Cruz, V. E., Guo, X., Engert, C., Schwartz, T. U. and Ploegh, H. L. (2016). The Caenorhabditis elegans protein FIC-1 is an AMPylase that covalently modifies heat-shock 70 family proteins, translation elongation factors and histones. PLoS Genet 12(5): e1006023.
  12. Truttmann, M. C. and Ploegh, H. L. (2017). rAMPing up stress signaling: Protein AMPylation in metazoans. Trends Cell Biol.
  13. Truttmann, M. C., Wu, Q., Stiegeler, S., Duarte, J. N., Ingram, J. and Ploegh, H. L. (2015). HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation. J Biol Chem 290(14): 9087-9100.
  14. Truttmann, M. C., Zheng, X., Hanke, L., Damon, J. R., Grootveld, M., Krakowiak, J., Pincus, D. and Ploegh, H. L. (2017). Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response. Proc Natl Acad Sci U S A 114(2): E152-E160.
  15. Woolery, A. R., Yu, X., LaBaer, J. and Orth, K. (2014). AMPylation of Rho GTPases subverts multiple host signaling processes. J Biol Chem 289(47): 32977-32988.
  16. Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L. and Orth, K. (2009). AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323(5911): 269-272.

简介

翻译后蛋白质修饰(PTM)协调各种蛋白质的活性并确保其功能正常。虽然诸如磷酸化或糖基化的修饰被很好地理解,但是更不寻常的修饰,包括亚硝基化或AMP化仍然比较差的表征。关于蛋白质AMP化的研究 - 其是将AMP部分共价加成到丝氨酸,苏氨酸或酪氨酸的侧链,已经经历了复兴(Yarbrough et al。,2009; Engel et al。 2012年; Ham等人,2014年; Woolery等人,2014年; Preissler等人,2015年; ; Sanyal等人,2015; Truttmann等人,2016; Truttmann等人,2017)。鉴定和表征含丝状(fic)结构域的AMPylases引起了对该PTM的新兴趣(Kinch等人,2009; Yarbrough等人,2009)。基于最近的体内和体外研究,我们现在知道分泌的细菌AMPylase共价连接AMP到Rho家族GTP酶的成员,而后生动物AMPylases修饰HSP70家族蛋白在细胞质和内质网(ER)(Itzen等人,2011; Hedberg和Itzen,2015; Truttmann和Ploegh,2017)。认为AMP化学将HSP70置于不能参与蛋白质重折叠反应的引发剂但瞬时失活的状态(Preissler等人,2015)。 AMP体外实验是评估由真核和真核酶催化的蛋白质AMPINK的活性,动力学和特异性的关键。这些简单的测定需要重组AMPylases,靶蛋白(Rho GTPases,HSP70s)以及ATP作为核苷酸来源。在这里,我们描述了在体外定性和定量研究蛋白质AMPTIV的策略。
【背景】Metazoan细胞信号传导是复杂的,需要严格控制。这种平衡良好的系统中的畸变威胁到细胞稳态,并引发几种恢复平衡的维护系统(Kim et al。,2013)。蛋白质AMPylation直接与细胞应激相关:细菌毒素对Rho GTP酶的AMP化反应导致GTPase依赖性信号传导,最终导致肌动蛋白细胞骨架和细胞死亡的崩溃(Yarbrough et al。,2009; Mattoo 等人,2011)。相比之下,ER中的Grp78 / BiP的AMPR化使得这个伴侣被引导而沉默的构象被唤醒,并且一旦ER中的未折叠蛋白质的负担超过某个阈值就会起作用(Preissler等人,,2015; Sanyal等人,2015)。我们和其他人已经在体外广泛使用AMP化学测定来研究Fic结构域的AMPylases的一般性质,靶选择性以及反应动力学。我们使用不同的体外药代动力学组合,i)鉴定复杂细胞裂解物中的新靶标,ii)验证疑似靶标,和iii)接近AMPases二聚化和自身修饰作为酶的先决条件的作用活动(Truttmann等人,2015; 2016和2017)。我们的努力旨在了解AMPylome对细胞信号传导的范围和影响。本文所述的AMP化学测定提供了实现该目标的方法。

关键字:AMP化, 腺苷酰化, Grp78/BiP, HSP70, GTP酶, 蛋白内稳态

材料和试剂

  1. 1.5ml管(1.5ml Snaplock Microcentrifuge Tube)(Corning,Axygen ,目录号:MCT-150-C-S)
  2. 移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:9400327,9401255,9401410)
  3. 微量离心管锁(Sorenson BioScience,目录号:11870)
  4. 放射自显影胶片(Carestream Health X-Omat TM LS Film)(Eastman Kodak,目录号:05-728-45)
  5. Saran包(通用)
  6. Whatman 3MM滤纸(GE Healthcare,目录号:3030-6185)
  7. 无菌,去离子水(通用)
  8. 冰隔离的遏制(通用)
  9. 乙醇(Sigma-Aldrich,目录号:362808)
  10. 1.0μg/μl或更高的纯化重组AMPC1(HIS6-HYPE aa187-437;自制的;参见Truttmann等人,2015)
  11. 纯化的重组靶蛋白为1.0μg/μl或更高(即,HIS Histone H3;自制的;参见Truttmann等人,2015 )
  12. 适当的TRIS-甘氨酸凝胶(标准品 TGX TM / sup> Precast Midi Protein Gel)(Bio-Rad Laboratories,目录号:5671023)
  13. 分子量标记(Precision Plus TM Protein Dual Color Standard)(Bio-Rad Laboratories,目录号:1610374)
  14. 2-氨基-2-(羟甲基)-1,3-丙二醇(Tris-碱)(Sigma-Aldrich,目录号:252859)
  15. 11.8M盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  16. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  17. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  18. [α-P] ATP,10mCi / ml; 3,000 Ci / mmol(Hartman Analytic,目录号:SRF-207)
    注意:使用[Alpha - 33 P] ATP是非常重要的,而不是[Gamma - p> ATP,用于研究激酶依赖性磷酸化事件。
  19. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  20. 氯化钾(KCl)(EMD Millipore,目录号:PX1405)
  21. 甘油(Sigma-Aldrich,目录号:G5516)
  22. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:74255)
  23. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  24. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  25. 聚苯基恶唑(PPO)(Sigma-Aldrich,目录号:216984)
  26. 二甲基亚砜(DMSO)Sigma-Aldrich,目录号:276855)
  27. 1M Tris-HCl(pH7.5)(参见食谱)
  28. 1 M DTT(见配方)
  29. 1 M MgCl 2 (见配方)
  30. 100 mM ATP(参见食谱)
  31. 5 M NaCl(见食谱)
  32. 蛋白质储存缓冲液(参见食谱)
  33. 反应缓冲液(见配方)
  34. SDS-PAGE 6x样品缓冲液(参见食谱)
  35. DMSO / PPO溶液(参见食谱)

设备

  1. 移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4600170,46000040和4600250)
  2. -20°C冰箱(通用)
  3. 4°C冰箱(通用)
  4. 盖革柜台(通用)
  5. 用于1.5ml Eppendorf管的冷藏台式离心机(Eppendorf,型号:5810 R)
  6. 10μlHamilton注射器(Hamilton,目录号:80075)
  7. 辐射安全装备和个人防护装备(通用)
  8. 真空干燥机(Bio-Rad Laboratories,型号:583型)
  9. 玻璃托盘(通用型;至少5 x 10英寸)
  10. 定时器(报警定时器)(Grainger,目录号:8RLR2)
  11. pH计(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Orion Star TM A111)
  12. 平衡(Sartorius,型号:Cubis ®精密天平)
  13. 涡旋(Vortex-Genie 2 Vortexer)(VWR,目录号:VWR-VG3)
  14. SDS-PAGE系统(Bio-Rad Laboratories,型号:Criterion TM Cell and PowerPac TM 基本电源,目录号:1656019)
  15. 放射自显影盒(FisherBiotech Electrophoresis Systems Autoradiography Cassette,8 x 10 in)(Fisher Scientific,型号:FBAC 810)
    注意:此产品不可用。

软件

  1. 斐济/ ImageJ图像分析软件( https://fiji.sc/

程序

注意: 33 P-ATP是放射性的。请向您的无线电安全主任咨询您所在研究所的无线电废弃物处理和无线电保护措施,并确保获得所有必需的防护设备,包括但不限于实验室外套,双手套和安全护目镜 - 开始实验之前。使用盖革计数器连续监测工作表面,如果需要,按照研究所的去污方案进行净化。

  1. AMP体外反应
    1. 在10μl蛋白质储存缓冲液(参见食谱)中混合5μg重组AMPCy酶,并加入补充有0.5μl/μL样品的P-ATP的20μl反应缓冲液(参见食谱)。
      注意:
      1. 在体外反应中使用的AMPC1的量取决于各个酶的相对活性;非常有效的酶(即VopS,IbpA)将体外修饰摩尔过量的靶蛋白(即Rac1,Cdc42);因此,1μg/反应就足够了。相比之下,野生型版本的后生动物AMPylases通常在体外表现不佳;因此优选5-10μg/反应。
      2. 使用盖革计数器,在此步骤中佩戴适当的保护设备并监控工作表面。
    2. 在20℃下孵育反应60分钟 注意:
      1. 这个步骤预加载或引发酶,这将最大限度地提高目标AMPTIV。
      2. 在整个方案中,建议的20℃孵育步骤也可以在室温(约20℃)下进行。
    3. 以7,000 x g离心样品30秒。
    4. 在10μl蛋白质储存缓冲液中加入2μg目标蛋白至底漆酶(最终反应体积:40μl)
    5. 在适当温度下孵育反应60分钟(VopS,IbpA:20℃; FIC-1:30℃; HYPE:37℃)。
      注意:反应可以在20°C进行;然而,在较高温度(37℃)下孵育可增强目标AMPylation
    6. 通过加入8μl6x SDS-PAGE加载缓冲液来淬灭反应(参见食谱)。
    7. 妥善关闭1.5毫升的管子并戴上管子锁,以防止盖子在步骤A9期间无意中打开。
    8. 在95℃下煮沸10分钟。
      注意:管锁是必不可少的,因为它们会最大限度地减少煮沸时管子爆裂的机会(见下一步);应该意外打开管子,使用盖革计数器确定可能的[ P] - 污染物,并根据您的辐射安全办公室的指导。
    9. 进行凝胶电泳或将样品储存在-80°C。

  2. 通过凝胶电泳和放射自显影进行样品分析
    1. 在冰上平衡样品10分钟
    2. 同时,选择合适的凝胶:如果可用,应使用TRIS-甘氨酸梯度凝胶(即,4-16%);或者,根据您的目标蛋白质质量选择聚丙烯酰胺百分比:即HSP70(约70kDa)为12%,组蛋白为15%(约15kDa)(丙烯酰胺:双丙烯酰胺= 30: 0.8);根据凝胶制造商的说明制备凝胶运行缓冲液。
    3. 以7,000 x g离心样品30秒。
    4. 加载凝胶:向SDS-PAGE凝胶的每个孔中加入分子量标记物和单个样品 注意:建议使用凝胶加载尖端,以尽量减少跨井溢出。或者,使用10μlHamilton注射器进行装载。仅加载总样本的50%;存储剩余的一半,以防您需要重新运行凝胶。
    5. 在堆叠阶段运行60-100 V凝胶,然后调节至120 V(恒定电压)〜2 h。
      注意:运行时间取决于使用的凝胶系统。

      注意:步骤B6-B17被称为荧光成像,Bonner和Laskey开发的方法(Bonner和Laskey,1974),以便能够检测 3 / sup> H在SDS-PAGE凝胶的放射自显影图中。该方法还提高了其他软发射体的灵敏度,对于较高能量的同位素,如

      另外的优点是,由于PPO的沉淀,该方法赋予低百分比凝胶的机械稳定性。丙烯酰胺百分比低于5%的凝胶不应进行氟摄影,因为这些凝胶的机械性能可能会产生不规则的最终产品。可用的用荧光团浸渍凝胶的商业解决方案通常比自制的DMSO-PPO溶液成本更高。 始终戴上手套,因为DMSO-PPO溶液可以穿透皮肤并导致PPO在组织中的局部沉淀 EM>


    6. 仔细将凝胶转移到金属或玻璃托盘(烤盘将用于此目的)。
    7. 加入500毫升的DMSO,用于100毫升体积的大凝胶,较小的凝胶可以用较少的处理 注意:
      1. 加入〜5倍于DMSO凝胶体积,完全覆盖凝胶。

      2. 在室温下孵育60分钟
    8. 丢弃DMSO
      注意:该DMSO必须被视为潜在的放射性;相应地处理。
    9. 加入DMSO的新鲜体积(参见步骤B7),并在室温下孵育60分钟
    10. 丢弃DMSO。
      注意:该DMSO必须被视为潜在的放射性;相应地处理。
    11. 在DMSO-PPO溶液(22.2g PPO和80ml DMSO;根据需要的体积)中覆盖凝胶(参见食谱)。
    12. 在室温下搅拌孵育2小时。
    13. 尽可能完全去除DMSO-PPO 注意:DMSO-PPO溶液可以循环使用并重复使用3次凝胶/实验。在每个循环后补充DMSO-PPO溶液中的PPO,其量约为现在在凝胶中沉淀的PPO量。
    14. 用玻璃托盘盖住2cm的ddH2O,并在室温下孵育60分钟(图1A和视频1)。
      注意:
      1. 向脱水的PPO浸泡的凝胶中加入ddH 2将导致PPO立即沉淀,并迅速转动透明凝胶 变成乳白色凝胶。
      2. 使用纸巾从托盘中移除任何沉淀的PPO。


        图1. PPO处理和最终放射自显影图。A.成功脱水和PPO加载后,SDS-PAGE凝胶的典型外观(详见视频1); 描述赋形酶的自动放大图(FIC-1(E274G),组成型活性FIC-1版本)以及目标修饰(HSP40)的代表性放射自显影图。

        Video 1. PPO precipitation in dehydrated SDS-PAGE gel. Dehydrated and PPO-loaded SDS-PAGE gel is kept in a glass tray and distilled water is added. Upon contact with water, PPO precipitates within the gel. Excess PPO still present in the glass tray precipitates, too. To achieve best results, incubate SDS-PAGE gel for 60 min at room temperature (step B14) before proceeding with washing (step B15).

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


    15. 丢弃ddH 2 O。
    16. 添加新的ddH 2 O。
    17. 在ddH 2 O中孵育凝胶10分钟。
    18. 丢弃ddH 2 O。
    19. 重复上一步5次。
    20. 具有ddH <2> O的预浸滤纸(Whatman 3MM)。
    21. 将凝胶面朝上转移到浸泡过的滤纸上。
    22. 将过滤器 - 凝胶堆叠转移到凝胶干燥单元。
    23. 用Saran包裹覆盖过滤器 - 凝胶堆叠。
      注意:添加单层Saran包装或类似物品可防止凝胶粘贴凝胶干燥器的橡胶盖,并有助于将干燥后的凝胶移除到过滤器上。
    24. 干凝胶在65℃下90分钟。必须保持强的真空度以避免凝胶的破裂。如有必要,请使用带有冷阱的冷冻干燥器式设置。
    25. 将凝胶转移到放射自显影盒中。
      注意:在这个阶段,有时候值得使用盖革计数器来获得信号强度的感觉;如果用敏感的盖革计数器听到,电影的过夜曝光通常会产生可解释的信号。
    26. 在黑暗的房间里,暴露在电影里。
    27. 根据预期的信号强度,将放射自显影曝光10分钟至10天 注意:这是一个相当任意的因素,往往必须经验性地评估; 33 P的半衰期约为30天。
    28. 如果胶片暴露时间超过12小时,将放射自显影盒存放在-80°C,以提高检测灵敏度。
    29. 根据制造商的说明制作影片(图1B)。

数据分析

  1. 本方案中提出的AMPylation测定将主要导致靶向AMP化学的定性评估。强烈建议在三个独立复制品中重复实验并验证目标AMPylation。
  2. 如果需要,可以如下量化蛋白质AMPylation:
    1. 扫描发展放射自显影影片。
    2. 在Fiji / ImageJ图像分析软件中打开扫描图像( https://fiji.sc/ )。
    3. 使用集成分析==>凝胶插件,并兑现描绘AMPR化蛋白质的个别条纹的强度
    4. 确定来自至少3个独立副本的数据的平均值和标准差。
    5. 使用适当的统计学方法来评估观察到的AMP化水平的显着性(例如,,Student's t检验)。
      注意:

      1. 适当的统计测试将取决于研究设计
      2. https://www.youtube.com/watch?v=JlR5v-DsTds br />

笔记

  1. 如果在相同的实验条件下进行,则在体外进行AMP化学测定是高度可重复的,并应提供类似的靶标修饰的定性评估。
  2. [ 33 P]的半衰期为25.3天;因此,从AMPylated靶标发射的颗粒的信号强度将随着每日增量而降低。如果需要量化信号强度,可能需要使用相对(与内部控制相比的强度)而不是绝对量化策略。

食谱

  1. 1M Tris-HCl(pH7.5)
    100 ml:
    12.1克Tris-base
    90毫升ddH 2 O O 用HCl调节至pH 7.5 调整体积与ddH <2> O至100 ml
    在室温下存放
  2. 1 M DTT
    100 ml:
    在100毫升ddH 2 O中的15克DTT 储存于-20°C等分试样
  3. 1 M MgCl 2
    100 ml:
    47.6g MgCl 2
    调整体积与ddH <2> O至100 ml
    储存于-20°C等分试样
  4. 100 mM ATP
    100 ml:
    5.51克ATP
    调整体积与ddH <2> O至100 ml
    储存于-20°C等分试样
  5. 5 M NaCl
    100 ml:
    29.22g NaCl的100ml ddH 2 O / / O
  6. 蛋白质储存缓冲液(50mM Tris-HCl pH7.5,150mM NaCl,10%甘油[v / v])
    10 ml:
    500μl1 M Tris-HCl pH 7.5
    300μl5 M NaCl
    1毫升甘油
    调整体积与ddH <2> O至10 ml
  7. 反应缓冲液(50mM TRIS-HCl pH7.5,10mM MgCl 2,150mM NaCl,2mM DTT)
    10 ml:
    500μl1 M Tris-HCl pH 7.5
    100μl1M MgCl 2
    300μl5 M NaCl
    20μl1 M DTT
    9.08ml ddH 2 O
  8. SDS-PAGE 6x样品缓冲液(375mM Tris-HCl pH 6.8,6%SDS [w / v],48%甘油[v / v],9%2-巯基乙醇[v / v],0.03%溴酚蓝[w / v])
    100 ml:
    5.91克Tris-HCl
    6克SDS
    48 ml 100%甘油 9 ml 14.7 M 2-巯基乙醇
    30毫克溴酚蓝
  9. DMSO / PPO溶液
    30 g PPO在120 ml DMSO中

致谢

作者感谢Ploegh实验室的成员进行有益的讨论和投入。这项工作得到了国家卫生研究院颁发给H.L.P. M.C.T.得到了翡翠基金会公司的青年研究者奖。该协议是从以前的工作中修改过的,如Truttmann等人,2016年所述。

参考

  1. Bonner,WM和Laskey,RA(1974)。&nbsp; A聚丙烯酰胺凝胶中氚标记的蛋白质和核酸的膜检测方法。 Eur J Biochem 46(1):83-88。
  2. Engel,P.,Goepfert,A.,Stanger,FV,Harms,A.,Schmidt,A.,Schirmer,T。和Dehio,C。(2012)。 Fic蛋白中分子内或分子间活性位点阻塞的腺相关控制。 em> 482(7383):107-110。
  3. Ham,H.,Woolery,AR,Tracy,C.,Stenesen,D.,Kramer,H.and Orth,K。(2014)。未折叠的蛋白质反应调节果蝇Fic(dFic)蛋白可逆地在内质网稳态期间AMPylates BiP伴侣。生物化学 289(52):36059-36069。
  4. Hedberg,C.和Itzen,A.(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/25486069”target =“_ blank”关于蛋白质腺苷酰化的分子观点。 10(1):12-21。
  5. Itzen,A.,Blankenfeldt,W.和Goody,RS(2011)。 Adenylylation:遗忘的翻译后修饰的复兴。 Trends Biochem Sci 36(4):221-228。
  6. Kim,YE,Hipp,MS,Bracher,A.,Hayer-Hartl,M.and Hartl,FU(2013)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm .nih.gov / pubmed / 23746257“target =”_ blank“>蛋白质折叠和蛋白质沉淀中的分子伴侣功能。 Annu Rev Biochem 82:323-355。
  7. Kinch,LN,Yarbrough,ML,Orth,K.and Grishin,NV(2009)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/ 19503829“target =”_ blank“> Fido,fic,doc和AvrB共同的新型AMPylation域。 PLoS One 4(6):e5818。
  8. Mattoo,S.,Durrant,E.,Chen,MJ,Xiao,J.,Lazar,CS,Manning,G.,Dixon,JE和Worby,CA(2011)。&lt; a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/21795713”target =“_ blank”>具有其他fic的免疫球蛋白结合蛋白A(IbpA)的Histophilus somni 的比较分析含有结构域的酶显示底物和核苷酸特异性的差异。生物化学 286(37):32834-32842。
  9. Preissler,S.,Rato,C.,Chen,R.,Antrobus,R.,Ding,S.,Fearnley,IM and Ron,D。(2015)。&nbsp; AMPylation将BiP活性与内质网中的客户蛋白质负载相匹配。 4:e12621。
  10. Sanyal,A.,Chen,AJ,Nakayasu,ES,Lazar,CS,Zbornik,EA,Worby,CA,Koller,A。和Mattoo,S。(2015)。 Fic(由cAMP引起的丝状丝氨酸)介导的腺相关/ AMP化和展开的蛋白质反应之间的一个新的联系。 a> J Biol Chem 290(13):8482-8499。
  11. Truttmann,MC,Cruz,VE,Guo,X.,Engert,C.,Schwartz,TU and Ploegh,HL(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 27138431“target =”_ blank“>秀丽隐杆线虫蛋白FIC-1是共价修饰热休克70家族蛋白,翻译延伸因子和组蛋白的AMPCyase。 PLoS Genet 12(5):e1006023。
  12. Truttmann,MC和Ploegh,HL(2017)。&nbsp; ramping上调应激信号:后生动物中的蛋白质AMPylation。趋势细胞生物
  13. Truttmann,MC,Wu,Q.,Stiegeler,S.,Duarte,JN,Ingram,J.and Ploegh,HL(2015)。&lt; a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/25678711“target =”_ blank“> HypE特异性纳米抗体作为调节HypE介导的靶AMP磷酸化的工具。生物化学 290(14) :9087-9100。
  14. Truttmann,MC,Zheng,X.,Hanke,L.,Damon,JR,Grootveld,M.,Krakowiak,J.,Pincus,D.and Ploegh,HL(2017)。&lt; a class =“ke-insertfile “href =”http://www.ncbi.nlm.nih.gov/pubmed/28031489“target =”_ blank“>无限制的AMPylation靶向胞质分子伴侣并激活热休克反应。 Proc Natl Acad Sci USA 114(2):E152-E160。
  15. Woolery,AR,Yu,X.,LaBaer,J.and Orth,K。(2014)。 Rho GTPases的AMP化颠覆多个宿主信号传导过程。 J Biol Chem 289(47):32977-32988。
  16. Yarbrough,ML,Li,Y.,Kinch,LN,Grishin,NV,Ball,HL和Orth,K。(2009)。&lt; a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 19039103“target =”_ blank“> Vibrio VopS对Rho GTP酶的AMPylation破坏了效应物结合和下游信号传导。 323(5911):269- 272.
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Truttmann, M. C. and Ploegh, H. L. (2017). In vitro AMPylation Assays Using Purified, Recombinant Proteins. Bio-protocol 7(14): e2416. DOI: 10.21769/BioProtoc.2416.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。