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Nov 2019

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Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ
嗜肺军团菌效应蛋白SidJ体外谷氨酰化活性的放射性测定   

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Abstract

The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ-carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U-14C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This procedure is useful for the identification of substrates for glutamylation, characterization of substrate and glutamylase activities due to mutations, and identification of proteins with glutamylation activity. Some studies have assayed glutamylation with the use of [3H] glutamate (Regnard et al., 1998) and the use of the GT335 antibody (Wolff et al., 1992). However, the use of [U-14C] glutamate requires a shorter radioactive exposure time with no dependence on antibody specificity.

Keywords: SidJ ( SidJ), SdeA ( SdeA), Glutamylation (谷氨酰化), 14C-Glutamate (14C-谷氨酸), Legionella (军团菌), Pseudokinase (假激酶)

Background

Legionella pneumophila is an infectious bacteria that causes Legionnaires’ disease (McDade et al., 1977), a potentially fatal form of pneumonia. During infection, Legionella utilizes an arsenal of over 300 effector proteins, many having unusual, unidentified biochemical functions that act to hijack host cellular functions (Hubber and Roy, 2010). One process co-opted by Legionella is the ubiquitination system. Studies have demonstrated that the SidE family of proteins can perform phosphoribosyl ubiquitination of substrate proteins independent of E1 and E2 enzymes (Bhogaraju et al., 2016; Qiu et al., 2016; Kotewicz et al., 2017). Some studies have implicated the importance of SidJ in the regulation of the SidE family of proteins, but the mechanism of regulation was not identified (Havey and Roy, 2015; Jeong et al., 2015; Urbanus et al., 2016). It was previously suggested that SidJ may act as a phosphoribosyl deubiquitinase (Qiu et al., 2017) using Legionella purified SidJ (Qiu and Luo, 2019); however, recent studies do not replicate these results (Bhogaraju et al., 2019; Wan et al., 2019; Shin et al., 2020). Our group (Sulpizio et al., 2019), and others (Bhogaraju et al., 2019; Black et al., 2019; Gan et al., 2019), have recently demonstrated that SidJ can polyglutamylate the SidE family member SdeA. For verification of this activity, it was important to recapitulate these findings in an in vitro reaction.

SidJ has a C-Terminal IQ helix, that can bind the eukaryotic protein calmodulin in a calcium-independent manner. Using this binding ability, the structure of SidJ in complex with calmodulin was determined by X-ray crystallography. SidJ contains a kinase-like domain with structural homology to many conserved features found in kinases. This kinase domain is positioned in an active conformation through interactions with calmodulin. Based on these features of SidJ, reaction components were identified and used for in vitro glutamylation assays. Other assays have been developed using radioactive glutamate and gel extraction of modified substrates with liquid scintillation to detect modification (Black et al., 2019). Liquid scintillation and mass spectrometry may provide more precise quantification of the amount of modified substrate and for the number of glutamates attached to a substrate side-chain, respectively. The assay described in this protocol allows for visualization of activity by autoradiogram and relative quantitation of activity. This assay can be used to identify SidJ substrates and analyze the effect of point mutations on activity. In addition, this protocol may also be used to identify other proteins or pseudokinases that can function as glutamylases.

Materials and Reagents

  1. Kim wipes (Kimberly-Clark Professional, catalog number: 34120 )
  2. Gloves (VWR, catalog number: 89038-270 )
  3. Film wrap (Spring Grove, catalog number: 405618 )
  4. Filter paper (GE Healthcare Life Sciences, Whatman GB003, catalog number: 10547922 )
  5. Laboratory tape (VWR, catalog number: 89098-062 )
  6. Pipette tips:
    10 μl XL Graduated Tips (USA Scientific, Tip One, catalog number: 1110-3700 )
    200 μl Graduated Quick Rack (Laboratory Products Sales, catalog number: 130430 )
    1,250 μl Pipette tips (Laboratory Product Sales, catalog number: L134770 )
  7. 1.7 ml Microtubes (Corning Incorporated, Axygen, catalog number: MCT-175-C)
  8. 50 ml Centrifuge tubes (VWR, catalog number: 525-0637 )
  9. Recombinant proteins: SidJ 89-853 truncation, SdeA 211-1152 truncation, and human calmodulin 2. Proteins were expressed with an N-terminal 6xHis Sumo tag in Escherichia coli Rosetta cells and purified as described previously (Sulpizio et al., 2019). Final purified proteins were stored in a buffer (20 mM Tris pH 7.5, 150 mM NaCl) without glycerol, aliquoted, flash-frozen, and stored at -80 °C
  10. Glutamic acid, L-[14C(U)] 50 μCi (Perkin Elmer, catalog number: NEC290E050UC ), stored at -20 °C, the manufacturer suggests 4 °C
  11. 2-Mercaptoethanol (Sigma, catalog number: M3148-100ML )
  12. 2-Propanol (J.T.Baker, catalog number: 9079-03 )
  13. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad, catalog number: 1610158 )
  14. Acetic acid, glacial (J.T. Baker, catalog number: 9508-06 )
  15. Adenosine 5′-triphosphate disodium salt hydrate (Sigma, catalog number: A2382-10G )
  16. Ammonium persulfate (APS) (Amresco, catalog number: 0486-100G )
  17. Brilliant Blue R-250 (Fisher, catalog number: BP101-50 )
  18. Bromophenol Blue sodium salt (Fisher, catalog number: BP114-25 )
  19. DL-Dithiothreitol (DTT) (Amresco, catalog number: M109-25g )
  20. Ethanol 200-proof (Koptec, catalog number: V1001 )
  21. Glycerol (Mallinckrodt Chemicals, catalog number: 5092-16 )
  22. Glycine (VWR, catalog number: 0167-5KG )
  23. Magnesium chloride, 6-hydrate (Mallinckrodt Chemicals, catalog number: 5958-04 )
  24. Methanol (Fisher, catalog number: A454SK-4 )
  25. N,N,N’,N’-tetramethylethylene-diamine (TEMED) (Bio-Rad, catalog number: 161-0800 )
  26. Precision Plus Protein All Blue Standards Protein Ladder (Bio-Rad, catalog number: 161-0373 )
  27. Sodium chloride (VWR, catalog number: 0241-10KG )
  28. Sodium dodecyl sulfate (SDS) (VWR Life Sciences, catalog number: 0227-1KG )
  29. Tris (VWR, catalog number: 0497-5KG )
  30. Reaction Buffer (see Recipes)
  31. 1 M MgCl2 Solution (see Recipes)
  32. 100 mM ATP Solution, pH 7.5 (see Recipes)
  33. 10x SDS-PAGE Running Buffer (see Recipes)
  34. SDS Sample Buffer (see Recipes)
  35. Coomassie Stain (see Recipes)
  36. Coomassie Destaining Solution (see Recipes)
  37. SDS-PAGE Gel (see Recipes)
    12% Resolving Gel
    4% Stacking Gel

Note: Products were stored as suggested by manufacture except where listed.

Equipment

  1. -80 °C freezer (So-Low, model: PV85-21 )
  2. Exposure cassette (GE Healthcare Life Sciences, model: 63003545 )
  3. Fixed speed centrifuge (Benchmark, model: myFugeTM mini c entrifuge, Type: C1008-C)
  4. Fluorescent image analyzer (FujiFilm Corporation, GE Healthcare Biosciences, model: Typhoon FLA 7000 )
  5. Offset Flat-Tip Forceps (Fisher, model: 16-100-116 )
  6. Gel dryer (Bio-Rad, model: 583 )
  7. Gel electrophoresis apparatus (Bio-Rad, model: Mini-PROTEAN Tetra System )
  8. Gel electrophoresis power supply (Bio-Rad, model: PowerPac Basic )
  9. Gel imager (Bio-Rad, model: Chemidoc MP Imaging System )
  10. Ice bucket
  11. Imaging plate (Fuji Film, model: FUJI BAS-IP MS 2025 )
  12. Labcoat (VWR, catalog number: 10141-306 )
  13. Lightbox (Laboratory Supplies Company Inc., model: G129A )
  14. Microwave (Sharp, model: R230KW )
  15. Pipettes (Gilson Pipetman classic P2, P20, P200, P1000, catalog numbers: F144801, F123600, F123601, F123602)
  16. Pyrex container (secondary containment)
  17. Refrigerate benchtop centrifuge (International Equipment Company, model: Micromax RF )
  18. Rocking shaker (Reliable Scientific, model: 55D 12x16 )
  19. Scissors
  20. Sheet protectors (Clear file, Archival Plus 5x7 Print, catalog number: 370100B)
  21. Vacuum pump (Bio-Rad, model: HydroTech Vacuum Pump )
  22. Vortex mixer 120V (Corning LSE, model: 6775 )
  23. Water bath incubator (PolyScience, model: WB05 )

Software

  1. Fiji Version 1.0 (Citation Schindelin et al., 2012, http://fiji.sc)
  2. Typhoon FLA 7000 Control Software Version 1.2 (General Electric Company, www.gelifesciences.com/contact/)
  3. GraphPad Prism 7 for Mac OS X (GraphPad Software, Inc., www.graphpad.com)

Procedure

  1. SidJ in vitro glutamylation reaction
    1. Review the flowchart of the experimental outline for SidJ radioactive glutamylation before beginning procedure (Figure 1).


      Figure 1. Experimental Outline for SidJ Radioactive Glutamylation. A flowchart of general experimental steps described in this procedure beginning with the SidJ radioactive glutamylation assay and concluding with data analysis.

    2. Thaw recombinantly purified SidJ 89-853, SdeA 211-1152, and calmodulin on ice. Thaw radioactive glutamic acid stock at room temperature in a radioactive workspace.
      Note: When working with radioactive materials, ensure that all regulations are followed. Use proper PPE, maximize distance from the sample, and minimize exposure time.
    3. Warm water bath to 37 °C and chill centrifuge to 4 °C (if possible).
    4. Prepare stock solutions, on ice, listed in Table 1 by diluting in reaction buffer (20 mM Tris pH 7.5, 50 mM NaCl).

      Table 1. Stock solution concentrations for SidJ in vitro glutamylation reaction


    5. Pipette the volumes of stock solution for a single 12.5 μl reaction listed in Table 2 into a chilled 1.7 ml microcentrifuge tube on ice. Move to radioactive workspace before the addition of [U-14C] glutamic acid. Pipette ATP last to initiate the reaction. Immediately vortex reaction gently, centrifuge briefly (approximately 10 s max speed), and incubate samples at 37 °C in a water bath for 30 min.
    6. Optional: Prepare a master mix for Step A5. Combine components contained in all reactions by pipetting stock solutions and 3 μl of reaction buffer per reaction. Prepare approximately 10% more reaction mix than needed for samples. The addition of reaction buffer for the master mix dilutes components to maintain protein stability. If a master mix is prepared, for each reaction, subtract the volumes of reaction components included in the mix and 3 μl of reaction buffer from the amount used in Table 2.

      Table 2. SidJ in vitro glutamylation reaction components and concentrations

      Note: For comparison of samples that may have minor differences in activities, the final protein concentration in the reaction of SidJ can be reduced to 50 nM. If studying the impact of calmodulin-binding on SidJ activity, reducing the final molar concentration of calmodulin to the concentration of SidJ may be beneficial. The reaction time can also be shortened to 15 min. Reducing SidJ concentration may make it difficult to accurately compare protein amounts and therefore relative activity using Coomassie staining.

    7. Stop the reaction with the addition of 3 μl of SDS sample buffer. Vortex to mix and centrifuge briefly (approximately 10 s at max speed).

  2. Gel electrophoresis and preparation for radiation exposure
    1. Electrophorese 2.5 μl of protein ladder and 13 μl of each reaction using a SDS-PAGE gel (4% separating gel, 12% resolving gel) at 80 V. Once samples have migrated through stacking gel, increase the voltage to 150 V and electrophorese until the dye front reaches the bottom of the gel. The running buffer will likely be contaminated with radioactive material.
    2. Quickly remove the gel from casting glass and remove the stacking gel. Transfer the gel to a plastic container with a lid in secondary containment. Microwave Coomassie stain, enough to cover gel, in a covered microwave-safe container until boiling. Pour heated Coomassie stain into the plastic container with the gel, ensuring not to inhale fumes, and quickly cover container.
    3. Stain gel by rocking using rocking shaker for a few hours, to overnight, at room temperature.
      Note: There was some difficulty visualizing the calmodulin band using Coomassie staining. Staining temperature and time may be decreased if calmodulin can be adequately stained.
    4. After staining, discard stain into a radioactive liquid waste container. Rinse with the destaining solution and then incubate in destaining solution for approximately 30 min to 1 h. Then discard the solution and repeat incubation. Repeat until protein bands are visible. If some background staining persists, allow longer water destaining in Step B5.
    5. Rehydrate gel and destain further by incubation in ddH2O while rocking for 1-2 h. Remove water and repeat if necessary. Adding a Kim wipe can assist in destaining and provide cleaner gel images.
    6. After the gel is rehydrated, transfer the gel to a sheet protector and image using a gel imager. Wiping dust and staining imperfections with a Kim wipe may help obtain clearer images (Figure 2A)
    7. Cut a sheet of filter paper in half with scissors. Gently transfer gel to two stacked layers of filter paper, by touching one side of the gel to the paper, and then smoothly allowing it to lay flat. Avoid bubbles and imperfections. Cover with a layer of plastic wrap and smooth out any imperfections. If there are lines in the plastic wrap, this may impact exposure efficiency.
    8. Dry the gel using a vacuum gel dryer at 80 °C for 1 h or until the gel is completely dried. Ensure gel is dried before removing vacuum to prevent gel cracking.
    9. While drying the gel, erase the image plate by exposing it on the lightbox for 30 min.
    10. Wrap plastic wrap around the filter paper to prevent damage to the image plate. Tape the gel and two filter paper sheets to the bottom side of the exposure cassette. Avoid using thick plastic sheet protectors as they will reduce signal.
    11. Place image plate over gel with the white side facing the gel. Record the positioning of the notched edge of the image plate relative to the gel. Expose for 3 to 4 days to obtain optimal signal intensity.

  3. Image acquisition
    1. After exposure, minimize the ambient light in the imaging room to prevent the alteration of the image signal. Attach the black magnetic section of the image plate to the phosphor stage and attach the stage to the imager.
    2. Open the software and select the phosphorimaging option. Select the following parameters for image acquisition: Laser: 650 nm, Filter: [IP], PMT: 1000, Pixel Size: 25 μM, Latitude: L5, Stage: Phosphor Stage, Mode: All.
    3. Click the “Start Scan” button and allow the instrument to scan the image plate. Once, the area containing your gel has been scanned acquisition can be stopped.
      If a manual selection was chosen, ensure that the area containing the gel was accurately selected. Partial scanning of gels will prevent accurate comparison between portions of the gel scanned separately.
    4. A gel file will then be saved in the area chosen upon scanning. This can be saved to a flash drive and transported to a personal computer (Figure 2B).


      Figure 2. Reaction components required for SidJ mediated glutamylation of SdeA. A. SidJ was incubated with the indicated reaction components with concentrations listed in Table 2. The SdeA E/A is a SdeA E860A mutant of the residue identified to be glutamylated by mass spectrometry. Reactions were conducted for 30 min at 37 °C. Proteins were electrophoresed by SDS-PAGE and stained with Coomassie stain. CaM is an abbreviation for calmodulin. B. An autoradiogram of the gel in A that was exposed for 4 days. This figure is from the original research article (Sulpizio et al., 2019).

Data analysis

  1. Open Fiji, or ImageJ, software. In the menu bar, select “File -> Open” and browse to the location of the .gel file.
    If the gel is not horizontally level, select “Image -> Transform -> Rotate”. Select the “Preview” checkbox and manually input a degree rotation and alter until gel preview is level. Then click “OK”.
  2. In the toolbar, select the rectangle selection tool. In the image window, click and drag to create a rectangular selection. This selection area should be large enough to fit the largest band. Then move this selection to cover the first band you would like to measure. In the menu bar, click “Analyze -> Measure.” A Results pop up window will appear with intensity data. Click in the center of this rectangle and drag it to surround the next band to be analyzed and measure intensity. Repeat process, recording which measurements correspond to which samples until the intensity of all bands has been measured. Measure the intensity of a background selection, ideally on the area where the gel was exposed but no samples were electrophoresed. All rectangular selections should be identical for an accurate comparison of intensities. Click on the Results window and in the menu bar select “File -> Save As…” and choose a file name and location.
  3. Measurements of relative radiographic intensities of bands can then be calculated by multiplying the area of selection by the mean, to get total intensity, for all measurements. Subtract the background measurement from all samples. Relative intensities can be compared by dividing one sample’s total intensity by another sample’s total intensity. Calculation of relative intensities should only be made for samples exposed on the same image plate, and preferably electrophoresed within the same gel. For example, the intensity of protein mutants can be divided by the wild type total radiographic intensity to ascertain the effect of these mutants on activity. Removal or alteration of reaction components can also be compared.
  4. Relative radiographic intensities can then be displayed using a bar graph. Assays and quantification should be repeated in triplicate. Error bars can be represented as a standard deviation, and P-values were calculated with a single-tailed t-test. For examples of data used for graphing and statistical calculations see “Additional Files, Source Data 1” (Sulpizio et al., 2019).


    Figure 3. Quantification of SidJ kinase-like domain mutants. A. SidJ and SidJ mutants were incubated with wild type SdeA and reaction components, with concentrations listed in Table 2, in the presence or absence of ATP for 15 min at 37 °C. The lane labeled Empty contained SDS-Sample Buffer with reaction buffer substituted for sample. Top panel: Coomassie stained gel, Bottom Panel: Autoradiogram with an example of a region used for WT signal quantification and the region for background subtraction shown as red and cyan boxes, respectively. WT and CaM are abbreviations for wild type and calmodulin, respectively. B. Quantitation of relative radioactive intensity of reactions of the bottom panel of A. Bar graphs are averages of three separate experiments with error bars depicted as standard deviation. The P-values were calculated from a t-test and ns, not significant, **, P < 0.01, ***, P < 0.001. The graph was generated using GraphPad Software. This figure is from original research article (Sulpizio et al., 2019).

Recipes

  1. Reaction Buffer
    50 mM Tris pH 7.5
    50 mM NaCl
    Stored at room temperature
  2. 1 M MgCl2 Solution
    Stored at room temperature
  3. 100 mM ATP Solution, pH 7.5
    Adjust pH to 7.5, aliquot and store at -80 °C
  4. 10x SDS-PAGE Running Buffer
    Store at room temperature and dilute 10-fold with ddH2O for use
    Component
    1 L
    Tris-Base
    30 g
    Glycine
    140 g
    SDS
    10 g
    ddH2O
    To 1 L
  5. SDS Sample Buffer
    Store at room temperature, freeze aliquots -20 °C for extended storage
    Component
    Concentration
    Bromophenol Blue
    0.25%(w/v)
    DTT
    0.5 M
    Glycerol
    50% (v/v)
    SDS
    10% (w/v)
  6. Coomassie Stain
    Store at room temperature
    Component
    500 ml
    Methanol
    225 ml
    ddH2O
    225 ml
    Glacial Acetic Acid
    50 ml
    Brilliant Blue R250
    1.25 g
  7. Coomassie Destaining Solution
    Store at room temperature
    Component
    1 L
    Ethanol
    450 ml
    ddH2O
    450 ml
    Glacial Acetic Acid
    100 ml
  8. SDS-PAGE Gel
    1. 12% Resolving Gel
      Component
      8 gels
      Final Conc.
      ddH2O
      13.3 ml

      30% Acrylamide/Bis Solution
      16 ml
      12%
      1.5 M Tris pH 8.8
      10 ml
      375 mM
      10% SDS
      400 μl
      0.1%
      10% APS
      300 μl
      0.075%
      TEMED
      24 μl
      0.06%
    2. 4% Stacking Gel
      Component
      8 gels
      Final Conc.
      ddH2O
      11.2 ml

      30% Acrylamide/Bis Solution
      2.16 ml
      4.14%
      1.0 M Tris pH 6.8
      2 ml
      128 mM
      10% SDS
      160 μl
      0.1%
      10% APS
      110 μl
      0.07%
      TEMED
      16 μl
      0.1%

Acknowledgments

This work was supported by National Institute of Health (NIH) grant 5R01GM116964 (to YM), the Cornell University Harry and Samuel Mann Outstanding Graduate Student Award (to AGS) and by the NIH under Ruth L Kirschstein National Research Service Award (6T32GM008267) from the NIGMS (to MEM).
   Protocol from original research article: Sulpizio, A., M. E. Minelli, M. Wan, P. D. Burrowes, X. Wu, E. J. Sanford, J.-H. Shin, B. C. Williams, M. L. Goldberg, M. B. Smolka and Y. Mao (2019). "Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ." eLife 8: e51162.

Competing interests

The authors declare no competing interests.

References

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  19. Wolff, A., de Nechaud, B., Chillet, D., Mazarguil, H., Desbruyeres, E., Audebert, S., Edde, B., Gros, F. and Denoulet, P. (1992). Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335. Eur J Cell Biol 59(2): 425-432.

简介

[摘要]在军团菌效应蛋白SidJ最近被确定为另一个执行polyglutamylation军团效应,SdeA,烧蚀SdeA的活动。SidJ是一种激酶样蛋白,需要小的真核蛋白钙调蛋白才能进行谷氨酰化。谷氨酰化是翻译后修饰的相对不常见的类型,其中游离谷氨酸氨基酸的氨基与底物蛋白质中谷氨酸侧链的γ-羧基共价连接。该协议描述了使用放射性[U- 14]的SidJ谷氨酰化反应 C]谷氨酸及其底物SdeA,通过凝胶电泳分离蛋白质,制备用于放射暴露的凝胶,以及相对定量的谷氨酰化活性。该方法可用于鉴定底物进行谷氨酰化,表征底物和由突变引起的谷氨酰胺酶活性,以及​​鉴定具有谷氨酰化活性的蛋白质。一些研究已经使用[ 3 H]谷氨酸(Regnard等,1998)和使用GT335抗体(Wolff等,1992)来分析谷氨酰化。但是,使用[U- 14 C] g谷氨酸盐需要更短的放射性暴露时间,而不依赖于抗体特异性。


[背景]嗜肺军团菌是感染细菌,造成军团病(麦克达德等人,1977) ,肺炎的一个潜在的致命的形式。在感染期间,Legionell a使用了300多种效应蛋白,其中许多蛋白具有不寻常的,无法识别的生化功能,可劫持宿主细胞的功能(Hubber和Roy,2010)。军团菌选择的一种方法是泛素化系统。研究表明,SidE蛋白家族可以对独立于E1和E2酶的底物蛋白进行磷酸核糖基泛素化(Bhogaraju等,2016; Qiu等,2016; Kotewicz等,2017)。一些研究暗示了SidJ在调节SidE蛋白质家族中的重要性,但尚未确定调节机制(Havey和Roy,2015; Jeong等,2015; Urbanas等,2016)。先前曾有人建议使用Legionella纯化的SidJ (Qiu和Luo,2019),将SidJ用作磷酸核糖基泛素化酶(Qiu等人,2017 ); 然而,最近的研究并未复制这些结果(Bhogaraju等人,2019; Wan等人,2019; Shin等人,2020)。我们的团队(苏睿德等人,2019)和其他(Bhogaraju等,2019;黑色。等,2019;甘等人,2019) ,最近表明,SidJ可以polyglutamylate侧家人SdeA。为了验证这种活性,重要的是在体外反应中概括这些发现。

SidJ具有C末端IQ螺旋,可以以钙非依赖性方式结合真核蛋白钙调蛋白。利用这种结合能力,通过X射线晶体学测定与钙调蛋白复合的SidJ的结构。SidJ包含一个激酶样结构域,与在激酶中发现的许多保守特征具有结构同源性。该激酶结构域通过与钙调蛋白的相互作用而处于活性构象。基于SidJ的这些特征,确定了反应成分并将其用于体外谷氨酰化测定。已经开发了使用放射性谷氨酸和通过液体闪烁的凝胶提取修饰的底物以检测修饰的其他测定法(Black等,2019)。液体闪烁和质谱可以分别提供对修饰的底物的量和附着在底物侧链上的谷氨酸盐数量的更精确定量。该协议中所述的测定方法可通过放射自显影和相对定量的活性显示活性。该测定法可用于鉴定SidJ底物并分析点突变对活性的影响。此外,该协议还可用于鉴定其他可充当谷氨酰胺酶的蛋白质或假激酶。

关键字:SidJ, SdeA, 谷氨酰化, 14C-谷氨酸, 军团菌, 假激酶

材料和试剂
1. Kim湿巾(Kimberly-Clark Professional,目录号:34120);
2.手套(VWR,目录号:89038-270);
3.薄膜包装(Spring Grove,目录号:405618);
4.滤纸(GE Healthcare Life Sciences,Whatman GB003,目录号:10574922);
5.实验胶带(VWR,目录号:89098-062);
6.移液器提示:;
10微升XL Gradua泰德提示(美国科学,提示ö NE,目录号:1110-3700)
200 μ升毕业连结架(实验室产品销售,目录号:130430)
1 ,250微升移液管头(实验室产品销售,目录号:L134770)
7. 1.7 ml微型管(Corning Incorporated,Axygen,目录号:MCT-175-C);
8. 50 ml离心管(VWR,目录号:525-0637);
9.重组蛋白:SidJ 89-853截短,SdeA 211-1152截短和人钙调蛋白2。蛋白质在N-末端6xHis Sumo标签中表达于大肠杆菌Rosetta细胞中,并如先前所述纯化(Sulpizio等人,2019年) )。将最终纯化的蛋白质存储在不含甘油的缓冲液(20 mM Tris pH 7.5,150 mM NaCl)中,分装,速冻,并保存在-80 °C;
10. Gluta麦克风酸,L- [ 14 C(U)] 50 μ次仪(Perkin Elmer,目录号:NEC290E050UC),储存在-20 ℃下,制造商建议4℃   
11. 2-巯基乙醇(Sigma,目录号:M3148-100ML)   
12. 2-丙醇(JTBaker,目录号:9079-03)   
13. 30%丙烯酰胺/双酚37.5:1(Bio - Rad,目录号:1610158)   
14.冰醋酸(JT Baker,目录号:9508-06)   
15.腺苷5 ' -triphosp讨厌二钠盐水合物(Sigma,目录号:A2382-10G)   
16.过硫酸铵(APS)(Amresco,目录号:0486-100G)   
17. Brilliant Blue R-250(Fis她,目录号:BP101-50)   
18.溴苯酚蓝钠盐(Fis她,目录号:BP114-25)   
19. DL-二硫苏糖醇(DTT)(Amresco,目录号:M109-25g)   
20.耐乙醇200(Koptec,目录号:V1001)   
21.甘油(Mallinckrodt Chemicals,目录号:5092-16)   
22.甘氨酸(VWR,目录号:0167-5KG)   
23.六水合氯化镁(Mallinckrodt Chemicals,目录号:5958-04)   
24.甲醇(Fis她,目录号:A454SK-4)   
25. N,N,N',N'-四甲基乙二胺(TEMED)(Bio - Rad,目录号:161-0800)   
26. Precision Plus蛋白质全蓝标准蛋白阶梯(Bio - Rad,目录号:161-0373)   
27.氯化钠(VWR,目录号:0241-10KG)   
28.十二烷基硫酸钠(SWR)(VWR Life Sciences,目录号:0227-1KG)   
29. Tris(VWR,目录号:0497-5KG)   
30.反应缓冲液(请参见食谱)   
31. 1 M MgCl 2溶液(请参阅配方)   
32. 100 mM ATP溶液,pH 7.5 (请参见食谱)   
33. 10x SDS-PAGE运行缓冲区(请参阅食谱)   
34. SDS样品缓冲液(请参阅配方)   
35.考马斯染色(见食谱)   
36.考马斯脱色溶液(请参阅食谱)   
37. SDS-PAGE凝胶(请参阅食谱)   
12%分解凝胶
4%堆积凝胶
注意:产品按照制造商的建议进行存储(除非列出)。
 
设备
 
-80 °C冷冻室(低温,型号:PV85-21)
曝光盒(GE Healthcare Life Sciences,型号:63003545)
定速离心机(基准,型号:myFuge TM mini c离心机,型号:C1008-C)             
荧光图像分析仪(富士胶片公司,GE Healthcare Biosciences,型号:Typhoon FLA 7000)
偏置式平头镊子(Fisher,型号:16-100-116)
凝胶干燥机(Bio - Rad,型号:583)
凝胶电泳仪(Bio - Rad,型号:Mini-PROTEAN Tetra System)
凝胶电泳电源(Bio - Rad,型号:PowerPac Basic)
凝胶成像仪(Bio - Rad,型号:Chemidoc MP Imaging System)
冰桶
成像板(富士胶片,型号:FUJI BAS-IP MS 2025)
Labcoat(VWR,目录号:10141-306)
灯箱(实验室用品公司,型号:G129A)
微波炉(锋利,型号:R230KW)
移液器(Gilson Pipetman classic P2,P20,P200,P1000,目录号:F144801,F123600,F123601,F123602)
Pyrex容器(辅助容器)
冷冻台式离心机(国际设备公司,型号:Micromax RF)
摇床(可靠科学公司,型号:55D 12x16)
剪刀
纸张保护贴(透明文件,Archival Plus 5x7打印,目录号:370100B)
真空泵(Bio - Rad,型号:HydroTech真空泵)
涡旋混合器120V(Corning LSE,型号:6775)
水浴培养箱(PolyScience,型号:WB05)
 
软件
 
斐济1.0版(Citation Schindelin等人,2012 ,http://fiji.sc)
Typhoon FLA 7000控制软件1.2版(通用电气公司,www.gelifesciences.com / contact / )
适用于Mac OS X的GraphPad Prism 7(GraphPad Software,Inc.,www.graphpad.com)
 
程序
 
SidJ体外谷氨酰化反应
审查的流程图的的实验概要SidJ放射性克lutamylation开始过程之前(图1) 。
D:\ Reformatting \ 2020-7-1 \ 2003114--1507 Alan Sulpizio 889051 \ Figs jpg \图1.jpg
图1. SidJ放射性谷氨酰化实验概述。此过程中描述的一般实验步骤的流程图,从SidJ放射性谷氨酰化测定开始,最后进行数据分析。
 
在冰上解冻重组纯化的SidJ 89-853,S deA 211-1152和钙调蛋白。在放射性工作区中于室温解冻放射性谷氨酸储备液。
注意:使用放射性物质时,请确保遵守所有规定。使用适当的PPE,与样品的距离最大,并减少暴露时间。
将水浴温热至37 ° C,然后离心分离至4 ° C(如果可能)。
通过在反应缓冲液(20 mM Tris pH 7.5,50 mM NaCl)中稀释,在冰上制备表1所列的储备溶液。
 
表1. SidJ体外谷氨酰化反应的原液浓度
零件
浓度
盛捷89-853
5 μ中号
需求211-1152
20微米
钙调蛋白
50微米
氯化镁2
62.5毫米
ATP pH 7.5
12.5毫米
 
移液管的储备溶液的单个12.5体积微升表2中列出到反应冷却1.7毫升microce在冰上ntrifuge管。在添加[U- 14 C]谷氨酸之前,先移至放射性工作区。最后用移液器ATP引发反应。立即轻轻涡旋反应,短暂离心(最大速度约10 s),并在37 ° C的水浴中孵育样品30分钟。
选项a1:为步骤A5准备预混料。通过移取储备溶液和每个反应3μl反应缓冲液,合并所有反应中包含的组分。准备比样品所需多约10%的反应混合物。为预混液添加反应缓冲液会稀释成分,以保持蛋白质稳定性。如果准备了预混合物,则对于每个反应,从表2中使用的量中减去混合物中所含反应组分的体积和3μl反应缓冲液。
 
表2. SidJ体外谷氨酰化反应的成分和浓度
组件(库存)
反应浓度
库存量(μl )
SidJ 89-853(5 μ M)
0.5 μ中号
1.25
钙调蛋白(50 μ M)
5微米
1.25
SdeA 211-1152(20μM)
2微米
1.25
氯化镁2 (62.5 mM)
5毫米
1个
谷氨酸(355 μ M)
50 μ M(1.76 NCI)
1.76
ATP pH 7.5(12.5毫米)
1毫米
1个
反应缓冲液
12.5微升
5.11
注意:为了比较活性可能有微小差异的样品,可以将SidJ反应中的最终蛋白质浓度降低至50 nM。如果研究钙调蛋白结合对SidJ活性的影响,将钙调蛋白的最终摩尔浓度降低到SidJ的浓度可能是有益的。反应时间也可以缩短到15分钟。降低SidJ浓度可能会导致难以准确比较蛋白质含量,因此难以使用考马斯亮蓝染色法比较相对活性。
 
圣运算通过加入3的反应μ SDS样品缓冲液的升。涡旋混合并短暂离心(最大速度下约10 s)。
 
凝胶电泳和辐射暴露准备
Electrophorese 2.5 μ蛋白梯升和13微升使用SDS-PAGE凝胶的每个反应在80V一旦样品已经通过堆叠凝胶迁移(4%分离胶,12%分离胶),增加电压至150 V和electrophorese直到染料前沿到达凝胶底部。运行中的缓冲液可能会被放射性物质污染。
从浇铸玻璃中快速除去凝胶,然后除去堆积的凝胶。将凝胶转移至带盖子的塑料容器中,该容器位于第二容器中。微波考马斯亮斑足以覆盖凝胶,置于有盖微波安全的容器中直至沸腾。将加热的考马斯污渍倒入装有凝胶的塑料容器中,以确保不会吸入烟气,并迅速覆盖容器。
在室温下,使用摇床摇动凝胶数小时至过夜,以对凝胶染色。
注意:使用考马斯染色在可视化钙调蛋白条带方面存在一些困难。如果钙调蛋白可以被充分染色,则可以降低染色温度和时间。
染色后,将污物丢弃到放射性废液容器中。用脱色溶液冲洗,然后在脱色溶液中孵育大约30分钟至1小时。然后丢弃溶液并重复孵育。重复直到可见蛋白带。如果仍然有一些背景污渍,请在步骤B5中进行更长的脱色处理。
重新水化凝胶并通过在ddH 2 O中孵育1-2小时同时进一步脱色。除去水,并在必要时重复。添加Kim擦拭布可以帮助脱色并提供更清洁的凝胶图像。
凝胶重新水化后,将凝胶转移到薄板保护器上,并使用凝胶成像仪成像。用Kim擦拭布擦拭灰尘和污点可帮助获得更清晰的图像(图2A)
用剪刀将滤纸切成两半。通过将凝胶的一侧与纸张轻轻接触,将其轻轻地转移至两叠滤纸层上,然后平稳地使其平放。避免气泡和瑕疵。用保鲜膜盖住并清除所有瑕疵。如果保鲜膜中有线条,这可能会影响曝光效率。
使用真空凝胶干燥器在80 ° C下干燥凝胶1小时或直到凝胶完全干燥。在去除真空之前,请确保凝胶已干燥,以防止凝胶破裂。
干燥凝胶时,将其在灯箱上暴露30分钟以擦去图像板。
用塑料纸包住滤纸,以免损坏图像板。将凝胶和两张滤纸用胶带粘在曝光盒的底部。避免使用较厚的塑料薄板保护器,因为它们会减少信号。
将图像板放在凝胶上,白色的一面朝向凝胶。记录图像板的缺口边缘相对于凝胶的位置。暴露3至4天以获得最佳信号强度。
 
图像采集
曝光后,请尽量减少成像室内的环境光,以防止图像信号改变。将成像板的黑色磁性部分连接到荧光粉台上,然后将其连接到成像仪上。
打开软件,然后选择磷光成像选项。选择用于图像采集参数如下:激光:650nm的过滤器:[IP],PMT:1000,像素尺寸:25 μ男,纬度:L5,阶段:磷阶段,模式:全部。
单击“开始扫描”按钮,并允许仪器扫描印版。一旦扫描完包含凝胶的区域,就可以停止采集。
如果选择了手动选择,请确保正确选择了包含凝胶的区域。凝胶的部分扫描将阻止在分别扫描的凝胶部分之间进行准确比较。
凝胶文件将保存在扫描时选择的区域中。可以将其保存到闪存驱动器并传输到个人计算机(图2B)。
 
D:\ Reformatting \ 2020-7-1 \ 2003114--1507 Alan Sulpizio 889051 \ Figs jpg \图2.jpg
图2. SidJ介导的SdeA谷氨酰化所需的反应组分。A. SidJ与所示的反应组分以表2所示的浓度进行孵育。SdeA E / A是质谱鉴定为被谷氨酰化的残基的SdeA E860A突变体。反应在37 ° C进行30分钟。通过SDS-PAGE对蛋白质进行电泳,并用考马斯亮蓝染色。CaM是钙调蛋白的缩写。B.将凝胶的放射自显影图曝光4天。这个数字来自原始研究文章(Sulpizio et al。,2019)。
 
数据一nalysis
 
打开斐济或ImageJ软件。在菜单栏中,选择“文件->打开”,然后浏览到.gel文件的位置。
如果凝胶不是水平的,请选择“图像->变换->旋转”。选中“ Preview”复选框,然后手动输入角度旋转并进行更改,直到达到凝胶预览水平为止。然后单击“确定”。
在工具栏中,选择矩形选择工具。在图像窗口中,单击并拖动以创建矩形选择。该选择区域应足够大以适合最大频段。然后移动此选择以覆盖您要测量的第一个波段。在菜单栏中,单击“分析->度量”。结果弹出窗口将显示强度数据。单击此矩形的中心,然后将其拖动以包围下一个要分析的波段并测量强度。重复该过程,记录哪些测量对应于哪些样本,直到测量完所有频段的强度为止。测量背景选择的强度,最好是在凝胶暴露但没有电泳的区域上进行。所有矩形选择均应相同,以便准确比较强度。单击结果窗口,然后在菜单栏中选择“文件->另存为...”,然后选择文件名和位置。
然后,可以通过将选择的面积乘以平均值以获得所有测量的总强度,来计算带的相对射线照相强度的测量值。从所有样本中减去背景测量值。可以通过将一个样品的总强度除以另一样品的总强度来比较相对强度。相对强度的计算仅应针对暴露在同一图像板上的样品进行,最好是在同一凝胶中进行电泳。例如,可以将蛋白质突变体的强度除以野生型总放射线照相强度,以确定这些突变体对活性的影响。还可以比较反应组分的去除或改变。
然后可以使用条形图显示相对射线照相强度。测定和定量应重复三次。误差棒可以表示为标准偏差,并且P值是通过单尾t检验来计算的。有关用于图形和统计计算的数据示例,请参见“其他文件,源数据1” (Sulpizio等,2019)。
 
D:\ Reformatting \ 2020-7-1 \ 2003114--1507 Alan Sulpizio 889051 \ Figs jpg \图3.jpg
图3. SidJ激酶样结构域突变体的定量。将A. SidJ和SidJ突变体与野生型SdeA和反应成分(表2中列出的浓度)在存在或不存在ATP的条件下于37 ° C孵育15分钟。标为“空”的泳道包含SDS-样品缓冲液,用反应缓冲液代替了样品。上图:考马斯染色凝胶,下图:放射自显影图,其中用于WT信号定量的区域和用于背景扣除的区域的示例分别显示为红色和青色框。WT和CaM分别是野生型和钙调蛋白的缩写。B.A.底部图的反应的相对放射性强度的定量。条形图是三个单独实验的平均值,误差条描述为标准偏差。该P -值是从一个计算吨-test和ns,不显著,**,P <0.01,***,P <0.001。该图是使用GraphPad软件生成的。这个数字来自原始研究文章(Sulpizio et al。,2019)。
菜谱
 
反应缓冲液
50 mM Tris pH 7.5
50毫米氯化钠
室温保存
1 M MgCl 2溶液
室温保存
100 mM ATP溶液,pH 7.5
将pH调节至7.5,等分并储存在-80 ° C
10 x SDS-PAGE运行缓冲区
室温保存,用ddH 2 O稀释10倍使用
零件
1升
Tris-Base
30克
甘氨酸
140克
安全数据表
10克
ddH 2 O
至1升
SDS样品缓冲液
在室温下储存,冷冻等分试样-20 ° C以延长储存时间
零件
浓度
溴酚蓝
0.25%(w / v)
DTT
50万
甘油
50%(v / v)
安全数据表
10%(w / v)
考马斯染色
室温保存
零件
500毫升
甲醇
225毫升
ddH 2 O
225毫升
冰醋酸
50毫升
艳蓝R250
1.25克
考马斯脱色溶液
室温保存
零件
1升
乙醇
450毫升
ddH 2 O
450毫升
冰醋酸
100毫升
 
SDS-PAGE凝胶
12%分解凝胶
零件
8凝胶
最终浓缩
ddH 2 O
13.3毫升
 
30%丙烯酰胺/双溶液
16毫升
12%
1.5 M Tris pH 8.8
10毫升
375毫米
10%SDS
400微升
0.1%
10%APS
300微升
0.075%
特美
24 μ升
0.06%
4%堆积凝胶
零件
8凝胶
最终浓缩
ddH 2 O
11.2毫升
 
30%丙烯酰胺/双溶液
2.16毫升
4.14%
1.0 M Tris pH 6.8
2毫升
128毫米
10%SDS
160 μ升
0.1%
10%APS
110 μ升
0.07%
特美
16 μ升
0.1%
 
致谢
 
这项工作得到了美国国立卫生研究院(NIH)授予5R01GM116964(授予YM),康奈尔大学哈里和塞缪尔·曼恩杰出研究生奖(授予AGS)以及NIH的Ruth L Kirschstein国家研究服务奖(6T32GM008267)的支持。 NIGMS(到MEM)。
  原始研究文章的协议:Sulpizio,A.,ME Minelli,M. Wan,PD Burrowes,X.Wu,EJ Sanford,J.-H. Shin,BC Williams,ML Goldberg,MB Smolka和Y.Mao(2019)。“由细菌钙调蛋白依赖性假激酶SidJ催化的蛋白质多谷氨酰化。” eLife 8 :e51162。
 
利益争夺
 
作者宣称没有利益冲突。
 
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Copyright Sulpizio et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sulpizio, A. G., Shin, J., Minelli, M. E. and Mao, Y. (2020). Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ. Bio-protocol 10(19): e3770. DOI: 10.21769/BioProtoc.3770.
  2. Sulpizio, A., Minelli, M. E., Wan, M., Burrowes, P. D., Wu, X., Sanford, E. J., Shin, J. H., Williams, B. C., Goldberg, M. L., Smolka, M. B. and Mao, Y. (2019). Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ. eLife 8: e51162.
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