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Nov 2019

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In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
嗜肺军团菌介导的泛素修饰和磷酸核糖泛素连接的体外谷氨酰化抑制   

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Abstract

Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA’s activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA’s modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA’s substrate Rab33b. Prior to the identification and publication of SdeA’s activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA’s activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.

Keywords: SidJ (SidJ蛋白), SdeA (SdeA酶), Glutamylation (谷氨酰化), PR-Ubiquitination (PR-Ubiquitination), Legionella (军团杆菌), Pseudokinase (假激酶), ADP-ribosylation (ADP-核糖基化)

Background

Legionella pneumophila is an infectious bacterium that opportunistically infects alveolar macrophages. This occurs through the inhalation of contaminated water aerosols, causing a potentially lethal form of pneumonia known as Legionnaires’disease (McDade et al., 1977). Legionella infects host cells by the secretion of over 300 effector proteins that are used to hijack many host cellular processes and prevent their lysosomal degradation (Hubber and Roy, 2010). One process hijacked by Legionella is the ubiquitination system (Hubber et al., 2013). Ubiquitination is a eukaryotic posttranslational modification, that regulates a variety of cellular processes (Hershko and Ciechanover, 1998; Chen and Sun, 2009; Hurley and Stenmark, 2011; Haglund and Dikic, 2012). This requires a concerted effort of E1, E2, and E3 enzymes to carefully regulate which proteins are ubiquitinated (Scheffner et al., 1995). However, Legionella have co-opted this modification with the SidE family of phosphoribosyl-ubiquitin ligases that act independently of E1 and E2 enzymes. This family uses two catalytic domains to ADP-ribosylate ubiquitin using the mono-ADP-ribosyl transferase (mART) domain, and then, ligates ADPR-Ub to a host protein serine residues using a phosphodiesterase (PDE) domain (Bhogaraju et al., 2016; Qiu et al., 2016; Kotewicz et al., 2017). These previously mentioned studies include means for assaying SidE activity. The SidE family includes a member, SdeA, which has been identified to be spatiotemporally regulated by SidJ (Havey and Roy, 2015; Jeong et al., 2015; Urbanus et al., 2016); although, the mechanism of this regulation was not completely understood. It was suggested that SidJ may act as a PR-deubiquitinase (Qiu et al., 2017) when using Legionella purified SidJ (Qiu and Luo, 2019); however, recent studies do not recapitulate these results (Bhogaraju et al., 2019; Wan et al., 2019; Shin et al., 2020).


Our group (Sulpizio et al., 2019), and several others (Bhogaraju et al., 2019; Black et al., 2019; Gan et al., 2019), have demonstrated that SidJ is a polyglutamylase that adds multiple glutamate amino acids to SdeA to inhibit SdeA’s function. These studies provided structural data that identified SidJ contains a kinase-like domain and binds the eukaryotic protein calmodulin. Furthermore, mass spectrometry studies have identified that the mechanism of SdeA’s inhibition is polyglutamylation of SdeA’s catalytic mART glutamate residue. Based on these results, an in vitro glutamylation assay was developed using the substrate SdeA, calmodulin, ATP/MgCl2, and glutamate. To demonstrate the inhibitory effect of glutamylation, in vitro glutamylation was followed by a SdeA activity assay. Other groups have also described SdeA inhibition using NAD hydrolysis (Bhogaraju et al., 2019), radioactive NAD (Black et al., 2019), Flag-tagged SdeA substrates (Gan et al., 2019), HA-Ub, and HA-Ub variants resistant to canonical ubiquitination by these groups. These alternative methods are suitable for identifying inhibition and may provide more quantitative detection. However, some of these experiments did not include in vitro inhibited SdeA, and those that monitor ubiquitin modification require the use of expensive reagents such as radioactive NAD. This protocol discusses a glutamylation assay developed for SidJ and assays in vitro inhibition of both SdeA PR-ubiquitin ligation and ubiquitin modification using standard gels and Coomassie staining. This can be used to identify the effects of mutations on activity, assay inhibition of both ubiquitin modification and PR-Ubiquitin ligation, and may more generally be adopted to determine inhibition of other ADP-ribosyl transferases by glutamylation.

Materials and Reagents

  1. 1.7 ml Microtubes (Corning Incorporated, Axygen, catalog number: MCT-175-C )

  2. 50 ml Centrifuge tubes (VWR, catalog number: 525-0637 )

  3. Gloves (VWR, catalog number: 89038-270 )

  4. Kim wipes (Kimberly-Clark Professional, catalog number: 34120 )

  5. 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)

  6. Recombinant proteins

    SidJ 89-853 truncation, SdeA 211-1152 truncation, human calmodulin 2, were expressed in Escherichia coli with an N-terminal 6XHis-SUMO tag and purified as described previously (Sulpizio et al., 2019). Rab33b (1-200) was also purified as proteins mentioned in (Sulpizio et al., 2019) and Ub was purified as described in Akturk et al. (2018). Final purified proteins were stored in buffer (20 mM Tris pH 7.5, 150 mM NaCl) without glycerol, aliquoted, flash-frozen, and stored at -80 °C.

  7. β-Nicotinamide adenine dinucleotide sodium salt (NAD) (Sigma, catalog number: N0632-1G )

  8. L-Glutamic Acid Monosodium Salt, Monohydrate (USB Corporation, catalog number: 16245 )

  9. 2-Mercaptoethanol (Sigma, catalog number: M3148-100ML )

  10. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad, catalog number: 1610158 )

  11. Acetic acid, glacial (J.T. Baker, catalog number: 9508-06)

  12. Adenosine 5’-triphosphate disodium salt hydrate (Sigma, catalog number: A2382-10G )

  13. Ammonium persulfate (APS) (Amresco, catalog number: 0486-100G )

  14. Brilliant Blue R-250 (Fisher, catalog number: BP101-50 )

  15. Bromophenol Blue sodium salt (Fisher, catalog number: BP114-25 )

  16. DL-Dithiothreitol (DTT) (Amresco, catalog number: M109-25g )

  17. Ethanol 200-proof (Koptec, catalog number: V1001 )

  18. Glycerol (Mallinckrodt Chemicals, catalog number: 5092-16 )

  19. Glycine (VWR, catalog number: 0167-5KG )

  20. Magnesium chloride, 6-hydrate (Mallinckrodt Chemicals, catalog number: 5958-04 )

  21. Methanol (Fisher, catalog number: A454SK-4 )

  22. N,N,N’,N’-tetramethylethylene-diamine (TEMED) (Bio-Rad, catalog number: 161-0800 )

  23. Precision Plus Protein All Blue Standards Protein Ladder (Bio-Rad, catalog number: 161-0373 )

  24. Sodium chloride (VWR, catalog number: 0241-10KG )

  25. Sodium dodecyl sulfate (SDS) (VWR Life Sciences, catalog number: 0227-1KG )

  26. Tris (VWR, catalog number: 0497-5KG )

  27. Reaction Buffer (see Recipes)

  28. MgCl2 1 M Solution (see Recipes)

  29. ATP 100 mM pH 7.5 Solution (see Recipes)

  30. Glutamic Acid 1 M Solution (see Recipes)

  31. 10x SDS-PAGE Running Buffer (see Recipes)

  32. 10x Native-PAGE Running Buffer (see Recipes)

  33. SDS Sample Buffer (see Recipes)

  34. Native Sample Buffer (see Recipes)

  35. Coomassie Stain (see Recipes)

  36. Coomassie Destaining Solution (see Recipes)

  37. 12% SDS-PAGE Resolving Gel (see Recipes)

  38. 4% SDS-PAGE Stacking Gel (see Recipes)

  39. 8% Native PAGE Resolving Gel (see Recipes)

  40. 3% Native PAGE Stacking Gel (see Recipes)

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

Equipment

  1. -80 °C freezer (So-Low, model: PV85-21 )

  2. Computer

  3. Dry Bath (Benchmark, model: BSH1001 )

  4. Fixed speed centrifuge (Benchmark, model: C1008-C )

  5. Forceps

  6. Gel electrophoresis apparatus (Bio-Rad, model: Mini-PROTEAN Tetra System )

  7. Gel electrophoresis power supply (Bio-Rad, model: PowerPac Basic )

  8. Gel imager (Bio-Rad, model: Chemidoc MP Imaging System )

  9. Ice bucket

  10. Labcoat (VWR, catalog number: 10141-306 )

  11. Laboratory tape (VWR, catalog number: 89098-062 )

  12. Microwave (Sharp, model: R230KW )

  13. Pipettes (Gilson, model: Pipetman classic P2, P20, P200, P1000, catalog numbers: F144801 , F123600 , F123601 , F123602 )

  14. Rocking shaker (Reliable Scientific, Inc., model: 55D 12 x 16 )

  15. Sheet protectors (Clear file, Archival Plus 5x7 Print, catalog number: 370100B )

  16. Vortex mixer 120V (Corning LSE, model: 6775 )

Procedure

  1. SidJ in vitro Glutamylation Reaction

    1. Review the flowchart of the experimental outline of SidJ glutamylation and SdeA activity assays before beginning the procedure (Figure 1).



      Figure 1. Experimental outline for SidJ in vitro Glutamylation and SdeA Inhibition assay. A flow chart of the general experimental steps described in this procedure. The left branch is to assay the inhibition of SdeA ADP-ribosylation or Phospho-Ribosylation of Ub. The right branch is the experiments to assay PR-Ub ligation to substrates.


    2. Thaw recombinantly purified SidJ 89-853, SdeA Core (truncation 211-1152), calmodulin, ubiquitin, and optionally, Rab33b (1-200) on ice.

      Note: Rab33b 1-200 was used in this assay due to the prominence of a single PR-Ubiquitination band and increased protein stability (Jon Wasilko, Mao Lab, unpublished results). Full-length Rab33b may also be used instead.

    3. Preheat dry bath to 37 °C.

    4. Prepare stock solutions, on ice, listed in Table 1 by diluting in reaction buffer (20 mM Tris pH 7.5, 50 mM NaCl). Prepare NAD solution fresh and use glutamic acid and ATP stocks stored at -80 °C.


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

    5. Pipette the volumes of stock solution listed in SidJ glutamylation reaction row for a 25 μl reaction listed in Table 2 into a chilled 1.7 ml microcentrifuge tube on ice. Pipette ATP last to initiate the reaction. Immediately vortex, centrifuge briefly (~10 s max speed), and incubate the samples at 37 °C in a dry bath for 30 min.

      Note: It is important that samples are mixed thoroughly and centrifuged. SdeA is very active and the reaction mixture needs to be homogenous for maximal inhibition.

    6. Optional: Prepare a master mix for Step A5. Combine components contained in all reactions by pipetting stock solutions and 10 μl of reaction buffer per reaction. Prepare approximately 10% more reaction mix than needed for samples. The addition of reaction buffer to 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 10 μl of reaction buffer from the amount used in Table 2.


      Table 2. SidJ in vitro glutamylation and SdeA modification reaction components and concentrations

    7. Pipette remaining reaction components for the SdeA activity portion of the assay into the tube. If assaying ubiquitin modification, replace Rab33b with reaction buffer. Immediately vortex, centrifuge briefly (~10 s max speed), and incubate samples at 37 °C in a dry bath for 30 min.

      Note: A master mix containing SdeA modification components and 3 μl reaction buffer per sample can be prepared to minimize pipetting inaccuracies. A negative control excluding NAD would be beneficial for the visualization of the absence of SdeA activity.

    8. If assaying ubiquitin modification, separate each reaction into two tubes by pipetting 12.5 μl into another 1.7 ml microcentrifuge tube. Label tubes and pipette 3 μl of native sample buffer into one tube for native-PAGE analysis, and 3 μl of SDS sample buffer into the other for SDS-PAGE analysis and visualization of protein loading. Vortex to mix and centrifuge briefly (10 s at max speed). If only assaying PR-ubiquitin ligation activity, the reaction can be halted by the addition of 6 μl of SDS sample buffer.

      Note: Do not include SDS in native sample buffer, native-PAGE gels, or native-PAGE running buffer.


  2. Detection of ubiquitin modification and PR-Ubiquitin ligation to Rab33b by gel electrophoresis

    1. For detection of ubiquitin modification, electrophorese 13 μl of the portion of each sample in native sample buffer using a native-PAGE gel and native-PAGE running buffer at 80 V. Once samples have migrated through stacking gel, increase the voltage to 120 V and electrophorese until dye front migrates 50-75% through the gel.

      Note: Native-PAGE is required to detect modification of ubiquitin. The use of cold native-PAGE running buffer, placement of gel apparatus on ice, and shortened electrophoresis distance may provide better separation and clarity of protein bands.

    2. For detection of PR-Ubiquitin ligation and visualization of protein loading for ubiquitin modification gel, electrophorese 2.5 μl of protein ladder and 13 μl of each reaction in SDS sample buffer using an SDS-PAGE gel (4% separating gel, 12% resolving gel) and SDS-PAGE running buffer at 80 V. Once samples have migrated through stacking gel, increase the voltage to 150 V and electrophorese until dye front reaches the bottom of the gel.

    3. Separate the gel from the casting glass and remove the stacking gel. Transfer the gel to a microwave-safe, plastic container with a lid. Pour Coomassie stain to cover gel and microwave briefly in a covered container, until boiling. Ensure not to inhale fumes when moving the container by maximizing distance from the container. Stain gel by rocking 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. Discard stain and rinse with the destaining solution and incubate in the destaining solution for approximately 30 min to 1 h. Discard 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. The addition of 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 the gel imager. Wiping dust and staining imperfections with Kim wipe may help obtain clearer images (Figure 2).

    7. Analyze results. The addition of phosphoribose or ADP-ribose to ubiquitin by SdeA causes a significant charge alteration relative to the overall size of the small ubiquitin protein. As a result, this alteration greatly shifts the electrophoretic mobility on a native gel, while this modification is not visualized if separating only by protein size as with SDS-PAGE. The inhibition of SdeA prevents the migration shift of ubiquitin (Figure 2A). This assay does not distinguish between the addition of ADP-ribose and further modification to PR-Ub. If assaying the PR-Ubiquitin ligation to Rab33b by SdeA, ligation is detected by incremental 8 kDa band shifts above the unmodified Rab33b band in the presence of NAD. This appearance of increased molecular weight bands corresponds to the attachment of one or multiple PR-Ub to Rab33b. Inhibition of SdeA’s PR-Ubiquitin ligase activity should decrease the intensity of the modified Rab33b compared to the uninhibited SdeA reaction (Figure 2B).



Figure 2. Reaction components required for SidJ mediated glutamylation and inhibition of SdeA. A. SidJ inhibition of the ability of SdeA to modify ubiquitin. SidJ glutamylation and SdeA modification were conducted with the reaction components and concentrations listed in Table 2, with Rab33b 1-200 replaced with reaction buffer. Reaction components were excluded as indicated. SidJ in vitro glutamylation assay was conducted for 30 min at 37 °C followed by SdeA modification for 30 min at 37 °C. Proteins were electrophoresed by native-PAGE and stained with Coomassie stain. B. SidJ inhibition of the ability of SdeA to PR-Ubiquitinate Rab33b 1-200. The reaction was conducted with concentrations listed in Table 2. SidJ in vitro glutamylation assay was conducted for 30 min at 37 °C followed by SdeA PR-Ubiquitination for 30 min at 37 °C. Proteins were electrophoresed by SDS-PAGE and the gel was stained with Coomassie stain. This figure is from the 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. MgCl2 1 M Solution

    Weigh 2.033 g of magnesium chloride, 6-hydrate and dilute to 10 ml with ddH2O

    Store at room temperature

  3. ATP 100 mM pH 7.5 Solution

    Weigh 551.14 mg adenosine 5′-triphosphate disodium salt hydrate and dissolve in 8 ml of ddH2O

    Adjust the pH to 7.5 with 10 M sodium hydroxide and dilute to a final volume of 10 ml with ddH2O

    Aliquot and store at -80 °C

  4. Glutamic Acid 1 M Solution

    Weigh 0.936 g of L-glutamic acid monosodium salt, monohydrate and dilute to 5 ml with ddH2O

    Aliquot and store at -80 °C or prepare fresh

  5. 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

  6. 10x Native-PAGE Running Buffer

    Store at room temperature and dilute 10-fold with ddH2O for use

    Component   1 L
    Tris-Base 35 g
    Glycine 144 g
    ddH2O         To 1 L

  7. 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)
    2-Mercaptoethanol     10% (v/v)

  8. 6x Native Sample Buffer

    Store at room temperature

    Component                50 ml
    ddH2O                         35 ml
    Glycerol                       15 ml
    Bromophenol Blue    0.125 g

  9. 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

  10. Coomassie Destaining Solution

    Store at room temperature

    Component                  1 L
    Ethanol                        450 ml
    ddH2O                         450 ml
    Glacial Acetic Acid      100 ml


    SDS-PAGE Gel

  11. 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%

  12. 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%


    Native-PAGE Gel

  13. 8% Resolving Gel

    Component                                                 20 ml (4 Gels)                            Final Conc.
    ddH2O                                                               9.34 ml
    1.5 M Tris, pH 8.8                                             5 ml                                        375 mM
    30% Acrylamide/Bis Solution                         5.34 ml                                   8%
    10% APS                                                            100 μl                                      0.05%
    TEMED                                                               20 μl                                        0.1%

  14. 3% Stacking Gel

    Component                                                 10 ml (4 Gels)                             Final Conc.
    ddH2O                                                           8.32 ml
    1.0 M Tris, pH 6.8                                         500 μl                                          50 mM
    30% Acrylamide/Bis Solution                    1.02 ml                                         3%
    10% APS                                                        50 μl                                            0.05%
    TEMED                                                           10 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.

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简介

[摘要]谷氨酰化是翻译后修饰,其中游离谷氨酸氨基酸的氨基与目标蛋白内谷氨酸侧链的羧基缀合。SidJ是一种军团菌激酶样蛋白,最近被发现可对军团菌SdeA磷酸核糖泛素(PR- Ub )连接酶进行蛋白多谷氨酰化,从而抑制SdeA的活性。SidJ将多个谷氨酸氨基酸附着到SdeA的催化谷氨酸残基上,从而抑制了SdeA对泛素的修饰(Ub )和结扎活动。在此协议中,我们将讨论使用其底物SdeA的SidJ非放射性,体外谷氨酰化测定。这也将包括一个第二反应以测定抑制SdeA通过使用免费的两个修改泛素和结扎ADP-核糖基化的泛素(ADPR-泛素),以SdeA的基板Rab33b。在鉴定和公布SidJ的活性之前,尚无SdeA抑制试验。我们的小组和其他小组演示了各种方法来抑制SdeA的活性。备选方案包括使用放射性NAD测量Ub的ADP核糖基化,NAD水解以及SdeA对HA- Ub连接的蛋白质印迹分析。该方案将描述使用廉价的标准凝胶和考马斯染色对SdeA的泛素修饰和PR- Ub连接的抑制。

[背景]嗜肺军团菌是感染性细菌,其机会性感染肺泡巨噬细胞。这是通过吸入被污染的水气溶胶而发生的,引起潜在的致命性肺炎,称为军团菌病(McDade et al。,1977)。军团菌通过分泌300多种效应蛋白感染寄主细胞,这些蛋白用于劫持许多宿主细胞过程并防止其溶酶体降解(Hubber和Roy,2010)。军团菌劫持的一个过程是泛素化系统(Hubber等,201 3 )。泛素化是一种真核翻译后修饰,可调节多种细胞过程(Hershko和Ciechanover ,1998; Chen和Sun,2009; Hurley和Stenmark,2011; Haglund和Dikic,2012)。这需要E1,E2和E3酶的一致努力来仔细调节哪些蛋白被泛素化(Scheffner等,1995)。但是,军团菌已将这种修饰与独立于E1和E2酶起作用的SidE磷酸核糖基泛素连接酶家族相提并论。该家族使用两个催化结构域使用单ADP-核糖基转移酶(mART )域对ADP-核糖基泛素进行修饰,然后使用磷酸二酯酶(PDE)域将ADPR- Ub连接至宿主蛋白丝氨酸残基(Bhogaraju等人, 2016; Qiu等,2016; Kotewicz等,2017)。在本质上前面提到的研究包括测定手段一侧活动。所述侧家族包括MEMB呃SdeA ,这已被确定要通过时空调控SidJ (Havey和Roy ,2015;郑等人,2015;都市实践等人,2016); 虽然,该法规的机制尚未完全了解。有人建议,当使用军团菌纯化的SidJ时,SidJ可能充当PR-泛素化酶(Qiu等人,2017年)(Qiu和Luo,2019年); 然而,最近的研究并未概括这些结果(Bhogaraju等人,2019; Wan等人,2019; Shin等人,2020)。

我们的研究小组(Sulpizio等人,2019)和其他几个研究者(Bhogaraju等人,2019; Black等人,2019; Gan等人,2019)证明SidJ是一种聚谷氨酰胺酶,可添加多个谷氨酸氨基酸到SdeA抑制SdeA的功能。这些研究提供了鉴定SidJ包含激酶样结构域并结合真核蛋白钙调蛋白的结构数据。此外,质谱研究还发现,SdeA抑制的机制是SdeA的催化性mART谷氨酸残基的多谷氨酰化。基于这些结果,使用底物SdeA ,钙调蛋白,ATP / MgCl 2和谷氨酸开发了体外谷氨酰化测定法。为了证明谷氨酰化的抑制作用,在体外谷氨酰化之后进行SdeA活性测定。其他基团也描述SdeA使用NAD水解抑制(Bhogaraju等人,2019) ,放射性NAD (黑色等人,2019),F滞后标记SdeA基板(甘等人,2019),HA-泛素,和这些群体对规范泛素化具有抵抗力的HA- Ub变体。这些替代方法适用于鉴定抑制作用,并可提供更多定量检测。但是,这些实验中的某些实验未包括体外抑制的SdeA ,而那些监测泛素修饰的实验需要使用昂贵的试剂,例如放射性NAD。该协议讨论了为SidJ开发的谷氨酰化测定法,以及使用标准凝胶和考马斯亮蓝染色法对SdeA PR泛素连接和泛素修饰的体外抑制作用。这可用于鉴定突变对活性的影响,对泛素修饰和PR-泛素连接的测定抑制,更普遍地可用于确定通过谷氨酰化对其他ADP-核糖基转移酶的抑制。

关键字:SidJ蛋白, SdeA酶, 谷氨酰化, PR-Ubiquitination, 军团杆菌, 假激酶, ADP-核糖基化

材料和试剂

 

1.7 ml微型管(Corning Incorporated,Axygen ,目录号:MCT-175-C)
50 ml离心管(VWR,目录号:525-0637)
手套(VWR,货号:89038-270)
金抹布(Kimberly-Clark Professional,货号:34120)
移液器提示:
10 μ升XL渐变提示(美国科学,提示一,目录号:1110-3700)

200 μ升毕业连结架(实验室制品销售,目录号:130430)

1250 μ升吸头(实验室产品销售,目录号:L134770)

重组蛋白
SidJ 89-8 53截短,SdeA 211-1152截短,人钙调蛋白2在大肠杆菌中表达,带有N端6XHis-SUMO标签,并按照先前的描述进行纯化(Sulpizio et al。,2019)。Rab33b(1-200)也按照(Sulpizio et al。,2019)中所述的蛋白质进行纯化,Ub如Akturk et al。(2018)。将最终纯化的蛋白质储存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下。

β-烟酰胺腺嘌呤二核苷酸钠盐(NAD)(Sigma,目录号:N0632-1G)
一水合L-谷氨酸一钠盐(USB Corporation,目录号:16245)
2-巯基乙醇(Sigma,目录号:M3148-100ML)
30%丙烯酰胺/双酚37.5:1(Bio - Rad,目录号:1610158)
冰醋酸(JT Baker,目录号:9508-06)
腺苷5'-三磷酸二钠盐水合物(Sigma,目录号:A2382-10G)
过硫酸铵(APS)(Amresco ,目录号:0486-100G)
艳蓝R-250(Fisher,货号:BP101-50)
溴酚蓝钠盐(Fisher,目录号:BP114-25)
DL-二硫苏糖醇(DTT)(Amresco ,目录号:M109-25g)
耐乙醇200(Koptec ,目录号:V1001)
甘油(Mallinckrodt Chemicals,目录号:5092-16)
甘氨酸(VWR,目录号:0167-5KG)
六水合氯化镁(Mallinckrodt Chemicals,目录号:5958-04)
甲醇(Fisher ,目录号:A454SK-4)
N,N ,N',N'-四甲基乙二胺(TEMED)(Bio - Rad,目录号:161-0800)
Precision Plus Protein All Blue Standards蛋白梯(Bio - Rad,目录号:161-0373)
氯化钠(VWR,目录号:0241-10KG)
十二烷基硫酸钠(SDS)(VWR Life Sciences,目录号:0227-1KG)
Tris(VWR,目录号:0497-5KG)
反应缓冲液(请参见配方)
MgCl 2 1 M溶液(请参阅配方)
ATP 100 mM pH 7.5溶液(请参阅食谱)
谷氨酸1 M溶液(请参阅食谱)
10x SDS-PAGE运行缓冲区(请参阅配方)
10x Native-PAGE运行缓冲区(请参阅食谱)
SDS样品缓冲液(请参阅配方)
本机样本缓冲区(请参阅食谱)
考马斯染色(见食谱)
考马斯脱色溶液(请参阅食谱)
12%SDS-PAGE分辨凝胶(请参阅食谱)
4%SDS-PAGE堆积凝胶(请参阅食谱)
8%天然PAGE解析凝胶(请参阅食谱)
3%天然PAGE堆积凝胶(请参阅食谱)
注意:除非另有列出,否则产品按制造商的建议进行存储。



设备

 

-80 °C冷冻室(低温,型号:PV85-21)
电脑
干澡(基准,米Odel等:BSH1001)
定速离心机(基准,型号:C1008-C)
钳子
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
Labcoat (VWR,目录号:10141-306)
实验室胶带(VWR,目录号:89098-062)
微波炉(锋利,型号:R230KW)
移液器(Gilson ,型号:Pipetman classic P2,P20,P200,P1000,目录号:F144801,F123600,F123601,F123602)
摇床(Reliable Scientific,Inc.,型号:55D 12 x 16)
纸张保护贴(透明文件,Archival Plus 5x7打印,目录号:370100B)
涡旋混合器120V(Corning LSE,型号:6775)
 

程序

 

SidJ体外谷氨酰化反应
在开始该程序之前,请查看SidJ谷氨酰化和SdeA活性测定实验大纲的流程图(图1)。
 



图1. SidJ体外谷氨酰化和SdeA抑制测定的实验概述。此过程中描述的一般实验步骤的流程图。左分支用于测定Ub的SdeA ADP-核糖基化或磷酸-核糖基化的抑制。右分支是测定PR- Ub与底物连接的实验。

 

在冰上解冻重组纯化的SidJ 89-853,SdeA Core(截断211-1152),钙调蛋白,泛素和可选的Rab33b(1-200)。
注意:由于单个PR-泛素化条带的突出和蛋白质稳定性的提高,Rab33b 1-200被用于该测定(Jon Wasilko ,Mao Lab,未发表的结果)。也可以使用全长Rab33b。

将干浴预热至37 ° C。
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中所列的储备溶液。新鲜制备NAD溶液,并使用储存在-80 ° C的谷氨酸和ATP储备液。
 

表1. SidJ体外谷氨酰化和SdeA抑制反应的原液浓度

零件

浓度

盛捷89-853

10 μ中号

钙调蛋白

100微米

SdeA核心

20微米

氯化镁2

125毫米

谷氨酸

125毫米

ATP pH 7.5

25毫米

泛素

625微米

Rab33b

250微米

NAD

25毫米

 

移液管的中列出原液的容积SidJ glutamylation反应行对于25 μ升表2中所列成冷冻1.7米反应升冰上微量离心管中。最后用移液器ATP引发反应。立即涡旋,短暂离心(约10 s最高速度),并在37 ° C的干浴中孵育样品30分钟。
注意:将样品彻底混合并离心很重要。SdeA非常活泼,反应混合物必须均匀才能获得最大抑制。

可选:为步骤A5准备预混料。通过移取储备溶液和每个反应10μl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。向预混液中添加反应缓冲液会稀释成分,以保持蛋白质的稳定性。如果准备了主混合物,则对于每个反应,从表2中使用的量中减去混合物中包含的反应组分的体积和10μl反应缓冲液。


表2. SidJ体外谷氨酰化和SdeA修饰反应成分和浓度

反应类型

组件(库存)

反应浓度

蓄积量(μ升)

 

SidJ 89-853(10 μ中号)

0.5 μ中号

1.25

 

钙调蛋白(100 μ中号)

5微米

1.25

SidJ谷氨酰化

SdeA核心(20μM )

1微米

1.25

反应(30分钟)

氯化镁2 (125毫米)

5毫米

1个

 

谷氨酸(125 m M)

5米中号

1个

 

ATP pH 7.5(25毫米)

1毫米

1个

 

泛素(625μM )

25微米

1个

SdeA活动

Rab33b 1-200(250μM )

10微米

1个

反应(30分钟)

NAD(25毫米)

1毫米

1个

 

反应缓冲液

到25 μ升

15.25

 

将测定法中SdeA活性部分的剩余反应成分移液到试管中。如果要测定ub Iquitin修饰,请用反应缓冲液替换Rab33b。立即涡旋,短暂离心(约10 s最大速度),并在37 ° C的干浴中孵育样品30分钟。
注意:一个主混合物含有SdeA修改组件和3 μ升每个样品反应缓冲液,可以制备以最小化移液误差。排除NAD的阴性对照将有助于可视化显示SdeA活性的缺失。

如果测定泛素修饰,通过吸取12.5各反应分成两个管μ升到另一个1.7米升微量离心管中。标签管和移液管3 μ升天然样品缓冲液为一个管,用于天然-PAGE分析,和3 μ升SDS样品缓冲液的成其他用于SDS-PAGE分析和蛋白质的可视化加载。涡旋混合并短暂离心(最大速度10 s)。如果仅测定PR-泛素连接活性,该反应可以通过加入6被停止μ升SDS样品缓冲液中。
注意:请勿在天然样品缓冲液,天然PAGE凝胶或天然PAGE运行缓冲液中包含SDS。

 

通过凝胶电泳检测泛素修饰和PR-泛素与Rab33b的连接
为了检测遍在蛋白修饰,electrophorese 13 μ升在80 V.天然样品中的每个样品的部分的缓冲器使用本机-PAGE凝胶和本机-PAGE电泳缓冲液一旦样品已通过积层凝胶迁移,增加电压至120伏电泳直至染料前沿在凝胶中迁移50-75%。
注意:需要Native-PAGE来检测泛素的修饰。使用冷的天然PAGE运行缓冲液,将凝胶仪器放置在冰上以及缩短的电泳距离可提供更好的蛋白质条带分离度和清晰度。

为了检测PR-泛素连接的和蛋白质负载的可视化的泛素修饰克EL,electrophorese 2.5 μ升蛋白梯和13的μ升缓冲液使用SDS-PAGE凝胶的SDS样品在每个反应中的(4%分离胶,12%溶解的凝胶)和80 V的SDS-PAGE运行缓冲液。一旦样品通过堆叠的凝胶迁移,将电压增加至150 V并电泳,直到染料前沿到达凝胶底部为止。
从流延玻璃中分离出凝胶,然后去除堆积的凝胶。将凝胶转移到带盖的微波安全塑料容器中。将考马斯染色剂倒入有盖容器中,短暂覆盖凝胶和微波,直到沸腾。通过与容器的最大距离,确保在移动容器时不要吸入烟雾。在室温下摇动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。

丢弃染色和漂洗用脱色溶液并孵育在d estaining溶液中约30分钟至1小时。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中进行更长的除水处理。
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。添加金氏擦拭布可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。用金擦拭布擦拭灰尘和污点可帮助获得更清晰的图像(图2)。
分析结果。通过SdeA向泛素中添加磷酸核糖或ADP-核糖相对于泛素小蛋白的整体大小会引起明显的电荷变化。结果,这种改变极大地改变了天然凝胶上的电泳迁移率,而如果仅像SDS-PAGE那样仅通过蛋白质大小进行分离,则这种改变是不可见的。SdeA的抑制作用可防止泛素迁移(图2A)。该测定法不区分添加ADP-核糖和进一步修饰PR- Ub 。如果通过SdeA分析PR-泛素与Rab33b的连接,则在存在NAD的情况下,通过在未修饰的Rab33b带上方增加8 kDa的带移来检测连接。分子量带增加的出现对应于一个或多个PR- Ub与Rab33b的连接。与未抑制的SdeA反应相比,抑制SdeA的PR-泛素连接酶活性应降低修饰的Rab33b的强度(图2B)。
 



图2. SidJ介导的谷氨酰化和抑制SdeA所需的反应组分。A. Sid J抑制SdeA修饰泛素的能力。用表2中列出的反应组分和浓度进行SidJ谷氨酰化和SdeA修饰,用Rab33b 1-200替换反应缓冲液。如所示排除反应组分。SidJ体外glutamylation测定在37 30分钟进行° C,随后SdeA修改为在30分钟时37 ° Ç 。通过天然PAGE电泳蛋白质,并用考马斯亮蓝染色。B. SidJ抑制SdeA对PR-泛素Rab33b 1-200的能力。将反应物用表2中所列的浓度进行SidJ体外在37 glutamylation测定30分钟内进行° C,随后SdeA PR-泛素化30分钟,在37 °下的蛋白质通过SDS-PAGE的进行电泳d的凝胶用考马斯蓝染色。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

MgCl 2 1 M溶液
称取2.033克的米agnesium酰氯,6-水合物和d ilute到10 ml的用的DDH 2 ö

小号撕在室温下

ATP 100 mM pH 7.5溶液
称量551.14毫克一个denosine 5 '三磷酸二钠盐水合物和溶解8 ml的的的DDH 2 ö

甲djust pH至7.5 -10 M小号ODI微米氢氧化物和稀到10的终体积毫升用的DDH 2 ö

分装并储存在-80 ° C

谷氨酸1 M溶液
称取0.936 g L-谷氨酸一钠盐,蛋清水并用ddH 2 O稀释至5 ml

分装并储存在-80 ° C或准备新鲜

10x 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

10x Native-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

35克

甘氨酸

144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)

2-巯基乙醇

10%(v / v)

6x原生样本缓冲区
室温保存

零件

50百万升

ddH 2 O

3500万升

甘油

1500万升

溴酚蓝

0.125克

考马斯染色
室温保存

零件

500百万升

甲醇

225米升

ddH 2 O

225米升

冰醋酸

50百万升

艳蓝R250

1.25克

考马斯脱色溶液
室温保存

零件

1升

乙醇

450百万升

ddH 2 O

450百万升

冰醋酸

1亿升

 

SDS-PAGE凝胶

12%分解凝胶
零件

8凝胶

最终浓缩

ddH 2 O

13.3百万升

 

30%丙烯酰胺/双溶液

1600万升

12%

1.5 M Tris pH 8.8

10百万升

375毫米

10%SDS

400 μ升

0.1%

10%APS

300 μ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

11.2百万升

 

30%丙烯酰胺/双溶液

2.16百万升

4.14%

1.0 M Tris pH 6.8

2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%

 

天然PAGE凝胶

8%分解凝胶
零件

20米升(4个凝胶)

最终浓缩

ddH 2 O

9.34百万升

 

1.5 M Tris,pH 8.8

5百万升

375毫米

30%丙烯酰胺/双溶液

5.34百万升

8%

10%APS

100 μ升

0.05%

特美

20 μ升

0.1%

3%堆积凝胶
零件

10米升(4个凝胶)

最终浓缩

ddH 2 O

8.32百万升

 

1.0 M Tris,pH 6.8

500 μ升

50毫米

30%丙烯酰胺/双溶液

1.02百万升

3%

10%APS

50 μ升

0.05%

特美

10 μ升

0.1%

 



致谢

 

这项工作是由美国国立卫生研究院(NIH)资助5R01GM116964(至YM),支持康奈尔大学哈利和塞缪尔·曼优秀研究生奖(AGS),并通过在露丝L时NIH Kirschstein国家研究服务奖(6T32GM008267)从NIGMS(到MEM)。

来自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威廉姆斯,ML Goldberg,MB Smolka和Y.Mao(2019)。“由细菌钙调蛋白依赖性假激酶SidJ催化的聚谷氨酰化作用。” 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., Minelli, M. E. and Mao, Y. (2020). In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors. Bio-protocol 10(21): e3811. DOI: 10.21769/BioProtoc.3811.
  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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