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Feb 2021

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Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteins in Malaria Parasites
基于活性的交联识别疟原虫中硫氧还蛋白结构域蛋白的底物   

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Abstract

Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity.

Graphic abstract:


Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.

Keywords: Plasmodium (疟原虫), Thioredoxin (硫氧还蛋白), Protein disulfide Iisomerase (蛋白质二硫键异构酶), Protein crosslinking (蛋白质交联), Redox (氧化还原)

Background

Disulfide bonds are crucial for the activity and regulation of many proteins; therefore, disulfide bond formation and reduction is a critical aspect of cellular life. Members of the thioredoxin (Trx) superfamily use the nucleophilic cysteines of their “CXXC” active sites to facilitate the formation and reduction of disulfide bonds (Lu and Holmgren, 2014). Given their importance for cell survival, thioredoxin-domain proteins and the thioredoxin system have been proposed as drug targets against apicomplexan parasites (Becker et al., 2000; Krnajski et al., 2001; Andricopulo et al., 2006; Kehr et al., 2010; Biddau et al., 2018; Biddau and Sheiner, 2019; Cobb et al., 2021). Despite being essential and their potential as drug targets, the substrates of these Trx-domain proteins are often ill-defined, as it can be difficult to discern specific substrates from other interacting partners.

One strategy for identifying the substrates of Trx domains relies on the active site mode of action: the first cysteine of the “CXXC'' active site forms a mixed disulfide bond with a client protein, and the sulhydryl group of the second cysteine is used to resolve that bond. Mutagenesis of the second cysteine, often to alanine or serine, prevents the reduction of the bond between the Trx-domain and client protein, trapping the two proteins together (Jessop et al., 2007; Oka et al., 2013). Subsequently, mass spectrometry can be used to identify the substrate. A major disadvantage to this approach is that it requires modification to the active sites of endogenous enzymes, that may be essential, or exogenous expression of the mutated enzymes, both of which may prove lethal for the organism. Particularly in organisms like Plasmodium falciparum, which is haploid for the majority of its lifecycle and carries single copies of genes for essential Trx-domain proteins, this approach may prove especially difficult.

Given the challenges inherent to a genetic approach, activity based chemical crosslinking of Trx-domains to their substrates is a straightforward and powerful option for identifying those substrates. Divinyl sulfone (DVSF) is a bifunctional, electrophilic, and cell permeable crosslinker shown to have remarkable specificity for nucleophilic cysteines, like those within Trx-domain active sites (West et al., 2011). Treatment of cells with DVSF results in irreversible crosslinking between redox-active cysteines, and it has been used to trap and identify substrates of Trx-domain proteins and other redox-active enzymes in yeast and human cells (Naticchia et al., 2013; Allan et al., 2016; Araki et al., 2017). Demonstrating the power of this activity-based technique for identifying Trx-domain substrates in a non-model organism, we used DVSF to trap and identify substrates of Trx-domain proteins in the endoplasmic reticulum (ER) of P. falciparum parasites (Cobb et al., 2021). We also demonstrated its specificity for redox-active nucleophilic cysteines, as other cysteine-containing proteins did not react with DVSF, and further validated our results by demonstrating ablation of crosslinking, by mutation of reactive cysteine residues to alanine residues (Cobb et al., 2021). In this protocol, we describe the use of DVSF to trap substrates of ER-localized Trx-domain proteins in P. falciparum and the identification of these trapped substrates via mass spectroscopy. Given the robust nature of this protocol, it will be of use for similar experiments in diverse organisms and within multiple cellular compartments. Because this approach can be utilized with minimal genetic modification, requiring only the addition of an affinity tag to the Trx-domain protein if a suitable antibody is not otherwise available, this protocol will allow others working with difficult organisms to study the interactions between Trx-domains and their substrates.

Materials and Reagents

  1. 15 mL centrifuge tubes (Genesee Scientific, catalog number: 28-103)

  2. 1.7 mL microtubes (Genesse Scientific, catalog number: 28-281)

  3. Filter tip pipette tips: 10 μL (Fisher Scientific, catalog number: 02-707-473), 200 μL (Fisher Scientific, catalog number: 02-707-478), 1 mL (Fisher Scientific, catalog number: 2707480)

  4. Serological pipettes: 5 mL (Genesee Scientific, catalog number: 12-102), 10 mL Genesee Scientific, catalog number: 12-104)

  5. TPP Tissue culture plate, 100 mm (MidSci, catalog number: TP92406)

  6. O+ AS-1 Packed Cells (Interstate Blood Bank, Memphis, TN)

  7. IRDye® 800CW anti-mouse (Li-Cor, catalog number: 926-32210 used at 1:20,000 for Western blot)

  8. IRDye® 680RD anti-rabbit (Li-Cor, catalog number: 926-68071, used at 1:20,000 for Western blot)

  9. Rabbit anti-HA antibody (Life Technologies, catalog number: 715500, used at 1:100 for Western blot)

  10. Mouse anti-PMV antibody (Gift from Daniel Goldberg, Washington University School of Medicine, used at 1:400 for Western blot)

  11. V5-Tag (D3H8Q) Rabbit mAb (Cell Signaling Tech, Inc., catalog number: 13202S, used at 1:1,000 for Western blot)

  12. Anti-HA Magnetic Beads (Thermo Fisher Scientific/PierceTM, catalog number: 88836)

  13. Divinyl sulfone, 97% stab. with 0.05% hydroquinone (Thermo Fisher Scientific/Alfa Aesar, catalog number: L12827)

  14. Saponin Quillaja sp., Sapogenin content 20-35% (Sigma, catalog number: S4521-10G)

  15. Protein sample loading buffer, 4× (Li-Cor, catalog number: 928-40004)

  16. Chameleon Duo pre-stained protein ladder (Li-Cor, catalog number:928-60000)

  17. 2-mercaptoethanol (Fisher Scientific, catalog number: 21985023)

  18. 10× Tris/Glycine/SDS buffer (Bio-Rad, catalog number: 161-0732)

  19. 4-20% Mini-PROTEAN® TGXTM precast protein gels (Bio-Rad, catalog number: 456-1093)

  20. InterceptTM blocking buffer (Li-Cor, catalog number: 927-70003)

  21. Odyssey® nitrocellulose membrane (Li-Cor, catalog number: 926-31092)

  22. Halt protease inhibitor cocktail (Thermo Fisher Scientific/Life Technologies, catalog number: 78429)

  23. HA synthetic peptide (Thermo Fisher Scientific/Life Technologies, catalog number: 26184)

  24. Simply Blue SafeStain (Thermo Fisher Scientific/Life Technologies, catalog number: LC6060)

  25. Tris-HCl (Fisher Bioreagents, catalog number: 1185-53-1)

  26. KCl (Sigma, catalog number: 7447-40-7)

  27. EDTA (Fisher Bioreagents, catalog number: 60-00-4)

  28. NP-40 (Sigma, catalog number: 9002-93-1)

  29. Na2HPO4 (Millipore Sigma, catalog number: 1065861000)

  30. KH2PO4 (Millipore Sigma, catalog number: 529568)

  31. NaCl (Fisher Scientific, catalog number: BP358212)

  32. TRIS base (Fisher Scientific, catalog number: YBP152500)

  33. Glycine (Fisher Scientific, catalog number: ALF-036435-A1)

  34. Methanol (Fisher Scientific, catalog number: A4081)

  35. Tween-20 (Fisher Scientific, catalog number: BP337-100)

  36. Immunoprecipitation extraction buffer (see Recipes)

  37. Immunoprecipitation binding buffer (see Recipes)

  38. Phosphate buffered saline (PBS), pH 7.4 (see Recipes)

  39. Protein loading dye (see Recipes)

  40. 10× Transfer buffer (see Recipes)

  41. 1× Transfer buffer (see Recipes)

  42. 0.1% Tween-20, in 1× PBS (see Recipes)

  43. 10× Tris-buffered saline (TBS) (see Recipes)

  44. 2 mg/mL HA peptide (see Recipes)

Equipment

  1. SorvallTM LegendTM XTR Centrifuge (Thermo Fisher Scientific, model: 75004506)

  2. Odyssey® CLx Imaging System (Li-Cor, model: 9140)

  3. DynaMagTM-2 Magnet (Life Technologies, model: 12321D)

  4. Branson S450D Digital Sonifier (Thermo Fisher Scientific, model: 101-063-593)

  5. Mini-PROTEAN Tetra Cell System (Bio-Rad, catalog number: 1658005EDU)

  6. PowerPacTM Basic Power Supply (Bio-Rad, catalog number: 1645050)

  7. FormaTM Series II Water-Jacketed CO2 incubator (ThermoFisher Scientific, catalog number: 3110)

Software

  1. Image Studio 5.x CLx (Li-Cor, https://www.licor.com/bio/image-studio-lite/)

Procedure

  1. Trap Trx-domain substrates with DVSF

    1. Incubate cells with DVSF

      1. Transfer cultures of red blood cells (RBCs) infected with P. falciparum parasites and expressing an epitope-tagged Trx-domain protein, into sterile 15 mL conical tubes. As a starting point, use 10 mL of culture at 2% hematocrit, with 10-15% parasitemia.

      2. Pellet the infected RBCs by centrifuging at 800 × g for 3 min.

      3. Remove the supernatant.

      4. Use a serological pipette to resuspend the RBCs in an equal volume of either 3 mM DVSF prepared in PBS, or PBS alone as a control (e.g., pellet 10 mL of culture, and resuspend the cells in 10 mL of 3 mM DVSF or PBS).

      5. Incubate the cells at 37°C, for 30 min in a CO2 incubator.

      6. Pellet the cells by centrifuging at 800 × g for 3 min.

      7. Remove the supernatant.

      8. Dispose of the DVSF supernatant according to your institution’s chemical waste policies.

      9. Wash the cells three times with PBS, using an equivalent volume of PBS (e.g., use 10 mL of PBS if 10 mL of DVSF was used), and performing spins at 800 × g for 3 min.

    2. Extract protein from the cells

      1. Prepare a solution of 0.04% (w/v) saponin in PBS and cool on ice, until the buffer is ice-cold.

      2. Isolate parasites by lysing RBCs in 0.04% saponin.

        1. Resuspend cells in 1 mL of ice-cold saponin per 4 mL of culture that was pelleted (e.g., resuspend cells pelleted from 10 mL of culture in 2.5 mL of saponin).

        2. Incubate on ice for 15 min.

        3. Pellet parasites by centrifuging at 2,000 × g for 5 min, at 4°C.

        4. Remove the supernatant and wash two times with ice-cold PBS, using the same volume of PBS as the volume of saponin used in Step A.2.a, performing spins at 2,000 × g for 5 min.

      3. Resuspend parasites in 50-100 μL of protein loading dye.

      4. Boil parasites at 95°C for 5 min.

      5. Pellet insoluble material, including parasite hemozoin, by centrifuging at full speed on a table-top centrifuge for 10 min, at room temperature.

      6. Transfer supernatant, which contains extracted proteins, to a clean tube.

      7. Demonstrate successful cross-linking by Western blot: compared to the PBS-only control sample, the DVSF-crosslinked sample should contain one or more bands at higher molecular weight, as the Trx-domain protein + substrate will migrate slower during SDS-PAGE than the Trx-domain protein alone (Figure 1).

        1. Load 30-50 μL of protein sample and a protein ladder into a polyacrylamide gel.

        2. Separate the proteins in the DVSF-crosslinked and PBS-only samples by SDS-PAGE, at 180 V for ~45 min, or until the bands of the protein ladder are sufficiently resolved.

        3. Transfer proteins from the gel to a nitrocellulose membrane, at 100 V for 1 h.

          Prepare the membrane by cutting it to the approximate size of the gel and pre-wetting in 1× transfer buffer before applying to the gel.

        4. Block the membrane for 1 h, at room temperature with gentle shaking, using 30 mL of 50% InterceptTM blocking buffer (v/v, prepared in 1× PBS).

        5. Remove the blocking buffer, and add 10 mL of primary antibody solution, prepared in 50% blocking buffer described above.

          Use primary antibodies directed against the Trx-domain protein of interest and a loading control.

        6. Incubate the membrane in the primary antibody solution at 4°C overnight, in a covered container, with gentle shaking.

        7. Remove the primary antibody solution and wash three times with 30 mL of 0.1% Tween-20 (v/v, prepared in 1× PBS), for 10 min, at room temperature with gentle shaking.

        8. Add 10 mL of secondary antibody solution prepared in 50% blocking buffer, as described in Step A.2.g.iv. above.

        9. Incubate for 1 h, at room temperature, in a covered container, with gentle rocking.

        10. Remove the secondary antibody solution and wash three times with 30 mL of 0.1% Tween-20 (v/v, prepared in 1× PBS), for 10 min, at room temperature with gentle shaking.

        11. Wash the membrane with 30 mL of 1× PBS for 5 min, at room temperature with gentle shaking.

        12. Image the membrane with the Odyssey® CLx Imaging System to visualize cross-linking of proteins to the Trx-domain protein of interest.



        Figure 1. DVSF crosslinking traps redox partners to P. falciparum Trx-domain proteins.

        Parasites were incubated with 3 mM DVSF for 30 min at 37°C, then proteins were extracted and analyzed via Western blot. The parasites expressed epitope-tagged A.) PfJ2 or B.) PfPDI8. Plasmepsin V (PfPMV) was used as a loading control, and as a non-Trx-domain protein, demonstrating the specificity of DVSF.


  2. Immunoprecipitate Trx-domain proteins with trapped substrates

    1. Extract protein from parasites

      1. Perform DVSF crosslinking and isolate parasites using 0.04% saponin, as described above in section A; importantly, include the PBS-only control parasites. For immunoprecipitation for mass spectroscopy protein identification, we start with 1 × 109 parasites and scale up or down as necessary.

      2. Resuspend the parasite pellet in 300 μL of extraction buffer, then add 15 μL of 10% NP-40, and 3 μL of HALT protease inhibitor.

      3. Incubate on ice for 15 min.

      4. Lyse parasites by sonication.

        1. Sonicate parasites with an amplitude of 10%, for 5 s.

        2. Let the sample rest on ice for 30 s.

        3. Repeat Steps B.1.d.i and -ii two more times, for a total of three rounds of sonication.

      5. Centrifuge the lysate at full speed on a tabletop centrifuge set to 4°C, for 15 min.

      6. Transfer the supernatant, containing extracted proteins, to a new tube.

      7. Perform a second extraction on the pellet from Step B.1.e.

        1. Add 300 μL of extraction buffer and 3 μL of HALT protease inhibitor to the pellet (no NP-40 is used for the second extraction) and proceed immediately to the sonication step without incubating on ice for 15 min.

        2. Sonicate and centrifuge as described in Steps B.1.a-e.

      8. Combine the supernatant with the extracted proteins from Step B.1.f.

      9. Transfer 60 μL of the combined supernatants to a clean tube as the “Input” sample and store at -20°C.

    2. Immunoprecipitate HA-tagged proteins

      1. Prepare Pierce anti-HA magnetic beads.

        1. Transfer 25 μL of the bead slurry to a clean 1.5 mL centrifuge tube.

        2. Add 175 μL of binding buffer to the beads, flick the tube to mix, and briefly centrifuge to collect all of the liquid at the bottom of the tube.

        3. Insert the tube into a magnetic rack and remove the buffer after the beads have collected to the side of the tube.

        4. Remove the tube and add 1 mL of binding buffer to the beads.

        5. Rotate the tube in your hand to mix beads and buffer, for 1 min.

        6. Use the magnetic rack to remove the buffer from the beads.

      2. Add the extracted proteins from Step B.1.h to the beads.

      3. Incubate with end-over-end rotation for 30 min at room temperature (or overnight at 4°C).

      4. Briefly centrifuge the tube to collect all of the liquid to the bottom, then use the magnetic rack to remove the unbound protein sample from the beads; save this sample in a tube labeled “Unbound”.

      5. Wash the beads three times with binding buffer, and save the buffer from each wash for downstream analysis if necessary.

        1. Resuspend the beads in 300 μL of binding buffer and use the magnetic rack to remove the buffer from the beads.

        2. Repeat Step B.2.e.i two more times, for a total of three washes.

      6. Elute proteins from the beads using the HA peptide.

        1. Resuspend the beads in 30 μL of 2 mg/mL HA peptide.

        2. Incubate at 37°C, shaking, for 5 min.

      7. Use the magnetic rack to separate the eluted proteins from the beads, and save the eluted proteins in a tube labeled “Elution”.


  3. Extract cross-linked proteins from acrylamide gels for protein identification

    1. Load 5 μL of protein ladder and the “Elution” samples of the immunoprecipitated proteins, collected in Step B.2.g above, into the wells of a polyacrylamide gel.

    2. Separate the proteins in the sample by SDS-PAGE, at 180 V, for approximately 45 min, or until the protein ladder has sufficiently resolved.

    3. Stain the proteins in the gel with SimplyBlue SafeStain, or another Coomassie stain (Figure 2).

      1. Rinse the gel three times with 100 mL of deionized water.

      2. Add approximately 20 mL of SimplyBlue SafeStain, or enough stain to completely cover the gel, and incubate at room temperature for 1 h, with gentle shaking.

      3. Remove the stain and wash the gel with 100 mL of deionized water for 1 h, at room temperature, with gentle rocking.

      4. After 1 h, add 20 mL of 20% NaCl (w/v, prepared in deionized water) and continue incubating for 2 h, or overnight, at room temperature with gentle shaking.



        Figure 2. Coomassie staining of DVSF-crosslinked proteins prior to extraction for mass spectroscopy protein identification.

        Parasites expressing epitope-tagged PfJ2 were incubated with 3 mM DVSF for 30 min at 37°C, then PfJ2 was immunoprecipitated from parasite lysates. The immunoprecipitated proteins were separated using SDS-PAGE and stained with SimplyBlue SafeStain. The black, dashed boxes indicate areas of the gel in the DVSF-treated lane extracted for protein identification. The red, dashed boxes indicate areas of the gel in the PBS-treated lane extracted as controls for the protein identification.


    4. Using a clean razor blade, extract the cross-linked protein bands from the gel (i.e., extract the bands that run higher in the gel than the non-crosslinked protein).

    5. Extract slices from equivalent areas of the gel in the PBS-only control sample (e.g., extract a cross-linked protein band that runs at 100 kDa, and extract an equivalently sized piece of gel at 100 kDa in the PBS-only sample).

    6. Transfer the gel slices into 1.5 mL centrifuge tubes.

    7. Destain the proteins in the gel slices.

      Incubate with 30% ethanol (v/v, prepared in deionized water) for 15 min, or until the gel slice is no longer stained, at room temperature, gently rotating.

    8. Identify the proteins present in each gel slice, either in-house or through a mass-spectroscopy service.

    9. Identify the potential Trx-domain substrates, by eliminating the proteins identified in the PBS-only gel slices from those identified in the DVSF-crosslinked samples.

Notes

We describe the use of DVSF for trapping, extracting, and identifying Trx-domain substrates in P. falciparum parasites, but the components of this protocol are easily adaptable for other organisms. Accordingly, the specifics of protein extraction, immunoprecipitation, and Western blotting should be adapted to the workflow that individual researchers have for their specific organisms.

Recipes

  1. Immunoprecipitation extraction buffer

    40  mM Tris-HCl pH 7.4

    150  mM KCl

    1 M EDTA

    1× HALT protease inhibitor

    0.5% NP-40 (v/v)

  2. Immunoprecipitation binding buffer

    20 mM Tris-HCl pH 7.4

    150 mM KCl

    1 mM EDTA

    1× HALT protease inhibitor

    0.1% NP-40 (v/v)

  3. Phosphate buffered saline (PBS), pH 7.4

    3.2 mM Na2HPO4

    0.5 mM KH2PO4

    1.3 mM KCl

    135 mM NaCl

  4. Protein loading dye

    120 μL of 4× Li-Cor Protein Sample Loading Buffer

    20 μL of 2-mercaptoethanol

  5. 10× Transfer buffer

    30.3 g of TRIS base

    144.1 g glycine

    Bring to 1 L with Milli-Q or deionized water.

  6. 1× Transfer buffer

    100 mL of 10× Transfer Buffer

    200 mL of methanol

    700 mL of Milli-Q or DI water

  7. 0.1% Tween-20, in 1× PBS

    Add 1 mL of Tween-20 to 1 L of 1× PBS

  8. 10× Tris-buffered saline (TBS)

    87.7 g NaCl

    100 mL of 1 M Tris-HCl, pH 7.5

    Milli-Q or deionized water to 1 L

    Dilute to 1× before using

  9. 2 mg/mL HA Peptide

    HA Synthetic Peptide was reconstituted in 1× TBS to a concentration of 2 mg/mL.

Acknowledgments

We thank Dan Goldberg for anti-PMV antibodies. This work was supported by awards from the ARCS Foundation to D.W.C., and the US National Institutes of Health to D.W.C. (T32AI060546) and to V.M. (R01AI130139).

Competing interests

The authors declare no conflicts of interest.

References

  1. Andricopulo, A. D., Akoachere, M. B., Krogh, R., Nickel, C., McLeish, M. J., Kenyon, G. L., Arscott, L. D., Williams, C. H., Jr., Davioud-Charvet, E. and Becker, K. (2006). Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents. Bioorg Med Chem Lett 16(8): 2283-2292.
  2. Allan, K. M., Loberg, M. A., Chepngeno, J., Hurtig, J. E., Tripathi, S., Kang, M. G., Allotey, J. K., Widdershins, A. H., Pilat, J. M., Sizek, H. J., et al. (2016). Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker. Free Radic Biol Med 101: 356-366.
  3. Araki, K., Ushioda, R., Kusano, H., Tanaka, R., Hatta, T., Fukui, K., Nagata, K. and Natsume, T. (2017). A crosslinker-based identification of redox relay targets. Anal Biochem 520: 22-26.
  4. Becker, K., Gromer, S., Schirmer, R. H. and Müller, S. (2000). Thioredoxin reductase as a pathophysiological factor and drug target: Thioredoxin reductase in medicine and parasitology. Eur J Biochem 267 (20): 6118-25.
  5. Biddau, M., Bouchut, A., Major, J., Saveria, T., Tottey, J., Oka, O., van-Lith, M., Jennings, K. E., Ovciarikova, J., DeRocher, A., et al. (2018). Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii. PLoS Pathog 14(2): e1006836.
  6. Biddau, M. and Sheiner, L. (2019). Targeting the apicoplast in malaria. Biochem Soc Trans 47(4): 973-983.
  7. Cobb, D. W., Kudyba, H. M., Villegas, A., Hoopmann, M. R., Baptista, R. P., Bruton, B., Krakowiak, M., Moritz, R. L. and Muralidharan, V. (2021). A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites. PLoS Pathog 17(2): e1009293.
  8. Jessop, C. E., Watkins, R. H., Simmons, J. J., Tasab, M. and Bulleid, N. J. (2009). Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins. J Cell Sci 122(Pt 23): 4287-4295.
  9. Krnajski, Z., Gilberger, T. W., Walter, R. D. and Muller, S. (2001). The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system. Mol Biochem Parasitol 112(2): 219-228.
  10. Kehr, S., Sturm, N., Rahlfs, S., Przyborski, J. M. and Becker, K. (2010). Compartmentation of redox metabolism in malaria parasites. PLoS Pathog 6(12): e1001242.
  11. Lu, J. and Holmgren, A. (2014). The thioredoxin superfamily in oxidative protein folding. Antioxid Redox Signal 21(3): 457-470.
  12. Naticchia, M. R., Brown, H. A., Garcia, F. J., Lamade, A. M., Justice, S. L., Herrin, R. P., Morano, K. A. and West, J. D. (2013). Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells. Chem Res Toxicol 26(3): 490-497.
  13. Oka, O. B., Pringle, M. A., Schopp, I. M., Braakman, I. and Bulleid, N. J. (2013). ERdj5 is the ER reductase that catalyzes the removal of non-native disulfides and correct folding of the LDL receptor. Mol Cell 50(6): 793-804.
  14. West, J. D., Stamm, C. E., Brown, H. A., Justice, S. L. and Morano, K. A. (2011). Enhanced toxicity of the protein cross-linkers divinyl sulfone and diethyl acetylenedicarboxylate in comparison to related monofunctional electrophiles. Chem Res Toxicol 24(9): 1457-1459.

简介

[摘要] 疟疾仍然是一个重大的公共卫生问题,每年感染近 2.2 亿人。恶性疟原虫耐药菌株在世界范围内的传播威胁到抗击这种疾病的进展。因此,确定恶性疟原虫寄生虫中的可药化和基本途径仍然是一个主要的研究领域。寄生虫生物学的一个鲜为人知的领域是二硫键的形成,这是许多蛋白质折叠的必要条件。具有硫氧还蛋白 ( Trx ) 结构域的专门伴侣催化氧化还原功能,这些功能是破坏蛋白质中不正确和形成正确二硫键所必需的。定义这些氧化还原伴侣的底物是困难的,并且基于免疫沉淀的测定不能区分底物和相互作用的伙伴。此外,底物或客户与氧化还原伴侣的相互作用通常是短暂的。基于活性的交联剂依赖于Trx结构域上的亲核半胱氨酸和客户端上的二硫键形成半胱氨酸,提供了一种易于扩展的方法来捕获和识别含有Trx结构域的伴侣的底物。细胞渗透性交联剂二乙烯基砜 (DVSF) 仅在蛋白质中存在亲核半胱氨酸的情况下才具有活性,因此,由于它们在催化活性过程中形成混合的二硫键,因此将Trx结构域与其底物一起捕获。这允许识别依赖Trx活性进行折叠的底物,以及发现干扰Trx结构域活性的小分子。

图文摘要:


二乙烯基砜交联和免疫沉淀质谱法鉴定硫氧还蛋白结构域底物。

关键词:疟原虫、硫氧还蛋白、蛋白质二硫键异构酶、蛋白质交联、氧化还原

【背景】二硫键对许多蛋白质的活性和调控至关重要;因此,二硫键的形成和还原是细胞生命的一个关键方面。硫氧还蛋白 ( Trx ) 超家族的成员使用其“CXXC”活性位点的亲核半胱氨酸来促进二硫键的形成和还原(Lu 和 Holmgren,2014 年)。鉴于它们对细胞存活的重要性,硫氧还蛋白结构域蛋白和硫氧还蛋白系统已被提议作为针对顶复门寄生虫的药物靶标(Becker等人,2000; Krnajski 等人,2001;安德里科普洛 等人,2006;克尔 等。 , 2010;比道 等人,2018 年;比道和希纳,2019; Cobb等人,2021 年)。尽管这些Trx域蛋白的底物是必不可少的并且它们具有作为药物靶点的潜力,但它们的底物通常是不明确的,因为很难从其他相互作用的伙伴中辨别出特定的底物。
Trx结构域底物的一种策略依赖于活性位点的作用模式:“ CXXC ”活性位点的第一个半胱氨酸与客户蛋白形成混合二硫键,第二个半胱氨酸的巯基用于解决该债券。第二个半胱氨酸的诱变,通常为丙氨酸或丝氨酸,可防止Trx结构域和客户蛋白之间的键减少,从而将两种蛋白质捕获在一起(Jessop等人,2007 年;Oka等人,2013 年)。随后,质谱可用于识别底物。这种方法的一个主要缺点是它需要修饰内源性酶的活性位点,这可能是必需的,或者突变酶的外源性表达,这两种酶都可能对生物体致命。特别是在恶性疟原虫等生物体中,其生命周期的大部分时间都是单倍体,并且携带必需的Trx域蛋白基因的单拷贝,这种方法可能特别困难。
鉴于遗传方法固有的挑战,基于活性的Trx结构域与其底物的化学交联是识别这些底物的直接且强大的选择。二乙烯基砜 (DVSF) 是一种双功能、亲电子和细胞可渗透的交联剂,显示出对亲核半胱氨酸具有显着的特异性,如Trx域活性位点中的那些(West等人,2011)。用 DVSF 处理细胞会导致氧化还原活性半胱氨酸之间发生不可逆的交联,它已被用于捕获和识别酵母和人类细胞中 Trx 结构域蛋白和其他氧化还原活性酶的底物( Naticchia 等。 , 2013;艾伦等人。 , 2016;荒木等人,2017)。我们使用 DVSF 来捕获和识别恶性疟原虫内质网 (ER) 中的Trx结构域蛋白底物,展示了这种基于活性的技术在非模式生物中识别Trx结构域底物的能力(Cobb等等人,2021 年)。我们还证明了它对氧化还原活性亲核半胱氨酸的特异性,因为其他含半胱氨酸的蛋白质不与 DVSF 反应,并通过将反应性半胱氨酸残基突变为丙氨酸残基证明交联消除进一步验证了我们的结果(Cobb等人, 2021)。在本协议中,我们描述了使用 DVSF 来捕获恶性疟原虫中 ER 定位的Trx域蛋白的底物,并通过质谱法识别这些被捕获的底物。鉴于该协议的稳健性,它将用于不同生物体和多个细胞隔间内的类似实验。因为这种方法可以用最少的基因修饰来使用,如果没有合适的抗体,只需向Trx域蛋白添加亲和标签,该协议将允许其他与困难生物体合作的人研究Trx之间的相互作用-域及其底物。

关键字:疟原虫, 硫氧还蛋白, 蛋白质二硫键异构酶, 蛋白质交联, 氧化还原

材料和试剂
1. 15 mL离心管(Genesee Scientific,目录号:28-103)
2. 1.7 mL微管( Geneesse Scientific,目录号:28-281)
3. 过滤器吸头移液器吸头:10 μL (Fisher Scientific,目录号: 02-707-473) 、200 μL (Fisher Scientific,目录号: 02-707-478) 、1 mL(Fisher Scientific,目录号:2707480)
4. 血清移液管:5 mL(Genesee Scientific,目录号:12-102),10 mL Genesee Scientific,目录号:12-104)
5. TPP 组织培养板,100 mm( MidSci ,目录号:TP92406)
6. O+ AS-1 包装细胞(州际血库,孟菲斯,田纳西州)
7. IRDye ® 800CW 抗小鼠(Li-Cor,目录号:926-32210,以 1:20,000 用于蛋白质印迹)
8. IRDye ® 680RD 抗兔(Li-Cor,目录号:926-68071,以 1:20,000 用于蛋白质印迹)
9. 兔抗HA抗体(Life Technologies,目录号:715500,以1:100用于蛋白质印迹)
10. 小鼠抗 PMV 抗体(来自华盛顿大学医学院 Daniel Goldberg 的礼物,用于蛋白质印迹的 1:400)
11. V5-Tag (D3H8Q) Rabbit mAb (Cell Signaling Tech, Inc.,目录号:13202S,以 1:1,000 用于蛋白质印迹)
12. 抗 HA 磁珠(Thermo Fisher Scientific/ Pierce TM ,目录号:88836)
13. 二乙烯基砜,97% 刺伤。含0.05%对苯二酚(Thermo Fisher Scientific/Alfa Aesar ,目录号: L12827)
14. 皂苷Quillaja sp.,皂苷元含量20-35%(Sigma,目录号: S4521-10G)
15. 蛋白质样品上样缓冲液,4 × (Li-Cor,目录号: 928-40004)
16. Chameleon Duo 预染色蛋白阶梯(Li-Cor,目录号:928-60000)
17. 2-巯基乙醇(Fisher Scientific,目录号:21985023)
18. 10 × Tris/甘氨酸/SDS缓冲液(Bio-Rad,目录号: 161-0732)
19. 4-20% Mini-PROTEAN ® TGX TM预制蛋白凝胶(Bio-Rad,目录号: 456-1093)
20. Intercept TM封闭缓冲液(Li-Cor,目录号: 927-70003)
21. Odyssey硝酸纤维素膜(Li-Cor,目录号: 926-31092 )
22. 停止蛋白酶抑制剂混合物(Thermo Fisher Scientific/Life Technologies,目录号: 78429)
23. HA合成肽(Thermo Fisher Scientific/Life Technologies,目录号: 26184)
24. Simply Blue SafeStain (Thermo Fisher Scientific/Life Technologies,目录号: LC6060)
25. Tris-HCl(Fisher Bioreagents,目录号: 1185-53-1)
26. KCl (Sigma,目录号: 7447-40-7)
27. EDTA(Fisher Bioreagents,目录号: 60-00-4)
28. NP-40(Sigma,目录号: 9002-93-1)
29. Na 2 HPO 4 (Millipore Sigma,目录号:1065861000)
30. KH 2 PO 4 (Millipore Sigma,目录号:529568)
31. NaCl(Fisher Scientific,目录号:BP358212)
32. TRIS 基础(Fisher Scientific,目录号:YBP152500)
33. 甘氨酸(Fisher Scientific,目录号:ALF-036435-A1)
34. 甲醇(Fisher Scientific,目录号:A4081)
35. Tween-20(Fisher Scientific,目录号:BP337-100)
36. 免疫沉淀提取缓冲液(见配方)
37. 免疫沉淀结合缓冲液(见配方)
38. 磷酸盐缓冲盐水 (PBS),pH 7.4(参见食谱)
39. 蛋白质加载染料(见食谱)
40. 10×传输缓冲区(见配方)
41. 1×传输缓冲区(见配方)
42. 0.1% Tween-20,在 1× PBS 中(见配方)
43. 10× Tris 缓冲盐水 (TBS)(参见食谱)
44. 2 mg/mL HA 肽(见配方)
设备
1. 索瓦尔TM Legend TM XTR 离心机(Thermo Fisher Scientific,型号: 75004506)
2. 奥德赛® CLx成像系统(Li-Cor,型号: 9140)
3. DynaMag TM -2 磁铁(Life Technologies,型号: 12321D)
4. Branson S450D 数字声纳器(Thermo Fisher Scientific,型号: 101-063-593)
5. Mini-PROTEAN Tetra 细胞系统(Bio-Rad,目录号:1658005EDU)
6. PowerPac TM基本电源(Bio-Rad,目录号:1645050)
7. Forma TM Series II 水套式 CO 2培养箱( ThermoFisher Scientific,目录号:3110)
软件
1. Image Studio 5.x CLx (Li-Cor, https: //www.licor.com/bio/image-studio-lite/ )


程序
A. 用 DVSF捕获Trx域底物
1. 用 DVSF 孵育细胞
a. 恶性疟原虫寄生虫并表达表位标记的Trx域蛋白的红细胞 (RBC)培养物转移到无菌 15 mL 锥形管中。作为起点,使用 10 mL 的培养物,血细胞比容为 2%,寄生虫血症为 10-15%。
b. 800 ×离心沉淀受感染的红细胞 g 3 分钟。
c. 去除上清液。
d. 使用血清移液器将红细胞重悬在等体积的 PBS 中制备的 3 mM DVSF 或单独的 PBS 作为对照(例如,沉淀 10 mL 培养物,并将细胞重悬在 10 mL 的 3 mM DVSF 或 PBS 中)。
e. 将细胞在 37 °C 下在 CO 2培养箱中孵育 30 分钟。
f. 800 ×离心沉淀细胞 g 3 分钟。
g. 去除上清液。
h. 处理DVSF 上清液。
i. 用 PBS 清洗细胞 3 次,使用等体积的 PBS(例如,如果使用 10 mL DVSF,则使用 10 mL PBS),并以 800 ×旋转 g 3 分钟。
2. 从细胞中提取蛋白质
a. 在 PBS 中制备 0.04% (w/v) 皂苷溶液并在冰上冷却,直到缓冲液冰冷。
b. 通过在 0.04% 皂苷中溶解红细胞来分离寄生虫。
i. mL沉淀的培养物在 1 mL冰冷的皂苷中重悬细胞(例如,在 2.5 mL的皂苷中重悬从 10 mL培养中沉淀的细胞)。
ii. 在冰上孵育 15 分钟。
iii. 以 2,000 ×离心沉淀寄生虫 g 5 分钟,在 4°C。
iv. 去除上清液并用冰冷的 PBS 洗涤两次,使用的 PBS 体积与步骤 A.2.a 中使用的皂苷体积相同,在 2 , 000 ×下进行旋转 g 5 分钟。
c. 在 50-100 μL的蛋白质加载染料中重新悬浮寄生虫。
d. 将寄生虫在 95°C 下煮沸 5 分钟。
e. 在室温下,在台式离心机上全速离心 10 分钟,使不溶性物质(包括寄生虫血红素)沉淀。
f. 将含有提取蛋白质的上清液转移到干净的试管中。
g. 通过蛋白质印迹证明成功的交联:与仅 PBS 的对照样品相比,DVSF 交联的样品应包含一条或多条更高分子量的条带,因为Trx结构域蛋白 + 底物在 SDS-PAGE 期间迁移速度比单独的Trx结构域蛋白(图 1 )。
i. 30-50 μL的蛋白质样品和蛋白质梯加载到聚丙烯酰胺凝胶中。
ii. 通过 SDS-PAGE 在 180 V 下分离 DVSF 交联和仅 PBS 样品中的蛋白质,持续约 45 分钟,或直到蛋白质梯的条带充分解析。
iii. 将蛋白质从凝胶转移到硝酸纤维素膜上,在 100 V 下 1 小时。
在涂抹到凝胶上之前在 1 ×转移缓冲液中预润湿来制备膜。
iv. mL的 50% Intercept TM封闭缓冲液(v/v,在 1 × PBS 中制备)在室温下轻轻摇动将膜封闭 1 小时。
v. 去除封闭缓冲液,加入 10 mL用上述 50% 封闭缓冲液制备的一抗溶液。
使用针对感兴趣的Trx结构域蛋白的一抗和上样对照。
vi. 在有盖的容器中,将膜在 4°C 的一抗溶液中孵育过夜,轻轻摇动。
vii. 取出一抗溶液,用 30 mL 的 0.1% Tween-20(v/v,在 1 × PBS 中制备)洗涤 3 次,在室温下轻轻摇晃 10 分钟。
viii. 加入 10 mL 在 50% 封闭缓冲液中制备的二抗溶液,如步骤 A.2.g.iv 中所述。多于。
ix. 在室温下,在有盖的容器中孵育 1 小时,轻轻摇动。
x. 取出二抗溶液,用 30 mL的 0.1% Tween-20(v/v,在 1 × PBS 中制备)洗涤 3 次,在室温下轻轻摇晃 10 分钟。
xi. mL的 1 × PBS清洗膜5 分钟,在室温下轻轻摇动。
xii. ®对膜进行成像 CLx成像系统可将蛋白质与感兴趣的Trx结构域蛋白质的交联可视化。


图 1. DVSF 交联将氧化还原伙伴捕获到恶性疟原虫 Trx结构域蛋白。
寄生虫在 37°C 下与 3 mM DVSF 孵育 30 分钟,然后通过蛋白质印迹提取和分析蛋白质。寄生虫表达表位标记的A.) Pf J2 或B.) Pf PDI8。 Plasmepsin V ( Pf PMV ) 用作上样对照,并用作非Trx结构域蛋白,证明了 DVSF 的特异性。
B. 免疫沉淀 具有捕获底物的Trx结构域蛋白
1. 从寄生虫中提取蛋白质
a. 使用 0.04% 皂苷执行 DVSF 交联和分离寄生虫,如上文 A 节所述;重要的是,包括 PBS-only 控制寄生虫。对于质谱蛋白质鉴定的免疫沉淀,我们从 1 × 10 9寄生虫开始,并根据需要放大或缩小。
b. 将寄生虫颗粒重新悬浮在 300 μL的提取缓冲液中,然后加入 15 μL的 10% NP-40 和 3 μL的 HALT 蛋白酶抑制剂。
c. 在冰上孵育 15 分钟。
d. 通过超声裂解寄生虫。
i. 以 10% 的振幅对寄生虫进行 5 秒的声波处理。
ii. 让样品在冰上停留 30 秒。
iii. 重复步骤 B.1.di 和 -ii 两次,共进行三轮超声处理。
e. 在设置为 4°C 的台式离心机上全速离心裂解物 15 分钟。
f. 将含有提取蛋白质的上清液转移到新试管中。
g. 对步骤 B.1.e 中的颗粒进行第二次提取。
i. 将 300 μL的提取缓冲液和 3 μL的 HALT 蛋白酶抑制剂添加到颗粒中(第二次提取时不使用 NP-40)并立即进行超声步骤,无需在冰上孵育 15 分钟。
ii. 如步骤 B.1.ae 中所述进行超声处理和离心。
h. 将上清液与从步骤 B.1.f 中提取的蛋白质混合。
i. 将 60 μL混合上清液转移到干净的试管中作为“输入”样品并储存在 -20°C。
2. 免疫沉淀 HA 标签蛋白
a. 准备 Pierce 抗 HA 磁珠。
i. 将 25 μL的珠浆转移到干净的 1.5 mL离心管中。
ii. 加入 175 μL结合缓冲液,轻弹试管进行混合,然后短暂离心以收集试管底部的所有液体。
iii. 将试管插入磁架,待珠子收集到试管一侧后取出缓冲液。
iv. 取出管子,在珠子中加入 1 mL的结合缓冲液。
v. 旋转您手中的管子以混合珠子和缓冲液,持续 1 分钟。
vi. 使用磁性架从珠子中取出缓冲液。
b. 将步骤 B.1.h 中提取的蛋白质添加到珠子中。
c. 在室温下颠倒旋转孵育 30 分钟(或在 4°C 下过夜)。
d. 将管子短暂离心以将所有液体收集到底部,然后使用磁力架将未结合的蛋白质样品从珠子中取出;将此样品保存在标有“未绑定”的试管中。
e. 用结合缓冲液清洗珠子 3 次,如有必要,将每次清洗的缓冲液保存起来用于下游分析。
i. 将珠子重新悬浮在 300 μL的结合缓冲液中,并使用磁架从珠子中取出缓冲液。
ii. 再重复步骤 B.2.ei 两次,总共洗涤 3 次。
f. 使用 HA 肽从珠子中洗脱蛋白质。
i. 将珠子重新悬浮在 30 μL的 2 mg/mL HA 肽中。
ii. 在 37°C 下摇动孵育 5 分钟。
g. 使用磁力架将洗脱的蛋白质从珠子中分离出来,并将洗脱的蛋白质保存在标有“洗脱”的管中。
C. 从丙烯酰胺凝胶中提取交联蛋白用于蛋白鉴定
1. 在上述步骤 B.2.g 中收集的5 μL蛋白质梯和免疫沉淀蛋白质的“洗脱”样品装入聚丙烯酰胺凝胶的孔中。
2. 通过 SDS-PAGE 在 180 V 下将样品中的蛋白质分离约 45 分钟,或直到蛋白质阶梯充分解析。
3. 用 SimplyBlue SafeStain或其他考马斯染料染色凝胶中的蛋白质(图 2 )。
a. mL去离子水冲洗凝胶 3 次。
b. 添加大约 20 mL的 SimplyBlue SafeStain或足够的染料以完全覆盖凝胶,并在室温下孵育 1 小时,同时轻轻摇动。
c. 去除污渍并在室温下用 100 mL去离子水清洗凝胶 1 小时,同时轻轻摇动。
d. 1 小时后,加入 20 mL的 20% NaCl(w/v,在去离子水中制备)并在室温下轻轻摇动继续孵育 2 小时或过夜。


图 2. DVSF 交联蛋白在提取之前对质谱蛋白进行鉴定的考马斯染色。
将表达表位标记的Pf J2 的寄生虫与 3 mM DVSF 在 37°C 下孵育 30 分钟,然后从寄生虫裂解物中免疫沉淀Pf J2。使用 SDS-PAGE 分离免疫沉淀的蛋白质并用 SimplyBlue SafeStain染色。黑色虚线框表示为蛋白质鉴定而提取的 DVSF 处理泳道中的凝胶区域。红色虚线框表示在 PBS 处理的泳道中提取的凝胶区域,作为蛋白质鉴定的对照。
4. 使用干净的剃须刀片,从凝胶中提取交联蛋白条带(即,提取凝胶中比非交联蛋白高的条带)。
5. 从仅含 PBS 的对照样品中凝胶的等效区域提取切片(例如,在仅含 PBS 的样品中提取一条 100 kDa的交联蛋白条带,并在 100 kDa处提取等量大小的凝胶片) .
6. 将凝胶切片转移到 1.5 mL离心管中。
7. 对凝胶切片中的蛋白质进行脱色。
用 30% 乙醇(v/v,在去离子水中制备)孵育 15 分钟,或直到凝胶切片不再染色,在室温下轻轻旋转。
8. 识别每个凝胶切片中存在的蛋白质,无论是在内部还是通过质谱服务。
9. 通过从 DVSF 交联样品中识别的蛋白质中消除仅 PBS 凝胶切片中识别的蛋白质,识别潜在的Trx结构域底物。
笔记
我们描述了使用 DVSF 来捕获、提取和识别恶性疟原虫寄生虫中的Trx域底物,但该协议的组件很容易适应其他生物体。因此,蛋白质提取、免疫沉淀和蛋白质印迹的细节应适应个别研究人员对其特定生物体的工作流程。
食谱
1. 免疫沉淀提取缓冲液
40 mM Tris-HCl pH 7.4
150 毫米氯化钾
1 M 乙二胺四乙酸
1 × HALT 蛋白酶抑制剂
0.5% NP-40 (v/v)
2. 免疫沉淀结合缓冲液
20 mM Tris-HCl pH 7.4
150 毫米氯化钾
1 毫米乙二胺四乙酸
1 × HALT 蛋白酶抑制剂
0.1% NP-40 (v/v)
3. 磷酸盐缓冲盐水 (PBS),pH 7.4
3.2 毫米钠2 HPO 4
0.5 毫米 KH 2 PO 4
1.3 毫米氯化钾 
135 毫米氯化钠
4. 蛋白质负载染料
120 μL 4 × Li-Cor 蛋白上样缓冲液
20 μL 2-巯基乙醇
5. 10 ×传输缓冲器
30.3克TRIS基础
144.1克甘氨酸
用 Milli-Q 或去离子水加至 1 L。
6. 1 ×传输缓冲器
100 mL 10 × Transfer Buffer
200 毫升甲醇
700 毫升 Milli-Q 或去离子水
7. 0.1% Tween-20,在 1 × PBS中
将 1 mL 的 Tween-20 添加到 1 L 的 1 × PBS中
8. 10 × Tris 缓冲盐水 (TBS)
87.7 克氯化钠
100 mL 1 M Tris-HCl,pH 7.5
Milli-Q 或去离子水至 1 L
使用前稀释至1 ×
9. 2 毫克/毫升 HA 肽
HA 合成肽在 1 × TBS 中重组为 2 mg/ mL 的浓度。 
致谢
我们感谢 Dan Goldberg 提供的抗 PMV 抗体。这项工作得到了 ARCS 基金会对 DWC 以及美国国立卫生研究院对 DWC (T32AI060546) 和 VM (R01AI130139) 的奖励的支持。
利益争夺
作者宣称没有利益冲突。
参考
1. Andricopolo , AD, Akoachere , MB, Krogh, R., Nickel, C., McLeish, MJ, Kenyon, GL, Arscott, LD, Williams, CH, Jr., Davioud-Charvet , E. 和 Becker, K. (2006 )。恶性疟原虫硫氧还蛋白还原酶的特异性抑制剂作为潜在的抗疟药。 Bioorg Med Chem Lett 16(8):2283-2292。
2. Allan,KM, Loberg ,MA, Chepngeno ,J., Hurtig ,JE,Tripathi,S.,Kang,MG, Allotey ,JK,Widdershins,AH, Pilat ,JM, Sizek ,HJ,等。 (2016 年)。用一种小的硫醇反应性交联剂捕获对氧化剂敏感的蛋白质中的氧化还原伙伴关系。 自由基生物学杂志101:356-366 。
3. Araki, K.、 Ushioda , R.、 Kusano , H.、Tanaka, R.、Hatta, T.、Fukui, K.、Nagata, K. 和Natsume , T. (2017)。基于交联剂的氧化还原中继目标识别。 肛门生物化学520:22-26。
4. Becker, K., Gromer , S., Schirmer, RH 和 Müller, S. (2000)。硫氧还蛋白还原酶作为病理生理因素和药物靶点:硫氧还蛋白还原酶在医学和寄生虫学中的应用。 Eur J Biochem 267 (20): 6118-25。
5. Biddau , M., Bouchut , A., Major, J., Saveria , T., Tottey , J., Oka, O., van-Lith, M., Jennings, KE, Ovciarikova , J., DeRocher , A.等。 _ (2018 年)。两种必需的硫氧还蛋白在弓形虫中介导顶体生物发生、蛋白质输入和基因表达。公共科学图书馆 病原体14(2):e1006836。
6. Biddau , M. 和Sheiner , L. (2019)。针对疟疾中的顶端质体。 Biochem Soc Trans 47(4):973-983。
7. Cobb, DW, Kudyba , HM, Villegas, A., Hoopmann , MR, Baptista, RP, Bruton, B., Krakowiak, M., Moritz, RL 和 Muralidharan, V. (2021)。一种氧化还原活性交联剂揭示了疟原虫内质网中一个重要且可抑制的氧化折叠网络。 公共科学图书馆 病原体17(2):e1009293。
8. Jessop, CE, Watkins, RH, Simmons, JJ, Tasab , M. 和Bulleid , NJ (2009)。蛋白质二硫化物异构酶家族成员表现出独特的底物特异性:P5 靶向 BiP 客户蛋白。 J 细胞科学122(Pt 23):4287-4295。
9. Krnajski , Z., Gilberger , TW, Walter, RD 和 Muller, S. (2001)。疟原虫恶性疟原虫具有功能性硫氧还蛋白系统。摩尔生化 寄生虫112(2):219-228。
10. Kehr , S., Sturm, N., Rahlfs , S., Przyborski, JM 和 Becker, K. (2010)。疟原虫氧化还原代谢的划分。 公共科学图书馆 病原体6(12):e1001242。
11. Lu, J. 和 Holmgren, A. (2014)。氧化蛋白质折叠中的硫氧还蛋白超家族。 抗氧化氧化还原信号21(3):457-470。
12. Naticchia ,MR,Brown,HA,Garcia,FJ, Lamade ,AM,Justice,SL,Herrin,RP, Morano ,KA 和 West,JD(2013 年)。双功能亲电子试剂将硫氧还蛋白与细胞中的氧化还原中继伙伴交联。 Chem Res Toxicol 26(3):490-497。
13. Oka, OB, Pringle, MA, Schopp , IM, Braakman , I. 和Bulleid , NJ (2013)。 ERdj5 是催化去除非天然二硫化物和正确折叠 LDL 受体的 ER 还原酶。摩尔细胞50(6):793-804。
14. West, JD, Stamm , CE, Brown, HA, Justice, SL 和Morano , KA (2011)。与相关的单功能亲电子试剂相比,蛋白质交联剂二乙烯基砜和乙炔二羧酸二乙酯的毒性增强。 Chem Res Toxicol 24(9):1457-1459。
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引用:Cobb, D. W., Woods, G. S. and Muralidharan, V. (2022). Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteins in Malaria Parasites. Bio-protocol 12(4): e4322. DOI: 10.21769/BioProtoc.4322.
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