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Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
通过代谢标记和脉冲追踪分析监测组织蛋白酶D对溶酶体的靶向作用   

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Journal of Cell Science
Jan 2017

Abstract

Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.

Keywords: Acid hydrolases (酸性水解酶), Cathepsin D (组织蛋白酶D), Mannose 6-phosphate receptors (6-磷酸甘露糖受体), MPR (MPR), Secretory pathway (分泌途径), Lysosomes (溶酶体), Metabolic labeling (代谢标签), Pulse-chase (脉冲追踪)

Background

Cathepsin D (catD) is a lysosomal aspartic protease sorted by Mannose 6-phosphate receptors (M6PRs) that transport it from the trans-Golgi network to endosomes/lysosomes in mammalian cells (Ghosh et al., 2003). CatD is synthesized as a precursor protein (~52 kDa), which is cleaved in lysosomes to generate an intermediate (~48 kDa) or the mature lysosomal form (~34 kDa). Trace amounts of the precursor protein are also secreted from the biosynthetic pathway (Benes et al., 2008). The abundance of catD can be determined using several approaches, such as immunofluorescence-based staining (Poole et al., 1972), Western blotting, fluorometric activity assay (Bewley et al., 2011) or metabolic labeling with pulse-chase analysis (Hirst et al., 2009; Kametaka et al., 2007; Tavares et al., 2017). The later is considered a highly sensitive and quantitative approach to monitor catD dynamics (post-translational processing, secretion and degradation) through the secretory pathway. Specifically it involves metabolically labeling newly synthesized proteins in cells followed by a chase, and then to immunoprecipitate catD from the cell lysate and media (secreted form). Therefore, this method provides a way to follow catD molecules from synthesis to lysosomal targeting or secretion with minimal disturbance of normal cell physiology in its natural environment. Herein we described metabolic labeling with [35S] methionine-cysteine in HeLa cells to monitor catD processing, secretion and degradation by using immunoprecipitation, SDS-PAGE and fluorography.

Materials and Reagents

  1. Filter pipette tips
  2. 6-well plate (Corning, catalog number: 3516 )
  3. Ice bucket
  4. Aluminum foil
  5. 1.5 ml micro-centrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  6. WhatmanTM 3030-347 Grade 3 MM Chr cellulose chromatography paper sheet (GE Healthcare, catalog number: 3030-347 )
  7. Minisart filters pore size 0.22 μm (Sartorius, catalog number: 16534-K )
  8. HeLa (ATCC, catalog number: CCL-2 )
  9. 10x PBS (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9624 )
  10. Penicillin-streptomycin solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
  11. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 12657029 )
  12. Dulbecco’s modified Eagle’s medium-high glucose-without L-methionine, L-cysteine and L-glutamine (Sigma-Aldrich, catalog number: D0422 )
  13. L-Methionine (Sigma-Aldrich, catalog number: M9625 )
  14. L-Cysteine (Sigma-Aldrich, catalog number: 168149 )
  15. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  16. EasyTagTM EXPRESS 35S protein labeling mix of both 35S-L-methionine and 35S-L-cysteine (Express Protein Label) (PerkinElmer, catalog number: NEG772007MC )
  17. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  19. Ethylenediamine tetraacetic acid (EDTA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  20. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  21. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  22. Protein A plus Ultralink resin (Thermo Fisher Scientific, catalog number: 53142 )
  23. 10% (v/v) bovine serum albumin (BSA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15561020 )
  24. Cathepsin D antibody (EMD Millipore, Calbiochem, catalog number: 219361 )
  25. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030149 )
  26. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  27. Glycerol (C3H8O3) (Sigma-Aldrich, catalog number: G5516 )
  28. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  29. Methanol (CH3OH) (Sigma-Aldrich, catalog number: 322415 )
  30. Acetic acid (CH3COOH) (Sigma-Aldrich, catalog number: 320099 )
  31. Amplify fluorographic reagent (GE Healthcare, catalog number: NAMP100 )
  32. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3701 )
  33. Dulbecco’s modified eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12800017 ) (see Recipes)
  34. 100x L-methionine/L-cysteine solution (see Recipes)
  35. Pulse medium (see Recipes)
  36. Chase medium (see Recipes)
  37. Lysis buffer (see Recipes)
  38. Wash buffer (see Recipes)
  39. 2x sample buffer (see Recipes)
  40. Fixation solution (see Recipes)
  41. Running buffer (see Recipes)

Equipment

  1. Pipettes
  2. Incubator
  3. Titer plate shaker (Thermo Fisher Scientific, catalog number: 4625Q )
  4. Centrifuge for 1.5 ml micro-centrifuge tubes (Thermo Fisher Scientific, model: SorvallTM ST 16 , catalog number: 75004380) with rotor for micro-centrifuge tubes (Thermo Fisher Scientific, catalog number: 75003652 )
  5. Tube rotator capable of end-over-end inversion of the tubes (Phoenix Luferco, catalog number: AP22 or equivalent)
  6. A chamber to run mini-gels (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell )
  7. Gel-dryer (Hoefer, model: GD2000 , Slab Gel Dryer) coupled with a vacuum pump
  8. Exposure cassette for unmounted screen, 20 x 25 cm (GE Healthcare, catalog number: 63-0035-44 )
  9. Pharos FX plus molecular imager (Bio-Rad Laboratories, catalog number: 1709450 )
  10. BAS storage phosphor screen (GE Healthcare, catalog number: 28-9564-82 )
  11. Appropriate receptacle to dispose of solid and liquid contaminated with 35S (according to the local radiation safety guidelines)

Software

  1. Quantity One 1-D Analysis Software (Bio-Rad Laboratories)

Procedure

  1. Radioactive pulse-chase
    1. Plate 5 x 105 HeLa CCL-2 cells/well (6-well plate) in 2 ml of complete DMEM and incubate for 24 h/37 °C/5% CO2 (Figure 1). Use one well for each time-point or condition.
    2. Tilt plate and aspirate media, wash cells twice with 1 ml pre-warmed (room temperature) PBS.
    3. Incubate the cells with 700 µl pre-warmed (37 °C) pulse medium (-Cys/-Met; supplemented with 10 µl/ml 100x L-glutamine, see Recipes) for 30 min/37 °C/5% CO2. Remember to check your local radiation safety guidelines. From this step on, the waste has to be collected separately and discarded according to your local safety guidelines.
    4. Transfer the plate to an ice bucket covered with aluminum foil.
    5. Remove the medium and add 700 µl/well ice-cold pulse medium (+Cys 35S/+Met 35S).
    6. Incubate for 15 min/37 °C/5% CO2.
      The length of time for pulse depends on the rate of catD biosynthesis, which may vary according to the cell line. For HeLa cells, 15 min/37 °C is adequate. For other cell lines, the user may have to optimize the time of the pulse.
    7. Transfer the plate to an ice bucket covered with aluminum foil.
    8. Tilt plate and aspirate media, wash cells twice with 1 ml ice-cold PBS.
    9. Add 800 µl pre-warmed (37 °C) chase medium (see Recipes).
    10. Incubate the cells for 5 h/37 °C/5% CO2.
    11. Transfer the plate to an ice bucket covered with aluminum foil.
    12. Transfer the culture media (hereafter called ‘Media’ sample) to micro-centrifuge tubes and place them on ice.
    13. Wash the cells twice with 1 ml ice-cold PBS.
    14. Remove PBS and add 800 µl ice-cold lysis buffer/well (supplemented with 1:100 protease inhibitor cocktail fleshly prior to use, see Recipes).
    15. Transfer the plate to an ice bucket and incubate for 15 min under gentle agitation on a titer plate shaker.
    16. After 15 min. incubation, gently homogenize the cell lysates (hereafter called ‘Cell lysate’ sample) on each well using a micropipette tip and transfer the lysates to micro-centrifuge tubes.
    17. Centrifuge the samples (Cell lysate or Media, see steps A12 and A16) for 20 min at 18,000 x g (or for 30 min at 14,000 x g) 4 °C.
    18. Transfer equal volume of cleared supernatant (~750 µl) from the tubes (cleared Cell lysate or Media samples) to new micro-centrifuge tubes, put them on ice and discard the pellets.
      1. Carryover of sedimented material into the immunoprecipitation steps may result in high nonspecific background.
      2. If necessary, the cleared Cell lysate and Media samples can be frozen at -80 °C until used for immunoprecipitation. However, it is preferable to harvest cells (from step A13) and freeze the cell pellet rather than the cell lysate (step A17) to avoid protein degradation.


        Figure 1. Schematic overview illustrating the metabolic labeling and pulse-chase analysis of Cathepsin D

  2. Immunoprecipitation
    1. Wash beads (30 μl of 50% Protein-A Ultralink resin/aqueous slurry per chase-time/condition point) 3 times with 500 μl of ice-cold PBS by centrifugation for 1 min at 3,000 x g, 4 °C (Figure 1). This step removes the aqueous slurry containing 0.02% sodium azide supplied with the beads, which might interfere with the antibody binding. Make sure to leave the initial volume of beads/slurry in the tube (~30 μl per chase-time/condition point).
      1. For large amounts of beads (more than 500 μl), a 15 ml conical polystyrene centrifuge tube may be used and washes are done with at least 1:3 volume of beads-slurry/PBS.
      2. It is important to start with around 10-20% extra volume of beads to compensate for loss during the wash and pipetting.
    2. After washing, resuspend 30 μl of beads (in 50% PBS slurry) in 500 μl of ice-cold PBS containing 5 μl of 10% (v/v) BSA and 2 μl Cathepsin D antibody. Mix the suspension thoroughly.
      When working with multiple samples, it is recommended to prepare a beads/antibody ‘master mix’. This is done by increasing the quantities indicated above proportionally to the number of samples, and incubating the suspension in a 15 ml conical polystyrene centrifuge tube.
    3. Incubate for 2-3 h/4 °C under slow rotation on a tube rotator (capable of end-over-end inversion). During this step, the antibody will bind to the beads.
    4. Pellet the beads by centrifugation for 1 min at 3,000 x g, 4 °C and remove the supernatant.
    5. Wash the beads 3 times with 1 ml ice-cold lysis buffer by repeating step B4.
    6. After the final washing step, centrifuge the tubes containing the beads and remove the supernatant making sure to leave the initial volume of beads/slurry in the tube (~30 μl per chase-time/condition point).
    7. For each immunoprecipitation sample, incubate in a microcentrifuge tube: 30 μl beads/antibody (50% PBS slurry), 750 µl of cleared Cell lysate or Media, sample, 10 μl 10% BSA and 200 μl lysis buffer (supplemented with 1:100 protease inhibitor cocktail).
    8. Incubate for 2-3 h/4 °C under slow rotation on a tube rotator.
    9. After the incubation step, centrifuge the tubes, remove the supernatant and wash the beads 3 times each with 1 ml wash buffer (see Recipes) and one final wash step with 1 ml 1x PBS.
    10. Remove all PBS from the microcentrifuge tubes and resuspend the beads in 20 µl of 2x sample buffer (see Recipes). At this point, samples can be stored at -80 °C.

  3. SDS-PAGE and fluorography
    1. Boil the samples for 5 min/95 °C.
    2. Spin the tubes and load the supernatant onto SDS-PAGE mini-gels (10%; 1 mm gels). Here you can also load a molecular weight standard on the gel.
    3. Run the electrophoresis for ~2 h at constant 120 V in running buffer (see Recipes). Note that running conditions and time will vary according to the electrophoresis system used.
    4. Fix gels with 25 ml/gel fixation solution (see Recipes) for 30 min at room temperature in a clean plastic container on titer plate shaker with slow shaking.
    5. Remove the fixation solution and incubate the gel with 25 ml/gel amplify fluorographic reagent for 30 min at room temperature on titer plate shaker with slow rotation.
    6. Dry gels onto chromatography paper in a gel dryer for 45 min/80 °C. Cover the gel with a plastic wrap to avoid sticking to the gel drier cover. The gel must be fully dehydrated (no longer sticky), 45 min at 80 °C is generally enough.
    7. Expose the BAS storage phosphor screen to the dried gel in an exposure cassette for generally 1 week at room temperature, protected from the light. The ideal exposure time will depend on the amount of labeled catD.
    8. Scan the screen using the Pharos FX plus molecular imager or equivalent and analyze the result by using the Quantity One 1-D Analysis Software (Bio-Rad Laboratories).

Data analysis

After revealing the screen, the image should show bands indicative of the precursor Cathepsin D (pro-catD, ~52 kDa), the intermediate form (int-catD, ~48 kDa) and the mature lysosomal form (m-catD, ~34 kDa) in cell lysates and traces of pro-catD only in the media/supernatant sample (Figure 2). Note that these molecular masses vary slightly in different cell types. You can analyze the alterations in lysosomal sorting of cathepsin D by densitometric quantification of the different catD species. One way is to compare the intracellular abundance of precursor and mature forms with the abundance of secreted precursor, as the ratio of secreted to total catD, or to compare the abundance of mature form with the total catD as a ratio (Hirst et al., 2009). The relative abundance of each catD species can also be normalized by the number of methionine and cysteine residues present in the corresponding species (Kametaka et al., 2007). You can check the number of methionine and cysteine residues predicted for each human catD species in Minarowska et al., 2008.


Figure 2. Representative image of a catD in vivo labeling/pulse-chase experiment. 24 h after plating into 6-well plates, HeLa cells were pulse-labeled with [35S] methionine-cysteine for 15 min at 37 °C, followed by incubation in chase buffer for 5 h at 37 °C. Cell lysate (lane 1) and Media (lane 2) samples were used for immunoprecipitation with an anti-cathepsin D antibody and the resulting immunoprecipitates were analyzed by SDS-PAGE and fluorography. The positions of pro-(pro-catD), intermediate (int-catD), and mature cathepsin D (m-catD) forms are indicated.

Recipes

  1. Dulbecco’s modified Eagle medium (DMEM) supplemented with 100 U of penicillin/ml and 0.1 mg/ml of streptomycin and10% (v/v) fetal bovine serum (FBS)
  2. 100x L-methionine/L-cysteine mix solution
    1. Dissolve 1.5 mg of both L-methionine and L-cysteine in 1 ml Milli-Q water
    2. Filter-sterilize in 0.22 µm syringe filter for storage
    3. Stored in a centrifuge tube at 4 °C
  3. Pulse medium
    Dulbecco’s modified Eagle’s medium methionine, cysteine and glutamine-free supplemented with:
    2 mM L-glutamine
    0.1 mCi/ml of [35S]-methionine-cysteine
  4. Chase medium
    Complete DMEM (Recipe 1) supplemented with 10 μl/ml of 100x L-methionine/L-cysteine mix solution
  5. Lysis buffer (store at 4 °C)
    50 mM Tris-HCl (pH 7.4)
    300 mM NaCl
    5 mM EDTA pH 8
    0.5% (v/v) Triton X-100
  6. Wash buffer (store at 4 °C)
    50 mM Tris-HCl (pH 7.4)
    300 mM NaCl
    5 mM EDTA pH 8
    0.1% (v/v) Triton X-100
  7. 2x sample buffer
    20 ml of 10% (v/v) SDS
    16 ml of 0.5 M Tris-HCl pH 6.8
    10 ml glycerol
    0.1% bromophenol blue
    4% β-mercaptoethanol
    Milli-Q water
    Note: This solution can be stored at room temperature but add β-mercaptoethanol fresh before use.
  8. Fixation solution
    50% methanol
    10% acetic acid
  9. Running buffer
    30 g Tris
    144 g glycine
    10 g SDS
    Complete with Milli-Q water for 1 L

Acknowledgments

This protocol has been adapted from a previously published paper (Tavares et al., 2017). This work was supported by the São Paulo Research Foundation (FAPESP) grant (2014/25812-0) to LLPdS and a PhD scholarship (2016/18207-9) to LAT. The authors declare no conflict of interest.

References

  1. Benes, P., Vetvicka, V. and Fusek, M. (2008). Cathepsin D--many functions of one aspartic protease. Crit Rev Oncol Hematol 68(1): 12-28.
  2. Bewley, M. A., Pham, T. K., Marriott, H. M., Noirel, J., Chu, H. P., Ow, S. Y., Ryazanov, A. G., Read, R. C., Whyte, M. K., Chain, B., Wright, P. C. and Dockrell, D. H. (2011). Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae. Mol Cell Proteomics 10(6): M111 008193.
  3. Ghosh, P., Dahms, N. M. and Kornfeld, S. (2003). Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol 4(3): 202-212.
  4. Hirst, J., Sahlender, D. A., Choma, M., Sinka, R., Harbour, M. E., Parkinson, M. and Robinson, M. S. (2009). Spatial and functional relationship of GGAs and AP-1 in Drosophila and HeLa cells. Traffic 10(11): 1696-1710.
  5. Kametaka, S., Moriyama, K., Burgos, P. V., Eisenberg, E., Greene, L. E., Mattera, R. and Bonifacino, J. S. (2007). Canonical interaction of cyclin G associated kinase with adaptor protein 1 regulates lysosomal enzyme sorting. Mol Biol Cell 18(8): 2991-3001.
  6. Minarowska, A., Gacko, M., Karwowska, A. and Minarowski, L. (2008). Human cathepsin D. Folia Histochem Cytobiol 46(1): 23-38.
  7. Poole, A. R., Dingle, J. T. and Barrett, A. J. (1972). The immunocytochemical demonstration of cathepsin D. J Histochem Cytochem 20(4): 261-265.
  8. Tavares, L. A., da Silva, E. M., da Silva-Januario, M. E., Januario, Y. C., de Cavalho, J. V., Czernisz, E. S., Mardones, G. A. and daSilva, L. L. (2017). CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking. J Cell Sci 130(2): 429-443.

简介

通过研究其配体组织蛋白酶D的加工和分泌的改变,可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定,并且包括新合成的蛋白质的代谢标记,其中[35 [S]甲硫氨酸半胱氨酸,以监测组织蛋白酶D处理,通过分泌途径和分泌使用免疫沉淀,SDS-PAGE和荧光。

【背景】组织蛋白酶d(CATD)是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体(M6PRs)排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体(戈什等人,2003 )。将CatD合成为前体蛋白(〜52kDa),其在溶酶体中被切割以产生中间体(〜48kDa)或成熟的溶酶体形式(〜34kDa)。微量的前体蛋白质也从生物合成途径分泌(Benes et al。,2008)。 catD的丰度可以使用几种方法来确定,例如基于免疫荧光的染色(Poole等人,1972),蛋白质印迹,荧光活性测定(Bewley等人, (Hirst等人,2009; Kametaka等人,2007; Tavares等人,2011)或用脉冲追踪分析进行代谢标记(Hirst等人,2009; Kametaka等人, 2017)。后者被认为是通过分泌途径监测catD动态(翻译后加工,分泌和降解)的高度敏感和定量的方法。具体来说,它涉及在细胞中代谢标记新合成的蛋白质,然后追踪,然后从细胞裂解物和培养基(分泌形式)免疫沉淀catD。因此,这种方法提供了一种方法来跟踪catD分子从合成到溶酶体靶向或分泌,在自然环境中对正常细胞生理学的干扰最小。在此我们描述了在HeLa细胞中用[35 S]甲硫氨酸 - 半胱氨酸进行代谢标记,以通过使用免疫沉淀,SDS-PAGE和荧光成像来监测catD处理,分泌和降解。

关键字:酸性水解酶, 组织蛋白酶D, 6-磷酸甘露糖受体, MPR, 分泌途径, 溶酶体, 代谢标签, 脉冲追踪

材料和试剂

  1. 过滤移液枪头
  2. 6孔板(Corning,目录号:3516)
  3. 冰桶
  4. 铝箔
  5. 1.5ml微量离心管(Corning,Axygen,目录号:MCT-150-C)
  6. Whatman TM 3030-347 Grade 3 MM Chr纤维素层析纸(GE Healthcare,目录号:3030-347)
  7. Minisart过滤孔径0.22μm(Sartorius,目录号:16534-K)
  8. (ATCC,目录号:CCL-2)
  9. 10x PBS(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9624)
  10. 青霉素 - 链霉素溶液(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
  11. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:12657029)
  12. Dulbecco改良的Eagle's中高葡萄糖 - 不含L-甲硫氨酸,L-半胱氨酸和L-谷氨酰胺(Sigma-Aldrich,目录号:D0422)
  13. L-甲硫氨酸(Sigma-Aldrich,目录号:M9625)
  14. L-半胱氨酸(Sigma-Aldrich,目录号:168149)
  15. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  16. EasyTag TM 35 SL蛋氨酸和35 SL-半胱氨酸(Express蛋白质)的35S蛋白质标记混合物标签)(PerkinElmer,目录号:NEG772007MC)
  17. Trizma base(Sigma-Aldrich,目录号:T1503)
  18. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  19. 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  20. Triton X-100(Sigma-Aldrich,目录号:T8787)
  21. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  22. 蛋白A加上Ultralink树脂(Thermo Fisher Scientific,目录号:53142)
  23. 10%(v / v)牛血清白蛋白(BSA)(Thermo Fisher Scientific,Invitrogen TM,目录号:15561020)
  24. 组织蛋白酶D抗体(EMD Millipore,Calbiochem,目录号:219361)
  25. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030149)
  26. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  27. 甘油(C 3 H 8 O 3)(Sigma-Aldrich,目录号:G5516)
  28. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  29. 甲醇(CH 3 OH)(Sigma-Aldrich,目录号:322415)
  30. 乙酸(CH 3 -COOH)(Sigma-Aldrich,目录号:320099)
  31. 放大荧光试剂(GE Healthcare,目录号:NAMP100)
  32. β-巯基乙醇(Sigma-Aldrich,目录号:M3701)
  33. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:12800017)(参见食谱)
  34. 100倍的L-蛋氨酸/ L-半胱氨酸溶液(见食谱)
  35. 脉冲媒介(见食谱)
  36. 追逐中等(见食谱)
  37. 裂解缓冲液(见食谱)
  38. 清洗缓冲液(见食谱)
  39. 2x样品缓冲液(见食谱)
  40. 固定解决方案(见食谱)
  41. 运行缓冲区(见食谱)

设备

  1. 移液器
  2. 孵化器
  3. 滴定板摇床(Thermo Fisher Scientific,目录号:4625Q)
  4. 用微型离心管(Thermo Fisher Scientific,目录号:75003652)的转子离心1.5ml微型离心管(Thermo Fisher Scientific,型号:Sorvall TM ST16,目录号:75004380) br />
  5. 管旋转器能够管端倒置(Phoenix Luferco,产品目录号:AP22或同类产品)
  6. 运行微型凝胶的室(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Cell)
  7. 凝胶干燥机(Hoefer,型号:GD2000,板式凝胶干燥机)与真空泵配套使用
  8. 用于未安装屏幕的曝光盒,20 x 25厘米(GE Healthcare,目录号:63-0035-44)
  9. Pharos FX plus分子成像仪(Bio-Rad Laboratories,目录号:1709450)
  10. BAS存储荧光屏(GE Healthcare,目录号:28-9564-82)
  11. 适当处理被35S污染的固体和液体的容器(根据当地的辐射安全指南)

软件

  1. Quantity One一维分析软件(Bio-Rad Laboratories)

程序

  1. 放射性脉冲追踪
    1. 在2ml完全DMEM中板/ 5×10 5 HeLa CCL-2细胞/孔(6孔板),孵育24小时/ 37℃/ 5%CO 2 / (图1)。每个时间点或条件使用一个井。
    2. 倾斜板和吸取介质,用1毫升预热(室温)PBS洗两次细胞。
    3. 用700μl预热(37℃)的脉冲培养基(-Cys / -Met;补充有10μl/ ml 100x L-谷氨酰胺,参见食谱)孵育细胞30分钟/ 37℃/ 5%CO 2 。请记得检查您当地的辐射安全指南。从这个步骤开始,废物必须根据您当地的安全指南单独收集和丢弃。
    4. 将板转移到覆盖着铝箔的冰桶。
    5. 去除培养基,加入700μl/孔冰冷的脉冲培养基(+ Cys 35 S / + Met 35S)。
    6. 孵育15分钟/ 37℃/ 5%CO 2。
      脉冲的时间长短取决于catD生物合成的速率,其可以根据细胞系而变化。对于HeLa细胞,15分钟/ 37°C就足够了。对于其他细胞系,用户可能必须优化脉冲的时间。
    7. 将板转移到覆盖着铝箔的冰桶。
    8. 倾斜板和吸取介质,用1毫升冰冷PBS洗两次细胞。
    9. 加入800μl预热(37℃)追逐培养基(见食谱)。
    10. 将细胞孵育5小时/ 37℃/ 5%CO 2。
    11. 将板转移到覆盖着铝箔的冰桶。
    12. 将培养基(以下称为“培养基”样品)转移到微量离心管中,并放置在冰上。
    13. 用1毫升冰冷的PBS洗细胞两次。
    14. 取出PBS并加入800μl冰冷的裂解缓冲液/孔(在使用前肉汁补充1:100蛋白酶抑制剂混合物,参见食谱)。
    15. 将平板转移到冰桶中,在滴定板摇床上温和搅拌下孵育15分钟。
    16. 15分钟后温育,使用微量移液管尖端将细胞裂解液(以下称为“细胞裂解液”样品)轻轻均质化并将裂解液转移到微量离心管中。
    17. 将样品(细胞裂解物或培养基,参见步骤A12和A16)在18,000×g(4℃,14000×gg)下离心20分钟。 />
    18. 将等体积的澄清上清液(约750μl)从管中(澄清的细胞裂解液或培养基样品)转移到新的微量离心管中,将它们放在冰上并丢弃颗粒。
      1. 沉淀物质进入免疫沉淀步骤可能会导致非特异性的高背景。
      2. 如有必要,可将澄清的细胞裂解液和培养基样品冷冻于-80°C直到用于免疫沉淀。然而,优选收获细胞(来自步骤A13)并冷冻细胞沉淀而不是细胞裂解物(步骤A17)以避免蛋白质降解。


        图1.图示组织蛋白酶D的代谢标记和脉冲追踪分析的示意图概述

  2. 免疫沉淀
    1. 用500μl冰冷的PBS通过在3000xg离心1分钟将珠子(30μl的50%蛋白-A Ultralink树脂/含水浆液每个追赶时间/条件点)洗涤3次, 4°C(图1)。该步骤除去含有0.02%叠氮化钠的含水浆液,其可能干扰抗体结合。确保在试管中留下初始体积的珠粒/浆液(每个追踪时间/条件点约30μl)。
      1. 对于大量珠子(超过500μl),可以使用15ml锥形聚苯乙烯离心管,并且用至少1:3体积的珠子浆液/ PBS进行洗涤。

      2. 。为了补偿洗涤和移液过程中的损失,开始使用大约10-20%额外量的珠子是很重要的
    2. 洗涤后,重悬在500μl含有5μl10%(v / v)BSA和2μl组织蛋白酶D抗体的冰冷的PBS中的30μl珠(在50%PBS浆液中)。彻底混合悬浮液。
      当处理多个样品时,建议制备珠/抗体“主混合物”。这是通过按照样品数量的比例增加上述数量,并将悬浮液在15ml锥形聚苯乙烯离心管中孵育来完成的。
    3. 在管旋转器上缓慢旋转下孵育2-3小时/ 4℃(能够颠倒颠倒)。在这个步骤中,抗体会结合珠子。
    4. 通过在4℃下3000g离心1分钟使珠粒团,并除去上清液。
    5. 用1ml冰冷的裂解缓冲液重复步骤B4洗珠3次。
    6. 在最后的洗涤步骤后,离心含有珠子的管并除去上清液,确保在试管中保留初始体积的珠/浆液(每个追踪时间/条件点约30μl)。
    7. 对于每个免疫沉淀样品,在微量离心管中孵育:30μl珠/抗体(50%PBS浆液),750μl澄清的细胞裂解物或培养基,样品,10μl10%BSA和200μl裂解缓冲液(补充有1:100蛋白酶抑制剂鸡尾酒)。

    8. 在缓慢旋转下在管子旋转器上孵育2-3小时/ 4℃
    9. 孵育步骤后,离心管,取出上清液,并用1毫升洗涤缓冲液(参见食谱)洗涤珠子3次,最后用1毫升1x PBS洗一次。
    10. 从微量离心管中取出所有的PBS,并重新悬浮在20μL的2x样品缓冲液(见食谱)的珠子。此时,样品可以保存在-80°C。

  3. SDS-PAGE和荧光成像
    1. 将样品煮沸5分钟/ 95°C。
    2. 旋转管并将上清液加载到SDS-PAGE微型凝胶(10%; 1mm凝胶)上。在这里,您还可以在凝胶上加载分子量标准。
    3. 在恒定的120V电泳缓冲液中运行约2小时的电泳(见食谱)。请注意,运行条件和时间会根据使用的电泳系统而有所不同。

    4. 在一个干净的塑料容器中,在室温下用25ml /凝胶固定溶液(见食谱)固定凝胶30分钟,
    5. 去除固定溶液,并在室温下在具有缓慢旋转的滴定板振荡器上用25ml /凝胶放大荧光试剂孵育凝胶30分钟。
    6. 在凝胶干燥器上将凝胶干燥到层析纸上45分钟/ 80℃。用保鲜膜覆盖凝胶,以避免粘在凝胶干燥器盖上。
      。凝胶必须完全脱水(不再有粘性),在80℃45分钟就足够了。
    7. 将BAS存储荧光屏暴露在暴露盒中的干燥凝胶上,在室温下通常保持1周,避光。理想的曝光时间将取决于标记的猫的数量。
    8. 使用Pharos FX plus分子成像仪或同等产品扫描屏幕,并使用Quantity One一维分析软件(Bio-Rad Laboratories)分析结果。

数据分析

在显示筛选后,图像应该显示指示前体组织蛋白酶D(pro-catD,〜52kDa),中间体形式(int-catD,〜48kDa)和成熟的溶酶体形式(m-catD,〜34 kDa)和仅在培养基/上清液样品中的痕量pro-catD(图2)。请注意,这些分子量在不同细胞类型中略有不同。您可以通过不同的catD物种的密度定量分析组织蛋白酶D的溶酶体分选中的改变。一种方法是比较前体和成熟形式的细胞内丰度与分泌前体的丰度,作为分泌总量与总catD的比率,或比较成熟形式的丰度与总catD作为比率(Hirst等人,2009)。每个catD物种的相对丰度也可以通过存在于相应物种中的甲硫氨酸和半胱氨酸残基的数量来标准化(Kametaka等人,2007)。您可以检查2008年Minarowska等人每种人类catD物种预测的蛋氨酸和半胱氨酸残基的数量。


图2.体内标记/脉冲追踪实验的代表性图像在6孔板中铺板24小时后,将HeLa细胞用[ 35 S]甲硫氨酸 - 半胱氨酸孵育15分钟,然后在37℃在追踪缓冲液中温育5小时。使用细胞裂解物(泳道1)和培养基(泳道2)样品用抗组织蛋白酶D抗体进行免疫沉淀,所得免疫沉淀物通过SDS-PAGE和荧光成像分析。指出pro-(pro-catD),中间(int-catD)和成熟组织蛋白酶D(m-catD)形式的位置。

食谱

  1. 补充100U青霉素/ ml和0.1mg / ml链霉素和10%(v / v)胎牛血清(FBS)的Dulbecco's改良Eagle培养基(DMEM)
  2. 100x L-蛋氨酸/ L-半胱氨酸混合溶液

    1. 溶解1毫升Milli-Q水中1.5毫克的L-蛋氨酸和L-半胱氨酸
    2. 用0.22μm注射器过滤器过滤消毒以保存
    3. 存放在4℃的离心管中。
  3. 脉冲媒体
    Dulbecco改良的Eagle's中等蛋氨酸,半胱氨酸和无谷氨酰胺补充:
    2mM L-谷氨酰胺
    0.1mCi / ml的35S-蛋氨酸半胱氨酸
  4. 追逐中等
    补充10μl/ ml 100x L-蛋氨酸/ L-半胱氨酸混合溶液的完全DMEM(方案1)
  5. 裂解缓冲液(4°C储存)
    50 mM Tris-HCl(pH 7.4)
    300 mM NaCl
    5mM EDTA pH 8
    0.5%(v / v)Triton X-100
  6. 清洗缓冲液(在4°C储存)
    50 mM Tris-HCl(pH 7.4)
    300 mM NaCl
    5mM EDTA pH 8
    0.1%(v / v)Triton X-100
  7. 2x样本缓冲区
    20毫升10%(v / v)SDS
    16毫升的0.5M Tris-HCl pH 6.8
    10毫升甘油
    0.1%溴酚蓝
    4%β-巯基乙醇
    Milli-Q水
    注意:该溶液可以在室温下储存,但在使用前加入新鲜的β-巯基乙醇。
  8. 固定解决方案
    50%甲醇
    10%醋酸
  9. 运行缓冲区
    30克Tris
    144克甘氨酸
    10克SDS
    用1毫升Milli-Q水完成

致谢

该协议已被改编自以前发表的论文(Tavares等人,2017年)。这项工作得到了圣保罗研究基金会(FAPESP)对LLPdS的资助(2014 / 25812-0)和LAT的博士奖学金(2016 / 18207-9)的支持。作者宣称没有利益冲突。

参考

  1. Benes,P.,Vetvicka,V.和Fusek,M。(2008)。 组织蛋白酶D - 一种天冬氨酸蛋白酶的许多功能 Crit Rev Oncol Hematol 68(1):12-28。
  2. (Bewley MA,Pham,TK,Marriott,HM,Noirel,J.,Chu,HP,Ow,SY,Ryazanov,AG,Read,RC,Whyte,MK,Chain,B.,Wright,PC和Dockrell,DH 2011)。 暴露于肺炎链球菌的巨噬细胞中组织蛋白酶D调节蛋白的蛋白质组学评估和验证。 Mol Cell Proteomics 10(6):M111 008193.
  3. Ghosh,P.,Dahms,N.M。和Kornfeld,S。(2003)。 甘露糖-6-磷酸受体:故事中的新曲折 Nat Rev Mol Cell Biol 4(3):202-212。
  4. Hirst,J.,Sahlender,D. A.,Choma,M.,Sinka,R.,Harbour,M.E.,Parkinson,M.and Robinson,M.S。(2009)。 果蝇中的GGAs和AP-1的空间和功能关系和HeLa细胞。 Traffic 10(11):1696-1710。
  5. Kametaka,S.,Moriyama,K.,Burgos,P.V.,Eisenberg,E.,Greene,L.E.,Mattera,R。和Bonifacino,J.S。(2007)。细胞周期蛋白G相关激酶与衔接蛋白1的典型相互作用调节溶酶体酶分选。
  6. Minarowska,A.,Gacko,M.,Karwowska,A。和Minarowski,L。(2008)。 人体组织蛋白酶D. Folia Histochem Cytobiol 46(1) ):23-38。
  7. Poole,A.R。,Dingle,J.T。和Barrett,A.J。(1972)。 组织蛋白酶D的免疫细胞化学证明 Histochem Cytochem 20(4):261-265。
  8. Tavares,L.A.,da Silva,E.M.,da Silva-Januario,M.E.,Januario,Y.C.,de Cavalho,J.V.,Czernisz,E.S。,Mardones,G.A。和daSilva,L.L.(2017)。 HIV-1蛋白Nef的CD4下调揭示了AP的γ1和γ2亚基的不同作用-1复合物在蛋白质运输中的作用。J Cell Sci 130(2):429-443。
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引用:Tavares, L. A. and daSilva, L. L. (2017). Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis. Bio-protocol 7(21): e2598. DOI: 10.21769/BioProtoc.2598.
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