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The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
长寿蛋白降解测定法:定量测定粘附细胞系中内源性蛋白自噬通量的有效方法   

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Autophagy
Oct 2013

Abstract

Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw conclusions regarding autophagic cargo flux. Here, we provide a protocol to quantitatively assess bulk autophagic flux by employing the long-lived protein degradation assay. Our procedure, which traces the degradation of 14C valine-labelled proteins, is simple and quick, allows for processing of a relatively large number of samples in parallel, and can in principle be used with any adherent cell line. Most importantly, it enables quantitative measurements of endogenous cargo flux through the autophagic pathway. As such, it is one of the gold standards for studying autophagic activity.

Keywords: Long-lived protein degradation (长寿蛋白降解), Autophagy (自噬), Autophagic flux (自噬通量), Endogenous cargo (内源性成分), Quantitative assay (定量测定法), Pulse-chase (脉冲追踪), Valine (缬氨酸), 14C radioactivity (14C放射性)

Background

Pulse-chase labelling approaches have been used to study protein turnover for decades. In the long-lived protein degradation (LLPD) assay described here, the proteome of cells in culture is radiolabelled with 14C valine and chased in order to follow the decline in radioactive proteins as readout of protein degradation. After an initial chase period, the cells are washed to eliminate the degradation products of short-lived proteins, which predominantly result from proteasomal activity. Thereafter, a second chase is initiated, and proper controls are included in order to monitor the autophagic degradation of long-lived proteins. We recently used this method to discover that the calcium-modulating compounds thapsigargin and A23187, which based on results with autophagic markers were previously widely believed to activate autophagy, actually completely block bulk autophagic flux (Engedal et al., 2013). The starting material used in that and previous protein degradation protocols was derived from cells grown in 6-well plates (Bauvy et al., 2009; Engedal et al., 2013) or more (Ronning et al., 1979; Seglen et al., 1979), or involved relatively high amounts of radioactivity (Mizushima et al., 2001; Fuertes et al., 2003a), which is expensive. Recently, we have downscaled and simplified the LLPD protocol to the validated time- and cost-efficient version that we present here.

An overview of the method is shown in Figure 1. To label proteins with radioactive valine, cells are seeded in 24-well plates in complete medium supplemented with 14C valine. As proteins are synthesised, they incorporate amino acids, including the 14C valine present in the medium, and therefore the amount of long-lived 14C valine-labelled proteins increases with time (Figure 1, first part of the curve). Valine is an optimal amino acid to use in the LLPD method, since it is a poorly metabolised amino acid, is well tolerated at high doses, and does not influence autophagy or protein degradation rates (Seglen et al., 1979). Moreover, free valine is readily exchanged over the plasma membrane (Seglen and Solheim, 1978), enabling efficient wash-out of released 14C valine. After 2-3 days of labelling, unincorporated 14C valine is removed by a simple wash procedure, and the cells receive new medium supplemented with a high concentration of non-radioactive (‘cold’) valine (‘chase medium’). The large surplus of cold valine prevents reincorporation of released 14C valine. Thus, from this point on the presence of free 14C valine is a result of endogenous protein degradation. After a chase period of 18 h, the free 14C valine that has been produced by degradation of short-lived proteins (predominantly due to proteasomal degradation) is washed out. Next, a second chase period, which we call the ‘sampling period’, is initiated along with experimental treatments and proper controls. Generally, we use a sampling period of 2-6 h to monitor the degradation of long-lived proteins. At the end of the sampling period, trichloroacetic acid (TCA) is added to precipitate intact proteins. The TCA-soluble fraction of degraded proteins (containing free amino acids and small peptides) is separated from the TCA-insoluble fraction (containing intact proteins) by centrifugation, and the radioactivity in each fraction is measured by liquid scintillation counting. This allows calculation of the rate of long-lived protein degradation in the sampling period, expressed as the percentage of radioactivity in the TCA-soluble fraction versus the total amount of radioactivity in the TCA-soluble and–insoluble fractions, divided by the duration of the sampling period (Figure 1).


Figure 1. Overview of the long-lived protein degradation (LLPD) assay. During the labelling period (2-3 days), the amount of radioactive long-lived proteins increases with time. Thereafter, an 18 h chase period allows for degradation of the short-lived proteins and subsequent elimination of the released 14C valine by a washing step. Consequently, only the degradation of long-lived proteins is followed in the 2-6 h sampling period. Compared to cells kept in complete, nutrient-rich medium (red line), incubating cells with EBSS starvation medium or the mTOR-inhibitor Torin1 produces a very strong degradation of long-lived proteins in the sampling period, due to enhanced bulk autophagy (green line). Autophagic-lysosomal LLPD can be revealed by treatment with the lysosomal inhibitor Bafilomycin A1 (Baf) or RNAi-mediated silencing of key ATGs (siATGs) (yellow line), whereas the contribution to LLPD from the proteasome can be assessed by treatment with proteasomal inhibitors like MG132 (purple line). Blocking both autophagic-lysosomal and proteasomal activity simultaneously will abrogate both main sources of LLPD, thus resulting in minimal loss of 14C-labelled intact protein (black line). Note that the rise and fall in the curves are schematic and purely intended for illustrative purposes–they are not intended to indicate exact details in the kinetics of long-lived protein labelling and/or degradation. See text for a more detailed description of each of the protocol steps and for representative examples of data.

Materials and Reagents

In the current protocol we use:

  1. Pipette tips (Thermo Fisher ART Barrier tips) (VWR, catalog numbers: 732-2223 (0.5-20 µl), 732-2207 (1-200 µl), and 732-2355 (100-1,000 µl))
  2. 75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353136 )
  3. 24-well tissue culture plates (Corning, Falcon®, catalog number: 353047 )
  4. Microcentrifuge tubes (VWR, catalog number: 211-2130 )
  5. Scintillation vials (PerkinElmer, catalog number: 6000292 )
  6. Nitrile gloves (VWR, catalog number: 112-2372 )
  7. 50 ml tube (VWR, catalog number: 525-0402 )
  8. 0.45 µm filter (VWR, catalog number: 514-0075 )
  9. LNCaP cells (ATCC, catalog number: CRL-1740 )
  10. U2OS cells (ATCC, catalog number: HTB-96 )
  11. VCaP cells (ATCC, catalog number: CRL-2876 )
  12. Huh7 cells (Nakabayashi et al., 1982) (Kindly provided by Dr. Line M. Grønning-Wang, Oslo, Norway)
  13. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 20012019 )
  14. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  15. RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 21875091 )
  16. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  17. [1-14C] L-valine, 45 mCi/mmol, 0.1 mCi/ml (Vitrax, catalog number: VC 308 )
  18. Poly-D-lysine (Sigma-Aldrich, catalog number: P6407-10X5MG )
  19. For RNAi reverse transfection:
    Lipofectamine RNAiMAX (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
    Ambion SilencerTM select siRNAs (negative control ‘siCtrl’, Thermo Fisher Scientific, InvitrogenTM, catalog number: 4390843 ; siULK1, s15964; siULK2, s18706)
    Opti-MEM reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11058021 )
  20. Earle’s balanced salt solution (EBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24010043 )
  21. Scintillation liquid (PerkinElmer, catalog number: 6013199 )
  22. DMSO (Sigma-Aldrich, catalog number: D2650 )
  23. Bafilomycin A1 (Enzo Life Sciences, catalog number: BML-CM110-0100 )
  24. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
  25. SAR-405 (Magento, ApexBio, catalog number: A8883 )
  26. Torin1 (Tocris Bioscience, catalog number: 4247 )
  27. Non-radioactive L-valine (Sigma-Aldrich, catalog number: V0513 )
  28. Bovine serum albumin (BSA) (VWR, catalog number: 422361V )
  29. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T0699 )
  30. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
  31. 200 mM cold L-valine (see Recipes)
  32. RPMI 1640/10%FBS (see Recipes)
  33. 1 mg/ml poly-D-lysine (see Recipes)
  34. 1 mM Torin1 (see Recipes)
  35. PBS/2%BSA (see Recipes)
  36. 25% TCA (see Recipes)
  37. 0.2 M KOH (see Recipes)
  38. 0.2 mM Bafilomycin A1 (see Recipes)
  39. 160 mM NH4Cl (see Recipes)

Equipment

  1. Pipettes (FinnpipetteTM F2 GLP Kit) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4701070 )
  2. Humidified incubator
  3. Tube rotator (VWR, catalog number: 444-0502 )
  4. Plate shaker (Grant Instruments, model: PMS-1000i )
  5. Magnetic stirrer (IKA, catalog number: 0003810001 )
  6. Autoflow IR Direct Heat CO2 incubator (NuAire, model: NU-5510E )
  7. Vortexer (Denville Scientific, catalog number: S7030 )
  8. Benchtop centrifuge with cooling (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75002430 )
  9. Scintillation counter (Liquid Scintillation Analyzer) (Packard, model: 1600 TR )

Procedure

  1. Radioactive labelling (standard 2-3 days)
    1. Culture adherent cells in a 75 cm2 tissue culture flask in a humidified incubator with 5% CO2 at 37 °C until a near confluent cell layer has been obtained. Wash once with 3 ml 37 °C PBS and incubate the cells with 3 ml 0.25% trypsin-EDTA at 37 °C until the cells have detached (typically 3-5 min).
    2. Collect the detached cells in 10 ml 37 °C RPMI 1640 supplemented with FBS to 10% final concentration (= Complete Medium, CM).
      Note: The concentrations of radioactive and cold valine in this protocol have been adapted specifically for use in RPMI 1640, based on the concentration of valine in this medium, and based on cost-efficiency with respect to the amount of radioactive valine used. When following this protocol, cells should therefore be resuspended in RPMI 1640 after trypsinization, even though they are not normally cultured in RPMI 1640. For the same reason, RPMI 1640 should be used for the remainder of the protocol. We have validated this assay in > 20 cell lines and have never observed signs of toxicity or altered phenotypes in cells that are normally not cultured RPMI 1640, as long as the cells do not require any special supplements. Other media can be used instead of RPMI 1640, but users should adjust valine concentrations throughout the protocol to account for differences in the valine concentration of RPMI 1640 versus the alternative medium used.
    3. Seed cells in a 24-well plate in 0.5 ml CM supplemented with 14C-valine to a final concentration 0.1 µCi/ml. Seed a cell number that will lead to the desired monolayer confluency at the time of experimental treatment. In general, we seed cells so that they reach 50-80% confluency after 2-3 days incubation in a humidified incubator with 5% CO2 at 37 °C.
      Notes:
      1. The protocol presented here is compatible with a 24-well plate format. Because of the ease of the procedure and its high reproducibility, we routinely process two 24-well plates (48 wells) per experiment with duplicate or triplicate wells per treatment condition. This makes it possible to test a relatively large number of conditions in a single experiment.
      2. For LNCaP cells, we seed 1 x 105 cells per well. For U2OS, VCaP and Huh7, we seed 3 x 104, 2.5 x 105, and 1.2 x 104 cells per well, respectively.
      3. When working with cell types that adhere poorly (e.g., LNCaP cells, HEK293 cells, and others), it is recommended to use tissue culture plates that have been coated to improve cell adherence (due to multiple wash steps in the protocol). We prefer to use poly-D-lysine (PDL), as it is relatively inexpensive and easy to use, and does not interfere with amino acid starvation (since it, unlike, e.g. poly-L-lysine, is not metabolised by cells). Per well in a 24-well tissue culture plate, add 300 µl sterile PDL (2.5 µg/ml). Incubate the plates in a sterile environment for 30 min at room temperature. Afterwards, remove the PDL using suction, and wash each well briefly with 0.5 ml sterile H2O. The plates are ready for use when dry. PDL-coated plates are stable for up to several weeks when stored at room temperature. Of note, for most cell types coating is not necessary.
      4. When doing siRNA transfection, we recommend using reverse transfection so that the cells can be transfected and radiolabelled simultaneously. This is both practical and gives a very high transfection efficiency. Combine Opti-MEM, siRNA, and Lipofectamine RNAiMAX according to the manufacturer’s protocol, and aliquot the transfection mix into a 24-well plate (50 µl per well). Incubate at room temperature for 20-45 min, and seed 0.5 ml cell suspension in CM containing 14C-valine in the wells containing the siRNA-Lipofectamine mixture. Carefully agitate the plate and place it in the incubator.
      5. Follow the rules of your institution with regard to work with radioactivity. For own protection, wear gloves with a thickness of ≥ 0.25 mm.
    4. Incubate the cells for 2-3 days in a humidified incubator at 37 °C and 5% CO2 to allow ample incorporation of 14C valine into long-lived proteins.

  2. Chase (standard 18 h)
    1. Aspirate the medium to remove unincorporated radioactivity.
    2. Wash cells once with 0.5 ml 37 °C CM supplemented with 10 mM cold valine (CM-V).
      Note: Since RPMI 1640 contains 0.17 mM L-valine, the final concentration of L-valine in CM-V will be 10.17 mM. The use of 10 mM cold L-valine to block re-incorporation of released 14C-valine is standard for this type of assay (Bauvy et al., 2009; Mizushima et al., 2001; Fuertes et al., 2003a). It is not known whether there is an upper limit of how high concentration of cold L-valine can be used, but in isolated rat hepatocytes it has been shown that up to 20 mM cold L-valine does not affect protein degradation activity (Seglen et al., 1979).
    3. Aspirate the medium and replace with 0.5 ml 37 °C CM-V.
    4. Incubate the cells for 18 h in a humidified incubator at 37 °C and 5% CO2.
      Notes: 
      1. The chase does not need to be 18 h. However, altering the duration of the chase will change the protein degradation rate during the sampling (Figure 2A), as well as the signal intensity during liquid scintillation counting (see Note 2 in the Notes section for comments on scintillation counting and the variability of assay measurements), and will also change the pool of proteins analysed (see Note 3 in the Notes section). One should keep this in mind when comparing results obtained with different chase durations.
      2. If doing experimental treatments longer than 6 h, add your treatments prior to or during the chase. This additionally gives an opportunity to sample treatment effects on LLPD at various time periods after initiation of the treatment.

  3. Sampling (standard 2-6 h)
    1. Remove released 14C valine (resulting from the degradation of short-lived proteins) by aspirating the medium.
    2. Wash cells once with 0.5 ml 37 °C of appropriate medium. If the subsequent treatment will be in complete medium (e.g., Torin1), wash with CM-V. If the subsequent treatment will be in amino acid starvation medium (e.g., EBSS), wash with EBSS supplemented with 10 mM cold valine (EBSS-V).
    3. Aspirate the medium and add your treatments in 0.25 ml 37 °C CM-V or EBSS-V.
    4. Incubate the cells for 2-6 h in a humidified incubator at 37 °C and 5% CO2.

  4. Harvest
    Day 1
    1. Remove the plate with cells from the incubator and cool it on ice for ~2 min.
    2. Add 50 µl ice-cold PBS/2%BSA per well.
      Note: BSA is added as a protein carrier to facilitate protein precipitation with TCA.
    3. Add 200 µl ice-cold 25% TCA per well to precipitate protein.
    4. Place the plate on a shaker at 4 °C and let it shake over night at 600 rpm.
      Note: Whereas overnight TCA precipitation is sufficient, the precipitation may be extended for several days without affecting the results.

    Day 2
    1. Transfer the precipitated protein (~500 µl) from each well to 1.5 ml microcentrifuge tubes.
      Note: Transfer the solution to microcentrifuge tubes without scraping. The protein precipitate remaining in the wells will be solubilised and collected in the protocol Steps D10-D12.
    2. Sediment precipitates by centrifugation at 5,000 x g for 10 min at 4 °C. After the centrifugation, the supernatant (TCA soluble fraction) contains amino acids and small peptides (including 14C valine released from proteins due to degradation during the sampling period) and the pellet (TCA insoluble fraction) contains intact proteins.
    3. After the centrifugation, place the tubes on ice.
    4. Transfer the supernatant (~500 µl) from each tube into 6 ml scintillation vials.
    5. Solubilise the pellet by adding 250 µl 0.2 M KOH per tube and rotating the tubes for at least 1 h at room temperature.
      Note: It is not necessary to wash the pellet prior to solubilising it in KOH.
    6. Solubilise remaining protein precipitate in the 24-well plate by adding 250 µl 0.2 M KOH per well.
    7. Rotate on a shaker at 600 rpm for 1 h at room temperature.
    8. Merge the solubilised protein from the respective wells and tubes (~500 µl) and transfer to 6 ml scintillation vials.
    9. Fill each vial from Steps D8 and D12 with 4 ml scintillation liquid. Cap the tubes, and vortex hard.
      Note: The scintillation liquid is an organic and toxic substance. For your own protection, add scintillation liquid, cap tubes, and vortex in a flow-cabinet, wearing protective gloves and lab coat.
    10. Measure the amount of radioactivity in all of the tubes by liquid scintillation counting (see Note 2 in the separate Notes section for comments on scintillation counting and the variability of assay measurements).

Data analysis

  1. Representative examples of data
    As detailed in the protocol above, we in general label proteins for 2-3 days followed by a chase for 18 h to enrich for radioactively labelled long-lived proteins. The chase can be reduced or extended to suit other experimental setups than shown here. It is, however, important to be aware that this will enrich for proteins with a different half-life, which in turn may alter the overall protein degradation rate during the sampling period, as well as the outcome of your experimental treatments (see further explanation in Note 3 in the Notes section). We therefore recommend keeping the labelling and chase periods as consistent as possible. As an example, decreasing the chase period from 18 h to 1 h increased the overall protein degradation rate from ≈ 1.05%/h to ≈ 1.3 %/h in LNCaP cells (Figure 2A).
    To identify the proportion of lysosomal protein degradation taking place under the experimental conditions examined, we recommend including an inhibitor of lysosomal degradation such as the vacuolar-type H+-ATPase-inhibitor Bafilomycin A1 (Baf). Baf deacidifies lysosomes and resultantly inhibits lysosomal degradation activity in a dose-dependent manner. In LNCaP cells, the Baf-mediated inhibition of long-lived protein degradation is saturated at 25-50 nM in complete medium (Figure 2B). The Baf-sensitive fraction represents lysosomal degradation, whereas the Baf-insensitive fraction represents non-lysosomal degradation, mainly proteasomal protein degradation. Confirming this, the lysosomotropic compound NH4Cl reduces LLPD to the same extent as Baf (Figure 2C). Lysosomal LLPD is caused by autophagy. In LNCaP cells, we find that treatment with the Phosphatidylinositol 3-Kinase Catalytic Subunit Type 3 (PIK3C3)-inhibitor SAR-405 or knockdown of the autophagy-related proteins Unc-51 like Autophagy Activating Kinase 1 (ULK1) and ULK2 inhibit LLPD to the same extent as Baf or NH4Cl (Figure 2C). Thus, in LNCaP cells, basal lysosomal LLPD is due to PIK3C3/ULK-dependent autophagy, i.e., canonical autophagosome-mediated autophagy (macroautophagy), whereas putative contributions from micro-autophagy, endosomal micro-autophagy, or chaperone-mediated autophagy (all of which are PIK3C3/ULK-independent) are negligible. The contribution of the proteasome to LLPD may be determined by applying proteasomal inhibitors such as MG132 (Fuertes et al., 2003a; Engedal et al., 2013). Lysosomal and proteasomal inhibitors can be used to infer whether treatment effects on LLPD are caused by alterations in either lysosomal and proteasomal activity. For example, when combining autophagy-inhibitory calcium modulators (A23187 and thapsigargin) with either Baf or MG132, we found additive reducing effects on LLPD with MG132, but not with Baf (Engedal et al., 2013), indicating that the calcium modulators inhibit autophagic-lysosomal LLPD rather than proteasomal LLPD. Of note, one should be aware that crosstalk between proteasomal and autophagic-lysosomal pathways may occur (Dikic, 2017). However, within the time frame (2-6 h) and conditions (basal conditions, or conditions of amino acid starvation, mammalian target of rapamycin (mTOR)-inhibition, or endoplasmic reticulum stress) that we have tested, we have not observed signs of such crosstalk at the level of LLPD. In LNCaP and U2OS cells we found the effects of Baf and MG132 to be perfectly additive (Engedal et al., 2013), thus indicating absence of any measurable effect of autophagy-proteasome crosstalk on LLPD.
    mTOR suppresses autophagy in a manner that is sensitive to the presence of amino acids and growth factors (Meijer et al., 2015). Thus, upon removal of amino acids and serum from the medium, mTOR activity is inhibited, and autophagy is induced. Figure 2D shows the typical response of LNCaP cells to treatment with the mTOR-inhibitor Torin1 or acute amino acid- and serum-starvation; total LLPD is increased by about two-fold, in a manner that in both cases is strongly sensitive to Baf, i.e., dependent on lysosomal activity. The increase in LLPD observed upon acute starvation differs from cell line to cell line. For example, the effect is weaker in VCaP and Huh7 cells compared to U2OS (Figure 2E) and LNCaP cells (Figure 2D). Importantly, the starvation-induced LLPD is sensitive to Baf in all cell lines, demonstrating that it mainly induces lysosomal LLPD. Of note, however, Baf does not completely abolish the effects of mTOR-inhibition or starvation (Figures 2D-2E), likely because these conditions slightly elevate proteasomal activity as well as autophagy (Fuertes et al., 2003a; Zhao et al., 2015 and our unpublished results). Also note that the degradation rate, and the degree of Baf-sensitive degradation, varies substantially between cell types in both complete medium and starvation medium (Figures 2D-2E). This reflects different degrees of basal autophagy (Baf-sensitive degradation in complete medium) and autophagic capacity (Baf-sensitive degradation in EBSS) in different cell lines.


    Figure 2. Quantitatively measuring autophagic flux of endogenous proteins using the long-lived protein degradation assay. A-D. LNCaP and (E) U2OS, VCaP, and Huh7 cell lines were labelled with 14C valine for 2-3 days, chased for (A, left bar) 1 h or (A, right bar, B-E) 18 h, and subjected to the indicated treatments in chase medium (the ‘sampling period’). Long-lived protein degradation (LLPD) was subsequently measured. A. LLPD was measured during a 6 h sampling period, subsequent to either 1 h or 18 h chase. B. LLPD is inhibited by Baf in a dose-dependent manner. The red, dotted line shows where Baf-mediated inhibition of LLPD is saturated. The remaining level of protein degradation is predominantly caused by the proteasome. C. Cells were reverse transfected with 10 nM non-targeting siRNA (‘siCtrl’) or 5 nM siULK1 in combination with 5 nM siULK2, and treated with DMSO (0.1%), Baf (50 nM), NH4Cl (10 mM), or 3 µM SAR-405 for 4 h. The red line indicates where LLPD inhibition is saturated. The remaining level of protein degradation is predominantly caused by the proteasome. D. Cells were subjected to the indicated treatments for 4 h. Baf and Torin1 were used at 50 nM. E. U2OS (left), VCaP (centre), and Huh7 (right) were subjected to the indicated treatments for 4 h. Baf was used at 50 nM in all cell lines. A-E. One representative of two independent experiments is shown (mean ± SD of 3 biological replicates). Baf, Bafilomycin A1; CM, complete medium; EBSS, Earle’s balanced salt solution.

  2. Data analysis
    The scintillation counter generates a text file where the sample numbers are listed with corresponding ‘counts per minute’ (CPM). The CPM values reflect the quantity of 14C in the individual samples. The degradation rate is calculated by dividing the amount of radioactivity in the TCA-soluble fraction by the total amount of radioactivity (the sum of radioactivity in the TCA-soluble and –insoluble fractions), divided by the duration of the sampling period. The degradation rate is expressed as ‘long-lived protein degradation (%/h)’.

Notes

  1. In general, we perform the experiment with duplicate or triplicate wells per treatment condition (i.e., two or three biological replicates in each experiment), whereas the scintillation counting is performed on the whole samples (i.e., no technical replicates for the measurement of radioactivity). The deviation between biological replicates is generally very low when following this protocol. From 23 independent experiments across 6 different cell lines and covering a total of 292 experimental conditions performed in triplicate, we have calculated the coefficient of variation (CV) of the LLPD rates of treatment replicates to be 2.3% ± 1.5% (mean CV ± standard deviation). More specifically, the CV values ranged from 0.1-8.9%, with 5%, 25%, 50%, 75%, and 95% percentile values of 0.4%, 1.2%, 2.0%, 3.2%, and 5.1%, respectively.
  2. Liquid scintillation counters can be set to record counts either for a specific time period, or until a specific number of counts has been accumulated. The uncertainty in the measurements is inversely correlated with the total number of counts. We recommend setting up the counter to accumulate at least 10,000 counts, whereby the standard deviation will be ≤ 1.0%. Following the current protocol with 2-3 days labelling with 0.1 µCi/ml 14C valine, these settings typically require less than a minute of counting for the TCA-insoluble fractions and a few minutes of counting for the TCA-soluble fractions. The background level is set during standard 14C calibration, and is very low (≤ 22 CPM). In the standard measurement protocol, this background is automatically subtracted from each sample. There is therefore no need to include additional background sample measurements (which would require many hours of sampling time for accurate measurement). As a general note, we recommend contacting your local service engineer for maintenance, calibration, and service questions.
  3. Different durations of the labelling, chase, and sampling periods than those presented here can be used. For example, some previous protocols used an 18-24 h labelling period, followed by a 1 h chase period (Mizushima et al., 2001; Bauvy et al., 2009). The advantage of increasing the labelling period to 2-3 days (as here) is that it can be conveniently combined with 2-3 days of transfection, e.g., with the reverse siRNA transfection described here. Moreover, the longer the labelling period, the more long-lived proteins (which are slowly synthesized and slowly degraded) will be labelled. It should be noted that in human fibroblasts, a pool of short-lived proteins that is predominantly degraded by the proteasome and not by macroautophagy has been described (Fuertes et al., 2003b). This pool of proteins showed a half-life of ~1.1-1.3 h, and an ~8 h chase period was required to fully eliminate them (Fuertes et al., 2003b). It was found that during 1-4 h of chase, 5-7.5% of the initial TCA-insoluble radioactivity of short-labelled proteins reflected the cellular release of non-degraded TCA-insoluble proteins to the medium. This release decreased to about 1/10th of the above values after 8 h of chase (Fuertes et al., 2003b). In summary, we advise a chase period of > 8 h, to minimize the influence of degradation and/or secretion of short-lived proteins. To be on the safe side, we routinely use an 18 h chase period. Such a long chase period has the additional advantage that when wishing to analyse effects of long-term experimental treatments on LLPD, the treatments can be initiated during the chase period (see Step B4b). Moreover, since the chase period is usually followed by a sampling period of 2-6 h, it is in relation to working hours more convenient with an 18 h chase period than an 8 h chase period. The sampling period can be longer than 6 h if desired, but if combining the assay with control treatments such as lysosomal and proteasomal inhibitors (as we recommend), it is advisable to keep the period shorter than 6 h to minimize secondary effects of the inhibitors. If wishing to analyse effects of experimental treatments on LLPD after more than 18 h of treatment, one may either increase the duration of the chase period, or even start the treatments during the labelling period, as alternatives to increasing the duration of the sampling period. Finally, the assay can also be done with shorter sampling periods than 2 h, but then great care must be taken to assure even time points for each experimental sample, and one may have to increase the amount of radioactive 14C-valine to achieve a higher degree of labelling and thus obtain an adequate level of counts in the scintillation counter (see Note 2 above).

Recipes

  1. 200 mM cold L-valine (Mw 117.15)
    1. Dissolve 1.172 g cold (i.e., non-radioactive) L-valine in 45 ml RPMI 1640 (without serum) or EBSS using a magnetic stirrer
    2. Adjust the volume to 50 ml with RPMI 1640 or EBSS, respectively
    3. Filter through a 0.45 µm filter into a sterile 50 ml tube
    4. This solution is stable for up to several months when stored at 4 °C. Do not preheat before use
  2. RPMI 1640/10% FBS (Complete medium; CM) supplemented with 10 mM cold L-valine (CM-V)
    1. Add 1 part of the 200 mM cold L-valine solution (Recipe 1 above) to 19 parts of CM
    2. Make a new solution for each experiment
  3. 1 mg/ml poly-D-lysine
    1. Dissolve 5 mg poly-D-lysine (PDL) in 5 ml sterile H2O
    2. Aliquot in Eppendorf tubes and store at -20 °C
    3. The stock solution is stable for years when stored at -20 °C, and can be thawed and re-frozen several times
  4. 1 mM Torin1 (Mw 607.62)
    1. Dissolve 10 mg Torin1 in 16.46 ml DMSO
    2. Aliquot in microcentrifuge tubes and store at -20 °C
    3. The stock solution is stable for years when stored at -20 °C, and can be thawed and re-frozen several times
  5. PBS/2% BSA
    1. Dissolve 5 g BSA + in 200 ml PBS
    2. Add PBS to a total volume of 250 ml
    3. Filter through a 0.45 µm filter into a sterile glass flask
    4. The stock solution is stable for up to several months when stored at 4 °C
  6. 25% TCA
    1. For 10 ml, mix 2.5 ml 100% TCA with 7.5 ml Milli-Q (MQ) H2O
      Caution: Add acid to the water, not vice-versa.
    2. Make the solution fresh prior to harvesting
  7. 0.2 M KOH (Mw 56.11)
    1. Dissolve 1.12 g KOH in 90 ml MQ H2O
    2. Add MQ H2O to a total volume of 100 ml
    3. The solution is stable for up to several months when stored at room temperature
  8. 0.2 mM Bafilomycin A1 (Mw 622.84)
    1. Dissolve 0.1 mg Bafilomycin A1 in 803 µl DMSO
    2. Aliquot in microcentrifuge tubes and store at -20 °C
    3. The stock solution is stable for years when stored at -20 °C, and can be thawed and re-frozen several times
  9. 160 mM NH4Cl (Mw 53.49)
    1. Dissolve 256.8 mg NH4Cl in 25 ml MQ H2O
    2. Adjust to pH 7.74 at room temperature (= pH 7.4 at 37 °C)
    3. Adjust the final volume to 30 ml using MQ H2O
    4. Filter through a 0.45 µm filter into a sterile 50 ml tube
    5. This solution is stable for years when stored at -20 °C

Acknowledgments

This work was financially supported by the Research Council of Norway, the University of Oslo, the Anders Jahre Foundation, the Nansen Foundation, and the Legacy in the memory of Henrik Homan. This protocol is a modified version of that described in the methods section of Engedal et al., 2013, which in turn was adapted from Bauvy et al., 2009. We also would like to acknowledge the original work of Per O. Seglen, on which much of the method is based (Seglen and Solheim, 1978; Ronning et al., 1979; Seglen et al., 1979). The authors declare that they have no conflicts of interest.

References

  1. Bauvy, C., Meijer, A. J. and Codogno, P. (2009). Assaying of autophagic protein degradation. Methods Enzymol 452: 47-61.
  2. Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu Rev Biochem 86: 193-224.
  3. Engedal, N., Torgersen, M. L., Guldvik, I. J., Barfeld, S. J., Bakula, D., Sætre, F., Hagen, L. K., Patterson, J. B., Proikas-Cezanne, T., Seglen, P. O., Simonsen, A. and Mills, I. G. (2013). Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy 9(10): 1475-1490.
  4. Fuertes, G., Martin De Llano, J. J., Villarroya, A., Rivett, A. J. and Knecht, E. (2003a). Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions. Biochem J 375(Pt 1): 75-86.
  5. Fuertes, G., Villarroya, A. and Knecht. E. (2003b). Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions. Int J Biochem Cell Biol 35: 651-664.
  6. Meijer, A. J., Lorin, S., Blommaart, E. F. and Codogno, P. (2015). Regulation of autophagy by amino acids and MTOR-dependent signal transduction. Amino Acids 47(10): 2037-2063.
  7. Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y. and Yoshimori, T. (2001). Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 152(4): 657-668.
  8. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. and Sato, J. (1982). Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42(9): 3858-3863.
  9. Ronning, O. W., Pettersen, E. O. and Seglen, P. O. (1979). Protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in vitro. Exp Cell Res 123(1): 63-72.
  10. Seglen, P. O., Grinde, B. and Solheim, A. E. (1979). Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem 95(2): 215-225.
  11. Seglen P. O. and Solheim, A. E. (1978). Valine uptake and incorporation into protein in isolated rat hepatocytes. Nature of the precursor pool for protein synthesis. Eur J biochemi 85: 15-25
  12. Zhao, J., Zhai, B., Gygi, S. P. and Goldberg, A. L. (2015). mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy. Proc Natl Acad Sci U S A 112(52): 15790-15797.

简介

自噬是维持真核生物细胞稳态的关键因素,包括癌症和神经退行性疾病在内的许多疾病都与自噬的改变有关。研究自噬的兴趣在过去的二十年里急剧增长,并且还有用于研究这种高度动态和复杂过程的方法库。通常通过Western印迹和各种显微镜技术来评估自噬相关蛋白的表达和/或定位中的变化。这样的分析可能表明自噬相关过程的改变,并且关于正在研究的特定标记物的信息。然而,由于这些蛋白质是自噬机制的一部分,而不是自噬性货物,所以它们不能用于得出关于自噬载货流量的结论。在这里,我们提供了一个协议,通过使用长寿命的蛋白质降解测定来定量评估体积自噬流量。我们的程序追踪14 C缬氨酸标记的蛋白质的降解是简单和快速的,允许并行处理相对大量的样品,并且原则上可以与任何贴壁细胞一起使用线。最重要的是,它可以通过自噬途径定量测量内源货物流量。因此,它是研究自噬活动的黄金标准之一。

【背景】脉冲追踪标记方法已用于研究蛋白质周转数十年。在此处描述的长寿命蛋白质降解(LLPD)测定中,培养细胞的蛋白质组用14 C缬氨酸放射性标记并追踪以追踪放射性蛋白质的下降,作为蛋白质降解的读数。在最初的追逐期后,清洗细胞以消除主要由蛋白酶体活性导致的短寿命蛋白质的降解产物。之后,开始第二次追踪,并且包括适当的对照以监测长寿命蛋白质的自噬降解。我们最近使用这种方法发现钙调节化合物thapsigargin和A23187,其基于自噬标记物的结果先前被广泛认为能够激活自噬,实际上完全阻断了大量的自噬流(Engedal等人 ,2013)。用于该蛋白质降解方案和之前的蛋白质降解方案中的起始材料来源于在6孔板中生长的细胞(Bauvy等人,2009; Engedal等人,2013) (Ronning等,1979; Seglen等,1979),或涉及相对高量的放射性(Mizushima et al。 >,2001; Fuertes等人,2003a),这是昂贵的。最近,我们将LLPD协议缩减并简化为经过验证的时间和成本效益版本。

该方法的概述如图1所示。为了用放射性缬氨酸标记蛋白质,将细胞接种在24孔板中的完全培养基中,该培养基补充有14 C缬氨酸。当合成蛋白质时,它们掺入氨基酸,包括存在于培养基中的14 C缬氨酸,因此长寿命14 C缬氨酸标记的蛋白质的量随着时间(图1,曲线的第一部分)。缬氨酸是用于LLPD方法的最佳氨基酸,因为它是低代谢的氨基酸,在高剂量下耐受性良好,并且不影响自噬或蛋白质降解速率(Seglen等人 ,1979)。此外,游离缬氨酸易于在质膜上交换(Seglen和Solheim,1978),从而能够有效洗脱释放的14 C缬氨酸。标记2-3天后,通过简单的洗涤程序除去未掺入的14 C缬氨酸,细胞接受补充有高浓度非放射性('冷')缬氨酸('追逐媒体“)。大量过量的冷缬氨酸可防止释放的14 C缬氨酸重新结合。因此,从这一点来说,游离14 C缬氨酸的存在是内源性蛋白质降解的结果。经过18小时的追踪期后,由短寿命蛋白质降解(主要由于蛋白酶体降解引起)产生的游离14 C缬氨酸被洗掉。接下来,第二个追逐时期,我们称之为“抽样期”,随着实验处理和适当的控制而开始。通常,我们使用2-6小时的采样时间来监测长寿命蛋白质的降解。在采样期结束时,加入三氯乙酸(TCA)以沉淀完整的蛋白质。通过离心从TCA不溶性部分(含有完整蛋白质)中分离降解蛋白质(含有游离氨基酸和小肽)的TCA可溶部分,并通过液体闪烁计数测量每部分中的放射性。这允许计算取样期间长寿命蛋白质降解的速率,表示为TCA可溶部分中的放射性百分比相对于TCA可溶性和不溶性部分中的放射性总量除以采样周期(图1)。


图1.长寿命蛋白质降解(LLPD)测定的概述在标记期(2-3天)期间,放射性长寿命蛋白质的量随时间增加。此后,追踪18小时可以使短寿命蛋白质降解并随后通过洗涤步骤消除释放的14 C缬氨酸。因此,在2-6小时的采样期间只能观察到长寿命蛋白质的降解。与保存在完整,富含营养物的培养基(红线)中的细胞相比,由于增强的体细胞自噬作用,将细胞与EBSS饥饿培养基或mTOR抑制剂Torin1孵育,可在采样期内产生非常强的长寿命蛋白质降解(绿色线)。通过用溶酶体抑制剂Bafilomycin A1(Baf)或RNAi介导的关键ATG(siATG)沉默(黄线)可以揭示自噬 - 溶酶体LLPD,然而可以通过用蛋白酶体抑制剂处理来评估来自蛋白酶体的LLPD的贡献像MG132(紫色线)。同时阻断自噬 - 溶酶体和蛋白酶体活性将消除LLPD的两个主要来源,因此导致14 C标记的完整蛋白(黑线)的损失最小。请注意,曲线的上升和下降是示意性的,纯粹用于说明目的 - 它们并非旨在指示长寿命蛋白质标记和/或降解动力学的确切细节。有关每个协议步骤和数据代表性示例的更详细说明,请参阅文本。

关键字:长寿蛋白降解, 自噬, 自噬通量, 内源性成分, 定量测定法, 脉冲追踪, 缬氨酸, 14C放射性

材料和试剂

在我们使用的当前协议中:


  1. 移液器吸头(Thermo Fisher ART Barrier tips)(VWR,产品目录号:732-2223(0.5-20μl),732-2207(1-200μl)和732-2355(100-1,000μl))
  2. 75cm 2组织培养瓶(Corning,Falcon,目录号:353136)。
  3. 24孔组织培养板(Corning,Falcon ,目录号:353047)
  4. 微量离心管(VWR,目录号:211-2130)
  5. 闪烁瓶(PerkinElmer,目录号:6000292)
  6. 丁腈手套(VWR,产品目录号:112-2372)
  7. 50毫升管(VWR,目录号:525-0402)
  8. 0.45μm过滤器(VWR,目录号:514-0075)
  9. LNCaP细胞(ATCC,目录号:CRL-1740)
  10. U2OS细胞(ATCC,目录号:HTB-96)
  11. VCaP细胞(ATCC,目录号:CRL-2876)
  12. Huh7细胞(Nakabayashi等人,1982)(由挪威奥斯陆的Line M.Grønning-Wang博士提供)
  13. PBS(Thermo Fisher Scientific,Gibco TM,目录号:20012019)
  14. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  15. RPMI 1640(Thermo Fisher Scientific,Gibco TM,目录号:21875091)
  16. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
  17. [1 -14 C] L-缬氨酸,45mCi / mmol,0.1mCi / ml(Vitrax,目录号:VC308)
  18. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P6407-10X5MG)
  19. 对于RNAi逆转录:
    Lipofectamine RNAiMAX(Thermo Fisher Scientific,Invitrogen TM,目录号:13778150)
    Ambion Silencer TM选择siRNA(阴性对照'siCtrl',Thermo Fisher Scientific,Invitrogen TM,目录号:4390843; siULK1,s15964; siULK2,s18706) Opti-MEM减少血清培养基(Thermo Fisher Scientific,Gibco TM,目录号:11058021)
  20. Earle平衡盐溶液(EBSS)(Thermo Fisher Scientific,Gibco TM,目录号:24010043)
  21. 闪烁液体(PerkinElmer,目录号:6013199)
  22. DMSO(Sigma-Aldrich,目录号:D2650)
  23. 巴弗洛霉素A1(Enzo Life Sciences,目录号:BML-CM110-0100)
  24. 氯化铵(NH4Cl)(Sigma-Aldrich,目录号:A4514)
  25. SAR-405(Magento,ApexBio,目录号:A8883)
  26. Torin1(Tocris Bioscience,目录号:4247)
  27. 非放射性L-缬氨酸(Sigma-Aldrich,目录号:V0513)
  28. 牛血清白蛋白(BSA)(VWR,目录号:422361V)
  29. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T0699)
  30. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60377)
  31. 200 mM冷L-缬氨酸(见食谱)
  32. RPMI 1640/10%FBS(见食谱)
  33. 1毫克/毫升聚-D-赖氨酸(见食谱)
  34. 1 mM Torin1(见食谱)
  35. PBS / 2%BSA(见食谱)
  36. 25%TCA(见食谱)
  37. 0.2 M KOH(见食谱)
  38. 0.2 mM Bafilomycin A1(见食谱)
  39. 160mM NH 4 Cl(参见食谱)

设备

  1. 移液器(Finnpipette TM F2 GLP试剂盒)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:4701070)
  2. 加湿孵化器
  3. 管旋转器(VWR,目录号:444-0502)
  4. 平板摇床(Grant Instruments,型号:PMS-1000i)
  5. 磁力搅拌器(IKA,目录号:0003810001)
  6. 自动流动IR直接加热CO 2培养箱(NuAire,型号:NU-5510E)
  7. Vortexer(Denville Scientific,目录号:S7030)
  8. 带冷却台式离心机(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:75002430)
  9. 闪烁计数器(液体闪烁分析仪)(Packard,型号:1600 TR)

程序

  1. 放射性标签(标准2-3天)
    1. 在37℃下在具有5%CO 2的潮湿培养箱中的75cm 2组织培养瓶中培养贴壁细胞直至获得接近汇合的细胞层。用3 ml 37°C PBS洗涤一次,并在37°C用3 ml 0.25%胰蛋白酶-EDTA孵育细胞,直到细胞脱落(通常3-5分钟)。
    2. 将分离的细胞收集在补充有FBS的10ml 37℃RPMI 1640中至终浓度为10%(=完全培养基,CM)。
      注:本方案中放射性和冷缬氨酸的浓度已根据缬氨酸在该培养基中的浓度以及基于所用放射性缬氨酸量的成本效率而专门用于RPMI 1640中。当遵循这个协议时,细胞因此应该在胰蛋白酶消化后重新悬浮在RPMI 1640中,即使它们通常不在RPMI 1640中培养。出于同样的原因,RPMI 1640应该用于该方案的其余部分。我们已经在> 20细胞系,并且在通常不培养RPMI 1640的细胞中从未观察到毒性或改变的表型迹象,只要细胞不需要任何特殊的补充。可以使用其他培养基代替RPMI 1640,但用户应该在整个实验过程中调整缬氨酸浓度,以考虑RPMI 1640缬氨酸浓度与所用替代培养基的差异。
    3. 种子细胞在0.5ml CM中的24孔板中补充14 C缬氨酸至最终浓度为0.1μCi/ ml。在实验处理时将种子细胞编号导致期望的单层汇合。一般而言,我们在37℃下在含有5%CO 2的湿润培养箱中温育2-3天后使种子细胞达到50-80%融合。
      备注:
      1. 此处介绍的协议与24孔板格式兼容。由于操作简便且重现性高,我们每个实验常规处理两个24孔板(48个孔),每个处理条件有两个重复或三个重复的孔。这使得可以在单个实验中测试相对较多的条件。
      2. 对于LNCaP细胞,我们每孔接种1×10 5个细胞。对于U2OS,VCaP和Huh7,我们分别接种每孔3×10 4个,2.5×10 5个和1.2×10 4个细胞。
      3. 当使用粘附不良的细胞类型时(例如,LNCaP细胞,HEK293细胞和其他细胞),建议使用已涂布的组织培养板以改善细胞粘附(由于方案中的多次洗涤步骤) 。我们更喜欢使用聚-D-赖氨酸(PDL),因为它相对便宜并且易于使用,并且不干扰氨基酸饥饿(因为它不像细胞聚L-赖氨酸那样不被细胞代谢) 。在24孔组织培养板中每孔加入300μl无菌PDL(2.5μg/ ml)。在无菌环境中室温孵育30分钟。之后,使用抽吸去除PDL,并用0.5ml无菌H 2 O短暂冲洗每个孔。干燥后即可使用。当在室温下储存时,PDL包被的平板可稳定长达数周。值得注意的是,对于大多数细胞类型涂层是没有必要的。
      4. 在进行siRNA转染时,我们建议使用反转染,以便可以同时转染和放射性标记细胞。这既实用又具有非常高的转染效率。根据制造商的方案将Opti-MEM,siRNA和Lipofectamine RNAiMAX合并,并将转染混合物等分到24孔板(每孔50μl)中。在室温下孵育20-45分钟,并在含有siRNA-Lipofectamine混合物的孔中接种含有14 C- C-缬氨酸的CM中的0.5ml细胞悬液。仔细搅拌培养板并将其放入培养箱。
      5. 遵照贵机构关于放射性工作的规定。为了保护自己,请佩戴厚度≥0.25毫米的手套。
    4. 在37℃和5%CO 2下在潮湿的培养箱中孵育细胞2-3天,以允许将14 C缬氨酸充分掺入长寿命蛋白质中。

  2. 追逐(标准18小时)
    1. 吸取培养基去除未掺入的放射性物质。

    2. 用0.5毫升补充有10毫克冷缬氨酸(CM-V)的37℃CM清洗细胞一次 注:由于RPMI 1640含有0.17 mM L-缬氨酸,因此CM-V中L-缬氨酸的终浓度为10.17 mM。使用10mM冷的L-缬氨酸阻断释放的14 C-缬氨酸的重新掺入对于这种类型的测定是标准的(Bauvy等,2009; Mizushima等,2001; Fuertes等,2003a)。目前还不知道是否有一个可以使用的高浓度冷L-缬氨酸的上限,但是在分离的大鼠肝细胞中已经显示高达20mM的冷L-缬氨酸不影响蛋白质降解活性(Seglen等1979)。
    3. 吸入培养基并用0.5 ml 37°C CM-V代替。

    4. 在37°C和5%CO 2的潮湿培养箱中孵育细胞18小时。
      注意: 
      1. 追逐不需要18小时。然而,改变追踪的持续时间将改变采样期间的蛋白质降解速率(图2A)以及液体闪烁计数过程中的信号强度(参见注释部分关于闪烁计数和测定变异性的注释中的注释2测量),并且还会更改分析的蛋白质库(请参阅注释部分的注释3)。在比较不同追逐持续时间获得的结果时,应该牢记这一点。
      2. 如果进行实验性治疗时间超过6小时,请在追踪之前或期间添加治疗。此外,还有机会在治疗开始后的不同时间段对LLPD进行治疗效果取样。

  3. 取样(标准2-6小时)
    1. 通过吸取培养基去除释放的14 C缬氨酸(由短寿命蛋白质降解产生)。
    2. 用0.5ml 37℃的适当培养基洗涤细胞一次。如果后续治疗处于完全中度(例如,Torin1),则用CM-V清洗。如果随后的处理将在氨基酸饥饿培养基(例如EBSS)中,用补充有10mM冷缬氨酸(EBSS-V)的EBSS洗涤。
    3. 吸出培养基,并添加0.25毫升37°C CM-V或EBSS-V中的治疗。
    4. 在37℃和5%CO 2下在潮湿的培养箱中孵育细胞2-6小时。

  4. 收获
    第1天

    1. 从培养箱中取出细胞板,并在冰上冷却约2分钟。

    2. 每孔加入50μl冰冷的PBS / 2%BSA 注:BSA作为蛋白质载体加入,以促进TCA蛋白沉淀。

    3. 每孔加入200μl冰冷的25%TCA以沉淀蛋白质。
    4. 将培养板置于4°C摇床上,让其以600 rpm的速度摇晃过夜。
      注意:过夜TCA沉淀足够,降水可能会延长几天而不会影响结果。

      第2天

    5. 将每孔沉淀的蛋白质(〜500μl)转移到1.5 ml微量离心管中。
      注意:将溶液转移到微量离心管中,不要刮擦。保留在孔中的蛋白质沉淀物将溶解并收集在方案步骤D10-D12中。
    6. 通过在4℃下5,000gxg离心10分钟使沉淀物沉淀。离心后,上清液(TCA可溶部分)含有氨基酸和小肽(包括在采样期间由于降解而从蛋白质释放的14 C缬氨酸),并且沉淀(TCA不溶部分)含有完整的蛋白质。
    7. 离心后,将试管置于冰上。
    8. 将每管中的上清液(〜500μl)转移到6 ml闪烁瓶中。
    9. 通过每管加入250μl0.2M KOH溶解沉淀并在室温下旋转管至少1小时。
      注意:在将溶液溶解于KOH之前,没有必要清洗沉淀。
    10. 通过每孔加入250μl0.2M KOH溶解24孔板中剩余的蛋白质沉淀。

    11. 在室温下以600转/分的速度在振动筛上旋转1小时
    12. 合并来自各孔和管的溶解蛋白(〜500μl)并转移至6ml闪烁瓶中。
    13. 将步骤D8和D12中的每个小瓶用4ml闪烁液体填充。盖住管子,并狠狠地旋转。
      注:闪烁液是一种有机和有毒物质。为了您自己的保护,请添加闪烁液体,盖帽管,并在流动柜中旋转,戴上防护手套和实验室涂层。
    14. 通过液体闪烁计数来测量所有管中的放射性数量(关于闪烁计数和化验测量变化的评论,请参见备注部分的注释2)。

数据分析

  1. 数据的代表性例子
    如上述方案中详述的,我们通常在2-3天内标记蛋白质,然后追踪18小时以富集放射性标记的长寿命蛋白质。追逐可以减少或延长以适应其他实验设置,如此处所示。然而,重要的是要意识到这将丰富具有不同半衰期的蛋白质,这又可以改变采样期间的整体蛋白质降解速率以及实验处理的结果(参见进一步解释在注释部分的注3中)。因此,我们建议保持标签和追逐期限尽可能一致。例如,将追踪时间从18小时减少到1小时,LNCaP细胞中的整体蛋白质降解率从≈1.05%/ h增加到≈1.3%/ h(图2A)。
    为了鉴定在所研究的实验条件下发生的溶酶体蛋白质降解的比例,我们推荐包括溶酶体降解抑制剂,如空泡型H + -ATP酶抑制剂Bafilomycin A1(Baf)。 Baf以剂量依赖性方式使溶酶体脱酸并且最终抑制溶酶体降解活性。在LNCaP细胞中,完全培养基中Baf介导的长寿命蛋白降解抑制在25-50nM饱和(图2B)。 Baf敏感部分代表溶酶体降解,而Baf敏感部分代表非溶酶体降解,主要是蛋白酶体蛋白降解。证实这一点,溶酶体促渗剂化合物NH 4 Cl将LLPD减少至与Baf相同的程度(图2C)。溶酶体LLPD是由自噬引起的。在LNCaP细胞中,我们发现用磷脂酰肌醇3-激酶催化亚基3型(PIK3C3)抑制剂SAR-405或自噬相关蛋白Unc-51如自噬激活激酶1(ULK1)和ULK2的敲低抑制LLPD至与Baf或NH 4 Cl的程度相同(图2C)。因此,在LNCaP细胞中,基底溶酶体LLPD归因于PIK3C3 / ULK依赖性自体吞噬,即典型自噬体介导的自噬(巨自噬),而来自微自噬,内涵体微自噬的推定贡献,或分子伴侣介导的自噬(所有这些都是PIK3C3 / ULK-独立的)都可以忽略不计。蛋白酶体对LLPD的贡献可以通过应用蛋白酶体抑制剂如MG132来确定(Fuertes等人,2003a; Engedal等人,2013)。溶酶体和蛋白酶体抑制剂可用于推断对LLPD的治疗效果是否由溶酶体和蛋白酶体活性的改变引起。例如,当将自噬抑制性钙调节剂(A23187和thapsigargin)与Baf或MG132结合时,我们发现用MG132对LLPD具有添加剂减少作用,但与Baf无关(Engedal et al。,2013) ,表明钙调节剂抑制自噬 - 溶酶体LLPD而不是蛋白酶体LLPD。值得注意的是,应该意识到可能发生蛋白酶体和自噬 - 溶酶体途径之间的串扰(Dikic,2017)。然而,在我们测试的时间范围(2-6小时)和条件(基础条件,或氨基酸饥饿条件,哺乳动物雷帕霉素靶标(mTOR)抑制或内质网应激)中,我们没有观察到迹象的LLPD级别的这种串扰。在LNCaP和U2OS细胞中,我们发现Baf和MG132的作用是完全相加的(Engedal et al。,2013),因此表明自噬 - 蛋白酶体串扰对LLPD没有任何可测量的影响。 /> mTOR以对氨基酸和生长因子的存在敏感的方式抑制自噬(Meijer et al。,2015)。因此,从培养基中除去氨基酸和血清后,mTOR活性受到抑制,并诱导自噬。图2D显示了LNCaP细胞用mTOR抑制剂Torin1或急性氨基酸和血清饥饿治疗的典型响应;总LLPD增加约两倍,其方式是在两种情况下都对Baf强烈敏感,即依赖于溶酶体活性。急性饥饿后观察到的LLPD增加不同于细胞系与细胞系。例如,与U2OS(图2E)和LNCaP细胞(图2D)相比,VCaP和Huh7细胞的效应较弱。重要的是,饥饿诱导的LLPD对所有细胞系中的Baf都敏感,表明它主要诱导溶酶体LLPD。值得注意的是,然而,Baf并没有完全消除mTOR抑制或饥饿的影响(图2D-2E),可能是因为这些条件稍微提高了蛋白酶体活性以及自噬(Fuertes等人, 2003a;赵等人,2015年和我们未发表的结果)。还要注意,在完全培养基和饥饿培养基中细胞类型之间的降解速率和Baf敏感性降解程度基本不同(图2D-2E)。这反映了不同程度的基底细胞自噬(完全培养基中Baf敏感性降解)和自噬能力(EBSS中Baf敏感降解)。


    2.使用长效蛋白质降解测定法定量测量内源蛋白质的自噬通量。A-D。将LNCaP和(E)U2OS,VCaP和Huh7细胞系用14 C缬氨酸标记2-3天,追踪(A,左柱)1小时或(A,右柱,BE )18 h,并在追踪培养基中进行指定处理(“采样期”)。随后测量长寿命蛋白质降解(LLPD)。 A.LLPD在6小时的采样期间进行测量,随后1小时或18小时追踪。 B.LLPD以剂量依赖性方式被Baf抑制。红色虚线显示Baf介导的LLPD抑制是饱和的。蛋白质降解的剩余水平主要由蛋白酶体引起。 C.细胞用10nM非靶向siRNA('siCtrl')或5nM siULK1与5nM siULK2组合,并用DMSO(0.1%),Baf(50nM),NH 4 (10mM)或3μMSAR-405处理4小时。红线表示LLPD抑制是饱和的。蛋白质降解的剩余水平主要由蛋白酶体引起。 D.细胞进行指定的处理4小时。 Baf和Torin1以50nM使用。 E. U2OS(左),VCaP(中)和Huh7(右)经过指定的处理4小时。 Baf在所有细胞系中以50nM使用。 A-E。显示了两个独立实验的一个代表(3个生物学重复的平均值±SD)。 Baf,Bafilomycin A1; CM,完全中等; EBSS,厄尔的平衡盐解决方案。

  2. 数据分析
    闪烁计数器生成一个文本文件,其中样本编号以相应的“每分钟计数”(CPM)列出。 CPM值反映了各个样品中14 C的数量。通过将TCA可溶部分中的放射性量除以放射性总量(TCA可溶部分和不溶性部分中的放射性总和)除以采样周期的持续时间来计算降解速率。降解率表示为“长寿命蛋白质降解(%/ h)”。

笔记

  1. 一般来说,我们在每个处理条件下进行重复或三重孔实验(每个实验中两次或三次生物重复实验),而闪烁计数则在整个样品上进行( ie ,没有技术上的重复测量放射性)。遵循此协议时,生物重复之间的偏差通常非常低。从6个不同细胞系的23个独立实验并覆盖总共292个实验条件一式三份,我们已经计算出治疗重复的LLPD率的变异系数(CV)为2.3%±1.5%(平均CV±标准偏差)。更具体地说,CV值分别为0.1-8.9%,分别为0.4%,1.2%,2.0%,3.2%和5.1%的5%,25%,50%,75%和95%百分点值。
  2. 液体闪烁计数器可以设置为在特定时间段内记录计数,或者直到累计了特定数量的计数。测量中的不确定度与计数总数成反比。我们建议设置计数器累计至少10,000次计数,其中标准偏差将≤1.0%。按照目前的方案,用0.1μCi/ ml 14 C缬氨酸标记2-3天,这些设置通常需要少于1分钟的TCA不溶部分计数和数分钟计数TCA可溶部分。背景水平在标准<14> C校准期间设置,非常低(≤22 CPM)。在标准测量方案中,从每个样品中自动减去此背景。因此不需要包括额外的背景样本测量(这将需要许多小时的采样时间用于精确测量)。作为一般说明,我们建议您联系您当地的服务工程师进行维护,校准和服务问题。
  3. 可以使用不同的标签,追逐和采样周期持续时间。例如,一些先前的方案使用18-24小时标记期,接着1小时追踪期(Mizushima et al。,2001; Bauvy et al。,2009年)。将标记时间增加至2-3天(如本文所述)的优点是可以方便地将其与2-3天的转染组合,例如,以及此处描述的反向siRNA转染。而且,标记时间越长,将会标记更长寿命的蛋白质(其被缓慢合成并缓慢降解)。应该指出的是,在人成纤维细胞中,已经描述了主要由蛋白酶体降解而不是由大自噬引起的短寿命蛋白质库(Fuertes等人,2003b)。这个蛋白质库的半衰期约为1.1-1.3小时,需要约8小时才能完全消除它们(Fuertes et al。,2003b)。发现在追踪1-4小时期间,短标记蛋白质的初始TCA不溶性放射性的5-7.5%反映了非降解的TCA不溶性蛋白质向培养基的细胞释放。追逐8小时后,该释放减少至上述值的大约十分之一(Fuertes et al。,2003b)。总之,我们建议追逐时间段&gt; 8小时,以使短命蛋白质的降解和/或分泌的影响最小化。为了安全起见,我们经常使用18小时的追逐期。如此漫长的追逐时期还有另外一个优点,即当希望分析长期实验性治疗对LLPD的影响时,治疗可以在追逐期开始(参见步骤B4b)。此外,由于追逐期通常在2-6小时的采样期后进行,因此18小时追逐期比8小时追逐期更方便。如果需要,取样时间可以超过6小时,但如果将测定与对照处理如溶酶体和蛋白酶体抑制剂结合(如我们推荐的那样),建议保持时间少于6小时以使抑制剂的继发效应最小化。如果希望在治疗超过18小时后分析实验性治疗对LLPD的影响,可以增加追踪期的持续时间,或者甚至在标签期开始治疗,作为延长采样期的替代方案。最后,测定也可以采用比2小时更短的采样时间来完成,但是必须非常小心地确保每个实验样品的均匀时间点,并且可能必须增加放射性14 C-缬氨酸以实现更高程度的标记,从而在闪烁计数器中获得足够的计数水平(参见上面的注2)。

食谱

  1. 200mM冷的L-缬氨酸(Mw 117.15)
    1. 使用磁力搅拌器将1.172g冷(即,非放射性)L-缬氨酸溶解于45ml RPMI 1640(无血清)或EBSS中。

    2. 用RPMI 1640或EBSS分别调节体积至50 ml
    3. 通过一个0.45微米的过滤器过滤到一个无菌的50毫升管
    4. 这种解决方案在4°C储存时可以稳定长达数月。使用前不要预热
  2. 补充有10mM冷L-缬氨酸(CM-V)的RPMI 1640/10%FBS(完全培养基; CM)
    1. 将200毫升冷的L-缬氨酸溶液(上面的配方1)的一部分加入到CM
      19份中
    2. 为每个实验制作一个新的解决方案
  3. 1毫克/毫升聚-D-赖氨酸
    1. 将5mg聚-D-赖氨酸(PDL)溶解于5ml无菌H 2 O中。
    2. 分装在Eppendorf管中并储存在-20°C。
    3. 当储存在-20°C时,储备液可以稳定多年,可以解冻并重新冻结几次。
  4. 1mM Torin1(Mw607.62)
    1. 将10毫克Torin1溶解在16.46毫升DMSO中
    2. 分装于微量离心管中并储存于-20°C。
    3. 当储存在-20°C时,储备液可以稳定多年,可以解冻并重新冻结几次。
  5. PBS / 2%BSA
    1. 将5克BSA +溶解在200毫升PBS中
    2. 添加PBS至总量为250 ml

    3. 通过0.45μm过滤器过滤到无菌玻璃烧瓶中

    4. 储存在4℃条件下可稳定储存数月
  6. 25%TCA
    1. 对于10ml,将2.5ml 100%TCA与7.5ml Milli-Q(MQ)H 2 O混合 注意:将酸加入水中,反之亦然。

    2. 在收获之前将溶液新鲜
  7. 0.2M KOH(Mw 56.11)
    1. 将1.12g KOH溶解于90ml MQ H 2 O中
    2. 将MQ H 2 O添加到总量为100 ml的












  8. 0.2mM Bafilomycin A1(分子量622.84)

    1. 在803μlDMSO中溶解0.1 mg Bafilomycin A1
    2. 分装于微量离心管中并储存于-20°C。
    3. 当储存在-20°C时,储备液可以稳定多年,可以解冻并重新冻结几次。
  9. 160mM NH 4 Cl(Mw 53.49)
    1. 将256.8mg NH 4 Cl溶于25ml MQ H 2 O中。
    2. 在室温下调节至pH7.74(= 37°C时pH = 7.4)
    3. 使用MQ H 2 O
      将最终体积调整至30 ml
    4. 通过一个0.45微米的过滤器过滤到一个无菌的50毫升管


    5. 这种解决方案在-20°C储存时稳定多年

致谢

挪威研究委员会,奥斯陆大学,Anders Jahre基金会,Nansen基金会和Legacy在纪念Henrik Homan的资助下完成了这项工作。该协议是2013年Engedal et al。的方法部分描述的修改版本,该修订版本又改编自2009年的Bauvy 等人,我们也想要承认Per O. Seglen的原创性工作,其中很多方法都是基于此(Seglen和Solheim,1978; Ronning等人,1979; Seglen等人, 1979年)。作者声明他们没有利益冲突。

参考

  1. Bauvy,C.,Meijer,A.J。和Codogno,P。(2009)。 自噬蛋白降解的测定方法Enzymol 452:
  2. Dikic,I.(2017)。 蛋白酶体和自噬降解系统
  3. Engedal N. Torgersen ML Guldvik IJ Barfeld SJ Bakula D.SætreF. Hagen LK Patterson JB Proikas-Cezanne T. Seglen PO P. Simonsen A.和米尔斯,IG(2013)。 调节细胞内钙稳态阻断自噬体形成 自噬 9(10):1475-1490。
  4. Fuertes,G.,Martin De Llano,J. J.,Villarroya,A.,Rivett,A. J.和Knecht,E。(2003a)。 血清戒断产生的人成纤维细胞中蛋白酶体和溶酶体的蛋白水解活性的变化,氨基酸缺失以及融合条件。生物化学杂志375(Pt 1):75-86。
  5. Fuertes,G.,Villarroya,A.和Knecht。 E.(2003b)。 蛋白酶体在各种生长条件下在人成纤维细胞中降解短寿命蛋白质的作用 a> Int J Biochem Cell Biol 35:651-664。
  6. Meijer,A.J.,Lorin,S.,Blommaart,E.F和Codogno,P。(2015)。 通过氨基酸和MTOR依赖性信号转导调节自噬 氨基酸 47(10):2037-2063。
  7. Mizushima,N.,Yamamoto,A.,Hatano,M.,Kobayashi,Y.,Kabeya,Y.,Suzuki,K.,Tokuhisa,T.,Ohsumi,Y.和Yoshimori,T。(2001)。 使用Apg5缺陷小鼠胚胎干细胞解剖自噬体形成 J Cell Biol 152(4):657-668。
  8. Nakabayashi,H.,Taketa,K.,Miyano,K.,Yamane,T。和Sato,J。(1982)。 在化学成分确定的培养基中具有不同功能的人肝癌细胞系的生长。 癌症研究42(9):3858-3863。
  9. Ronning,O.W。,Pettersen,E.O.和Seglen,P.O.(1979)。 通过人NHIK 3025细胞体外细胞周期的蛋白质合成和蛋白质降解 em>。 Exp Cell Res 123(1):63-72。
  10. Seglen,P.O.,Grinde,B。和Solheim,A.E。(1979)。 氨,甲胺,氯喹和亮肽素抑制离体大鼠肝细胞蛋白降解的溶酶体途径。 Eur J Biochem 95(2):215-225。
  11. Seglen P. O.和Solheim,A. E.(1978)。 缬氨酸在离体大鼠肝细胞中的摄取和掺入蛋白质。蛋白质合成前体库的性质。 Eur J biochemi 85:15-25
  12. Zhao,J.,Zhai,B.,Gygi,S.P.and Goldberg,A.L。(2015)。 mTOR抑制通过泛素蛋白酶体系统以及自体吞噬来激活整体蛋白质降解。
    美国国立科学院美国科学院院士112(52):15790-15797。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luhr, M., Sætre, F. and Engedal, N. (2018). The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines. Bio-protocol 8(9): e2836. DOI: 10.21769/BioProtoc.2836.
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