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Insights into the relationship between the proteasome and autophagy in human and yeast cells
Accepted Manuscript Title: Insights into the relationship between the proteasome and autophagy in human and yeast cells Author: Athan´e Axel Buisson Anthony Challier Marion Beaumatin Florian Manon St´ephen Bhatia-Kiˇssˇov´a Ingrid Camougrand Nadine PII: DOI: Reference:
S1357-2725(15)00103-X http://dx.doi.org/doi:10.1016/j.biocel.2015.04.002 BC 4594
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
19-11-2014 13-2-2015 3-4-2015
Please cite this article as: Axel, A., Anthony, B., Marion, C., Florian, B., St´ephen, M., Ingrid, B.-K., and Nadine, C.,Insights into the relationship between the proteasome and autophagy in human and yeast cells, International Journal of Biochemistry and Cell Biology (2015), http://dx.doi.org/10.1016/j.biocel.2015.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Insights into the relationship between the proteasome and autophagy in human and yeast cells
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Athané Axel1,2, Buisson Anthony1,2, Challier Marion1,2, Beaumatin Florian1,2, Manon Stéphen1,2, Bhatia-Kiššová Ingrid3, Camougrand Nadine1,2,*
CNRS and 2Université de Bordeaux, IBGC, UMR5095, 1 rue Camille Saint-Saëns, F33000 Bordeaux, France 3 Comenius University, Faculty of Natural Sciences, Department of Biochemistry, Mlynská dolina CH1, 84215, Bratislava, Slovak Republic
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*Correspondence to Nadine Camougrand: [email protected] Tel: +33 (0) 556999045; Fax: +33 (0) 556999051
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Abstract In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions. Keywords: proteasome, autophagy, mitophagy, yeast, human cell lines
Running title: Relationship between proteasome and autophagy 1. Introduction The ubiquitin-proteasome system and the autophagy process are the two major proteolytic mechanisms in cells, required to maintain cellular homeostasis and protein balance. The proteasome is a nuclear multi-component complex responsible for the specific removal of ubiquitin (Ub) tagged proteins from multiple cellular compartments. Ubiquitylation of proteins occurs via three sequential steps: ubiquitin activation,
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conjugation to a carrier and ligation of Ub to the target mediated by a dedicated ligase. This system allows for the degradation of short-lived, abnormal or damaged proteins and in this way regulates the cellular proteome (Voges et al., 1999). Autophagy is an intracellular vacuolar or lysosomal degradation process targeting long-lived proteins or organelles. This process involves the formation of double-membrane vesicles termed autophagosomes that sequester portions of cytosol. Autophagy is orchestrated by a set of autophagic Atg(s) proteins acting at the different steps of the process. In some cases, when a specific component or organelle is targeted, this process becomes selective; for example mitophagy allows for the degradation of mitochondria (Kissova et al., 2007). Autophagy has been initially thought to protect cells from nutrient deprivation but it is also involved in cellular stress responses (Klionsky and Codogno, 2013). These two degradative processes must be tightly regulated and a crosstalk between them is of great importance for cell health. The UPS and autophagy are thought to cooperate, each regulating the other. It has been shown that proteasome inhibition induces autophagy, implying the role of autophagy as a compensatory mechanism upon an impairment of proteasomal degradation (Rideout et al., 2004; Iwata et al., 2005; Pandey et al., 2007; Suraweera et al., 2012). Regulation of the UPS by autophagy has been less studied. A possible link between the UPS and autophagy processes has been previously investigated by several groups. Cuervo et al pointed out the possibility that inhibition of autophagy might enhance the accumulation of proteasomes (Cuervo et al., 1995). In one of their works, Rubinsztein et al showed that autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates (Korolchuk et al., 2009). Recently, the same group investigated the UPS and autophagy-mediated tau clearance mechanisms and outlined the biochemical connection between these two processes (Lee et al., 2013). Recent work using human colon cancer cells unveiled a novel crosstalk between these two major protein degradation systems (Wang et al., 2013). The authors showed that the proteasome was activated in response to autophagy inhibition by a pharmacological agent or by inactivation of autophagy-related genes under nutrient-deficient conditions. As yeast has proved to be a good model for studying both autophagy and the proteasome, we investigated the relationship between these two degradative processes in different yeast strains grown under various physiological and stress conditions. In our work, we also show that proteasome activity in human cancer cells is, stimulated in response to impaired basal autophagy, in the absence of any additional stimulus. 2. Material and methods 2.1. Yeast strains, human cells lines and growth conditions
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The yeast strains expressing GFP-Atg8 fusion protein used in this study are listed in Table 1. W303-1B (MATα, ade2, his3, leu2, trp1, ura3, can1) was used as wild-type strain. Plasmid pRS416 GFP-ATG8 was a gift from Dr. Klionsky. Yeast cells were grown aerobically at 28°C in a minimal medium, YNB [0.175% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop-Mix (mixture of amino acids except auxotrophic requirements), 0.01% auxotrophic requirements, pH 5.5], supplemented with 2% lactate or 2% glucose as a carbon source. Growth of cells was followed by measurement of optical density (OD) at 600 nm. In some experiments, starved cells were prepared in nitrogen starvation medium [0.175% yeast nitrogen base without amino acids and ammonium sulfate and 2% lactate, pH 5.5]. When used, antimycin A (2 μg/ml) was added to the growth medium for 8 hours. HCT116 cells were grown in McCoy's 5A medium. HeLa cells were grown in RPMI (4.5 g/L glucose). All growth media were supplemented with 10% glutamax (from Gibco Invitrogen), penicillin (100 U/mL), streptomycin (100 mg/mL) and 1% fetal calf serum. Further, cells were pre-incubated with or without bafilomycin (0.1 μM) or 3methyladenine (10 mM) for 6 hours. Cells were harvested when confluence was around 90%.
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2.2. Preparation of protein extracts for western blots and proteasome activity measurements
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Yeast cells (2x108) were harvested by centrifugation, washed with water and resuspended in 0.65 M mannitol, 2 mM EGTA, 10 mM Tris-Maleate, pH 6.8. Cells were broken by homogenization with glass beads for 3 min at a frequency of 20/sec in a cell homogenizer (Retsch-MM400). Unbroken cells were removed by centrifugation at 1000 × g for 5 min and supernatants were used to prepare total cell protein extract. Human cells were scraped and washed twice in Dulbecco phosphate buffer by centrifugation for 2 min at 200 × g. The pellet was then solubilized in 400 µl of MB buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES pH 7.2–7.4). Cells were broken with 20 strokes using a 25-gauge syringe. The samples were centrifuged at 100 × g for 5 min at 4°C. The supernatants were used to prepare total cell protein extract. 2.3. Immunoblotting of LC3, GFP-Atg8, 20S proteasome, Pgk1 and actin 400 µg of proteins (total cell extract) were precipitated by TCA on ice. After centrifugation for 10 min at 10,000 x g, the pellet fraction was washed with 0.2 ml of acetone, dried and resolubilized in 40 µl of 5% SDS plus 40 µl of Laemmli buffer (2% mercaptoethanol, 2% SDS, 0.1 M Tris-HCl, pH 8.8, 20% glycerol, 0.02% bromophenol
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blue). Samples were incubated at 70°C for 5 min prior to loading on gels. Cell lysates equivalent to 50 µg protein were separated by electrophoresis in 12.5% SDS-PAGE and subjected to immunoblotting analysis with anti-LC3 antibody (Sigma, 1/5000 dilution), anti-GFP antibody (Roche, 1/5000 dilution), anti-20S proteasome alpha6 subunit polyclonal antibody (Viva Bioscience, 1/5000 dilution), anti-Pgk1 antibody (Invitrogen, 1/10000 dilution), or anti-actin antibody (Millipore, 1/5000 dilution). Mouse or rabbit secondary antibodies were used at dilution of 1/5000. Detection was made with ECL+ reagent (Amersham). When needed, for quantification of the western blots was used ImageJ software.
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2.4. Proteasome activity assay
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The chymotrypsin-like and trypsin-like activities of the proteasome were determined using the fluorogenic peptides succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVYAMC) or N-t-BOC-leu-ser-Thr—Arg7-76amido-4-Methyl coumarine (LSTR-AMC) respectively, as substrates (Bulteau et al., 2001). LLVY-AMC activity is fully inhibited by 20 μM MG132, a potent proteasome inhibitor while LSTR-AMC activity is partially inhibited. The mixture, containing from 20 µg to 100 µg of total protein extract in 25 mM Tris, pH 7.5, was incubated at 37°C with 50 µM LLVY-AMC in a final volume of 200 µl. Enzymatic kinetics were measured in a temperature-controlled microplate fluorimeter (Polarstar omega, BMG Labtech), at excitation/emission wavelengths of 360/460 nm, measuring fluorescence every 2 min for 30 min. Proteasome activity was determined as the slope of AMC accumulation over time per mg of total protein (pmoles/min/mg). 2.5. Statistical analysis For proteasome activity and the expression of proteasome subunits, results were expressed as the mean ± SEM from 5 to 14 independent experiments. P-values were assessed using paired Student's t tests, p <0.05 were considered statistically significant. 3. Results and discussion
3.1. The stationary phase induced autophagy and inhibited proteasome activity in yeast Autophagy and the UPS are conserved among all eukaryotes. Yeast is an established model organism for studying both processes, and permitted the identification of both Atg proteins and proteasome components as well as an understanding of the steps underlying both processes. In mammals and yeast, the proteasome has three distinct proteolytic activities, caspase-like, trypsin-like and chymotrypsin-like, which can be attributed to the
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β1, β2 and β5 subunits of the 20S core structure of the proteasome, respectively (Heinemeyer et al., 1997). In this study, the chymotrypsin-like and trypsin-like activities were assayed by a chemiluminescence-based method, while the expression of 20S proteasome core subunits was examined by immunoblotting. Autophagy was measured with the GFP-Atg8 processing assay, in which the relative level of free GFP generated by Atg8-proteolysis in the vacuole serves as an indicator of autophagic activity (Shintani and Klionsky, 2004). To explore the relationship between autophagy and the UPS in yeast, autophagic and proteasome activities were examined in wild-type cells grown under strict respiratory conditions in a medium supplemented with lactate as a carbon source. The marked increase of free GFP in wild-type cells entering into the stationary phase of growth indicates autophagy induction (Fig 1A). This autophagic activity likely reflects the increased need for housekeeping the recycling of altered biological material during the stationary phase. Under the same conditions, proteasome chymotrypsin-like and trypsin-like activities in cells decreased by three-fold compared to the early exponential growth phase (Fig 1B, 1C). The level of polyubiquitinated proteins tended to decrease when cells reached the stationary phase (Fig S1 A) but proteasome assembly was not altered on native gels (Fig S1B). The inhibition of the proteasome activity could be related to a decreased expression of proteasome subunits observed in the stationary phase (Fig 1D, 1E). This is further related to previous reports showing an age-related impairment of proteasome functions (Carrard et al., 2002; Bajorek et al., 2003; Hanna et al., 2012; Saunier et al., 2013). 3.2. Knockdown of ATG5 and ATG32 stimulated proteasome activity in yeast in nutrient-rich conditions To assess whether the inhibition of non-selective autophagy at an early stage and a selective mitochondrial degradation (mitophagy) could induce proteasome activity, chymotrypsin-like and trypsin-like activities of the proteasome were determined in Δatg5 (autophagy/mitophagy-deficient) and Δatg32 (mitophagy-deficient) mutants grown under respiratory conditions. As shown in Figure 1A, autophagy induced in the stationary growth phase was similar in Δatg32 mutant as in wild-type. As expected from the complete impairment of autophagy, no accumulation of free GFP was observed in the Δatg5 mutant under the same conditions. As in the parental strain, proteasome activity was reduced in both mutants entering into the stationary phase (Fig 1B,1C) and this decline correlated with a decrease in the expression of proteasome components (Fig 1D,1E). It should be noted that proteolytic activities of the proteasome in the Δatg32 mutant were slightly higher than those of the parental strain, both in the exponential and
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the stationary growth phase (Fig 1B, 1C). The same is true for the Δatg5 mutant, except for the trypsin-like activity in the exponential growth phase (Fig. 1C). A similar increase in proteasome activities was observed in both Δatg32 and Δatg5 mutants growing under fermentative conditions with glucose as a carbon source (Fig S2). It should also be noted that no significant differences were observed in the profiles of polyubiquitinated proteins and in the proteasome assembly in native gels between wild-type strain and Δatg32 and Δatg5 mutants, both in exponential and stationary growth phases (Fig S1A, S1B). These findings suggest that Δatg5 and Δatg32 mutants maintain a higher basal proteasome activity than the parental strain, thus compensating for the absence of autophagy or mitophagy. Nevertheless, the increase in proteasome activity is subtle, likely because the UPS has only limited capacity to stand as a substitute for other degradative pathways.
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3.3. The inactivation of ATG5 and ATG32 partly abolished nitrogen starvationinduced reduction of proteasome activity The shift of yeast cells from nutrient-rich to nitrogen starvation conditions in the media with a respiratory carbon source is well established to induce both autophagy and mitophagy (Kissova et al., 2004, 2007). As shown in Figure 2A and 2B, the same condition reduced proteolytic activities of the proteasome in wild-type cells. However, proteasome activities in Δatg5 and Δatg32 mutants were significantly higher than in the parental strain, showing that these mutants sustained this activity despite the starvation stress. Like in the stationary phase, the drop in proteasome activity induced by nitrogen starvation correlated with the level of polyubiquitinated proteins (Fig S1C) but not with a decrease in the expression of the proteasome components (Fig 2C, 2D). 3.4. The absence of Atg32 protein stimulated proteasome activity following antimycin A treatment It has been hypothesized that autophagy could play an alternative role as a stress-induced housekeeping mechanism involved in the general maintenance of cellular homeostasis, namely in the quality control of mitochondria. Indeed, it has been shown that, mitochondrial damage induced by the respiratory complex III inhibitor antimycin A leads to non-selective autophagy in respiratory conditions (Deffieu et al., 2013). Interestingly, a component that is specific and essential for mitophagy, Atg32p, was shown to be required for this process. As shown in Figure 3A, in wild-type strain, antimycin A treatment caused a slight decrease in chymotrypsin-like activity, while trypsin-like activity did not seem affected. Under the same conditions, proteasome activity in the Δatg5 strain was not significantly changed and was maintained at the level observed in the early exponential
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growth phase. On the other hand, chymotrypsin- and trypsin-like proteasome activities slightly increased in the antimycin A treated mitophagy-deficient Δatg32 mutant (Fig 3A). The stimulation of the activity did not correlate with an increase in the expression of proteasome components (fig 3C, 3D), any significant change in the profile of polyubiquitinated proteins (Fig S1C) or an altered proteasome assembly on native gels (Fig S1B). This finding is very interesting, because it could indicate that particularly in the absence of the mitophagy receptor Atg32p the inability of cells to induce autophagy following antimycin A-induced mitochondrial damage results in a stimulation of proteasome activity to compensate for this unfavorable situation.
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3.5. Inhibition of basal autophagy enhanced proteasome activity in human HeLa and HCT116 cancer cells
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To confirm data obtained with yeast as a model organism, we investigated how the inhibition of basal autophagy, which is thought to be important for normal turnover of cytoplasmic content within cells in nutrient-rich conditions, impacts proteasome activity in human cell lines. Bafilomycin is an inhibitor of the lysosomal V-ATPase proton pump and prevents the acidification of lysosomes and thus impairs the late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes. On the other hand, 3methyladenine (3-MA) is an inhibitor of the initial phase of the autophagic process, which blocks autophagosome formation via the inhibition of class III PI3K. Both of these inhibitors were used to prevent autophagy in two cancer cell lines HeLa and HCT116. To assess the efficacy of bafilomycin and 3-MA, the level of LC3-II was detected by immunoblotting in these two cell lines. LC3-II, the PE-conjugated form, is an autophagosomal surface protein marker that is ultimately degraded by acidic hydrolases after the formation of autolysosomes. Western blots showed that the LC3-II levels were dramatically increased by bafilomycin treatment in HeLa and HCT116 cells, indicating the impairment of the autophagic flux (Fig 4A). Pharmacological inhibition of basal autophagy by bafilomycin and 3-MA induced a slight but significant increase in proteasome proteolytic activity in HeLa and HCT116 cells (Fig 4A, 4C). 3Methyladenine or bafilomycin treatment did not significantly change the expression of certain proteasome subunits (Fig 4A, 4C). Our findings suggest that, contrary to a previous report (Wang et al., 2013), inhibition of basal autophagy flux could also stimulate proteasome activity. Our data supports the idea that the dialogue between these two protein degradation systems is continuous, and as soon as one of these processes is impaired, the other one takes over.
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4. Conclusion
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The fact that the proteasome and autophagy are both essential to maintain protein homeostasis and cell viability, invites speculation about a mutual regulation between these processes. The data obtained in this study reinforces the concept of a dialogue between the UPS and autophagy, both in yeast and human cells. In both models, stimulation of proteasome activity was observed following inhibition of basal autophagy by pharmacological agents as well as inactivation of the yeast autophagy-related genes, ATG5 and ATG32. The study also found that the increase in the proteasome activity was more subtle than that reported by Wang et al, which examined proteasome activity after autophagy inhibition in human cancer cells under nutrient-deficient conditions (Wang et al., 2013). Nevertheless, our work highlighted a need to overcome the loss of even a relatively little autophagic flux by enhanced compensatory proteasome activity, which may be relevant to cell physiology. In yeast, given that proteasome activity was altered by both activation and inhibition of autophagy, our findings point out the possibility of direct or mediated (by crosstalk with other signaling pathways) regulation of proteasome activity by autophagy independent of substrate delivery (non-selective portion of cytosol, mitochondria) in various cellular contexts (e.g. growth, aging, starvation, stress). Considering the important role played by autophagy and the UPS in disease development, it will be crucial for future studies to investigate the molecular events that mediate the mutual regulation of the two main protein degradation pathways in eukaryotes.
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ACKNOWLEDGMENTS
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This work was supported by grants from the CNRS, the Université Victor Ségalen, the Association Française contre les Myopathies (to N.C.), the Conseil Régional d’Aquitaine (to UMR5095). The authors wish to thank Drs. Anne Laure Bulteau (IPREM-LCABIE UMR5254) for technical advices and critical reading of the manuscript, Muriel Priault and Isabelle Sagot (CNRS UMR5095) for helpful discussions.
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The authors declare no conflict of interest.
Figure 1: Alterations in autophagy and proteasome activity during yeast growth. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFP-Atg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source and harvested at the early exponential phase (early expo), the late exponential phase (late expo) and the stationary phase (stat). A: Immunoblotting analysis with anti-GFP and anti-Pgk1 antibody. B and C: Chymotrypsin-like (n=9 for W303, n=8 for Δatg32 and n=7 for Δatg5) and trypsin-like (n=9 for W303, n=8 for Δatg32 and n=7 for Δatg5) activities of the proteasome measured at early and stationary phases and expressed as the percentage of activity of wild-type cells in the early exponential growth phase; *P<0.05. D: Immunoblotting analysis with anti-20S proteasome and anti-Pgk1 antibody. E: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=9). Figure 2: Inactivation of ATG5 and ATG32 partly reversed nitrogen starvation-
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induced reduction of proteasome activity. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFP-Atg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source, harvested at the early exponential phase (expo) and submitted to nitrogen starvation for 6 hours (-N 6h). A and B: Chymotrypsin-like (n=14 for W303, n=14 for Δatg32 and n=8 for Δatg5) and trypsin-like (n=9 for W303, n=5 for Δatg32 and n=9 for Δatg5) activities of the proteasome expressed as percentage of activity of wildtype cells in the early exponential growth phase; *P<0.05. C: Immunoblotting analysis with anti-20S proteasome and anti-Pgk1 antibody. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=5).
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Figure 3: The absence of Atg32 protein stimulated proteasome activity following antimycin A treatment. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFPAtg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source and harvested at the early exponential phase (expo) and treated with antimycin A (2 µg/ml) for 8 hours (anti A). A and B: Chymotrypsin-like (n=13 for W303, n=14 for Δatg32 and n=11 for Δatg5) and trypsin-like (n=8 for W303, n=10 for Δatg32 and n=7 for Δatg5) activities of the proteasome expressed as percentage of activity of wild-type cells in the early exponential growth phase; *P<0.05. C: Immunoblotting analysis with anti20S proteasome and anti-Pgk1 antibody. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=6).
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Figure 4: Autophagy inhibition enhanced proteasome activity in cultured human cancer cells. HCT116 and HeLa cells were grown in an adequate nutrient-rich medium and treated or left untreated with 3-MA (10 mM) or bafilomycin (0.1 µM) for 6 hours. A: Immunoblotting analysis with anti-LC3, anti-actin and anti-20S proteasome antibodies. B and C: Chymotrypsin-like (n= 6 to 8 for HeLa cells and n= 6 to 9 for HCT cells) and trypsin-like (n= 8 to 9 for HeLa cells and n= 6 to 16 for HCT cells) activities of the proteasome expressed as percentage of untreated control; *P<0.05. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n= 3 to 9).
Table 1: Strains used in this study W303-1B
Mat α ade2-1 his3-11 leu2-3_112 trpΔ2 ura3-52 can1-100 + pRS416 GFP-Atg8
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W303-1B atg5Δ::KAN + pRS416 GFP-Atg8
atg32
W303-1B atg32Δ::KAN + pRS416 GFP-Atg8
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atg5
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S1357-2725(15)00103-X http://dx.doi.org/doi:10.1016/j.biocel.2015.04.002 BC 4594
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
19-11-2014 13-2-2015 3-4-2015
Please cite this article as: Axel, A., Anthony, B., Marion, C., Florian, B., St´ephen, M., Ingrid, B.-K., and Nadine, C.,Insights into the relationship between the proteasome and autophagy in human and yeast cells, International Journal of Biochemistry and Cell Biology (2015), http://dx.doi.org/10.1016/j.biocel.2015.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Insights into the relationship between the proteasome and autophagy in human and yeast cells
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Athané Axel1,2, Buisson Anthony1,2, Challier Marion1,2, Beaumatin Florian1,2, Manon Stéphen1,2, Bhatia-Kiššová Ingrid3, Camougrand Nadine1,2,*
CNRS and 2Université de Bordeaux, IBGC, UMR5095, 1 rue Camille Saint-Saëns, F33000 Bordeaux, France 3 Comenius University, Faculty of Natural Sciences, Department of Biochemistry, Mlynská dolina CH1, 84215, Bratislava, Slovak Republic
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*Correspondence to Nadine Camougrand: [email protected] Tel: +33 (0) 556999045; Fax: +33 (0) 556999051
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Abstract In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions. Keywords: proteasome, autophagy, mitophagy, yeast, human cell lines
Running title: Relationship between proteasome and autophagy 1. Introduction The ubiquitin-proteasome system and the autophagy process are the two major proteolytic mechanisms in cells, required to maintain cellular homeostasis and protein balance. The proteasome is a nuclear multi-component complex responsible for the specific removal of ubiquitin (Ub) tagged proteins from multiple cellular compartments. Ubiquitylation of proteins occurs via three sequential steps: ubiquitin activation,
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conjugation to a carrier and ligation of Ub to the target mediated by a dedicated ligase. This system allows for the degradation of short-lived, abnormal or damaged proteins and in this way regulates the cellular proteome (Voges et al., 1999). Autophagy is an intracellular vacuolar or lysosomal degradation process targeting long-lived proteins or organelles. This process involves the formation of double-membrane vesicles termed autophagosomes that sequester portions of cytosol. Autophagy is orchestrated by a set of autophagic Atg(s) proteins acting at the different steps of the process. In some cases, when a specific component or organelle is targeted, this process becomes selective; for example mitophagy allows for the degradation of mitochondria (Kissova et al., 2007). Autophagy has been initially thought to protect cells from nutrient deprivation but it is also involved in cellular stress responses (Klionsky and Codogno, 2013). These two degradative processes must be tightly regulated and a crosstalk between them is of great importance for cell health. The UPS and autophagy are thought to cooperate, each regulating the other. It has been shown that proteasome inhibition induces autophagy, implying the role of autophagy as a compensatory mechanism upon an impairment of proteasomal degradation (Rideout et al., 2004; Iwata et al., 2005; Pandey et al., 2007; Suraweera et al., 2012). Regulation of the UPS by autophagy has been less studied. A possible link between the UPS and autophagy processes has been previously investigated by several groups. Cuervo et al pointed out the possibility that inhibition of autophagy might enhance the accumulation of proteasomes (Cuervo et al., 1995). In one of their works, Rubinsztein et al showed that autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates (Korolchuk et al., 2009). Recently, the same group investigated the UPS and autophagy-mediated tau clearance mechanisms and outlined the biochemical connection between these two processes (Lee et al., 2013). Recent work using human colon cancer cells unveiled a novel crosstalk between these two major protein degradation systems (Wang et al., 2013). The authors showed that the proteasome was activated in response to autophagy inhibition by a pharmacological agent or by inactivation of autophagy-related genes under nutrient-deficient conditions. As yeast has proved to be a good model for studying both autophagy and the proteasome, we investigated the relationship between these two degradative processes in different yeast strains grown under various physiological and stress conditions. In our work, we also show that proteasome activity in human cancer cells is, stimulated in response to impaired basal autophagy, in the absence of any additional stimulus. 2. Material and methods 2.1. Yeast strains, human cells lines and growth conditions
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The yeast strains expressing GFP-Atg8 fusion protein used in this study are listed in Table 1. W303-1B (MATα, ade2, his3, leu2, trp1, ura3, can1) was used as wild-type strain. Plasmid pRS416 GFP-ATG8 was a gift from Dr. Klionsky. Yeast cells were grown aerobically at 28°C in a minimal medium, YNB [0.175% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop-Mix (mixture of amino acids except auxotrophic requirements), 0.01% auxotrophic requirements, pH 5.5], supplemented with 2% lactate or 2% glucose as a carbon source. Growth of cells was followed by measurement of optical density (OD) at 600 nm. In some experiments, starved cells were prepared in nitrogen starvation medium [0.175% yeast nitrogen base without amino acids and ammonium sulfate and 2% lactate, pH 5.5]. When used, antimycin A (2 μg/ml) was added to the growth medium for 8 hours. HCT116 cells were grown in McCoy's 5A medium. HeLa cells were grown in RPMI (4.5 g/L glucose). All growth media were supplemented with 10% glutamax (from Gibco Invitrogen), penicillin (100 U/mL), streptomycin (100 mg/mL) and 1% fetal calf serum. Further, cells were pre-incubated with or without bafilomycin (0.1 μM) or 3methyladenine (10 mM) for 6 hours. Cells were harvested when confluence was around 90%.
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2.2. Preparation of protein extracts for western blots and proteasome activity measurements
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Yeast cells (2x108) were harvested by centrifugation, washed with water and resuspended in 0.65 M mannitol, 2 mM EGTA, 10 mM Tris-Maleate, pH 6.8. Cells were broken by homogenization with glass beads for 3 min at a frequency of 20/sec in a cell homogenizer (Retsch-MM400). Unbroken cells were removed by centrifugation at 1000 × g for 5 min and supernatants were used to prepare total cell protein extract. Human cells were scraped and washed twice in Dulbecco phosphate buffer by centrifugation for 2 min at 200 × g. The pellet was then solubilized in 400 µl of MB buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES pH 7.2–7.4). Cells were broken with 20 strokes using a 25-gauge syringe. The samples were centrifuged at 100 × g for 5 min at 4°C. The supernatants were used to prepare total cell protein extract. 2.3. Immunoblotting of LC3, GFP-Atg8, 20S proteasome, Pgk1 and actin 400 µg of proteins (total cell extract) were precipitated by TCA on ice. After centrifugation for 10 min at 10,000 x g, the pellet fraction was washed with 0.2 ml of acetone, dried and resolubilized in 40 µl of 5% SDS plus 40 µl of Laemmli buffer (2% mercaptoethanol, 2% SDS, 0.1 M Tris-HCl, pH 8.8, 20% glycerol, 0.02% bromophenol
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blue). Samples were incubated at 70°C for 5 min prior to loading on gels. Cell lysates equivalent to 50 µg protein were separated by electrophoresis in 12.5% SDS-PAGE and subjected to immunoblotting analysis with anti-LC3 antibody (Sigma, 1/5000 dilution), anti-GFP antibody (Roche, 1/5000 dilution), anti-20S proteasome alpha6 subunit polyclonal antibody (Viva Bioscience, 1/5000 dilution), anti-Pgk1 antibody (Invitrogen, 1/10000 dilution), or anti-actin antibody (Millipore, 1/5000 dilution). Mouse or rabbit secondary antibodies were used at dilution of 1/5000. Detection was made with ECL+ reagent (Amersham). When needed, for quantification of the western blots was used ImageJ software.
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2.4. Proteasome activity assay
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The chymotrypsin-like and trypsin-like activities of the proteasome were determined using the fluorogenic peptides succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVYAMC) or N-t-BOC-leu-ser-Thr—Arg7-76amido-4-Methyl coumarine (LSTR-AMC) respectively, as substrates (Bulteau et al., 2001). LLVY-AMC activity is fully inhibited by 20 μM MG132, a potent proteasome inhibitor while LSTR-AMC activity is partially inhibited. The mixture, containing from 20 µg to 100 µg of total protein extract in 25 mM Tris, pH 7.5, was incubated at 37°C with 50 µM LLVY-AMC in a final volume of 200 µl. Enzymatic kinetics were measured in a temperature-controlled microplate fluorimeter (Polarstar omega, BMG Labtech), at excitation/emission wavelengths of 360/460 nm, measuring fluorescence every 2 min for 30 min. Proteasome activity was determined as the slope of AMC accumulation over time per mg of total protein (pmoles/min/mg). 2.5. Statistical analysis For proteasome activity and the expression of proteasome subunits, results were expressed as the mean ± SEM from 5 to 14 independent experiments. P-values were assessed using paired Student's t tests, p <0.05 were considered statistically significant. 3. Results and discussion
3.1. The stationary phase induced autophagy and inhibited proteasome activity in yeast Autophagy and the UPS are conserved among all eukaryotes. Yeast is an established model organism for studying both processes, and permitted the identification of both Atg proteins and proteasome components as well as an understanding of the steps underlying both processes. In mammals and yeast, the proteasome has three distinct proteolytic activities, caspase-like, trypsin-like and chymotrypsin-like, which can be attributed to the
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β1, β2 and β5 subunits of the 20S core structure of the proteasome, respectively (Heinemeyer et al., 1997). In this study, the chymotrypsin-like and trypsin-like activities were assayed by a chemiluminescence-based method, while the expression of 20S proteasome core subunits was examined by immunoblotting. Autophagy was measured with the GFP-Atg8 processing assay, in which the relative level of free GFP generated by Atg8-proteolysis in the vacuole serves as an indicator of autophagic activity (Shintani and Klionsky, 2004). To explore the relationship between autophagy and the UPS in yeast, autophagic and proteasome activities were examined in wild-type cells grown under strict respiratory conditions in a medium supplemented with lactate as a carbon source. The marked increase of free GFP in wild-type cells entering into the stationary phase of growth indicates autophagy induction (Fig 1A). This autophagic activity likely reflects the increased need for housekeeping the recycling of altered biological material during the stationary phase. Under the same conditions, proteasome chymotrypsin-like and trypsin-like activities in cells decreased by three-fold compared to the early exponential growth phase (Fig 1B, 1C). The level of polyubiquitinated proteins tended to decrease when cells reached the stationary phase (Fig S1 A) but proteasome assembly was not altered on native gels (Fig S1B). The inhibition of the proteasome activity could be related to a decreased expression of proteasome subunits observed in the stationary phase (Fig 1D, 1E). This is further related to previous reports showing an age-related impairment of proteasome functions (Carrard et al., 2002; Bajorek et al., 2003; Hanna et al., 2012; Saunier et al., 2013). 3.2. Knockdown of ATG5 and ATG32 stimulated proteasome activity in yeast in nutrient-rich conditions To assess whether the inhibition of non-selective autophagy at an early stage and a selective mitochondrial degradation (mitophagy) could induce proteasome activity, chymotrypsin-like and trypsin-like activities of the proteasome were determined in Δatg5 (autophagy/mitophagy-deficient) and Δatg32 (mitophagy-deficient) mutants grown under respiratory conditions. As shown in Figure 1A, autophagy induced in the stationary growth phase was similar in Δatg32 mutant as in wild-type. As expected from the complete impairment of autophagy, no accumulation of free GFP was observed in the Δatg5 mutant under the same conditions. As in the parental strain, proteasome activity was reduced in both mutants entering into the stationary phase (Fig 1B,1C) and this decline correlated with a decrease in the expression of proteasome components (Fig 1D,1E). It should be noted that proteolytic activities of the proteasome in the Δatg32 mutant were slightly higher than those of the parental strain, both in the exponential and
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the stationary growth phase (Fig 1B, 1C). The same is true for the Δatg5 mutant, except for the trypsin-like activity in the exponential growth phase (Fig. 1C). A similar increase in proteasome activities was observed in both Δatg32 and Δatg5 mutants growing under fermentative conditions with glucose as a carbon source (Fig S2). It should also be noted that no significant differences were observed in the profiles of polyubiquitinated proteins and in the proteasome assembly in native gels between wild-type strain and Δatg32 and Δatg5 mutants, both in exponential and stationary growth phases (Fig S1A, S1B). These findings suggest that Δatg5 and Δatg32 mutants maintain a higher basal proteasome activity than the parental strain, thus compensating for the absence of autophagy or mitophagy. Nevertheless, the increase in proteasome activity is subtle, likely because the UPS has only limited capacity to stand as a substitute for other degradative pathways.
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3.3. The inactivation of ATG5 and ATG32 partly abolished nitrogen starvationinduced reduction of proteasome activity The shift of yeast cells from nutrient-rich to nitrogen starvation conditions in the media with a respiratory carbon source is well established to induce both autophagy and mitophagy (Kissova et al., 2004, 2007). As shown in Figure 2A and 2B, the same condition reduced proteolytic activities of the proteasome in wild-type cells. However, proteasome activities in Δatg5 and Δatg32 mutants were significantly higher than in the parental strain, showing that these mutants sustained this activity despite the starvation stress. Like in the stationary phase, the drop in proteasome activity induced by nitrogen starvation correlated with the level of polyubiquitinated proteins (Fig S1C) but not with a decrease in the expression of the proteasome components (Fig 2C, 2D). 3.4. The absence of Atg32 protein stimulated proteasome activity following antimycin A treatment It has been hypothesized that autophagy could play an alternative role as a stress-induced housekeeping mechanism involved in the general maintenance of cellular homeostasis, namely in the quality control of mitochondria. Indeed, it has been shown that, mitochondrial damage induced by the respiratory complex III inhibitor antimycin A leads to non-selective autophagy in respiratory conditions (Deffieu et al., 2013). Interestingly, a component that is specific and essential for mitophagy, Atg32p, was shown to be required for this process. As shown in Figure 3A, in wild-type strain, antimycin A treatment caused a slight decrease in chymotrypsin-like activity, while trypsin-like activity did not seem affected. Under the same conditions, proteasome activity in the Δatg5 strain was not significantly changed and was maintained at the level observed in the early exponential
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growth phase. On the other hand, chymotrypsin- and trypsin-like proteasome activities slightly increased in the antimycin A treated mitophagy-deficient Δatg32 mutant (Fig 3A). The stimulation of the activity did not correlate with an increase in the expression of proteasome components (fig 3C, 3D), any significant change in the profile of polyubiquitinated proteins (Fig S1C) or an altered proteasome assembly on native gels (Fig S1B). This finding is very interesting, because it could indicate that particularly in the absence of the mitophagy receptor Atg32p the inability of cells to induce autophagy following antimycin A-induced mitochondrial damage results in a stimulation of proteasome activity to compensate for this unfavorable situation.
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3.5. Inhibition of basal autophagy enhanced proteasome activity in human HeLa and HCT116 cancer cells
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To confirm data obtained with yeast as a model organism, we investigated how the inhibition of basal autophagy, which is thought to be important for normal turnover of cytoplasmic content within cells in nutrient-rich conditions, impacts proteasome activity in human cell lines. Bafilomycin is an inhibitor of the lysosomal V-ATPase proton pump and prevents the acidification of lysosomes and thus impairs the late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes. On the other hand, 3methyladenine (3-MA) is an inhibitor of the initial phase of the autophagic process, which blocks autophagosome formation via the inhibition of class III PI3K. Both of these inhibitors were used to prevent autophagy in two cancer cell lines HeLa and HCT116. To assess the efficacy of bafilomycin and 3-MA, the level of LC3-II was detected by immunoblotting in these two cell lines. LC3-II, the PE-conjugated form, is an autophagosomal surface protein marker that is ultimately degraded by acidic hydrolases after the formation of autolysosomes. Western blots showed that the LC3-II levels were dramatically increased by bafilomycin treatment in HeLa and HCT116 cells, indicating the impairment of the autophagic flux (Fig 4A). Pharmacological inhibition of basal autophagy by bafilomycin and 3-MA induced a slight but significant increase in proteasome proteolytic activity in HeLa and HCT116 cells (Fig 4A, 4C). 3Methyladenine or bafilomycin treatment did not significantly change the expression of certain proteasome subunits (Fig 4A, 4C). Our findings suggest that, contrary to a previous report (Wang et al., 2013), inhibition of basal autophagy flux could also stimulate proteasome activity. Our data supports the idea that the dialogue between these two protein degradation systems is continuous, and as soon as one of these processes is impaired, the other one takes over.
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4. Conclusion
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The fact that the proteasome and autophagy are both essential to maintain protein homeostasis and cell viability, invites speculation about a mutual regulation between these processes. The data obtained in this study reinforces the concept of a dialogue between the UPS and autophagy, both in yeast and human cells. In both models, stimulation of proteasome activity was observed following inhibition of basal autophagy by pharmacological agents as well as inactivation of the yeast autophagy-related genes, ATG5 and ATG32. The study also found that the increase in the proteasome activity was more subtle than that reported by Wang et al, which examined proteasome activity after autophagy inhibition in human cancer cells under nutrient-deficient conditions (Wang et al., 2013). Nevertheless, our work highlighted a need to overcome the loss of even a relatively little autophagic flux by enhanced compensatory proteasome activity, which may be relevant to cell physiology. In yeast, given that proteasome activity was altered by both activation and inhibition of autophagy, our findings point out the possibility of direct or mediated (by crosstalk with other signaling pathways) regulation of proteasome activity by autophagy independent of substrate delivery (non-selective portion of cytosol, mitochondria) in various cellular contexts (e.g. growth, aging, starvation, stress). Considering the important role played by autophagy and the UPS in disease development, it will be crucial for future studies to investigate the molecular events that mediate the mutual regulation of the two main protein degradation pathways in eukaryotes.
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ACKNOWLEDGMENTS
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This work was supported by grants from the CNRS, the Université Victor Ségalen, the Association Française contre les Myopathies (to N.C.), the Conseil Régional d’Aquitaine (to UMR5095). The authors wish to thank Drs. Anne Laure Bulteau (IPREM-LCABIE UMR5254) for technical advices and critical reading of the manuscript, Muriel Priault and Isabelle Sagot (CNRS UMR5095) for helpful discussions.
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The authors declare no conflict of interest.
Figure 1: Alterations in autophagy and proteasome activity during yeast growth. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFP-Atg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source and harvested at the early exponential phase (early expo), the late exponential phase (late expo) and the stationary phase (stat). A: Immunoblotting analysis with anti-GFP and anti-Pgk1 antibody. B and C: Chymotrypsin-like (n=9 for W303, n=8 for Δatg32 and n=7 for Δatg5) and trypsin-like (n=9 for W303, n=8 for Δatg32 and n=7 for Δatg5) activities of the proteasome measured at early and stationary phases and expressed as the percentage of activity of wild-type cells in the early exponential growth phase; *P<0.05. D: Immunoblotting analysis with anti-20S proteasome and anti-Pgk1 antibody. E: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=9). Figure 2: Inactivation of ATG5 and ATG32 partly reversed nitrogen starvation-
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induced reduction of proteasome activity. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFP-Atg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source, harvested at the early exponential phase (expo) and submitted to nitrogen starvation for 6 hours (-N 6h). A and B: Chymotrypsin-like (n=14 for W303, n=14 for Δatg32 and n=8 for Δatg5) and trypsin-like (n=9 for W303, n=5 for Δatg32 and n=9 for Δatg5) activities of the proteasome expressed as percentage of activity of wildtype cells in the early exponential growth phase; *P<0.05. C: Immunoblotting analysis with anti-20S proteasome and anti-Pgk1 antibody. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=5).
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Figure 3: The absence of Atg32 protein stimulated proteasome activity following antimycin A treatment. Wild-type strain, Δatg32 and Δatg5 mutants expressing GFPAtg8 were grown in a minimal YNB medium supplemented with lactate as a carbon source and harvested at the early exponential phase (expo) and treated with antimycin A (2 µg/ml) for 8 hours (anti A). A and B: Chymotrypsin-like (n=13 for W303, n=14 for Δatg32 and n=11 for Δatg5) and trypsin-like (n=8 for W303, n=10 for Δatg32 and n=7 for Δatg5) activities of the proteasome expressed as percentage of activity of wild-type cells in the early exponential growth phase; *P<0.05. C: Immunoblotting analysis with anti20S proteasome and anti-Pgk1 antibody. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n=6).
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Figure 4: Autophagy inhibition enhanced proteasome activity in cultured human cancer cells. HCT116 and HeLa cells were grown in an adequate nutrient-rich medium and treated or left untreated with 3-MA (10 mM) or bafilomycin (0.1 µM) for 6 hours. A: Immunoblotting analysis with anti-LC3, anti-actin and anti-20S proteasome antibodies. B and C: Chymotrypsin-like (n= 6 to 8 for HeLa cells and n= 6 to 9 for HCT cells) and trypsin-like (n= 8 to 9 for HeLa cells and n= 6 to 16 for HCT cells) activities of the proteasome expressed as percentage of untreated control; *P<0.05. D: Proteasome expression presented as the 20S/Pgk1 ratio in function of the individual condition (n= 3 to 9).
Table 1: Strains used in this study W303-1B
Mat α ade2-1 his3-11 leu2-3_112 trpΔ2 ura3-52 can1-100 + pRS416 GFP-Atg8
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W303-1B atg5Δ::KAN + pRS416 GFP-Atg8
atg32
W303-1B atg32Δ::KAN + pRS416 GFP-Atg8
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atg5
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Figure 1
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Figure 4
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