Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities
2007; Elsevier BV; Volume: 6; Issue: 11 Linguagem: Inglês
10.1074/mcp.m700187-mcp200
ISSN1535-9484
AutoresOliver Drews, Robert Wildgruber, Chenggong Zong, Ute Sukop, Mikkel Nissum, Gerhard Weber, Aldrin V. Gomes, Peipei Ping,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit β2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium. The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit β2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium. The half-life of many proteins in cell proliferation, apoptosis, and gene expression is dependent on the ubiquitin-proteasome system (UPS) 1The abbreviations used are: UPS, ubiquitin-proteasome system; BisTris, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane; CIAP, calf intestinal alkaline phosphatase; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid); FFE, free flow electrophoresis; PTM, posttranslational modification; Z, benzyloxycarbonyl; AMC, amino-4-methylcoumarin; Boc, t-butoxycarbonyl; Suc, succinyl; 2-DE, two-dimensional electrophoresis. 1The abbreviations used are: UPS, ubiquitin-proteasome system; BisTris, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane; CIAP, calf intestinal alkaline phosphatase; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid); FFE, free flow electrophoresis; PTM, posttranslational modification; Z, benzyloxycarbonyl; AMC, amino-4-methylcoumarin; Boc, t-butoxycarbonyl; Suc, succinyl; 2-DE, two-dimensional electrophoresis. 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These studies suggest that the current view on proteasome systems (the constitutive and inducible models) may no longer adequately describe the 20 S proteasome complexity. In the present study, the hypothesis whether proteasome subpopulations exist in other mammals was investigated in Mus musculus. With regard to current controversies in cardiovascular proteasome research, we addressed the question whether distinct proteasome complexes exist in cardiac tissue. Previous studies from our group and other groups indicated charge differences between subpopulations by different assembly and posttranslational modifications (PTMs). Therefore, separation according to the pI appeared to be essential. Native IEF at high resolution of multiprotein complexes in excess of 700 kDa, such as the 20 S proteasomes, is quite challenging. Free flow electrophoresis (FFE) was chosen for this task because of its proven ability to separate native proteins as well as organelles in a system without passing the analytes through a solid matrix (34Moritz R.L. Simpson R.J. Liquid-based free-flow electrophoresis-reversed-phase HPLC: a proteomic tool.Nat. Methods. 2005; 2: 863-873Crossref PubMed Scopus (41) Google Scholar, 35Weber G. Islinger M. Weber P. Eckerskorn C. Volkl A. Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing.Electrophoresis. 2004; 25: 1735-1747Crossref PubMed Scopus (50) Google Scholar, 36Zischka H. Braun R.J. Marantidis E.P. Buringer D. Bornhovd C. Hauck S.M. Demmer O. Gloeckner C.J. Reichert A.S. Madeo F. Ueffing M. Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.Mol. Cell. Proteomics. 2006; 5: 2185-2200Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In our study, a novel FFE protocol was used for IEF to effectively separate 20 S proteasome complexes. Proteasome complexes were purified from murine liver and hearts according to modified published protocols (6Zong C. Gomes A.V. Drews O. Li X. Young G.W. Berhane B. Qiao X. French S.W. Bardag-Gorce F. Ping P. Regulation of murine cardiac 20S proteasomes: role of associating partners.Circ. Res. 2006; 99: 372-380Crossref PubMed Scopus (121) Google Scholar, 37French S.W. Mayer R.J. Bardag-Gorce F. Ingelman-Sundberg M. Rouach H. Neve And E. Higashitsuji H. The ubiquitin-proteasome 26s pathway in liver cell protein turnover: effect of ethanol and drugs.Alcohol Clin. Exp. Res. 2001; 25: 225S-229SCrossref PubMed Google Scholar). Briefly hearts or livers were homogenized in 20 mm Tris (pH 7.8), 0.1 mm EDTA, 1 mm DTT, Complete protease inhibitors (Roche Applied Science), and phosphatase inhibitor mixtures 1 and 2 (Sigma) and centrifuged at 25,000 × g for 2 h. Next ammonium sulfate was added slowly to the supernatant at a final concentration of 40%, and then the sample was centrifuged at 12,000 × g for 1 h. The procedure was repeated with the supernatant to achieve a final concentration of 60% ammonium sulfate, and again the sample was centrifuged at 12,000 × g for 1 h. The pellet was dissolved in 20 mm Tris (pH 7.4), 5 mm MgCl2, and 1 mm DTT and dialyzed against the same solution overnight. Then the dialyzed sample was reconstituted to 45% B (A: 20 mm Tris (pH 7.4), 5 mm MgCl2, 0.5 mm DTT, and 10% glycerol; B: same as A plus 600 mm KCl) and loaded on an XK 26 40 column (GE Healthcare) packed with Q Sepharose (GE Healthcare), which was equilibrated with 45% B. After washing the column, B was increased to 75% and the eluant was collected and centrifuged at 205,000 × g for 19 h. The pellet was dissolved by homogenization in A and centrifuged at 3,000 × g to remove insoluble particles. Then the supernatant was loaded on a Mono Q 5/50 column (GE Healthcare) equilibrated with A. Finally purified 20 S proteasomes were eluted at ∼55% B by running a shallow gradient up to 100% B. All steps were performed at 4 °C or on ice. Partial dephosphorylation of 20 S proteasome complexes was achieved by calf intestinal alkaline phosphatase (CIAP, Promega, Madison, WI) treatment. In variation with the 10× buffer supplied by the provider, dephosphorylation was performed in buffer lacking spermidine, thus containing 0.5 m Tris (pH 9.3), 10 mm MgCl2, and 1 mm ZnCl2. In total, 60 μg of purified 20 S proteasomes were incubated in the presence of 250 units of CIAP for 30 min at 37 °C. Free flow electrophoresis was performed on the BD FFE System (BD Biosciences). For high resolution FFE, a 0.4-mm spacer was used, resulting in a total volume of 20 ml in the separation chamber. Accurate and reproducible determination of the isoelectric point of the 20 S proteasome complexes in narrow pH gradients was achieved by interval FFE. The separation chamber was tempered at 10 °C to prevent protein degradation and precipitation. Furthermore the device was operated in a cold room at 4 °C to keep the samples in their latent state for high reproducibility in subsequent analysis of fractions by proteolytic assays. The BD FFE System features in total seven inlets for the separation and stabilizing media. The outermost inlets were used for stabilization media as recommended by the manufacturer. Stabilizing media were prepared dependent on the pH gradient used for IEF. For pH 3–10, 100 mm H2SO4 and 100 mm NaOH were used as anodal and cathodal stabilizer, respectively. The remaining five inlets were used for the separation medium containing 1% Servalytes 3–10. For pH 4–6, the anodal stabilizer solution contained 100 mm H2SO4, 50 mm acetic acid, 200 mm 2-aminobutyric acid, and 50 mm glycylglycine. The cathodal stabilizer consisted of 100 mm NaOH, 50 mm Tris, 30 mm BisTris, 150 mm EPPS, 50 mm HEPES, and 30 mm MOPS. The separation medium on inlets 2–6 contained 1.2% Servalytes 4–6 and 0.3% Servalytes 3–10. All media used for separation, stabilization as well as counterflow, contained 0.2% (hydroxypropyl)methyl cellulose (average molecular weight 86,000, Aldrich) and 25% glycerol. In addition, 1 mm DTT was added to all media. Anodic and cathodic electrode solutions were prepared according to the manufacturer and contained 100 mm H2SO4 and 100 mm NaOH, respectively. Samples subjected to FFE were diluted 1:10 with separation medium. In general, 200 μl of diluted sample was applied containing typically 60 μg of purified 20 S proteasome complexes. For visualization of injection, separation, and fractionation, 1.5 μl of sulfanilic acid azochromotrop (1% (w/v), Sigma) was added to each sample. The diluted samples were injected into the separation chamber at the cathodic sample inlet (S4) using flow rates of 1 ml/h for continuous and 9 ml/h for interval mode with the improved separation protocol. Before and after sample entry, undiluted separation medium was injected into the separation chamber at the same flow rate as the sample flow rate. In continuous mode, the flow rate of the separation medium was set to 60 ml/h, and IEF conditions were limited to 1500 V and 50 mA. For high resolution separation in narrow pH gradients, the media flow rate during sample entry was 300 ml/h. Then prolonged IEF was performed at a maximal 1200 V and 30 mA in interval mode with intervals set to 5 min and a reduced flow rate of 50 ml/h. Operating the FFE can be visualized as virtual elongation of the separation chamber and thus extension of the residence time of the sample by alternating the pump direction of the media flow after each interval period between forward and backward. Accurate and reproducible IEF of 20 S complexes was achieved after 1 h in interval mode. Finally the media flow rate was set to 300 ml/min to move the sample to the fraction collector. In both the continuous and the interval modes, fractions were collected in 96-well plates when the red marker sulfanilic acid azochromotrop reached the fraction collector. Isoelectric focusing of the FFE fractions was performed on the IPGphor (GE Healthcare) in combination with the Manifold (GE Healthcare). The samples were separated on pH 3–10, 18-cm IPG strips (GE Healthcare) according to a modified protocol of Gorg et al. (38Gorg A. Drews O. Weiss W. Separation of proteins using two-dimensional gel electrophoresis.in: Simpson R.J. Purifying Proteins for Proteomics: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2004: 391-430Google Scholar). The IPG strips were rehydrated in 350 μl of buffer containing 8 m (w/v) urea, 1% (w/v) CHAPS, 0.2% (w/v) DTT, and 0.5% (v/v) Pharmalytes 3–10. Prior to IEF, FFE fractions were diluted at a ratio of 1:5 with buffer containing 9 m (w/v) urea, 4% (w/v) CHAPS, 2% (w/v) DTT, and 1% (v/v) Pharmalytes 3–10. Sample entry was facilitated by 30 min at 150 V, 30 min at 300 V, and 30 min at 600 V. After that, the voltage was increased to 8000 V using a 30-min voltage gradient, and IEF reached a steady state after a minimum of 18 kV-h. The second dimension was performed on 12% acrylamide gels after equilibration of the samples as published previously (39Gorg A. Postel W. Gunther S. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 1988; 9: 531-546Crossref PubMed Scopus (865) Google Scholar). Finally the separated proteins were visualized by silver staining. For one-dimensional SDS-PAGE, FFE fractions were diluted with SDS sample buffer and separated on 12.5% SDS gels. Gels were either stained with silver or SYPRO Ruby (Invitrogen) according to the manufacturer. Fluorescence was detected by laser densitometry using a Typhoon 9600 variable mode imager (GE Healthcare) and quantified by the ImageQuant 5.2 software (GE Healthcare). Blue native gel electrophoresis of FFE fractions was carried out according to a modified protocol of Schagger et al. (40Schagger H. Cramer W.A. von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1037) Google Scholar). FFE fractions were mixed with blue native sample buffer and separated on 6% acrylamide gels. Ferritin complexes (molecular mass, 440 kDa) and thyroglobulin (molecular mass, 669 kDa; GE Healthcare) were used as molecula
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