Contrasting proteome biology and functional heterogeneity of the 20 S proteasome complexes in mammalian tissues - PubMed (original) (raw)

Comparative Study

Contrasting proteome biology and functional heterogeneity of the 20 S proteasome complexes in mammalian tissues

Aldrin V Gomes et al. Mol Cell Proteomics. 2009 Feb.

Abstract

The 20 S proteasome complexes are major contributors to the intracellular protein degradation machinery in mammalian cells. Systematic administration of proteasome inhibitors to combat disease (e.g. cancer) has resulted in positive outcomes as well as adversary effects. The latter was attributed to, at least in part, a lack of understanding in the organ-specific responses to inhibitors and the potential diversity of proteomes of these complexes in different tissues. Accordingly, we conducted a proteomic study to characterize the 20 S proteasome complexes and their postulated organ-specific responses in the heart and liver. The cardiac and hepatic 20 S proteasomes were isolated from the same mouse strain with identical genetic background. We examined the molecular composition, complex assembly, post-translational modifications and associating partners of these proteasome complexes. Our results revealed an organ-specific molecular organization of the 20 S proteasomes with distinguished patterns of post-translational modifications as well as unique complex assembly characteristics. Furthermore, the proteome diversities are concomitant with a functional heterogeneity of the proteolytic patterns exhibited by these two organs. In particular, the heart and liver displayed distinct activity profiles to two proteasome inhibitors, epoxomicin and Z-Pro-Nle-Asp-H. Finally, the heart and liver demonstrated contrasting regulatory mechanisms from the associating partners of these proteasomes. The functional heterogeneity of the mammalian 20 S proteasome complexes underscores the concept of divergent proteomes among organs in the context of an identical genome.

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Figures

Fig. 1.

Analysis of purified murine cardiac and hepatic 20 S proteasomes. A, blue-native gel of purified murine cardiac and hepatic 20 S proteasomes. B, liquid chromatography of trypsin-digested heart and liver 20 S proteasome bands. The chromatograms for trypsin cleaved heart and liver 20 S proteasomes were similar, but distinct. Some of the major peaks in both samples are labeled.

Fig. 2.

Quantification of heart and liver 20 S proteasome subunits. A, quantification using the average of the two highest peaks from the same peptides in liver and heart. n = 3; *, p < 0.05. B, quantification using 18O:16O labeling of proteasome subunits in the heart and liver. n = 3; *, p < 0.05. The peaks are normalized to the median of all the ratios in the set and are the result of three experimental repetitions. Inset shows a representative spectrum of 18O: 16O labeling of a β1i peptide.

Fig. 3.

Comparison of free and assembled 20 S proteasome subunits in heart and liver by immunoblotting. A, comparison of constitutive and inducible proteasomes subunits in cytosolic fractions and purified 20 S from the heart and liver. Each lane contained 25 μg of cytosolic fraction or 1 μg of purified proteasome. B, comparison of proteasomes subunits in cytosolic fractions and purified 20 S from the heart and liver. Heart and liver 20 S proteasomes were run on SDS-PAGE, transferred to nitrocellulose, and probed with anti-proteasome antibodies. Each lane contained 1 μg of heart proteasome or 1 μg of liver proteasome. C, the assembly index for proteasome subunits in heart and liver cytosolic fractions. The assembly index is the ratio of the total amount of a proteasome subunit in the cytosolic fraction (free + partially assembled + assembled subunits) versus the amount of that proteasome subunit in the 20 S proteasome purified from the cytosolic fraction (only assembled subunits). The insert in Fig. 3_C_ is an enlarged view of the assembly index for β5 and β1i.

Fig. 4.

Comparison of post-translational modifications on purified 20 S proteasomes from heart and liver. A, comparison of the phosphoproteome of purified 20 S proteasomes from heart and liver. Each lane contained 2 μg of heart proteasome or 2 μg of liver proteasome. B, upper panel, mass spectra of a peptide from heart β6 proteasome subunit, which is mono-methylated on arginine; lower panel, a peptide from liver α2, which is dimethylated on lysine. # represents methylated arginine residue. The methylated peptide (β6) had an Xcorr of 4.114 and a precursor ion m/z of 1157.30 (3+ charge). @ represents dimethylated lysine residue. The dimethylated peptide (α2) had an Xcorr of 5.266 and a precursor ion m/z of 893.23 (3+ charge). Protein loading was controlled by Ponceau S stain (PS).

Fig. 5.

Validation of associating partners of cardiac and hepatic 20 S proteasomes. A, cardiac and hepatic 20 S proteasome was run on a native gel, transferred to nitrocellulose, and probed with polyclonal anti-NEDD8 and monoclonal anti-β7. B, identification of a peptide from NEDD8 present in the BN-PAGE 20 S band using LC-MS/MS analysis. C, immunoblot of native gel to verify interaction of PP1 with the heart 20 S proteasomes. D, identification of a peptide from PP1 present in the BN-PAGE 20 S band using LC-MS/MS analysis.

Fig. 6.

Confocal validation of the associating partners with proteasome complexes in mammalian cells. Panel 1, representative single confocal sections of cardiomyocytes immunostained with anti-20 S proteasome α3 (A1, green), anti-PP1γ (A2, red), and the overlay of α3 and PP1γ (A3). a1_–_a3 are the regions in the squares at higher display magnification. A4, CC histogram. PPI of 0.87 for α3 with proximity to PP1γ, indicating a high degree of association. A5, P significance test versus CC for α3 with proximity to PP1γ; the highlighted area has <0.05. A6, PPI as a function of pixel shift. Panel 2, immunostained cells with anti-20 S proteasome α3 (B1, green), anti-CKII (B2, red), and overlay (B3). b1_–_b3 are regions at higher magnification. B4, CC histogram. PPI of 0.47 for α3 proximity to CKII. B5, CC for α3 with proximity to CKII; the highlighted area has p < 0.05. B6, PPI as a function of pixel shift. Panel 3, immunostained cells with anti-20 S proteasome α3 (C1, green), anti-NEDD8 (C2, red), and overlay (C3). c1_–_c3 are regions at higher magnification. C4, CC histogram. PPI of 0.82 for α3 proximity to NEDD8. C5, CC for α3 with proximity to NEDD8; the highlighted area has p < 0.05. C6, PPI as a function of pixel shift. Panel 4, immunostained cells with anti-20 S proteasome α3 (D1, green), anti-ZFHX4 (D2, red), and overlay (D3). d1_–_d3 are regions at higher magnification. D4, CC histogram. PPI of 0.64 for α3 with proximity to ZFHX4. D5, CC for α3 proximity to CKII; the highlighted area has p < 0.05. D6, PPI as a function of pixel shift. Immunostained cells with anti-core 20 S proteasome (E1, green), anti-Rpt4 19 S proteasome (E2, red), and overlay of core and Rpt4 (E3). e1_–_e3 are regions in the squares at higher magnification. E4, CC histogram. PPI of 0.82 for core proximity to Rpt4. E5, Psign test versus CC for core proximity to Rpt4; the highlighted area has p < 0.05. E6, PPI as a function of pixel shift.

Fig. 7.

Proteolytic activities of the purified 20 S proteasomes from the heart and liver. A, comparison of the proteolytic activities of the 20 S proteasomes in cytosolic fractions from the heart and liver. *, p < 0.05; n = 4. B, comparison of the inhibition of 20 S proteasome activities in cytosolic fractions from the heart and liver. *, p < 0.05; n = 4. Heart and liver cytosol fractions (25 μg each) were assayed for β1, β2, and β5 activities in the presence of 100–500 m

of fluorescent substrate. *, p < 0.05; n = 5. C, comparison of the proteolytic activities of the purified 20 S proteasomes from the heart and liver. *, p < 0.05; n = 4. D, comparison of the inhibition of purified 20 S proteasome activities from the heart and liver.

Fig. 7.

Fig. 8.

Characterization of the effects of calyculin A, PP1, DMAT (CKII inhibitor), or CKII on the proteolytic activities of the purified proteasomes. A, proteolytic activities of purified 20 S proteasomes 50 min after the addition of 10 n

PP1 inhibitor calyculin A. Data is average over 3–6 experiments. B, β5 proteolytic activity 50 min after 36 n

recombinant PP1 added to purified 20 S proteasomes. Other proteolytic activities showed no detectable effect. Data is average of three experiments. *, p < 0.05. C, proteolytic activities of purified 20 S proteasomes 50 min after addition of 100 n

CKII inhibitor DMAT. D, β5 proteolytic activity 60 min after 15 units of recombinant CKII were added to purified 20 S proteasomes. *, p < 0.05; n = 3–6 for all experiments.

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References

1. Merchionne, F., Perosa, F., and Dammacco, F. ( 2007) New therapies in multiple myeloma. Clin. Exp. Med. 7, 83–97 - PMC - PubMed
1. Voortman, J., and Giaccone, G. ( 2006) Severe reversible cardiac failure after bortezomib treatment combined with chemotherapy in a non-small cell lung cancer patient: a case report. BMC Cancer 6, 129. - PMC - PubMed
1. Enrico, O., Gabriele, B., Nadia, C., Sara, G., Daniele, V., Giulia, C., Antonio, S., and Mario, P. ( 2007) Unexpected cardiotoxicity in haematological bortezomib treated patients. Br. J. Haematol. 138, 396–397 - PubMed
1. Hacihanefioqlu, A., Tarkun, P., and Gonulle, E. ( 2008) Acute severe cardiac failure in myeloma patient due to proteasome inhibitor bortezmib. Int. J. Hematol. 88, 219–222 - PubMed
1. Asher, G., Bercovich, Z., Tsvetkov, P., Shaul, Y., and Kahana, C. ( 2005) 20 S proteasomal degradation of ornithine decarboxylase is regulated by NQO1. Mol. Cell 17, 645–655 - PubMed

Contrasting proteome biology and functional heterogeneity of the 20 S proteasome complexes in mammalian tissues - PubMed (original) (raw)