Proteome dynamics and proteome function of cardiac 19S proteasomes

Abstract

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

Fig. 1.

Purification of distinct 19S subpopulations from the murine heart. Panel. A, MonoQ chromatography was applied as the final step of murine 19S complex purification. With a slow ramping of salt concentration (shown in the form of conductivity: mS/cm), two distinct subpopulations of 19S complexes (19S I and 19S II) were collected (UV280: mAU). B, The structural integrity of 19S complexes was probed with a panel of antibodies targeting subunits from the “base” (Rpn2, Rpt4) and the “lid” (Rpn7) substructures. Both 19S species were shown to be structurally intact. Densitometry measurement showed a consistent enrichment pattern for 19S subunits in support of the UV280 nm absorption traces (A). C, The biological function of murine cardiac 19S complexes was evaluated as their ability to stimulate 20S proteasome-dependent proteolysis. With ATP as a necessary cofactor, the activity of 19S complexes in selected fractions (C5-C14) was assayed. The relative activities among these fractions were in agreement with both the UV280 nm profile (A) and densitometry trace (B). All analyses were made with equal volume from each chromatographic fraction.

Fig. 2.

Variation in functional dynamics between distinct 19S subpopulations. A, The content of 19S complexes in two pooled subpopulations was normalized based on the results of quantitative immunoblotting. The content of 19S II was titrated to match the content of 19S I. All subsequent functional comparisons were made following careful normalization of the protein. B, Postnormalization, the content of 19S complexes in the two normalized pools was verified with a panel of antibodies (Rpt1, Rpt2, Rpt3, Rpt4, Rpt5, Rpn2 and Rpn7) via quantitative immunoblotting. C, The biological potency of 19S complexes was evaluated as the ability of each to stimulate 20S proteasome-dependent proteolysis. 19S II showed significantly higher (up to 40%) activity over that of 19S I.

Fig. 3.

Molecular composition of 19S subpopulations evaluated with quantitative proteomics. A, 2-D DIGE was applied for quantitative analysis of 19S proteasome heterogeneity. 19S I was labeled with Cy3 (green) and 19S II with Cy5 (red). The molecular identity of each spot was acquired via LC-MS/MS. Hsp90 was enriched in 19S I (green); S5b and eIF3 subunits were enriched in 19S II (red). B, The murine cardiac 19S complex subpopulations 19S I and 19S II were displayed by nondenaturing native PAGE, trypsin-digested and labeled with ¹⁸O/¹⁶O, respectively, at their C termini. The labeled peptides were then combined and quantitatively analyzed via LTQ-Orbitrap LC-MS/MS. C, The intensity ratio (19S I over 19S II) was indicative of relative protein abundance between the two subpopulations. S5b was the only conventional 19S subunit that was significantly different in abundance between the two subpopulations, with an enrichment in 19S II.

Fig. 4.

Differential recruitment of Hsp90 contributes to cardiac 19S complex heterogeneity. A, The molecular identity of 19S-associated Hsp90 was confirmed with LC-MS/MS following nondenaturing native PAGE. The spectrum for Hsp90 peptide ₂₉₂NPDDITQEEYGEFYK₃₀₆ was shown. B, Quantitative immunoblotting assays with Hsp90 specific antibodies demonstrated that Hsp90 was unique to subpopulation 19S I. The relative contents of 19S subunits Rpt2, Rpt4, Rpn2 and Rpn7 were also probed as loading controls. C, Two-dimensional native PAGE and SDS-PAGE was performed for 19S I, confirming the interaction of Hsp90 with 19S I under a native, nondenaturing state.

Fig. 5.

Hsp90 Suppressed the biological potency of 19S subpopulation I. A, The functional impact of Hsp90 on 19S complexes was evaluated with an Hsp90 inhibitor, BIIB021. Inhibition of 19S I-associated Hsp90 significantly enhanced the activating potency of 19S I on 20S-dependent proteolytic activity, indicating that Hsp90 is a negative regulator of 19S I (n = 4). B, The functional impact of Hsp90 on 19S complexes was validated with another Hsp90 inhibitor, geldanamycin, supporting Hsp90 as a specific negative regulator of 19S I (n = 4). C, Neither BIIB021 nor geldanamycin impacts 20S activity significantly (n = 3). D, As another negative control, the functional impact of Hsp90 on 20S proteasomes alone (without 19S) was examined. The proteolytic function of 20S proteasomes was not inhibited with exogenous Hsp90, suggesting that the Hsp90-induced functional inhibition of 20S proteolysis was transduced through 19S (n = 4). E, In vitro treatment with recombinant Hsp90 did not significantly alter the function of 19S I or 19S II (n = 4).