Abstract-The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species-and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key ␣ and ␤ subunits in cardiomyocytes. The expression of 14 constitutive ␣ and ␤ subunits in parallel with their three inducible subunits (␤1i, ␤2i, and ␤5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (␣2, ␣5, ␣7, ␤3, and ␤4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, ␣7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium. T he proteasome is a key proteolytic enzymatic system governing the degradation of majority intracellular proteins. 1 Recent studies implicate proteasomes in cardiac diseases. [2][3][4][5][6][7][8][9][10][11] Proteasome inhibitors are widely used in cancer therapy, however, their impact on cardiac function remains unclear; investigations in the heart have reported conflicting results. 2-4,12 A major contributing factor to these controversies is the lack of information pertaining to the structural organization and protein composition of cardiac proteasomes, therefore prohibiting the identification of mol...

Abstract-Our recent studies have provided a proteomic blueprint of the 26S proteasome complexes in the heart, among which 20S proteasomes were found to contain cylinder-shaped structures consisting of both ␣ and ␤ subunits. These proteasomes exhibit a number of features unique to the myocardium, including striking differences in post-translational modifications (PTMs) of individual subunits and novel PTMs that have not been previously reported. To date, mechanisms contributing to the regulation of this myocardial proteolytic core system remain largely undefined; in particular, little is known regarding PTM-dependent regulation of cardiac proteasomes. In this investigation, we seek to elucidate the function and regulation of 20S proteasome complexes in the heart. Functionally viable murine cardiac 20S proteasomes were purified. Tandem mass spectrometry analyses, combined with native gel electrophoresis, immunoprecipitation, and immunoblotting, revealed the identification of 2 previously unrecognized functional partners in the endogenous intact cardiac 20S complexes: protein phosphatase 2A (PP2A), and protein kinase A (PKA). Furthermore, our results demonstrated that PP2A and PKA profoundly impact the proteolytic function of 20S proteasomes: phosphorylation of 20S complexes enhances the peptidase activity of individual subunits in a substrate-specific fashion. Moreover, inhibition of PP2A or the addition of PKA significantly modified both the serine-and threonine-phosphorylation profile of proteasomes; multiple individual subunits of 20S (eg, ␣1 and ␤2) were targets of PP2A and PKA. Taken together, these studies provide the first demonstration that the function of cardiac 20S proteasomes is modulated by associating partners and that phosphorylation may serve as a key mechanism for regulation.