Regulation of Murine Cardiac 20S Proteasomes

Abstract

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.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

“…[16][17][18][19][20][21][22] Phosphorylation of proteasome subunits is currently being under intense investigation, as this modification is important for regulation of various cellular processes. 20,[23][24][25][26][27] Detection of phosphorylation sites in a number of core and regulatory subunits of 26S proteasome strongly suggests that this post-translational covalent modification is involved in regulation of proteasomal activity. 17,20,28 Multiple data not shown).…”

Circadian proteomics of the mouse retina

“…Several components of the proteasome complex are modified posttranslationally, and PSMA1 is phosphorylated by protein kinase A (PKA) in murine cardiac muscle [48], suggesting that spot 1747 and 1762 may reflect a difference in phosphorylation state of this protein. Since cAMP level shows circadian variation in the chicken retina with a peak at late night [49], the activity of PKA is predicted to be high during the subjective night, being consistent with an increase of more acidic spot 1747 at late night.…”

Proteomic insights into cardiac cell death and survival

“…How has proteomics contributed to our understanding of proteasome regulation in the normal and diseased heart? First, proteomic maps of cardiac proteasomes (both the core 20S particle as well as the 26S holoeznyme) provided a cell/organ type-specific parts list [21,22]. Without such insights, functional analyses related to the contribution of individual proteins could not move forward.…”

“…Interestingly, these studies found that cardiac proteasomes harbor distinct subunit compositions, including participation of so-called "inducible subunits" thought from studies in other cell types to be involved in proteasome function only under specific stimuli. Moreover, proteasome complexes were found to accommodate signaling molecules (specifically protein kinase A and protein phosphatase 2A) that regulate the function of the degradation machinery [21]. Gone is the notion that cardiac proteasomes are static protein-disposal organelles without the capacity for discrete manipulation.…”

“…MS was used to map out the specific modified residues (on the alpha-7 subunit) and to determine the degree to which occupation of these residues by phosphates alters protein breakdown by the complex. MS analyses also revealed acetylation of multiple subunits [21], adding further complexity to the manner in which the complex is regulated.…”