Oliver Drews

Regulation of Murine Cardiac 20S Proteasomes. Role of Associating Partners

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 α and β 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, α1 and β2) 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.

Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities

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.

Differential Regulation of Proteasome Function in Isoproterenol-Induced Cardiac Hypertrophy

Rationale: Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation. Objective: The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart. Method and Results: Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by &bgr;-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible &bgr; subunits. Conclusions: Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.

Contrasting Proteome Biology and Functional Heterogeneity of the 20 S Proteasome Complexes in Mammalian Tissues

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.

Revealing the Dynamics of the 20 S Proteasome Phosphoproteome A Combined CID and Electron Transfer Dissociation Approach

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO2), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential β5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

An image based two‐dimensional (2‐D) reference map of very alkaline yeast cell proteins was established by using immobilized pH gradients (IPG) up to pH 12 (IPG 6–12, IPG 9–12 and IPG 10–12) for 2‐D electrophoresis and by using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry peptide mass fingerprinting for spot identification. Up to now 106 proteins with theoretical isoelectric points up to pH 11.15 and molecular mass between 7.5 and 115 kDa were localized and identified. Additionally, due to the improved resolution of steady‐state isoelectric focussing with IPGs, even low copy number proteins with codon bias below 0.02 were detected and identified.

Naturally Presented Peptides on Major Histocompatibility Complex I and II Molecules Eluted from Central Nervous System of Multiple Sclerosis Patients

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.

Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes

PTMs serve as key regulatory mechanisms for 20S proteasome functions. Alterations in 20S PTMs have been previously observed with changes in modified protein degradation patterns and altered cellular phenotypes. Despite decades of investigation, our knowledge pertaining to the various PTMs of 20S complexes and their biological significance remain limited. In this investigation, we show that 2‐DE offers an analytical tool with high resolution and reproducibility. Accordingly, it has been applied for the characterization of PTMs including glycosylation, phosphorylation, oxidation, and nitrosylation. The PTMs of murine cardiac 20S proteasomes and their associating proteins were examined. Our 2‐DE analyses displayed over 25 spots for the 20S complexes (17 subunits), indicating multiply modified subunits of cardiac proteasomes. The identification of specific PTM sites subsequent to 2‐DE was supported by MS. These PTMs included phosphorylation and oxidation. Most of the PTMs occurred in low stoichiometry and required enrichment to enhance the detection sensitivity. In conclusion, our studies support 2‐DE as a central tool in the analyses of 20S proteasome PTMs. The approaches utilized in this investigation demonstrate their application in mapping the PTMs of the 20S proteasomes in cardiac tissue, which are applicable to other samples and biological conditions.

Identification of natural MHC class II presented phosphopeptides and tumor-derived MHC class I phospholigands.

MHC molecules present protein-derived peptides to T lymphocytes. By developing TiO2-based microcentrifugation columns, we identified the first phosphorylated MHC class I ligands from tumor tissue (renal cell carcinoma) and, by comparison to healthy renal tissue, found one Brf1-derived ligand as potentially tumor-associated. We further discovered the first natural phosphorylated MHC class II ligands. They revealed several novel phosphorylation sites of significant transmembrane receptors, such as Frizzled 6, CXCR4 and CD20.