Proteome dynamics and proteome function of cardiac 19S proteasomes

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.     

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.     

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (²H₂O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with ²H₂O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level ²H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d⁻¹ and 0.163 d⁻¹, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (∼60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.Mitochondrial dysfunctions are observed in disorders such as neurodegeneration, cardiovascular diseases, and aging (1–3). It is postulated that the failure to contain or replenish mitochondrial proteins damaged by reactive oxygen species directly underlies many pathological phenotypes (4). The development of effective treatments for these diseases therefore relies on understanding the molecular basis of protein dynamics. Outstanding questions are how the processes of mitochondrial proteome dynamics are regulated in different systems, and how their perturbations could progress to pathological remodeling of the organelle. Thus far, quantitative proteomics efforts have been predominated by steady-state measurements, which often provide fragmentary snapshots of the proteome that are difficult to comprehend in the context of other cellular events.To further understand mitochondrial dynamics in vivo, we examined the turnover rates of individual heart and liver mitochondrial proteins on a proteome scale. Both the liver and the heart contain large numbers of mitochondria, but cardiac and hepatic mitochondria differ in their protein composition, oxygen consumption, substrate utilization, and disease manifestation. However, these differences are often interpreted only by protein compositions and steady-state abundance, without the consideration of protein kinetics in the temporal dimension. Abnormal protein kinetics may indicate dysfunctions in protein quality control, the accumulation of damaged proteins, misfolding, or other proteinopathies. Protein dynamics itself is an important intrinsic property of the proteome, the disruption of which could be causal of cellular etiologies.At minimum, a kinetic definition of the proteome requires knowledge of the rate at which individual proteins are being replaced. Isotope tracers are particularly useful for tracking such continual renewal of the proteome in living systems, because they allow differentiation between preexisting and newly synthesized proteins (5). Among the available stable isotope precursors, heavy water (²H₂O) labeling offers several advantages with respect to safety, labeling kinetics, and cost (6, 7). First, ²H₂O administration to animals and humans at low enrichment levels is safe for months or even years (8). Second, maintaining constant ²H enrichment levels in body water following the initial intake of ²H₂O is easily achieved, because administrated ²H₂O rapidly equilibrates over all tissues but decays slowly (9, 10). Third, ²H₂O labeling is more cost effective than other stable isotope labeling methods. Importantly, ²H₂O intake induces universal ²H incorporation into biomolecules. Systematic insights into protein turnover in vivo could therefore be correlated to that of nucleic acids, carbohydrates, or lipids, enabling broad applications for this technology in studying mammalian systems, including humans.A variety of methodologies have been developed to analyze the extent of ²H incorporation in proteins following ²H₂O labeling, including GC-MS measurements of hydrolyzed target proteins (11–14) and peptide analysis in MALDI-TOF MS (15) and LC-MS (16, 17). More recently, Price et al. described an approach for measuring protein turnover by calculating the theoretical number of ²H-labeling sites on a peptide sequence (18) and reported the turnover rates of ∼100 human plasma proteins. Here we describe another novel strategy to determine protein turnover rates on a proteomic scale using ²H₂O labeling. By computing the parameters needed to deduce fractional protein synthesis using software we developed, we were able to obtain protein half-life data without relying on the asymptotic isotopic abundance of peptide ions. Our approach also has the unique benefit of automating all steps of isotopomer quantification and postcollection data analysis, and it does not require knowledge of the exact precursor enrichment or labeling sites of peptides. We observed diverse kinetics from 458 liver and heart mitochondrial proteins that inform essential characteristics of mitochondrial dynamics and intragenomic differences between the two organs.     

HUPO 2011: The new Cardiovascular Initiative - integrating proteomics and cardiovascular biology in health and disease

A newly reorganized HUPO Cardiovascular Initiative was announced at the HUPO 2011 Cardiovascular Initiative Workshop at Geneva. The new initiative is now part of the biology- and disease-driven component of the HUPO Human Proteome Project (B/D-HPP). Here we report the recent achievements and future directions of the initiative, and offer a perspective on the present challenges of cardiovascular proteomics and its integration with the cardiovascular biology community at large.