Substrate- and Isoform-Specific Proteome Stability in Normal and Stressed Cardiac MitochondriaNovelty and Significance
Rationale: Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart.
Objective: To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms.
Methods and Results: Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography–tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities.
Conclusions: The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.
The cardiac mitochondria are primary recipients of oxidative damage and thus have a considerable need for protein quality control. Dysregulation of the mitochondrial proteome is thought to be a fundamental concurrence of the mitochondrial function perturbations observed in many cardiac diseases. Nevertheless, factors that regulate the dynamics and homeostasis of this proteome remain obscure, especially regarding the rates and mechanisms by which proteins are degraded in the mitochondria (Figure 1A). Endogenous proteolysis in the mitochondria has primarily been associated with several intramitochondrial AAA+ proteases, particularly the Lon protease homolog (LONP1). LONP1 degrades mildly oxidized aconitase in vitro1 and impedes protein carbonyl accumulation in cultured cells.2 Nevertheless, data on physiological LONP1 substrates are limited, and proteome-wide generalizations are yet to be substantiated.
The cytosolic proteasomes conduct >70% of intracellular proteolysis3 and recently were described as contributing to mitochondrial protein homeostasis through poorly understood pathways.4,5 Mitochondrial morphology and functions are disrupted by proteasome inhibitors, including MG1324 and bortezomib.6 The inner-membrane uncoupling protein 2 (UCP2) is stable in mitochondria isolated from cultured mammalian cells and requires cytosolic proteasomes to restore its normal turnover.5 Although the lack of a known protein export mechanism argues against proteasomal degradation of intramitochondrial proteins, at least 3 indirect evidences support the occurrence of protein retrotranslocation into the cytosol. First, mitochondrial proteins accumulate in extramitochondrial spaces in the postischemic myocardium and under calcium stress in vitro.7 Second, ubiquitin ligases and proteasome-recruitment proteins, including VCP and NPL4, are known to associate with mitochondria.8,9 Third, incubation with proteasomes restores UCP2 turnover kinetics in intact mitochondria but not extracted mitoplasts,5 which implies active transport.
Similar reconstitution of proteolysis could compensate for the turnover of other intramitochondrial components. Accordingly, we used a targeted proteomics approach to examine protein degradation in purified mitochondria, either by their endogenous proteolytic activity or by cytosolic proteasomes. The measurement of proteolytic rates in an isolated system circumvents any confounding protein synthesis and translocation. This strategy thus enables direct assessments of proteolytic perturbations and presents a straightforward approach for defining the dynamics of multiple proteins under uniform contexts.
Experiments were conducted in accordance with guidelines published by the Guide for the Care and Use of Laboratory Animals published by the National Research Council. Hsd:ICR(CD-1) mouse cardiac mitochondria and 20S proteasomes were isolated as described previously.7,10 The mitochondrial proteome was allowed to degrade either by its endogenous proteases or exogenous, active 20S proteasomes. Relative protein abundances after proteolysis were determined by 2-dimensional differential gel electrophoresis. Detailed descriptions are provided in the online-only Data Supplement.
The relative abundances of cardiac mitochondrial proteins were measured by staining with the ruthenium-based fluorescent dye SYPRO Ruby after isoelectric focusing–polyacrylamide gel electrophoresis separation and in parallel by mass spectrum counts. Both independent methods indicated that AAA+ proteases are not highly abundant in the cardiac mitochondria (Figures 1B and 1C). LONP1 was the only species with appreciable concentration, representing ≈0.1% of total detected mitochondrial proteins. We then examined the endogenous proteolytic activity of cardiac mitochondria in vitro. The isolated mitochondria were competent in degrading fluorescein-labeled casein, an ability effectively attenuated by a protease inhibitor cocktail (Roche, Indianapolis, IN; Online Figure I). After incubation of cardiac mitochondria in isolation to promote endogenous proteolysis, 111 unique proteins were identified by liquid chromatography–tandem mass spectrometry. The majority of the proteins showed an observable but minute decrement in abundance (Figures 2A and 2B). On average, >80% of each protein species remained intact, which indicates the isolated mitochondrial proteome exhibited remarkable stability in vitro. This observation is in accordance with similar reports in yeast11,12 and reflects a limited intrinsic capacity for proteolysis in the mitochondria under basal conditions. Consistently, functional ablation of the yeast Lon homologue PIM1 resulted in the accumulation of few detectable proteins,13 and proteolysis assays generally did not indicate the respiratory complexes to be efficient substrates of mitochondrial proteases.12,13
Despite the overall stability of the proteome, different degrees of degradation were discernible from individual protein spots. A number of mitochondrial proteins existed in charge variants that were readily resolved by isoelectric focusing. Mitochondrial proteases were found to favor the degradation of the more basic isoforms of multiple proteins, which suggests a possible recognition mechanism for proteolysis that was otherwise masked (Figures 2B and 2C). The charge specificity remained observable in reverse fluorescence labeling and in silver staining and was absent from proteolysis conducted by proteasomes (Online Figure I). The reported preference of LONP1 to degrade mildly oxidized aconitase1 suggested oxidative damage as a potential cause of the observed degradation profile; however, exposure of mitochondrial lysate to H2O2 shifted the isoelectric pattern toward the anode (Figure 2D) and decreased the general ability of isolated mitochondria to degrade fluorescein-labeled casein (Figure 2E).
The observed isoform preference was limited to specific protein species. A number of proteins susceptible to oxidative damage, including the NADH:ubiquinone oxidoreductase iron-sulfur cluster subunits, did not appear to be efficient substrates of mitochondrial proteases. These proteins could conceivably be degraded by extramitochondrial factors, provided there is accessibility. When the endogenous proteolytic activity was contrasted with that of an exogenous proteolytic effector, the 20S proteasome, the different substrate preferences of the 2 were apparent (Figures 3A through 3C; Online Table I). The protein degradation profiles were correlated with functional categories and multiprotein complex association. The tricarboxylic acid cycle and the NADH:ubiquinone oxidoreductase complex contained more proteins susceptible to the 20S proteasome (Figure 3D). The identified NADH:ubiquinone oxidoreductase components had a median half-life of 7.1 hours in vitro (5th–95th percentiles, 3.4–17.1 hours). In comparison, proteins belonging to other respiratory chain complexes had more than twice the median half-life, at 15.7 hours, in the same experiments (5th–95th percentiles, 9.2–44.4 hours). The discrepancy in degradation rates could not be satisfactorily correlated to any examined biophysical parameters, including hydrophobicity, abundance, isoelectric point, and molecular weight (Online Figure II). The data, therefore, favor the hypothesis that biological properties confer substrate selectivity in proteolysis.
The stability and dynamics of the cardiac mitochondrial proteome constitute a previously unappreciated aspect of homeostasis in the myocardium. To the best of our knowledge, the present study is the first global investigation of autonomous proteolysis of mitochondrial proteins in the heart. Likewise, the substrate isoform specificity of mitochondrial proteases has not been observed previously. The protein isoelectric variants are explainable by small, charge-conferring posttranslational modifications and may represent a recognition mechanism that pertains to protein removal. Notwithstanding this, the stability of the isolated mitochondrial proteome indicates incomplete autonomy of mitochondria in protein degradation. Moreover, oxidative damage diminished the proteolytic capacity of cardiac mitochondria (Figures 2D and 2E), which suggests the need for exogenous factors to maintain protein homeostasis amid elevated oxidative stress.
Recent studies have accumulated on alternative mechanisms of mitochondrial turnover, including autophagous removal. The diverse turnover rates of mitochondrial proteins observed from in vivo isotope-labeling experiments by us and others,14 however, dispute the indispensability of indiscriminate mitophagy in individual protein homeostasis. Proteolysis occurs continuously in every mitochondrion under physiological and pathological conditions, whereas mitophagy could remove damaged mitochondria under stress. Because misfolded proteins typically are degraded through multiple pathways, several effectors may act in concert to modulate mitochondrial proteome turnover. The 20S proteasome presents another candidate contributor of extramitochondrial degradation mechanisms and here has been shown to independently degrade mitochondrial proteins in the absence of ubiquitination. The present data, therefore, reinforce the important roles of proteasomes in cardiac mitochondrial dynamics. In particular, the 20S proteasomes may act as an oxidative stress response to modulate mitochondrial protein dynamics under different physiological and pathological conditions. After ischemia-reperfusion in mice, we additionally observed that 20S proteasome activities were intricately modulated during oxidative stress (Online Figure III). Consistent with this, transgenic overexpression of the 11S particle in mice has been shown to promote protein carbonyl removal on H2O2 stress in cardiomyocytes15 and to improve ventricular function after ischemia-reperfusion.16 Ultimately, therapeutic designs in cardioprotection17 should benefit from further insights into protein-removal mechanisms. The stability of protein targets will directly influence the efficacy and pharmacokinetics of cardioprotective agents. The half-life of crucial cardioprotection mediators could conceivably be prolonged if their primary removal mechanisms are simultaneously inhibited while the drug is active. Alternatively, parallel mechanisms could be exploited to intervene in cell death pathways and minimize injury.
Sources of Funding
This study was supported in part by the National Institutes of Health (NIH-R37-63901 and NHLBI-HHSN-268201000035C).
In February 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.77 days.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.268359/-/DC1.
- Received March 2, 2012.
- Revision received March 13, 2012.
- Accepted March 19, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Abnormalities in mitochondrial function and structure are observed in most cardiomyopathies.
The mitochondrial proteome is altered because of elevated oxidative stress in the diseased heart.
What New Information Does This Article Contribute?
Mitochondrial proteases degrade proteins in isolated cardiac mitochondria minimally but show preferences for selected protein charge variants.
Oxidative damage affects protein charge variants and intrinsic proteolytic activities of mitochondria; likewise, the activities of 20S proteasomes are modulated in in vivo ischemic injury.
Mitochondrial proteases and cytosolic proteasomes together mediate the turnover of the mitochondrial proteome and its major metabolic pathways.
Mitochondrial proteins are primary sources and vulnerable targets of oxidative damage in the heart, but the regulation of their turnover and degradation is virtually unknown. We isolated cardiac mitochondria from mice and subjected them to proteolysis under various conditions. Unexpectedly, cardiac mitochondria remained largely stable on isolation, whereas the 20S proteasomes efficiently and specifically degraded selected mitochondrial proteins. Thus, the preference of mitochondrial proteases for degrading selected protein isoforms may be of therapeutic significance. Further investigations are required to delineate the role of alternative protein degradation mechanisms in maintenance of mitochondrial homeostasis in the heart under normal and stressed conditions.