Substrate- and Isoform-specific Proteome Stability in Normal and Stressed Cardiac Mitochondria

Abstract

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 using two-dimensional differential gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mitochondrial proteases demonstrated a substrate preference for basic protein variants, indicating 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.

Introduction

The cardiac mitochondria are primary recipients of oxidative damage and thus under considerable needs 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 (Fig 1A). Endogenous proteolysis in the mitochondria has primarily been associated with several intra-mitochondrial AAA⁺ proteases, particularly the Lon protease homolog (LONP1). LONP1 degrades mildly oxidized aconitase in vitro,¹ 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.

Fig. 1. Protein degradation and abundance of endogenous proteases in cardiac mitochondria.

A. Potential proteolytic fates of a hypothetical mitochondrial protein (center). B. Percentage abundance of mitochondrial AAA+ proteases. SYPRO Ruby stain intensity is based on measured fluorescence of the identified protein spot divided by total fluorescence intensity. Spectral abundance is based on the number of assigned mass spectra in an independent LC-MS/MS experiment (see Online Supplements) CLPP and CLPX: proteolytic subunits of mitochondrial matrix protease ClpXP; AFG3L2 and SPG7: proteolytic subunits of mitochondrial inner membrane m-AAA protease facing the matrix; YME1L1: proteolytic subunit of mitochondrial inner membrane i-AAA protease facing the inter-membrane space. C. Mitochondrial proteins were categorized by their ranks from highest to lowest abundance. The top 100 species represented 76.9% of total protein content.

The cytosolic proteasomes conduct >70% of intracellular proteolysis,³ and are recently described to contribute to mitochondrial protein homeostasis through poorly understood pathways.^4,5 Mitochondrial morphology and functions are disrupted by proteasome inhibitors including MG132⁴ and bortezomib⁶. 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 intra-mitochondrial proteins, at least three indirect evidences support the occurrence of protein retrotranslocation into the cytosol. First, mitochondrial proteins accumulate in extra-mitochondrial spaces in the post-ischemic 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 implicating active transport.

Similar reconstitution of proteolysis could compensate the turnover of other intra-mitochondrial 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 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.

Methods

Experiments were conducted in accordance with guidelines by the NRC. Hsd:ICR(CD-1) mouse cardiac mitochondria and 20S proteasomes were isolated as described.^7,10 The mitochondrial proteome was allowed to degrade either by its endogenous proteases or exogenous, active 20S proteasomes. Relative protein abundances following proteolysis were determined by two-dimensional differential gel electrophoresis (2D-DIGE). Detailed descriptions are provided in the Online Supplement.

Results

The relative abundances of cardiac mitochondrial proteins were measured by staining with the ruthenium-based fluorescent dye SYPRO Ruby after isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) separation, and in parallel by mass spectrum counts. Both independent methods indicated that AAA⁺ proteases are not highly abundant in the cardiac mitochondria (Fig. 1B–C). 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, a capacity effectively attenuated by a protease inhibitor cocktail (Roche) (Online Figure I). Following incubation of cardiac mitochondria in isolation to promote endogenous proteolysis, 111 unique proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority of the proteins showed observable but minute decrement in abundance (Fig. 2A–B). On average, >80% of each protein species remained intact, indicating the isolated mitochondrial proteome exhibited remarkable stability in vitro. This observation corroborates with similar reports in yeast^11,12 and reflects a limited intrinsic capacity for proteolysis in the mitochondria under basal conditions. Consistently, functional ablation of the yeast Lon homolog PIM1 resulted in the accumulation of few detectable proteins¹³ and proteolysis assays generally did not indicate the respiratory complexes to be efficient substrates of mitochondrial proteases.^12,13

Fig. 2. Endogenous mitochondrial proteases display isoform-specific substrate preferences.

A. Experimental design. B. 2D-DIGE image of mitochondrial proteins after incubation reflects the stability of the mitochondrial proteome in isolation. The most basic isoelectric isoform of OGDH (2-oxoglutarate dehydrogenase E1) was more susceptible to degradation by mitochondrial proteases (indicated by outline and arrowhead). pI: isoelectric point; MW: molecular weight. C. Heat map of normalized degree of degradation of adjacent isoelectric isoforms of proteins. The most intense isoelectric isoform is designated as 0; the adjacent isoform toward the cathode, +1, etc. LRPPRC: leucine-rich PPR motif-containing protein; PDHB: pyruvate dehydrogenase E1 component subunit beta; SUCLA2: succinyl-CoA ligase [ADP forming] subunit beta; SDHA: succinate dehydrogenase [ubiquinone] flavoprotein subunit. D. Exposing isolated mitochondria to 1 mmol·L⁻¹ H₂O₂ for 30 min (Cy3) caused acidic isoelectric shift in OGDH isoforms. E. The effect of H₂O₂ on the proteolytic activity of 20S proteasome (20S) and mitochondria (Mito). Error bar: SEM; *: p<0.05 vs. no H₂O₂, Student’s t-test; n=4 for 20S proteasome, n=10 for mitochondrial proteases.

Despite the overall stability of the proteome, different degrees of degradation were discernible from individual protein spots. A number of mitochondrial proteins exist in charge variants that were readily resolved by IEF. Mitochondrial proteases were found to favor the degradation of the more basic isoforms of multiple proteins, suggesting a possible recognition mechanism for proteolysis that was otherwise masked (Fig. 2B–C). 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, exposing mitochondrial lysate to H₂O₂ shifted the isoelectric pattern toward the anode (Fig. 2D) and decreased the general capacity of isolated mitochondria to degrade fluorescein-labeled casein (Fig. 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 extra-mitochondrial 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 two were apparent (Fig. 3A–C; Online Table I). The protein degradation profiles were correlated with functional categories or multi-protein complex association. The tricarboxylic acid cycle and the NADH:ubiquinone oxidoreductase complex contained more proteins susceptible to the 20S proteasome (Fig. 3D). The identified NADH:ubiquinone oxidoreductase components had a median half-life of 7.1 h in vitro (5^th–95^th percentile: 3.4–17.1 h). In comparison, proteins belonging to other respiratory chain complexes had over twice the median half-life at 15.7 h in the same experiments (5^th–95^th percentile: 9.2–44.4 h). 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.

Fig. 3. Mitochondrial proteins are differentially susceptible to proteolysis by 20S proteasome in vitro.

A. Experimental design. Mitochondria were maintained at 37°C for ~9 h with (Cy5) or without (Cy3) murine cardiac 20S proteasome (20S) in 50:1 molar ratio. B. 2D-DIGE image of mitochondrial proteins after incubation and examples of different susceptibility to 20S proteasomes in vitro. ATP5B (ATP synthase subunit beta) was unsusceptible and appeared yellow in the overlaid Cy3 and Cy5 fluorescence image; SUCLA2 was degraded in the Cy5 sample and appeared green. C. The residual fraction of each protein was fit to an exponential decay equation to approximate experimental half-life under different proteolysis conditions in vitro. Box: 25^th–75^th percentile; whiskers: 5^th–95^th percentile; 20S: 20S proteasome experiment, 83 quantified proteins; Mito: mitochondrial protease experiment, 111 quantified proteins. D: The degradation susceptibility of metabolic proteins categorized by biological processes or complex association. C-I to C-V: respiratory chain complex I to V; ETF: electron transport flavoproteins; TCA: tricarboxylic acid cycle. Gaps denote non-detection or obscuration by proteasome subunits in the 20S proteasome experiment.

Discussion

The stability and dynamics of the cardiac mitochondrial proteome constitute a previously unappreciated aspect of homeostasis in the myocardium. To our knowledge, this 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 previously observed. The protein isoelectric variants are explainable by small, charge-conferring post-translational modifications and may represent a recognition mechanism pertaining to protein removal. Notwithstanding, 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 (Fig. 2D–E), suggesting the needs 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. As misfolded proteins are typically degraded through multiple pathways, several effectors may act in concert to modulate mitochondrial proteome turnover. The 20S proteasome presents another candidate contributor of extra-mitochondrial degradation mechanisms, and is here shown to independently degrade mitochondrial proteins in the absence of ubiquitination. Our 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. Following 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 seen to promote protein carbonyl removal upon H₂O₂ stress in cardiomyocytes,¹⁵ and to improve ventricular functions following 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.

Novelty and Significance.

What is known?

• Abnormalities in mitochondrial functions 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 proteasome 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 upon 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 significances. Further investigations are required to delineate the role of alternative protein degradation mechanisms in maintain mitochondrial homeostasis in the heart under normal and stressed conditions.

Non-standard Abbreviations and Acronyms

2D-DIGE

Two-dimensional differential gel electrophoresis

IEF

Isoelectric focusing

PAGE

Polyacrylamide gel electrophoresis

LC-MS/MS

liquid chromatography-tandem mass spectrometry