Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.Mitochondria are the primary energy-generating systems in eukaryotes. They play a crucial role in oxidative metabolism, including carbohydrate metabolism, fatty acid oxidation, and urea cycle, as well as in calcium signaling and apoptosis (1, 2). Mitochondrial dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, Parkinson disease, and cancer (3). The most prevalent form of cellular protein post-translational modifications (PTMs),1 reversible phosphorylation (4–6), is emerging as a central mechanism in the regulation of mitochondrial functions (7, 8). The steadily increasing numbers of reported mitochondrial kinases, phosphatases, and phosphoproteins imply an important role of protein phosphorylation in different mitochondrial processes (9–11).Mass spectrometry (MS)-based proteome analysis is a powerful tool for global profiling of proteins and their PTMs, including protein phosphorylation (12, 13). A variety of proteomics techniques have been developed for specific enrichment of phosphorylated proteins and peptides and for phosphopeptide-specific data acquisition techniques at the MS level (14). Enrichment methods based on affinity chromatography, such as titanium dioxide (TiO₂) (15–17), zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) (18), immobilized metal affinity chromatography (IMAC) (19, 20), and ion exchange chromatography (strong anion exchange and strong cation exchange) (21, 22), have shown high efficiencies for enrichment of phosphopeptides (14). Recently, we demonstrated that calcium phosphate precipitation (CPP) is highly effective for enriching phosphopeptides (23). It is now generally accepted that no single method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide data sets to enhance the overall results in phosphoproteome analysis (24, 25). Phosphopeptide sequencing by mass spectrometry has seen tremendous advances during the last decade (26). For example, MS/MS product ion scanning, multistage activation, and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylated peptides (14, 26).A “complete” mammalian mitochondrial proteome was reported by Mootha and co-workers (27) and included 1098 proteins. The mitochondrial phosphoproteome has been characterized in a series of studies, including yeast, mouse and rat liver, porcine heart, and plants (19, 28–31). To date, the largest data set by Deng et al. (30) identified 228 different phosphoproteins and 447 phosphorylation sites in rat liver mitochondria. However, the in vivo phosphoproteome of human mitochondria has not been determined. A comprehensive mitochondrial phosphoproteome is warranted for further elucidation of the largely unknown mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.The percutaneous muscle biopsy technique is an important tool in the diagnosis and management of human muscle disorders and has been widely used to investigate metabolism and various cellular and molecular processes in normal and abnormal human muscle, in particular the molecular mechanism underlying insulin resistance in obesity and type 2 diabetes (32). Skeletal muscle is rich in mitochondria and hence a good source for a comprehensive proteomics and functional analysis of mitochondria (32, 33).The major aim of the present study was to obtain a comprehensive overview of site-specific phosphorylation of mitochondrial proteins in functionally intact mitochondria isolated from human skeletal muscle. Combining an efficient protocol for isolation of skeletal muscle mitochondria with several different state-of-the-art phosphopeptide enrichment methods and high performance LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, many of which have not been reported before. We characterized this mitochondrial phosphoproteome by using bioinformatics tools to classify functional groups and functions, including kinase substrate motifs.     

Ceramide Stimulates ABCA12 Expression via Peroxisome Proliferator-activated Receptor �� in Human Keratinocytes

ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C₂-Cer and C₆-Cer), but not C₈-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C₆-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARδ expression (but not other PPARs or liver X receptors), whereas PPARδ knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARδ is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARδ-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.The outermost layer of mammalian epidermis, the stratum corneum, is essential for permeability barrier function and critical for terrestrial life. The stratum corneum consists of terminally differentiated, anucleate keratinocytes, or corneocytes, surrounded by lipid-enriched lamellar membranes composed of three major lipids, ceramides, cholesterol, and free fatty acids (1). These lipids are delivered to the extracellular spaces of the stratum corneum through exocytosis of lamellar body contents from outermost stratum granulosum cells (2). Mature lamellar bodies contain primarily cholesterol, phospholipids, and glucosylceramides (3). Following lamellar body secretion, the secreted phospholipids and glucosylceramides are converted to free fatty acids and ceramides by phospholipases and β-glucocerebrosidase, respectively (1, 4). ABCA12 (ATP binding cassette transporter, family 12), a lipid transporter predominantly expressed in epidermis, has been shown to play a vital role in the formation of mature lamellar bodies (5, 6), although how this transporter is regulated remains unresolved.ABCA12 is a member of the ABCA subfamily of transporters, which are involved in the transport of a variety of lipids (7). Mutations in ABCA1 cause Tangier disease, which is due to a defect in transporting cholesterol and phospholipids from intracellular lipid stores to apolipoproteins, particularly apolipoprotein A-I (8–11). Mutations in ABCA3 cause neonatal respiratory failure due to a defect in surfactant transport from alveolar type II cells into the alveolar space (12). Mutations in ABCA4 cause Stargardt''s macular degeneration, with visual loss due to a defect in transporting phosphatidylethanolamine-retinylidene out of retinal pigment cells (13).Recently, mutations in ABCA12 have been shown to cause harlequin ichthyosis and a subgroup of lamellar ichthyosis, two disorders of keratinization (5, 14, 15). ABCA12 mutations lead to an abnormality in lamellar body formation, a decrease in lamellar membranes in the extracellular spaces of the stratum corneum, an accumulation of glucosylceramide in the epidermis with a reduction in ceramide (16), and ultimately loss of permeability barrier function (17), which in harlequin ichthyosis can result in neonatal lethality (5, 15). Strikingly, genetic correction of ABCA12 deficiency in patients'' keratinocytes by gene transfer normalized loading of glucosylceramides into lamellar bodies (5). These studies demonstrate a critical role for ABCA12 in epidermal physiology, specifically in the formation of mature lamellar bodies and subsequent permeability barrier homeostasis. Hence, it is crucial to understand how ABCA12 is regulated.Our laboratory recently demonstrated that activation of peroxisome proliferator-activated receptor (PPARδ and PPARγ) or liver X receptor (LXR) stimulates ABCA12 expression in cultured human keratinocytes (18). Both PPARs and LXR are important lipid sensors that stimulate keratinocyte differentiation and enhance permeability barrier function (19). Additionally, PPARα and -δ as well as LXR activators stimulate ceramide synthesis in keratinocytes (20, 21). Likewise, ceramide synthesis increases in keratinocytes during differentiation, foreshadowing the formation of lamellar bodies (22, 23).In addition to serving as structural membrane components, ceramides are also important signaling molecules that can induce growth arrest, differentiation, and apoptosis in various cells, including keratinocytes (24–26). Moreover, distal ceramide metabolites, sphingosine and sphingosine-1-phosphate (Fig. 1), are also important signaling molecules (27).Open in a separate windowFIGURE 1.The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.It is well established that the expression of ABCA1 is regulated by cellular cholesterol levels in many cell types, including keratinocytes (28). Cholesterol, if metabolized to certain oxysterols, can activate LXR, which then stimulates ABCA1 expression and the transport of cholesterol out of cells (29). This example of feed-forward regulation leads us to hypothesize that either ceramide or a metabolite of ceramide might stimulate ABCA12 expression, thereby leading to an increase in the transport of glucosylceramides into maturing lamellar bodies. Here, we provide evidence that ceramide stimulates ABCA12 expression in keratinocytes via a mechanism involving PPARδ signaling.     

Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains: IMPLICATIONS FOR THE STRUCTURE AND DYNAMICS OF MAMMALIAN INTRACELLULAR AND PLASMA MEMBRANES*

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.Cholesterol (Chol)³ is the most abundant sterol in mammalian plasma membrane and has unique biophysical properties (1, 2). Chol interacts with the high melting temperature (T_m) sphingolipids (SL) in the membrane, leading to the formation of SL/Chol-enriched microdomains (so-called lipid rafts). These domains are in a more ordered state (usually referred to as liquid-ordered (l_o) phase) than the bulk membrane (liquid-disordered phase (l_d)) (3, 4). Ceramide (Cer) is an SL formed in stress situations either from sphingomyelin (SM) in rafts or synthesized de novo by serine palmitoyltransferase and ceramide synthase. Both of these processes can be induced by diverse stimuli (5). Cer-induced membrane alterations (e.g. raft fusion into large signaling platforms (6)) were proposed to be the mechanism by which this lipid mediates diverse cellular processes, namely apoptosis (7–10). Cer presents an unusually small polar headgroup and in general very high gel-fluid T_m (e.g. for palmitoyl-Cer (PCer) it is ∼90 °C) (11). Membrane Cer levels are usually very low, although in cells undergoing apoptosis it can reach values up to 12 mol % total lipid (7), a percentage that in model membranes leads to Cer-rich gel domain formation (12–17). It was suggested that the formation of these domains might also be involved in Cer biological action (8, 18, 19).However, Cer effects on membrane properties are extremely dependent on membrane lipid composition, especially on Chol amounts (13, 20–23). For instance, in raft-forming model membranes (i.e. ternary mixtures of phosphocholines (PC), sphingomyelin (SM), and Chol), Cer-rich gel domains are formed at low but not at high Chol content (23). This result was explained by the competition between the two small headgroup molecules, Chol and Cer, for the bulkier headgroup, SM, to minimize acyl chain exposure to water. In fact, it is suggested that Cer selectively displaces Chol molecules from rafts, both in model (24–27) and in cell membranes (28, 29). However, a recent study showed that Cer-generated from SM hydrolysis leads to the formation of gel domains in these ternary mixtures only when Chol levels are low, suggesting that even for SM-depleted mixtures Chol is still able to modulate Cer effects (30). Therefore, to fully disclose the conditions that lead to the activation/regulation of Cer-mediated processes, further knowledge about Cer effects on membrane properties and their modulation by Chol is required.It is important to clarify the relation between Cer threshold for gel formation, cholesterol amount, and the properties of the remaining lipids (namely their propensity to form gel phases, which depends mainly on their gel-fluid transition temperature). This is because of the fact that each organelle membrane has its own specific composition. For example, there is a gradient of cholesterol concentration from the endoplasmic reticulum (ER) to the plasma membrane (PM) (31). In addition, there is a close relation between intracellular Cer levels, Ca²⁺ release from the ER, and Cer-induced permeability increase of the mitochondrial outer membrane (but not the inner membrane) (32, 33).The application of a uniquely sensitive method for Cer-rich gel detection allowed us to study for the first time Chol-Cer interactions in detail for high Chol and low Cer concentrations, i.e. a composition similar to mammalian plasma membranes. In addition, low Chol membranes were also studied. Our results clearly show that in a fluid matrix of representative mammalian membranes lipids, Cer-rich gel domains are destroyed by high amounts of Chol in the absence of SM and even in the absence of an l_o phase. We show that this outcome is a consequence of the higher solubility of Cer in Chol-rich membranes than in poor ones, the low solubility of Chol in Cer domains, and that it depends on the average T_m of the remaining lipids. These solubility differences offer a unified rationale for all Cer-Chol biophysical studies that can be translated into a ternary phase diagram, and the biological implications of the results are discussed.     

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting, and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkage of oxidative stress to culture degeneration was evident in A. nidulans. Taken together with the verifications of cell biology and biochemical data, a comparative mitochondrial proteome analysis revealed that, unlike the healthy wild type, a spontaneous fluffy sector culture of A. nidulans demonstrated the characteristics of mitochondrial dysfunctions. Relative to the wild type, the features of cytochrome c release, calcium overload and up-regulation of apoptosis inducing factors evident in sector mitochondria suggested a linkage of fungal degeneration to cell apoptosis. However, the sector culture could still be maintained for generations without the signs of growth arrest. Up-regulation of the heat shock protein chaperones, anti-apoptotic factors and DNA repair proteins in the sector could account for the compromise in cell death. The results of this study not only shed new lights on the mechanisms of spontaneous degeneration of fungal cultures but will also provide alternative biomarkers to monitor fungal culture degeneration.Culture degeneration, also called colony deterioration, of filamentous fungi can frequently occur during subsequent maintenance of fungal culture on artificial media by showing morphological changes, such as colony sectorization, loss or impaired ability of sporulation, fruiting, and sexuality (1, 2). It was first called as “woolly degeneration” in the model fungus Neurospora crassa (3). These morphological variations are also accompanied with the loss or reduction in secondary metabolites production, thus resulting in great commercial losses (4–7). Different from the mutation of genes involved in conidiation producing fluffy phenotypes (8–10), fungal culture degeneration spontaneously occurs and is usually irreversible. The rate of colony deterioration varies considerably among fungal species/strains and correlates with the growth environments, especially the composition of nutrient medium (11, 12). In addition, unlike the phenotype of senescence observed in the model fungus Podospora anserina (13), morphological variants of other filamentous fungi could be subcultured for many generations without growth arrest (14, 15). The mechanism(s) underlying fungal culture degeneration is poorly understood. Genetic mutations were not evident in the degenerated fungal isolates of Penicillium chrysogenum (6). Otherwise, methylation of genomic DNA was reported in a sector of the plant pathogenic fungus Fusarium oxysporum after successive subculturing (16). In some fungi, degeneration was linked to chromosome instability (4, 17).Our previous studies on the insect pathogenic fungus Metarhizium anisopliae revealed that fungal culture degeneration showed the signs of aging such as cellular accumulation of reactive oxygen species (ROS)¹, mitochondrial (mt) dysfunctions, and mtDNA glycation (14, 15). According to the vicious cycle theory of aging, mitochondria are the primary source of intracellular ROS production and also one of the important targets of ROS damage, which leads to generation of additional ROS (18, 19). Mitochondrial proteomics analyses have been frequently performed for studies on human diseases, and the physiologies of plants and yeasts (20, 21). Proteins localized in the mitochondria control mt dynamics, morphology, and function and their dysregulation or damage may induce abnormality in mitochondrial function (22). However, it is not known whether these changes occur in degenerated fungal cultures.In this study, cell biology, biochemical and comparative mitochondrial proteomic analyses were performed by using the model fungus Aspergillus nidulans to better understand the features and mechanisms of fungal culture degeneration. We found a significant difference in mitochondrial protein profiles between the wild type (WT) and nonsporulation sector culture. Many of the altered proteins fall into the functional categories of energy metabolism, stress responses and cell death. Functional consequences of these changes are supported by our experimental data. The observed features such as cellular oxidative stress, mitochondrial dysfunctions, accelerated autophagy and releases of apoptotic factors in degenerated A. nidulans resemble the apoptotic process observed in mammalian cells.     

Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.Mitochondria are the source of energy to sustain life. In addition to their evolutionary origin as an energy-producing organelle, their functionality has integrated into every aspect of life, including the cell cycle, ROS¹ production, apoptosis, and ion balance (1, 2). Our understanding of mitochondrial biology is still growing. Several systems biology approaches have been dedicated to exploring the molecular infrastructure and dynamics of the functional versatility associated with this organelle (3–5).To meet tissue-specific functional demands, mitochondria acquire heterogeneous properties in individual organs, a first statement of their plasticity in function and proteome composition (1, 6). The heterogeneity is evident even in an individual cardiomyocyte (7). A catalogue of the cardiac mitochondrial proteome is emerging via a joint effort (3–5). The dynamics of the mitochondrial proteome manifest at multiple levels, including post-translational modifications, such as phosphorylation. Our investigative goal is to decode this organellar proteome and its post-translational modification in a biological and functional context. In cardiomyocytes, mitochondria are also constantly exposed to fluctuation in energy demands and in ionic conditions. The capacity of mitochondria to cope with such a dynamic environment is essential for the functional role of mitochondria in normal and disease phenotypes (8–10). Unique protein features enabling the mitochondrial proteome to adapt to these biological changes can be interrogated by proteomics tools (10–12). Protein phosphorylation as a rapid and reversible chemical event is an integral component of these protein features (12–14).It has been estimated that one-third of cellular proteins exist in a phosphorylated state at least one time in their lifetime (15). However, only a handful of phosphorylation events have been identified to tune mitochondrial functionality (13, 14, 16) despite the fact that the first demonstration of phosphorylation was reported on a mitochondrial protein more than 5 decades ago (17). Kinases and phosphatases comprise nearly 3% of the human genome (18, 19). In mitochondria, ∼30 kinases and phosphatases have been identified thus far within the expected organellar proteome of a few thousand (3–5, 16). The number of identified mitochondrial phosphoproteins is far below one-third of its proteome size (20). Thus, it appears that the current pool of reported phosphoproteins represents only a small fraction of the anticipated mitochondrial phosphoproteome. The seminal studies from several groups (12–14, 16) demonstrated the prevalence as well as the dynamic nature of phosphorylation in cardiac mitochondria, suggesting that obtaining a comprehensive map of the mitochondrial phosphoproteome is feasible.In this study, we took a systematic approach to tackle the phosphorylation of murine cardiac mitochondrial pathways. We applied the unique strengths of both electron transfer dissociation (ETD) and collision-induced dissociation (CID) LC-MS/MS to screen phosphorylation events in a site-specific fashion. A total of 236 phosphorylation sites in 203 unique phosphopeptides were identified and mapped to 181 phosphoproteins. Novel phosphorylation modifications were discovered in diverse pathways of mitochondrial biology, including ion balance, proteolysis, and apoptosis. Consistent with the role of mitochondria as the major source of energy production under delicate control, metabolic pathways claimed one-third of phosphorylation sites captured in this analysis. To study molecular players steering mitochondrial phosphorylation, we probed the effects of calcium loading on phosphorylation. In addition, a number of kinases with previously unappreciated mitochondrial residence are suggested as potential players modulating mitochondrial pathways. Taken together, the cohort of novel phosphorylation events discovered in this study constitutes an essential step toward the full delineation of the cardiac mitochondrial phosphoproteome.     

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D₂ synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands.The bioactive sphingolipid ceramide is involved in the regulation of a wide variety of cellular processes, including apoptosis, autophagy, and cell cycle progression in cancer (1–3). Ceramide has also been implicated in a number of disease states, including inflammation and inflammatory disorders (4) and neurodegenerative diseases (5).Despite the wide range of processes regulated by ceramide, the precise molecular mechanisms by which ceramide acts as a signaling molecule are not clear. It has been suggested that plasma membrane ceramide acts to stabilize lipid rafts, which act as platforms for the concentration of signaling molecules (6, 7). Another possible mechanism of ceramide signaling is through direct interaction with target proteins. However, relatively few direct protein interactions with ceramide have been described. Examples of proteins that are regulated by direct ceramide binding include KSR (8), Raf-1 (9), protein kinase C-ζ (10), PP2A inhibitor SET (11), and cathepsin D (12). Thus, the identification of additional ceramide-binding proteins could lead to a better mechanistic understanding of how ceramide functions as a signaling molecule.Although various techniques have been used previously, in general, efforts to systematically screen for protein-lipid interactions have proved challenging (13–15). The commonly used yeast two-hybrid system is ineffective when the bait cannot be expressed inside the yeast cell and phage and bacterial display is limited due to prokaryotic expression of eukaryotic proteins. Column-based affinity purification (16, 17) and protein chip methods (18, 19) have been utilized, but they also have drawbacks, including the difficulty in recovering low abundance proteins and cost of setup and quality control (14).We have previously described the generation and application of yeast surface cDNA display libraries to novel protein-ligand discovery (13, 15, 20–24). Here, we describe their application for proteome-wide identification of human ceramide-binding proteins. Utilizing deep sequencing to comprehensively interrogate enriched selection outputs, we have identified a large number of ceramide-binding proteins, many of which represent novel interactions. For example, we have identified and validated EF-hand and STI1 domain-containing proteins as ceramide-specific binding proteins, suggesting that ceramide may regulate cellular pathways by interacting directly with those proteins.     

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.Post-translational modifications (PTMs)¹ are complex and fundamental mechanisms modulating diverse protein properties and functions, and have been associated with almost all known cellular pathways and disease processes (1, 2). Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3–6), malonylation (7, 8), crotonylation (9, 10), propionylation (11–13), butyrylation (11, 13), and succinylation (7, 14–16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonyl-CoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17, 18).Among these lysine PTMs, lysine succinylation is a highly dynamic and regulated PTM defined as transfer of a succinyl group (-CO-CH₂-CH₂-CO-) to a lysine residue of a protein molecule (8). It was recently identified and comprehensively validated in both bacterial and mammalian cells (8, 14, 16). It was also identified in core histones, suggesting that lysine succinylation may regulate the functions of histones and affect chromatin structure and gene expression (7). Accumulating evidence suggests that lysine succinylation is a widespread and important PTM in both eukaryotes and prokaryotes and regulates diverse cellular processes (16). The system-wide studies involving lysine-succinylated peptide immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS/MS) have been employed to analyze the bacteria (E. coli) (14, 16), yeast (S. cerevisiae), human (HeLa) cells, and mouse embryonic fibroblasts and liver cells (16, 19). These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19, 20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19–22).Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major cause of mortality worldwide and claims more human lives annually than any other bacterial pathogen (23). About one third of the world''s population is infected with Mtb, which leads to nearly 1.3 million deaths and 8.6 million new cases of TB in 2012 worldwide (24). Mtb remains a major threat to global health, especially in the developing countries. Emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mtb, and also the emergence of co-infection between TB and HIV have further worsened the situation (25–27). Among bacterial pathogens, Mtb has a distinctive life cycle spanning different environments and developmental stages (28). Especially, Mtb can exist in dormant or active states in the host, leading to asymptomatic latent TB infection or active TB disease (29). To achieve these different physiologic states, Mtb developed a mechanism to sense diverse signals from the host and to coordinately regulate multiple cellular processes and pathways (30, 31). Mtb has evolved its metabolic network to both maintain and propagate its survival as a species within humans (32–35). It is well accepted that metabolic network is a central mediator and defining feature of the pathogenicity of Mtb (23, 36–38). Knowledge of the regulation of metabolic pathways used by Mtb during infection is therefore important for understanding its pathogenicity, and can also guide the development of novel drug therapies (39). On the other hand, increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells (14, 19–22). It is tempting to speculate that lysine succinylation may play an important regulatory role in metabolic processes in Mtb. However, to the best of our knowledge, no succinylated protein in Mtb has been identified, presenting a major obstacle to understand the regulatory roles of lysine succinylation in this life-threatening pathogen.In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the succinylated protein in Mtb. Because Mtb H37Rv is the first sequenced Mtb strain (40) and has been extensively used for studies in dissecting the roles of individual genes in pathogenesis (41), it was selected as a test case. We analyzed the succinylome of Mtb H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and render particular enrichment to metabolic process. A large proportion of the succinylation sites are present on proteins in the central metabolism pathway. We further dissected the regulatory role of succinylation on acetyl-CoA synthetase (Acs) via site-specific mutagenesis analysis and molecular dynamics (MD) simulations showed that reversible lysine succinylation could inhibit the activity of Acs. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a deacetylase and as a desuccinylase of Acs in in vitro assays. Together, our findings provide significant insights into the range of functions regulated by lysine succinylation in Mtb.     

Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca²⁺ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca²⁺ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca²⁺ responses because of the release of Ca²⁺ from both ER and acidic stores. Ca²⁺ responses to menadione were accompanied by elevation of Ca²⁺ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca²⁺ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca²⁺ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca²⁺ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca²⁺ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca²⁺ with pericam. We conclude that elevated Ca²⁺ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.Apoptosis, a mechanism of programmed cell death, usually occurs through intrinsic or extrinsic apoptotic pathways. The caspases involved in apoptosis can be split into two groups, the initiator caspases such as caspase-9 and effector caspases such as caspase-3. Effector caspases are activated by initiator caspases and mediate many of the morphological cellular changes associated with apoptosis (1).Calcium is an important signaling ion involved in the regulation of many physiological as well as pathological cellular responses (2). In the pancreas, we have shown that Ca²⁺ signals elicit enzyme secretion (3), apoptosis (4–6), and pathological intracellular activation of digestive enzymes (7). As such, there must be mechanisms in place by which the cell can differentiate between apoptotic and non-apoptotic Ca²⁺ signals.The spatiotemporal pattern of calcium signaling is crucial for the specificity of cellular responses. For example, repetitive cytosolic calcium spikes confined to the apical region of the pancreatic acinar cell are elicited by physiological stimulation with acetylcholine (ACh) or cholecystokinin (CCK) and result in physiological secretion of zymogen granules (8, 9). However, a sustained global increase in free cytosolic Ca²⁺ induced by supramaximal stimulation with CCK, which resembles prolonged hyperstimulation of pancreatic acinar cells in the pathophysiology of acute pancreatitis, can lead to premature activation of digestive enzymes and vacuole formation within the cell (10–12). Alternatively, global repetitive calcium spikes induced in the pancreatic acinar cell in response to oxidant stress can lead to induction of the mitochondrial permeability transition pore (mPTP)⁴ and apoptosis (4, 5, 13).To understand the role of calcium in apoptosis, several investigators have examined the influence of intracellular stores on the molding of calcium signals that lead to cell death (14–16). It has been well established in a range of cell types that the endoplasmic reticulum (ER) is the major intracellular calcium store required for induction of apoptosis. Pinton et al. (17) have shown that decreasing ER Ca²⁺ concentration with tBuBHQ increased HeLa cell survival in response to oxidant stress induced by ceramide. Scorrano and Korsmeyer (18) also observed that double knock-out Bax and Bak (pro-apoptotic proteins) mouse fibroblasts displayed a reduced resting concentration of ER Ca²⁺ compared with wild type and were resistant to induction of apoptosis by various stimulants, including ceramide. These important studies strongly suggest that the concentration of Ca²⁺ in the ER is a critical determinant of cellular susceptibility to apoptotic stimuli in the cell types studied.A key event in early apoptosis is permeabilization of the mitochondrial membrane. The mPTP is a pore whose molecular composition is still debated (19). Activation of an open pore state can result in swelling of the mitochondrial matrix and release of the apoptogenic proteins from the intermembrane space (20).One important activator of the mPTP is Ca²⁺ (20–22), a function which implicates Ca²⁺ in the initiation of apoptosis (23, 24). Once Ca²⁺ is released from the ER into the cytoplasm, mitochondria take up part of the released Ca²⁺ to prevent propagation of large calcium waves (25–27). This influx is followed by calcium efflux from the mitochondria back into the cytosol (28, 29). An increase in mitochondrial Ca²⁺ concentration in response to physiological stimuli induces increased activity of the mitochondrial respiratory chain and the synthesis of ATP to meet with increasing energy demands on the cell. When mitochondria are exposed to a pathological overload of calcium, opening of the mPTP is triggered, leading to mitochondrial dysfunction and eventually cell death. The mechanism through which calcium can trigger mPTP opening is still unclear and may involve cyclophilin D (30) and voltage-dependent anion channel (31). The mitochondria are endowed with selective and efficient calcium uptake (a calcium-selective uniporter) and release mechanisms (Ca²⁺/Na exchanger, Ca²⁺/H⁺ exchanger, and mPTP) (16, 29, 32, 33).Oxidant stress is a well known inducer of apoptosis in several cell types (34) and is thought to play an important role in the pathogenesis of acute pancreatitis (35). We have used the quinone compound menadione to induce oxidative stress in the pancreatic acinar cell. Menadione is metabolized by flavoprotein reductase to semiquinone and then is oxidized back to quinone, resulting in generation of superoxide anion radicals, hydrogen peroxide, and other reactive oxygen species (ROS) (36). In vivo, menadione causes depolarization and swelling of the mitochondria (37). In pancreatic acinar cells, treatment with menadione not only produces an increase in ROS, but has also been found to evoke cytosolic Ca²⁺ responses, mPTP opening, activation of caspases and apoptotic cell death (4, 5). When cells were pretreated with the calcium chelator BAPTA-AM, menadione was unable to induce apoptosis, indicating that oxidant stress-induced apoptosis in the pancreatic acinar cell is highly calcium-dependent. Here we show that in pancreatic acinar cells, oxidative stress-induced apoptosis is strongly dependent on the Ca²⁺ concentration within mitochondria at the time of ROS production.