Unraveling the Phosphoproteome Dynamics in Mammal Mitochondria

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Unraveling the Phosphoproteome Dynamics in Mammal Mitochondria from a Network Perspective Ana Isabel Padraõ ,† Rui Vitorino,*,† José Alberto Duarte,‡ Rita Ferreira,† and Francisco Amado§ †

QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal CIAFEL, Faculty of Sports, University of Porto, 4200-450 Porto, Portugal § QOPNA, Health School of Sciences, University of Aveiro, 3810-193 Aveiro, Portugal ‡

S Supporting Information *

ABSTRACT: With mitochondrion garnering more attention for its inextricable involvement in pathophysiological conditions, it seems imperative to understand the means by which the molecular pathways harbored in this organelle are regulated. Protein phosphorylation has been considered a central event in cellular signaling and, more recently, in the modulation of mitochondrial activity. Efforts have been made to understand the molecular mechanisms by which protein phosphorylation regulates mitochondrial signaling. With the advances in mass-spectrometry-based proteomics, there is a substantial hope and expectation in the increased knowledge of protein phosphorylation profile and its mode of regulation. On the basis of phosphorylation profiles, attempts have been made to disclose the kinases involved and how they control the molecular processes in mitochondria and, consequently, the cellular outcomes. Still, few studies have focused on mitochondrial phosphoproteome profiling, particularly in diseases. The present study reviews current data on protein phosphorylation profiling in mitochondria, the potential kinases involved and how pathophysiological conditions modulate the mitochondrial phosphoproteome. To integrate data from distinct research papers, we performed network analysis, with bioinformatic tools like Cytoscape, String, and PANTHER taking into consideration variables such as tissue specificity, biological processes, molecular functions, and pathophysiological conditions. For instance, data retrieved from these analyses evidence some homology in the mitochondrial phosphoproteome among liver and heart, with proteins from transport and oxidative phosphorylation clusters particularly susceptible to phosphorylation. A distinct profile was noticed for adipocytes, with proteins form metabolic processes, namely, triglycerides metabolism, as the main targets of phosphorylation. Regarding disease conditions, more phosphorylated proteins were observed in diabetics with some distinct phosphoproteins identified in type 2 prediabetic states and early type 2 diabetes mellitus. Heart-failure-related phosphorylated proteins are in much lower amount and are mainly involved in transport and metabolism. Nevertheless, technical considerations related to mitochondria isolation and protein separation should be considered in data comparison among different proteomic studies. Data from the present review will certainly open new perspectives of protein phosphorylation in mitochondria and will help to envisage future studies targeting the underlying regulatory mechanisms. such as phosphorylation, acetylation, or ubiquitination.10 Protein phosphorylation is the most widespread type of PTM in signal transduction and at present is the most studied PTM.11−15 The role of mitochondrial phosphorylated proteins in the regulation of cellular and physiological events has been known for decades; however, the extent of this mode of regulation is just beginning to be disclosed. Moreover, abnormal protein phosphorylation is known to cause or be the consequence of many pathophysiological conditions, such as cancer, diabetes mellitus, and neurodegenerative diseases.16−18 Proteins of eukaryotic cells are mainly phosphorylated on serine (Ser), threonine (Thr), or tyrosine (Tyr) amino acid residues by the enzymatic activities of protein kinases that

1. INTRODUCTION Cell survival critically depends on the integrity and functionality of mitochondria, which house biosynthetic pathways and oxidative phosphorylation machinery for ATP production. Besides providing cellular energy, mitochondria are involved in multiple cell-signaling cascades and in the regulation of cellular metabolism, calcium homeostasis and apoptosis.1,2 In agreement with this multitude of cellular functions, mitochondrial dysfunction is thought to contribute to cellular aging and to a series of diseases, such as diabetes mellitus, cancer, and neurodegenerative disorders.3−7 The involvement of mitochondria in such a wide range of cellular processes requires an integrated system of signaling to coordinate the various molecular messages that enter or exit the mitochondrion according to the diverse needs of the cells.8 Growing evidence suggests that mitochondrial plasticity relies on a range of reversible protein post-translational modifications (PTMs),9 © 2013 American Chemical Society

Received: April 26, 2013 Published: August 22, 2013 4257

dx.doi.org/10.1021/pr4003917 | J. Proteome Res. 2013, 12, 4257−4267

Journal of Proteome Research

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catalyze the transfer of γ-phosphate group from adenosine 5′triphosphate (ATP) to side chains of these specific amino acids in proteins.15 Indeed, the dynamic protein phosphorylation is tightly regulated by kinases and phosphatases systems.16,19 There are more than 500 kinases encoded from the human genome,20 and around 700 000 phosphorylated sites are predicted for any given kinase.15 Phosphorylation also occurs on histidine residues; however, phosphohistidine is highly labile and therefore hardly identified.21 Protein phosphorylation has the potential to induce functional changes in a protein,22 through modification of protein’s activity, location, or interaction with other cellular components.23 These functional changes typically occur as a consequence of conformational changes to protein tertiary or quaternary structures.10 The extent and complex nature of phosphorylation events arise from the possibility of multiple amino acid residues being phosphorylated on a single protein.24,25 Moreover, it is predicted that more than 30% of cellular proteins are phosphorylated at any given time.26 While some amino acid residues are always quantitatively phosphorylated, others may only be transiently phosphorylated up to 0.5%. Especially regarding signaling pathways, phosphorylation rates and thereby phosphoprotein abundance are very low (with only 1 to 2% of a given protein present in a phosphorylated form). The analysis of phosphoproteins is also complicated by the complexity of phosphorylation patterns and the sheer number of phosphorylation sites.11 Nevertheless, one decade after the introduction of proteomics and following the advances in mass spectrometry (MS), there is a substantial hope and expectation in the increasing knowledge of protein profile analysis and in the understanding of the molecular mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.10,27 Some authors argue that the analysis of mitochondrial phosphoproteome may lead to the identification of novel therapeutic targets and biomarkers.18,26 Considering the central regulatory role of mitochondrial phosphorylation in the maintenance of living cells’ dynamics, here we review the main findings in the characterization of mitochondrial phosphoproteome using mass-spectrometry-based proteomics, the potential kinases involved in its regulation, and the alterations in the phosphorylation pattern related to pathophysiological conditions. To integrate data from distinct studies focused on mitochondrial phosphoproteome profiling, we used the bioinformatic tools Cytoskape (Bingo28 and ClueGo29 plugins), PANTHER,30 and STRING.31

and human) and pathophysiological conditions studied. Eighty eight percent of these phosphoproteins were identified in liver mitochondria, but no known study was performed with human liver samples, unlike that observed for skeletal muscle. In heart, 184 distinct phosphoproteins were identified in isolated mitochondria from rodents, and only 8 were reported in human tissue (Table S1 in the Supporting Information). Regarding animal species, most of the phosphorylated proteins identified in mitochondria correspond to mouse (57%) and rat (45%) proteins (Table S2 in the Supporting Information). The orthology prediction analysis of the 3817 proteins reported to be phosphorylated in isolated mitochondria retrieved 3465 ortholog groups (Table S3 in the Supporting Information). This high number suggests that few protein sequences are shared by two or more species/genomes. We evaluated the distribution of phosphoproteins per tissue by performing an integrated analysis of the unique proteins in each tissue with Cytoscape v2.8.3, an open-source software platform for visualizing complex networks. The protein−protein interaction network presented in Figure S1 in the Supporting Information was imported from the databases Intact (http://www.ebi.ac.uk/ intact/) and STRING 9.1 (http://string-db.org) and contains 3284 proteins and 3693 protein−protein interactions. These proteins are distributed in nine clusters centered in distinct organs/cells. The highest number of phosphopeptides, phosphorylation sites, and phosphoproteins was observed in liver, followed by heart, without any direct relation to the methodology used (with and without phosphopeptide enrichment). Most of the studies on mitochondrial phosphoproteome profiling were performed with phospho-enriched and nonenriched samples obtained from different species. In brain, only two studies targeting mitochondrial phosphoproteome are known, and both used gel-free approaches without using phosphoenrichment procedures (Table S1 in the Supporting Information), which might justify the considerable lower number of phosphoproteins reported in this tissue in comparison with heart and liver. To the best of our knowledge, only one study performed mitochondrial phosphoprotein profiling in skeletal muscle. The comparison of the mitochondrial phosphoproteome among tissues (Figure 1) revealed only two common phosphoproteins, specifically aconitase and ADP/ATP translocase 1 (ANT1), with no evident tissue-specific phosphosites. Hexokinase protein 2 was

2. MITOCHONDRIAL PHOSPHOPROTEOME SURVEY The importance of phosphorylation/dephosphorylation in the regulation of mitochondrial protein activities is supported by the findings that mitochondria contain protein kinases, phosphatases, and phosphorylated proteins.32 In mitochondria, more than 50 kinases were already identified33 within the expected organellar proteome of a few thousand, and the number of identified mitochondrial phosphoproteins is far below the one-third of proteome size.34 With the development of mass-spectrometry-based approaches, a growing number of studies have provided evidence that phosphorylation of mitochondrial proteins is much more frequent than expected.9,35,36 Studies focused on mitochondrial protein phosphorylation profiling retrieved more than 3817 distinct phosphoproteins identified in mitochondria (although 72 proteins have no known annotation at Uniprot), considering all tissues/cells from all specie (rat, mouse, cow, bovine, rabbit,

Figure 1. Venn diagram of phosphorylated mitochondrial proteins identified in different tissues (brain, liver, heart, and skeletal muscle) (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Data here analyzed were retrieved from several studies: 2 for brain, 17 for heart, 11 for liver, and 1 for skeletal muscle, independently of the pathophysiological condition and methodology strategy used. 4258



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Figure 2. Categorical analysis based on molecular function of phosphorylated proteins in several tissues/organs performed with the Gene Ontology Annotation after Bonferroni correlation.

only found phosphorylated in mitochondria isolated from brain. Sixty-four common phosphorylated mitochondrial proteins

were observed when comparing heart with liver, most of which belong to metabolic processes (28%), generation of 4259



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Figure 3. Categorical analysis based on biological process of phosphorylated proteins in different tissues/cells performed with the Gene Ontology Annotation after Bonferroni correlation.

precursor metabolites and energy (14%), and cellular processes (11%) according to PANTHER. Only seven phosphoproteins

were identified when comparing skeletal muscle with heart, the majority of which are involved in metabolic processes. Despite 4260



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Figure 4. Venn diagram of phosphorylated mitochondrial proteins identified in liver (A) and heart (B) under distinct pathophysiological conditions (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Data related to pathophysiological conditions were retrieved from two studies performed with liver and three with heart mitochondria. Regarding heart mitochondria, one of the studies targeted the inhibition of PDH phosphorylation under energized and de-energized conditions (with DCA), other characterized εPKC targets in ischemia, and the third used tissue from heart failure patients (Obese/lean, obese/lean type 2 diabetes mellitus; DCA, dichloroacetate; D, de-energization; E, energization; Pre-DM, prediabetes mellitus type 2; Early-D2M, early diabetes mellitus type 2).

density gradients (e.g., Percoll, Ficoll, or Nycodenz). Despite yielding highly purified preparations, this approach is expensive and demands a large amount of sample.49 To overcome this limitation, some authors use pools of tissue from different animals.34 Using MS-based approaches, many researchers have studied the mitochondrial phophoproteome profile in heart,34,38,50−65 liver,9,36,50,58,65−72 skeletal muscle,27 adipocytes,73,74 brain,75,76 INS-1 β cells,33 and T98G gliobastoma cells,77 aiming to understand the molecular and physiological role of phosphorylation in mitochondria biology. Putative mitochondrial phosphoproteins identified by MS are summarized in Table S1 in the Supporting Information. On the basis of the published studies focused on protein phosphorylation in mitochondria isolated from different organs (specifically liver, heart, brain, and skeletal muscle), we performed categorical analysis based on molecular function and biological processes with the Gene Ontology Annotation after Bonferroni correlation. Regarding molecular function (Figure 2), most of the phosphorylated mitochondrial proteins in liver present binding, catalytic, and transcription activity, whereas in heart the prevalent cluster is motor activity. In skeletal muscle mitochondria, most of the phosphorylated proteins have catalytic, binding, and transport activity, and in the brain all of the identified phosphorylated proteins belong to these three clusters. Considering biological processes (Figure 3), the majority of phosphoproteins are involved in metabolism, cellular processes, and transport, with similar distribution profiles observed for heart, liver, and skeletal muscle mitochondria. In brain, mitochondrial phosphorylated proteins are distributed per three clusters: metabolism, generation of precursor metabolites and energy, and transport. Independently of the tissue/cell used for mitochondria isolation, the most prevalent phosphorylated amino acid residue is serine, with 9416 phosphoserines identified (82%), followed by phosphothreonine (15%) and phosphotyrosine (3%) (Table S1 in the Supporting Information).

technical issues underlying tissue-specific proteomic data, this variety seems to reflect differences in complexity and in intracellular signaling within each tissue, possibly involving distinct kinases, to make these biological processes more organefficient.35,37,38 2.1. Mitochondrial Phosphoproteins Identified by MS-Based Approaches

The analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research.39 Phosphoproteomic analyses are more difficult than protein identification for several reasons. First, highly sensitive methods are required for detection due to a low stoichiometry. Because only 5−10% of a protein kinase substrate is phosphorylated, methods that detect the modified protein at very low levels (

Unraveling the Phosphoproteome Dynamics in Mammal Mitochondria

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