Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle

Journal Pre-proof Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle

B. Sunitha, Manish Kumar, Niya Gowthami, Sruthi Unni, Narayanappa Gayathri, T.S. Keshava Prasad, Atchayaram Nalini, Kiran Polavarapu, Seena Vengalil, Veeramani Preethish-Kumar, B. Padmanabhan, M.M. Srinivas Bharath PII:

S1874-3919(19)30328-8

DOI:

https://doi.org/10.1016/j.jprot.2019.103556

Reference:

JPROT 103556

To appear in:

Journal of Proteomics

Received date:

29 April 2019

Revised date:

8 October 2019

Accepted date:

17 October 2019

Please cite this article as: B. Sunitha, M. Kumar, N. Gowthami, et al., Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle, Journal of Proteomics (2019), https://doi.org/10.1016/ j.jprot.2019.103556

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© 2019 Published by Elsevier.

Journal Pre-proof

Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle B. Sunitha1*#, Manish Kumar2,3*, Niya Gowthami4, Sruthi Unni5, Narayanappa Gayathri1, T.S. Keshava Prasad3,6, Atchayaram Nalini7, Kiran Polavarapu7,8, Seena Vengalil7, Veeramani Preethish-Kumar7,8, B. Padmanabhan5, M. M. Srinivas Bharath4@

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From the 1Department of Neuropathology, NIMHANS, Bangalore-560029, Karnataka, India; 2 Manipal University, Madhav Nagar, Manipal-576104, Karnataka, India; 3Institute of Bioinformatics, Whitefield, Bangalore-560066, Karnataka, India; 4Department of Clinical Psychopharmacology and Neurotoxicology, NIMHANS, Bangalore- 560029, Karnataka, India; 5Department of Biophysics, NIMHANS, Bangalore- 560029, Karnataka, India; 6YUIOB Centre for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore575018, Karnataka, India; 7Department of Neurology, NIMHANS, Bangalore-560029, Karnataka, India; 8Department of Clinical Neurosciences, NIMHANS, Bangalore-560029, Karnataka, India #

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Present address: John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, United Kingdom.

@

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*Both contributed equally to this work

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To whom correspondence should be addressed: M.M. Srinivas Bharath, PhD, Department of Clinical Psychopharmacology and Neurotoxicology, NIMHANS, No. 2900, Hosur Road, Bangalore-560029, Karnataka, India. Tel: +91-80-26995113; Fax: +91-80-26564830; Email: [email protected] Running title: Mitochondrial phosphoprotein profile in healthy and diseased muscle

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Journal Pre-proof Abstract Analysis of human muscle diseases highlights the role of mitochondrial dysfunction in the skeletal muscle. Our previous work revealed that diverse upstream events correlated with altered mitochondrial proteome in human muscle biopsies. However, several proteins showed relatively unchanged expression suggesting that post-translational modifications, mainly protein phosphorylation could influence their activity and regulate mitochondrial processes. We conducted mitochondrial phosphoprotein profiling, by proteomics approach, of

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healthy human skeletal muscle (n=10) and three muscle diseases (n=10 each):

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Dysferlinopathy, Polymyositis and Distal Myopathy with Rimmed Vacuoles. Healthy human

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muscle mitochondrial proteins displayed 253 phosphorylation sites (phosphosites), which

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contributed to metabolic and redox processes and mitochondrial organization etc. Electron transport chain complexes accounted for 84 phosphosites. Muscle pathologies displayed 33

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hyperphosphorylated and 14 hypophorphorylated sites with only 5 common proteins,

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indicating varied phosphorylation profile across muscle pathologies. Molecular modelling revealed altered local structure in the phosphorylated sites of Voltage-Dependent Anion

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Channel 1 and complex V subunit ATP5B1. Molecular dynamics simulations in complex I subunits NDUFV1, NDUFS1 and NDUFV2 revealed that phosphorylation induced structural alterations thereby influencing electron transfer and potentially altering enzyme activity. We propose that altered phosphorylation at specific sites could regulate mitochondrial protein function in the skeletal muscle during physiological and pathological processes.

Key words: Skeletal muscle; mitochondria; Dysferlinopathy; Polymyositis; Distal Myopathy with Rimmed Vacuoles; molecular dynamics

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Journal Pre-proof Introduction Muscle diseases, especially the inherited pathologies are often associated with considerable mortality and morbidity in humans [1, 2]. Assessment of muscle pathology in these diseases indicates direct involvement of mitochondrial damage [3]. Mitochondrial abnormalities are evident in Duchenne muscular dystrophy (DMD) [4], collagen VI myopathies, congenital myopathies, calpainopathy, inflammatory myopathies [5, 6], facioscapulohumeral dystrophy [7], epidermolysis bullosa simplex with MD, sarcoglycan

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deficient MDs and others [8, 9]. Mitochondrial dysfunction has been noted in the skeletal

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muscle of the DMD mouse model [10-12]. Mitochondrial damage in muscle pathologies

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entails release of pro-apoptotic proteins [13], altered calcium homeostasis [14], oxidative

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stress [15] and abnormal mitochondrial permeability transition pore [16, 17] among others . We assessed the mitochondrial dysfunction in muscle pathologies by employing a

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cardiotoxin (CTX) mouse model of muscle degeneration [18]. Muscle mitochondria from the

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CTX model showed depolarization, altered cristae, decreased mitochondrial activities and down-regulation of proteins involved in energy metabolism. Similar analysis in muscle

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biopsies from patients with established muscle diseases revealed that diverse upstream events such as loss of sarcolemmal proteins, myoinflammation, intracellular morphological alterations and abnormalities in lipid storage showed altered mitochondrial function as indicated by morphological and biochemical analysis [19, 20]. At the cellular level, mitochondrial dysfunction in these diseases correlated with significantly altered mitochondrial proteome. Mitochondrial proteins from the CTX model and human muscle pathologies also displayed higher tryptophan oxidation compared to controls, with structural implications [19] linking mitochondrial dysfunction with oxidative protein damage [21]. Our proteomic study in human muscle pathologies [19] revealed that although 80 mitochondrial proteins were commonly down-regulated among Dysferlinopathy (dysfy)

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Journal Pre-proof polymyositis (PM) and distal myopathy with rimmed vacuoles (DMRV) compared to control, a substantial number of proteins displayed relatively unchanged expression. It is possible that expression-independent alterations, such as post-translational modifications (PTMs) could influence the activity of these proteins thereby contributing to altered mitochondrial function. PTMs regulate the structure-function relationship of mitochondrial proteins [22]. These include oxidative PTMs [19, 23] and non-oxidative PTMs mainly including phosphorylation. Protein phosphorylation/ dephosphorylation dynamics are reported to regulate

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mitochondrial processes [24, 25]. Our recent study revealed extensive phosphorylation of

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mammalian mitochondrial complex I (CI) subunits [26]. Although advances in

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phosphoproteomic technologies have increased the identification of mitochondrial

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phosphorylation sites (phosphosites) and phosphoproteins, their functional importance and the role of specific Kinases/ Phosphatases are unclear [27]. Such analysis is crucial to

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understand their role in human diseases. While global proteomic approaches [28-30] are

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employed to characterize the mitochondrial phosphoproteome across different species, phosphoproteomics in human muscle mitochondria is limited [27]. Further, the role of

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specific phosphorylation events on the structure function relationship of mitochondrial proteins has not been explored.

In the current study, we conducted a comprehensive phosphoprotein profiling of healthy human skeletal mitochondria and compared the same with three muscle pathologies with varied clinical and pathological features. These include muscle-intrinsic pathologies, viz., muscular dystrophy (MD) (dysfy) with proximal, distal and proximo-distal weakness, inflammatory myopathy (PM) with proximal muscle involvement and DMRV with distal involvement. We have also assessed the structural implications of protein phosphorylation of selected mitochondrial proteins by molecular modelling and molecular dynamics simulations.

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Journal Pre-proof Materials and methods All chemicals utilized in the study were of analytical grade. Bulk chemicals and solvents were obtained from Merck (Whitehouse Station, NJ, USA) and Sisco Research Laboratories Pvt. Ltd. (Mumbai, Maharashtra, India). Fine chemicals and Nagarse were obtained from Sigma (St. Louis, MO, USA). Proteomic grade Trypsin was obtained from Promega (Madison, WI, USA). Tandem mass tag (TMT) isobaric labelling kit was obtained from Thermofisher Scientific (Waltham, MA, USA). Consumables for phosphopeptide

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enrichment were obtained from GL Sciences (Tokyo, Japan). Antibodies against Dystrophin

[α-sarc

β-sarc

(RRID:AB_442041),

(RRID:AB_564136),

γ-sarc

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Sarcoglycans

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[dys1 (RRID:AB_442080), dys2 (RRID:AB_442081) and dys3 (RRID:AB_442082)],

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(RRID:AB_442096) and ẟ-sarc (RRI D:AB_442079] and Merosin (RRID:AB_442108) and

Study design

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(Newcastle Upon Tyne, UK).

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Dysferlin (RRID:AB_442097) were procured from Novocastra Laboratories Limited (NCL)

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Muscle biopsies from confirmed cases of dysfy (n=10), DMRV (n=10) and PM (n=10) and age-matched healthy controls (n=10) were utilized in the current study. The cases were characterized by clinical evaluation followed by confirmation of diagnosis by biochemical and histopathological analysis. Total mitochondria from muscle biopsies of dysfy, DMRV and PM and controls [n=10 samples for each disease and n=10 samples (biological replicates) as control], were subjected to tryptic digestion and TMT labelling followed by total proteomic and phosphoproteomic analysis (carried out in n=2 technical replicates for each sample). The complete mitochondrial phosphoprotein profile in healthy muscle was analyzed followed by its comparison across different disease samples to understand the molecular basis of mitochondria dysfunction in these pathologies. The

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Journal Pre-proof structural implications of the phosphorylation events were then tested by structural bioinformatics and molecular dynamics simulations.

Human tissue samples Ethics statement: The study protocol conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964) and was approved by the NIMHANS Institutional Ethics Committee (Approval

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reference number: NIMH/91st IEC/2014 Item No. XII, Sl. No. 12.01, Clinical

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Neurosciences). In all cases, samples were procured after obtaining written informed consent.

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Samples: Patients with muscle diseases evaluated at the Neuromuscular Disorders Clinic, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India from

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2007-2016 were selected following diagnostic procedures. The study included confirmed

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cases [based on histology/ immunohistochemistry (IHC)/ molecular genetic analysis/ western blot] of Dysferlinopathy (dysfy) (n =10), DMRV (n =10), and PM (n =10) [19]. Sample size

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calculation was not performed regarding the samples assessed in this study. The clinical details of all patients were recorded. This included demographic details (age, gender, age at onset and presentation), initial symptoms and severity, Creatine Kinase levels and assessment of muscle strength (both upper and lower limbs). After obtaining informed consent, open muscle biopsies were conducted by a neurologist, under local anaesthesia. A moderately weak muscle (either from biceps or quadriceps) free from previous trauma was selected for uniformity and submitted for routine diagnosis to the Department of Neuropathology, NIMHANS. A fragment of the fresh biopsy was snap-frozen in isopentane that was pre-cooled in liquid nitrogen and stored at -80 °C [19, 31]. From the shortlisted cases, following diagnostic procedures and confirmation of specific muscle disease, fresh

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Journal Pre-proof muscle biopsies of 10 cases from each disease were considered for the proteomics experiment. No randomization methods were used during allocation of subjects to different experimental groups in the study. Further, no blinding was performed in the study. Inclusion and exclusion criteria considered for selection of the patient samples have been described previously [19].

Controls: Paraspinal muscle samples from age-matched patients (n=20) undergoing spinal

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surgery were collected from the Department of Neurosurgery, NIMHANS as controls and 10

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samples from these were carefully selected for the proteomics experiment. Comparison of

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human post-mortem paraspinal, biceps and quadriceps muscle types was carried out earlier

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Preparation of mitochondria

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[19] to ascertain the comparable mitochondrial features across different tissues.

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Muscle mitochondria were prepared as described [32] with minor modifications [19]. Briefly, muscle tissue (100 mg) was minced and incubated with 10 % Nagarse in ionic

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medium (100 mM Sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46 mM KCl, pH 7.4) containing phosphatase inhibitors [Sodium fluoride (1 mM), Sodium orthovandate (2 mM), Sodium pyrophosphate (2 mM) and β glycerophosphate (2 mM)] for 5 min, washed with ionic medium containing 0.5 % bovine serum albumin (BSA), homogenized and centrifuged (500 Xg, 10 min; 4 °C). The supernatant was centrifuged (12,000 g, 10 min; 4 °C), and the mitochondrial pellet was washed twice with 1X phosphate buffer saline (PBS). Mitochondrial enrichment was confirmed by Electron Microscopy (EM) observation and Rotenone sensitivity [18].

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Journal Pre-proof Mitochondrial Proteomics Sample preparation: Mitochondrial pellet from control muscle tissue (C) (n=10) and muscle pathologies [dysfy (n=10), DMRV (n=10) and PM (n=10)] (mitochondria were pooled in all groups to get a representative profile from statistically significant number of samples) were resuspended in extraction buffer [1 mM triethylammonium bicarbonate (TEABC) buffer, pH 8.5 containing 9 M urea and phosphatase inhibitors], sonicated (30 s) at 37 % amplitude on ice, followed by centrifugation at 14,000 rpm for 30 min. The supernatant was collected and

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subjected to protein estimation using Bicinchoninic acid (BCA) assay [33] (Pierce, Waltham,

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MA, USA). An equal amount of protein from each case (28 μg) was reduced using DTT

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(final concentration= 5 mM) at 60 ˚C for 30 min and alkylated using 10 mM iodoacetamide

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for 10 min at room temperature in dark. Trypsin was added to the extract in a ratio of 1 unit of trypsin per 20 unit of sample and incubated for overnight digestion at 37˚C. Following

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confirmation of tryptic digestion on SDS-PAGE, the peptides were lyophilized and subjected

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to Tandem mass tag (TMT) isobaric labelling method [20]. Control sample was labelled with 127 TMT channels, while Dysfy, DMRV and PM were labelled with 128, 129 and 130 TMT

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channels. TMT labelling was carried out using a commercial kit (Thermofisher Scientific) as per the manufacturer’s instructions. The labelling efficiency for each sample was checked by analyzing 2 μg equivalent peptides on mass spectrometer (MS). The labelled samples were pooled in equal amounts (250 μg) from control and disease groups and lyophilized [34, 35].

MS analysis of total mitochondrial proteome: One tenth of the labelled peptide mixture was utilized for comparative analysis of total mitochondrial proteome across different groups as described [19, 20, 35]. The peptides were subjected to bRPLC fractionation using XBridge column (C18, 5μm 250 × 4.6mm column) (Waters Corporation), which was coupled to an Agilent 1200 series HPLC system. For fractionation, 7 mM TEABC at pH 8.4 and 7 mM

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Journal Pre-proof TEABC in 90% ACN, pH 8.4 were used as solvent A and B, respectively. A flow rate of 1 ml/min was used with a gradient of 5 % B to 60 % B over 60 min. A total of 96 fractions were collected, which were further pooled and lyophilized into 12 fractions.

LC-MS/MS analysis: Fractionated samples were analyzed in MS3 mode for accurate quantitation. For MS3, synchronous precursor selection was enabled and 10 precursor ions were selected for fragmentation with 55 % HCD collision energy. The resolution was set to

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60,000 with AGC target value of 50,000 and 150 ms ion injection time. Internal calibration

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was carried out using lock mass option (m/z 445.1200025) from ambient air.

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Titanium dioxide (TiO2) based enrichment of phosphopeptides: The remaining 90 % of the peptide mixture was processed for TiO2-based enrichment of phosphopeptides as described

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[36]. The sample was reconstituted in 200 μl of 5 % dihydroxybenzoic acid in 80% ACN and

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3% trifluoro acetic acid (TFA). Titansphere beads (GL Sciences, Japan) were mixed with peptides in 1:1 ratio and incubated on a rotary shaker for one hour at room temperature.

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Peptides were then washed with 80 % ACN in 3 % TFA and eluted using 4 % ammonia solution followed by neutralization with 3 % TFA. The eluted peptides were then fractionated into six fractions using SCX-StageTip (Empore Solid Phase Extraction Disk). The fractionated samples were then dried and desalted using C18 stage tips and then subjected to LC-MS/MS analysis.

LC-MS/MS analysis: Phosphopeptides enriched TMT labelled fractions were analyzed in technical duplicates on Orbitrap Fusion Tribrid MS interfaced with Proxeon Easy-nLC 1000 system (Thermo Scientific, Bremen, Germany) [37]. Each fraction was reconstituted in 20 μl of 0.1% formic acid. The sample was then loaded on to a 2 cm long pre-column packed in-

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Journal Pre-proof house with magic C18 AQ (Michrom Bioresources, Auburn, CA, USA). A linear gradient of 5 % to 30 % of solvent B (0.1 % formic acid in 95 % ACN) over 100 min was then used to resolve the sample on an analytical column (75 μ x 25 cm, 3 μ particle and 100 Å pore size). Quadrupole isolation was enabled and full scans were acquired with scan range of 400-1600 m/z at a resolution of 120,000 at 200 m/z. AGC target was set to 2 million with ion injection time of 55 ms and dynamic exclusion was set to 30 s. Most intense precursor ions were selected at top speed data dependent mode with 3 s cycle and were fragmented using higher-

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energy collisional dissociation (HCD). Fragment ions in mass range of 110-2000 were

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detected in Orbitrap with a resolution of 30,000. AGC target value was set to 50,000 with

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maximum ion injection time of 150 ms.

Data analysis: The MS/MS data was searched against Human RefSeq database version 75

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(with common contaminants) using Sequest and Mascot search engines (version 2.4.1)

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through Proteome Discoverer (Version 2.1.0) software (Thermo Fisher Scientific, Bremen, Germany). The search parameters included trypsin as the proteolytic enzyme, precursor ion

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mass tolerance was set to 10 ppm and fragment ion mass tolerance was set to 0.05 Da. Oxidation of met was considered as dynamic modification, whereas, carbamidomethylation of cys, TMT labelling at peptide N-terminus and lys were considered as static modification. Data was searched with precursor mass tolerance of 20 ppm, fragment mass tolerance of 0.1 Da and 1 missed cleavage site were allowed for the searches. Peptide-spectrum match (PSM) level False Discovery Rate (FDR) of 1 % was employed for protein identification. The relative quantitation was carried out using the reporter ions quantifier node of Proteome Discoverer [20]. For phosphoproteomic data, phosphorylation at ser, thr and tyr residues was considered as dynamic modification. Probability of phosphorylation was calculated based on

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Journal Pre-proof ptmRS score. A PSM level FDR of 1 % was applied using Percolator. The relative abundance of the reporter ions from MS scans was calculated by using reporter ions quantifier node in Proteome Discoverer. Filtering strategies included reliability of each data point from both replicates. Proteins in the phosphoproteome data which were not detected during total proteome analysis were excluded. Peptides exhibiting ≥1.5 fold increase in phosphorylation status compared to controls were considered to be hyperphosphorylated, while hypophosphorylation corresponded to phosphorylation level of ≤0.6 compared to control

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(=1.0). Further, Phosphopeptides with phosphoRS score (which predicts the best

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phosphorylation site probabilities) of ≥ 70% were considered [36].

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Online bioinformatics tools: Gene enrichment, pathway analysis and functional annotation clustering of the identified proteins were carried out using Database for Annotation,

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Visualization and Integrated Discovery (DAVID) v6.7. Differentially regulated protein lists

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were analyzed in the overall protein list and potentially interesting clusters were manually examined [38]. KinasePhos tool was used to locate the motifs and respective kinases acting

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on different sites of various differentially regulated proteins. The presence of novel and reported phosphorylation sites of various mitochondrial proteins were confirmed by uniport (www.uniprot.org) [39], PhosphoSitePlus (www.phosphosite.org) [40] and Phosida (www.phosida.com) [41] databases. Network analysis was carried out using Stringdb functional protein association networks (http://string-db.org) [42].

Molecular modelling Among the phosphorylated mitochondrial proteins, VDAC1 and ATP5B1 were selected for structural analysis by molecular modelling. The corresponding PDB structures of these peptides were visually analyzed by Coot [19, 43] and PyMol software

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Journal Pre-proof (http://www.pymol.org). In each PDB structure, the secondary structures, the interacting amino acids and domains containing the phosphorylated residue were visually analyzed. By using Coot software [43], specific ser, thr and tyr residues were labelled and the potential structural implications were carefully inspected.

Molecular Dynamics Simulations (MDS) Generation of phosphorylated structure: The mitochondrial complex I (CI) structure (PDB

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code: 5XTB) was downloaded from PDB (https://www.rcsb.org) followed by MDS analysis

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of three subunits NDUFS1, NDUFV1 and NDUFV2 which interact with each other in the

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peripheral arm of the complex. The MDS were performed using the MD package, Desmond

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4.4 software [44] on the phosphorylated and unmodified subunits of the complex. Based on the MS data, phosphorylated residues in these three subunits (T599, S650 and S478/S479 in

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NDUFS1, S31 in NDUFV1, S145 and T163 in NDUFV2) were introduced by replacing

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unmodified residues with phosphorylated amino acid. The proteins were processed using the protein preparation wizard module of the Schrodinger Drug Discovery Suite. The protein

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structures were reviewed for the presence of any important water molecules for the simulation. All the water molecules were deleted before the simulation considering that none of them established any important bonds with the protein in its vicinity. Further, the H-bonds were optimized to the neutral pH and performed restrained minimization converging the heavy atoms close to 0.30 Å.

Molecular Dynamics Production: The final molecular dynamics production was carried out for a simulation time of 50 ns by passing the default relaxation of system at a temperature of 300 K and pressure of 1.01 bar. The trajectory files were recorded for every 4.8 ps. The final simulation trajectories were analyzed using other Desmond operations including Root mean

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Journal Pre-proof square deviation (RMSD), Root mean square fluctuation (RMSF) and radius of gyration (Rg) of the proteins. PyMOL (The PyMOL Molecular Graphics System) was used for viewing trajectories and exhibition of the important aspects as illustrations.

Analysis of trajectories: The 50 ns trajectories of the control (unmodified) and phosphorylated CI subunits were analyzed for protein backbone parameters such as the RMSD, RMSF, secondary structure and Rg throughout the duration of the simulation. The

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RMSD and Rg values were calculated against the simulation time and expressed as the

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deviation or radius of the selected group of atoms, respectively, in Å. The RMSF values of

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the protein backbone were calculated over the range of residues and expressed as summation

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throughout the simulation for each residue and was expressed in Å. Although the RMSD and Rg values were calculated for the protein backbone, the same parameters were also calculated

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for the three individual subunits of CI to describe the detailed effects translated over the

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timeline. The distances between electron transfer groups involving the FMN and iron-sulfur [Fe-S] clusters were calculated for the control and phosphorylated form. The Desmond

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module was used for the calculation of parameters. Maestro and PyMOL were used for the generation of illustrations as evidences.

Statistical analysis

For the MS experiment, data regarding the list of proteins and corresponding fold changes across the replicates compared with the corresponding controls was uploaded on Perseus software for calculation of p-value [45].

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Journal Pre-proof Results Characteristics of human muscle tissues For the current study, we chose muscle biopsies from dysfy (representing dystrophic pathology), PM (representing inflammatory pathology) and DMRV. The muscle morphology of these pathologies compared with control biceps/quadriceps (n=3) [with No Diagnostic Pathology (NDP)] was confirmed, as described [19, 21] (data not shown). Immunostaining, western blotting and genetic analysis based confirmation of loss of dysferlin protein in dysfy

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cases has been described [19].

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Since the NDP samples were limited in number with inadequate tissue in each case,

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we utilized paraspinal muscle from non-neuromuscular cases as control for the proteomics

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experiment. Control experiments from our previous study on health muscle [19] demonstrated that paraspinal, biceps and quadriceps muscle displayed similar mitochondrial

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function. A pilot study revealed that the mitochondrial yield required for the

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phosphoproteomics experiment from individual muscle biopsy of pathological conditions especially dysfy was poor prompting us to pool the samples from each group.

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Muscle biopsies from confirmed cases of dysfy (n=10), DMRV (n=10) and PM (n=10) and age-matched healthy controls (n=10) were utilized in the current study (Table 1). Following confirmation of diagnosis total mitochondria from muscle biopsies of dysfy, DMRV and PM and controls, were subjected to tryptic digestion and TMT labelling followed by total proteomic and phosphoproteomic analysis (Figure 1). The complete mitochondrial phosphoprotein profile in healthy muscle was analyzed followed by its comparison across different disease samples to understand the molecular basis of mitochondria dysfunction in these pathologies. Before comparing the phosphoproteome between healthy and diseased tissue, we chose to carry out a comprehensive phosphoprotein profiling in control muscle mitochondria.

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Journal Pre-proof Phosphoproteomic analysis of muscle mitochondria from healthy controls We first analyzed the mitochondrial phosphoproteome in control muscle tissue (n=10) (Figure 1). The enrichment of phosphopeptides in control muscle mitochondria was followed by stringent data filtering strategy to include only the reliable duplicates in both total and phosphoproteomics data. Preliminary analysis of the raw data did not find any confident hit exclusive to either the disease or control samples. This is possible because we had acquired our data in MS2 mode due to low amount of sample, which potentially leads to

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underrepresentation of such candidates. Although acquiring data in MS3 mode (which

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provides better quantitation) might help in identifying such unique phosphopeptides, it

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requires significantly higher quantity of mitochondria. Hence mitochondrial proteins that

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were commonly detected in controls and disease samples were considered for further analysis. Further, proteins not detected in the total mitochondrial proteome data were

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excluded. Total proteome analysis identified 1715 cellular proteins including 581

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mitochondrial proteins across all the groups (Figure 2A) indicating ~ 50 % coverage of mitochondrial proteins as per the Human MitoCarta2.0 database (www.broadinstitute.org/

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files/ shared/ metabolism/ mitocarta/ human.mitocarta2.0. html). Phosphoproteomics data revealed a total of 950 phosphopeptides from 506 proteins (Figure 2A and Supplementary Figure S1). This corresponds to 888 singly phosphorylated phosphopeptides (93.5 % of total phosphopeptides), 57 doubly phosphorylated phosphopeptides (6 % of total phosphopeptides) and 5 triply phosphorylated phosphopeptides (0.52 % of total phosphopeptides). The 950 phosphopeptides corresponded to 928 unique phosphosites, with 731 phosphorylated serine (p-ser) residues, 156 phosphorylated threonine (p-thr) residues and 41 phosphorylated tyrosine (p-tyr) residues. Among the 950 phosphopeptides, 253 unique phosphosites (+25 ambiguous sites= 278 sites) corresponded to 119 mitochondrial proteins and 264 phosphopeptides and the remaining 686 phosphopeptides corresponded to 387 organellar and

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Journal Pre-proof cytosolic proteins (Figure 2A, Tables 2-3 and Supplementary Tables S1-S2). The 264 mitochondrial

phosphopeptides

from

119

proteins,

corresponded

to

259

singly

phosphorylated peptides (98.1 % of total phosphopeptides) and 5 doubly phosphorylated peptides (1.9 % of total phosphopeptides). The 253 mitochondrial unique phosphosites entails 194 p-ser, 37 p-thr and 22 p-tyr residues. Ser was the most phosphorylated residue both in total and mitochondrial phosphoproteome. Interestingly, 151 mitochondrial phosphosites are previously reported (Uniprot, PhosphoSitePlus and Phosida databases), while the rest of 102

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mitochondrial phosphosites were novel (Figure 2D). Functional classification of the

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phosphoproteins indicated that 56 % of phosphorylation events were in proteins localized to

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the inner mitochondrial membrane, 21 % proteins to the mitochondrial matrix, 17 % proteins

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to the outer membrane and 5.6 % to the inter-membrane space (Figure 2B). In a previous study on human muscle mitochondrial proteome, 155 distinct

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phosphorylation sites in 76 mitochondrial phosphoproteins, including 116 p-ser, 23 p-thr, and

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16 p-tyr residues were reported [46]. Comparison of these numbers with our study indicated that 44 phosphoproteins were common with the previous study, while 32 were unique to that

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study, while 73 proteins were unique in our study (Figure 2C-D). Analysis of the phosphorylation profile indicated that phosphorylated proteins were part of metabolic pathways, redox proteins and redox processes, mitochondrial organization, structure, fusion and mitophagy, apoptosis, chaperone function, membrane transport among others including phosphoproteins with unknown function (Figure 2B). Assessment of functional protein-protein interaction based on string database analysis among the phosphoproteins indicated that all the proteins shared a strong link (Figure 3). Among these, majority of the phorphorylated proteins contributed to metabolic pathways and mitochondrial organization (Figure 4). These mainly included electron transport chain complexes with 36 phosphoproteins corresponding to 82 phosphosites. This included mitochondrial complex I

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Journal Pre-proof with 10 proteins (with 14 phosphosites), complex II with 2 proteins (5 sites) complex III with 7 proteins (17 sites) and complex IV with 7 proteins (13 sites) and complex V with 10 proteins and most number of phosphosites (35 sites) among the complexes (Figure 5A, Table 2). ATP5A1 subunit of complex V with 10 sites had the most phosphosites among the respiratory complexes. The tricarboxylic acid (TCA) cycle included 13 phosphorylated proteins corresponding to 24 sites (Figure 5A). Among the other mitochondrial proteins, Trifunctional enzyme subunit alpha

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contributing to fatty acid oxidation (HADHA) (7 sites), prohibitin (PHB) (9 sites)

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contributing to mitochondrial organization, Voltage-dependent anion-selective channel

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protein 1 (VDAC1) (8 sites) contributing to anion transport, MICOS complex subunit MIC60

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isoform 1 (IMMT) (11 sites) contributing to cristae structure contained relatively higher number of phosphosites compared to other mitochondrial proteins (Table 2).

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The phosphopeptide sequences obtained from the phosphoproteome data provided a

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source for assessing the kinase families involved in phosphorylation and their substrates. We used Kinasephos tool to generate a list of predicted potential kinases of 250 distinct

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mitochondrial phosphosites based on reported consensus sequences. Our data revealed 18 kinases to act on the specific motifs in different mitochondrial phosphosites. Among these, Protein Kinase G (PKG) was found to act on majority of the peptide motifs (n=30) followed by ATM Ser/Thr Kinase, Protein Kinase C (PKC), MAP Kinase (MAPK), Protein Kinase A (PKA) among others (Figure 5B, Supplementary Table S3).

Phosphorylation profile of mitochondrial proteins in muscle pathologies Analysis of the phosphoproteome from different pathologies vs. control revealed a total of 135 phosphopeptides corresponding to 110 proteins to be differentially phosphorylated (Peptides with ≥1.5 fold increase in phosphorylation= hyperphophorylation; peptides with

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Journal Pre-proof ≤0.6 fold phosphorylation= hypophosphorylation compared to control (=1.0)]. This corresponds to 144 unique phosphosites, with 113 p-ser, 23 p-thr and 8 p-tyr residues. These phosphopeptides

predominantly

included

hyperphosphorylation

events

with

77

hyperphosphorylated peptides containing 81 unique phosphosites (in 64 proteins) and 58 hypophosphorylated peptides with 63 unique phosphosites (in 46 proteins). Among these differentially phosphorylated peptides, 45 were mitochondrial, corresponding to 47 unique phosphosites in 38 mitochondrial proteins. Again, these predominantly included

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hyperphosphorylation events with 31 hyperphosphorylated peptides containing 33 unique

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phosphosites (in 25 proteins) and 14 hypophorphorylated peptides with 14 unique

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phosphosites (in 13 proteins) (Figure 6A-D).

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Among the hyperphosphorylated proteins, 9 sites in 9 peptides of 9 proteins were noted in Dysfy, 7 sites in 7 peptides of 7 proteins in DMRV, while 32 sites in 30 peptides of

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23 proteins were noted in PM (Figure 6A-D and Table 4). Among the hypophosphorylated

na

proteins, 8 sites in 8 phosphopeptides of 8 proteins were noted in Dysfy, 5 sites in 5 peptides of 4 proteins in DMRV and 7 sites in 7 peptides of 6 proteins were noted in PM (Figure 6A-D

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and Table 5). Only 4 hyperphosphorylated and 1 hypophosphorylated protein was common among the different diseases (Figure 6E) indicating varied phosphorylation profile across different

pathologies.

Interestingly,

Ser138

of

TOMM20

protein

which

was

hyperphosphorylated in PM was hypophosphorylated in DMRV while Ser 73 of COX6C protein which was hyperphosphorylated in Dysfy was hypophosphorylated in PM (Tables 45). The differentially phosphorylated proteins displayed significant functional interaction as indicated by string db analysis (Figure 6F). The kinase profiling revealed Creatine Kinase II (CKII) to contribute the maximum towards differential phosphorylation of mitochondrial proteins in muscle pathologies, followed by PKG (Figure 6G).

18

Journal Pre-proof Structural implications of phosphorylation of mitochondrial proteins Protein phosphorylation regulates mitochondrial function probably by influencing the structure-function relationship of specific proteins. To understand this, we investigated the structural influence of phosphorylation on selected mitochondrial proteins by structural bioinformatics approach. Accordingly, Voltage-Dependent Anion Channel (VDAC-1), ATP synthase and mitochondrial complex I (NADH-Ubiquinone oxidoreductase) were chosen for structural analysis. VDAC-1 displayed phosphorylation of 7 residues. Structural modelling of

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165, T167, S234 and S240 in human VDAC 1 (PDB id: 5XDO) showed that the distance

ro

between these residues and the neighbouring amino acids was significantly changed

-p

following phosphorylation (Figure 7A-D), which could induce potential alterations in the

re

local structure. Similar analysis in the ATP5B 1 subunit of bovine ATP synthase (PDB id: 5FIK) revealed that the distance between residues T60, T116, S327 and S415 and the

lP

neighbouring amino acids was significantly changed following phosphorylation (Figure 7E-I)

na

indicating potential alterations in the local structure. To understand whether altered local structure could potentially impinge on the overall

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structure of mitochondrial proteins, we chose mitochondrial complex I (CI), whose phosphorylation status has been associated with altered mitochondrial function [26]. Fourteen phosphosites were detected in 10 subunits of CI (Table 2). This included 5 sites in three important subunits NDUFS1 (T613, S664), NDUFV1 (S31) and NDUFV2 (S145, T163) that interact with each other. These possess FMN and Fe-S cluster sites making them physiological relevant to the enzyme activity of CI. The location of these subunits in human complex I structure (PDB id: 5XTD and 5XTB) and the phosphosites in these subunits are indicated in Figure 8. Molecular dynamics simulation (MDS) of unphosphorylated forms of NDUFS1, NDUVF1 and NDUFV2 vs. phosphorylated proteins was carried out. All the three subunits

19

Journal Pre-proof are located at the distal end of the peripheral arm of CI. NDUFV1 (MW: 51 kDa) contains the NADH and FMN binding sites along with the Fe-S cluster N3. NDUFV2 (MW: 24 kDa) contains one Fe-S cluster N1a, while NDUFS1 has three Fe-S clusters N1b, N4 and N5. All three subunits participate in the electron transfer activity from FMN to the Fe-S clusters [47]. The structural parameters investigated in CI subunits are represented by Root mean square deviation (RMSD), Root mean square fluctuation (RMSF) and radius of gyration (Rg), in addition to visual inspection of trajectory data.

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Subunit-wise assessment revealed significant RMSF in NDUFV1 and NDUFS1 and

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to a relatively lesser extent in NDUFV2 in the phosphorylated form compared to control

-p

(Figure 9A). The RMSD of the phosphorylated subunits exhibited significant differences

re

compared with control (Figure 9B). The backbone of the three chains exhibited higher RMSD from 0-10 ns compared to control. RMSD of NDUFV1 did not show any difference between

lP

the phosphorylated form and control (Figure 9C). On the other hand, RMSD of

control (Figure 9D).

na

phosphorylated NDUFS1 was relatively higher throughout the simulation compared to

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The RMSD of NDUFV2 remained unchanged in the phosphorylated form compared to control until 30 ns, by significantly decreased from 30 ns up to 50 ns (Figure 9E). These data indicate significantly altered structure of the three subunits following phosphorylation, which could affect CI function. To test whether electron transfer through the three phosphorylated subunits could be affected, we measured the distance between two consecutive electron transferring units including FMN and Fe-S clusters in the phosphorylated form compared to control. Our data revealed that the distances between FMN and N3 and between N3 and N1b were significantly higher in the phosphorylated form compared to control, while the distance between FMN and N1a was altered to a small extent indicating that phosphorylation could affect the efficiency of electron transfer and

20

Journal Pre-proof consequently the activity of CI (Figure 9F-H). However, neither the overall backbone of all three chains nor the individual subunits in the phosphorylated form revealed any significant differences in Rg values compared with the control (Supplementary Figure S2 A-D). This data indicates that the functional alterations in CI could be due to subtle local structural

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na

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change without affecting the entire complex.

21

Journal Pre-proof Discussion Phosphoproteomics of normal muscle provides insights into mitochondrial dynamics The mitochondrial phosphoproteome has been reported in plants, yeast and mammalian tissues [27]. The rat mitochondrial phosphoproteome displays tissue specificity [48]. Accordingly, muscle and heart shared 39 %, while liver shared 19 and 25 % of phosphosites with muscle and heart respectively. While OXPHOS and the TCA cycle proteins were phosphorylated in heart and muscle, liver displayed phosphorylation of proteins

of

involved in fatty acid and amino acid metabolism [48]. Mouse liver mitochondria displayed

ro

strain-specific and biological condition-specific phosphorylation [49]. These data indicate

-p

that the phosphoprotein profile is influenced by tissue/cell type and other physiological

re

paraments. Similarly, muscle health and disease could also correlate with muscle-specific alterations in the mitochondrial phosphoproteome.

lP

The first phosphoproteomic study in human skeletal muscle identified 306

na

phosphosites in 127 proteins, with only a few mitochondrial phosphosites [50]. Mitochondrial phosphoproteomics in human skeletal muscle identified 155 phosphosites in 77 mitochondrial

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proteins [46]. The largest study in human muscle mitochondria reported 207 phosphosites in 95 proteins [51]. However, mitochondrial phosphoproteomics of the skeletal muscle from healthy subjects vs. patients with varied muscle pathologies are non-existent, highlighting the novelty of our study. We identified 253 phosphosites in 264 phosphopeptides of 119 mitochondrial proteins in human skeletal muscle. Comparison with the previous muscle mitochondrial studies [46, 50-52] revealed 151 to be common phosphosites, while 102 sites were novel. The phosphosite numbers could be improved by including proteins not covered by MitoCarta [53] and employing complementary fragmentation strategies [54]. Most of the phosphoproteins identified in our study are involved in metabolic pathways, redox processes, mitochondrial organization, fusion, mitophagy, apoptosis,

22

Journal Pre-proof chaperone function and membrane transport among others (Figure 2B) consistent with previous studies [46, 51, 55]. Among these, respiratory complexes, TCA cycle and fatty acid metabolism enzymes displayed robust phosphorylation with respiratory complexes accounting for 84 phosphosites in 36 phosphoproteins. Additional sites could be identified by targeted phosphoproteomics of each complex. Complex V with 35 sites in 10 subunits was the most phosphorylated complex (Table 2). This is significantly higher than the previous study [51], which detected 13 phosphosites across 8 subunits (Supplementary Table S4).

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Conversely, some phosphosites reported earlier were undetected in this study. CISD1

ro

(CDGSH iron-sulfur domain-containing protein 1; MitoNeet), an outer mitochondrial

-p

membrane protein that regulates oxidative phosphorylation with implications in human

re

disease [56-58] was reported to be phosphorylated at ser2 and ser7 [46, 59]. While we did not detect these sites, we noted phosphorylation at Thr94 (Table 2). VDAC 1-3 proteins which

lP

form membrane channels [60] were reported to be phosphorylated at 19 distinct sites [46],

na

while we recorded 13 sites (Table 2). Our recent bioinformatics study [26], predicted 450 ser, 352 thr and 187 tyr residues to be potentially phosphorylated in CI. The vast difference

Jo ur

between the experimental and bioinformatics data could be due to the transient nature of phosphorylation, tissue specificity, loss of phosphopeptides during MS, limitations of enrichment techniques etc.

The functional significance, mole fractions of phosphopeptides and role of the kinases/ phosphatases are unknown for most phosphosites. Many sites could be less specific without any functional role indicating that each site has to be carefully studied [27, 51, 55]. We chose to assess these by structural biology approach. Molecular modelling revealed significant alteration in the local structure in VDAC1 and ATP5B1 (Figures 7-8). MDS analysis concluded that phosphorylation could alter the efficiency of electron transfer from FMN, ultimately influencing CI activity (Figures 9-10). We are tempted to speculate that the

23

Journal Pre-proof mitochondria sense the metabolic flux across the respiratory complexes and utilize transient phosphorylation to fine tune the efficiency of electron transfer, proton pumping and the rate of ATP production.

Role of kinases in healthy muscle mitochondria The distribution of p-ser/p-thr/p-tyr residues and phosphopeptide motifs reflects the mitochondrial kinases [52]. Mammalian skeletal muscle [50, 52] and human skeletal muscle

of

mitochondria display higher fraction (4-5%) of p-tyr residues although this might not specific

ro

to muscle mitochondria [46, 48, 51, 61-64].

-p

Our study predicted Protein Kinase G (PKG) as the most predominant kinase

re

followed by ATM and in healthy muscle mitochondria (Figure 5B), in contrast with previous studies [46, 48, 51], which reported protein kinase A (PKA), protein kinase C (PKC), and

lP

glycogen synthase kinase (GSK), followed by CKII and DNAPK as predominant kinases.

na

This disparity should be interpreted with caution since many kinase motifs could be nonspecific and without physiological importance [55]. PKG potentially influences

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mitochondrial function. PKG protects mitochondria by stimulating mitochondrial biogenesis via regulation of PGC1-α expression [65-67]. Activation of PKG protects heart mitochondria by inhibiting the opening of mitochondria permeability transition pore via opening mitochondria KATP channel [68, 69]. Similarly, PKA and PKC have been reported to have mitochondrial localization and phosphorylates respiratory complexes and other mitochondrial proteins with effects on mitochondrial function, cell survival among others [27].

Potential role of differentially phosphorylated mitochondrial proteins in muscle diseases Studies correlating phosphoproteome with diseases have focused on metabolic disorders such as diabetes [54]. Insulin-dependent phosphorylation of respiratory complexes

24

Journal Pre-proof and other mitochondrial proteins in the muscle [70, 71] leading to switch in fuel preference [72] indicate disease-specific transient phosphorylation. Studies on disease specific mitochondrial phosphoproteome in the skeletal muscle are not available making this as one of the first studies. We considered muscle disease to be represented by three varied pathologies that differ in their manifestation, disease course, and severity of the symptoms. Previously [19], we noted that all three pathologies shared commonly down-regulated mitochondrial proteins. We hypothesized that PTMs could offer

of

disease-specific variations in the mitochondrial function. This was substantiated by

ro

phosphoprotein profiling since the number of common hypo and hyperphoshorylated proteins

-p

were not substantially common among the conditions (Figure 6E). Altered phosphorylation

re

could have significant functional implications. NDUFS4 subunit of CI showed altered phosphorylation in muscle pathology (Table 7). The mitochondrial import of NDUFS4 and

lP

assembly of a functional CI is dependent on phosphorylation [73].

na

The mitochondrial contact site and cristae organizing system (MICOS) complex regulates inner mitochondrial membrane structure via interaction with the respiratory

Jo ur

complexes and membrane lipids [74, 75]. MIC60, the central component of MICOS is potentially targeted for phosphorylation by PKA. Out study showed hypophosphorylation of MIC60 in all the conditions. PKA-mediated phosphorylation of MIC60 is associated with the mitochondrial translocation of proteins and mitochondrial function [76]. The phosphorylation status of Prohibitin 1 (PHB1) is associated with cell apoptosis, survival, and differentiation and could influence the mitochondria genome, mitochondrial dynamics, morphology and biogenesis [77]. Our study showed hypophosphorylation of PHB2 at S151 in dysfy (Table 7). The preprotein translocase of the outer mitochondrial membrane (TOM) facilitates import of nuclear-encoded proteins into mitochondria and consists of three receptors Tom20, Tom22, Tom70 and Tom40. Cytosolic kinases regulate the biogenesis and activity of the Tom

25

Journal Pre-proof receptors, with TOMM20 being influenced by Casein Kinase 2 dependent phosphorylation [78]. We noted that ATP synthase subunits displayed robust phosphorylation with implications for structural alterations with two subunits ATP5A1 and TAP5B displayed hyperphosphorylation in muscle pathologies (Table 6). The ATP5B subunit is demonstrated to have abnormal phosphorylation at specific sites in insulin-resistant muscle with hyperphosphorylation of selected sites [50]. Boja et al (2009) demonstrated altered

of

phosphorylation of the ATP5A1 subunit in response to physiological stimuli [79]. The same

ro

study also showed that the S337 site hyper-phosphorylated in BCKDH protein in all the

-p

conditions in our study (Table 6), displayed hyperphosphorylation at the same site with

re

implications for altered activity.

Proteins such as COX6C and TOMM20 showed contrasting phosphorylation status in

lP

different pathologies indicating disease-specific alterations (Table 6 and 7). While COX6C

na

was hyperphosphorylated at S73 in Dysfy, the same site was hypophosphorylated in PM. On the other hand, while TOMM20 was hyperphosphorylated at S138 in PM, the same site was

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hypophosphorylated in DMRV.

In conclusion, protein phosphorylation potentially plays a key role in mitochondrial physiology. However, the kinase/phosphatases involved in mitochondrial protein phosphorylation have yet to be determined for most of the phosphosites reported. Although MS allows us to identify an increasing number of mitochondrial phosphosites, their functional importance is largely unknown. Combination of proteomics with structural biology could provide additional information about the structural role of phosphorylation. Taken together, study of phosphosites will improve our understanding of the mitochondrial metabolism in health and disease with therapeutic implications.

26

Journal Pre-proof Abbreviations: Dysfy, dysferlinopathy; PM, polymyositis; DMRV, distal myopathy with rimmed vacuoles; CI, mitochondrial complex I; CII, mitochondrial complex II; CIII, mitochondrial complex III; CIV, mitochondrial complex IV; TMT, tandem mass tag; LCMS/ MS, Liquid chromatography-tandem mass spectrometry; ETC, electron transport chain; MDS, molecular dynamics simulations; RMSD, root mean square deviation; RMSF, root mean square fluctuation; Rg, radius of gyration.

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Acknowledgements: This study was financially supported by grants from the Indian Council

ro

of Medical Research (ICMR) (Grant no. 5/4-5/144/Neuro/2014-NCD-I to NG and MMSB).

-p

The funding agency did not have any role in the design and execution of the study. BS was

re

supported by a Senior Research Fellowship from ICMR. NG is supported by a Junior Research Fellowship from ICMR. The MS proteomics data have been deposited to the

na

identifier PXD013632.

lP

ProteomeXchange Consortium via the PRIDE partner repository with the dataset

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Author contributions: MMSB designed and supervised the experiments. BS and MK carried out the experiments and analyzed data along with MMSB. TSKP contributed to the proteomics experiment. AN, SV, KP, VPK contributed to the clinical evaluation and biopsy of the patients. NG contributed to the histopathology of the biopsies. Niya G carried out the molecular modeling. Niya G, SU and BP contributed to the molecular dynamics simulation study. BS and MMSB wrote the manuscript. The final draft of the manuscript was edited and approved by all the authors.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this article.

27

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mitochondrial complex I assembly in health and disease. Biochimica et biophysica acta 2012; 1817(6): 851-62. [48] S. Bak, I.R. Leon, O.N. Jensen, K. Hojlund. Tissue specific phosphorylation of mitochondrial proteins isolated from rat liver, heart muscle, and skeletal muscle. J Proteome Res 2013; 12(10): 4327-39. [49] P.A. Grimsrud, J.J. Carson, A.S. Hebert, S.L. Hubler, N.M. Niemi, D.J. Bailey, A. Jochem, D.S. Stapleton, M.P. Keller, M.S. Westphall, B.S. Yandell, A.D. Attie, J.J. Coon, D.J. Pagliarini. A quantitative map of the liver mitochondrial phosphoproteome reveals posttranslational control of ketogenesis. Cell Metab 2012; 16(5): 672-83. [50] K. Hojlund, B.P. Bowen, H. Hwang, C.R. Flynn, L. Madireddy, T. Geetha, P. Langlais, C. Meyer, L.J. Mandarino, Z. Yi. In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS. J Proteome Res 2009; 8(11): 4954-65. [51] X. Zhao, S. Bak, A.J. Pedersen, O.N. Jensen, K. Hojlund. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. J Proteome Res 2014; 13(5): 2359-69. [52] A. Lundby, A. Secher, K. Lage, N.B. Nordsborg, A. Dmytriyev, C. Lundby, J.V. Olsen. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. Nat Commun 2012; 3: 876. [53] S.E. Calvo, K.R. Clauser, V.K. Mootha. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res 2016; 44(D1): D1251-7. [54] W.J. Deng, S. Nie, J. Dai, J.R. Wu, R. Zeng. Proteome, phosphoproteome, and hydroxyproteome of liver mitochondria in diabetic rats at early pathogenic stages. Mol Cell Proteomics 2010; 9(1): 100-16. [55] R. Covian, R.S. Balaban. Cardiac mitochondrial matrix and respiratory complex protein phosphorylation. Am J Physiol Heart Circ Physiol 2012; 303(8): H940-66. [56] S.E. Wiley, A.N. Murphy, S.A. Ross, P. van der Geer, J.E. Dixon. MitoNEET is an ironcontaining outer mitochondrial membrane protein that regulates oxidative capacity. Proc Natl Acad Sci U S A 2007; 104(13): 5318-23. [57] S.E. Wiley, M.L. Paddock, E.C. Abresch, L. Gross, P. van der Geer, R. Nechushtai, A.N. Murphy, P.A. Jennings, J.E. Dixon. The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster. J Biol Chem 2007; 282(33): 23745-9. [58] W.J. Geldenhuys, T.C. Leeper, R.T. Carroll. mitoNEET as a novel drug target for mitochondrial dysfunction. Drug Discov Today 2014; 19(10): 1601-6. [59] M.L. Paddock, S.E. Wiley, H.L. Axelrod, A.E. Cohen, M. Roy, E.C. Abresch, D. Capraro, A.N. Murphy, R. Nechushtai, J.E. Dixon, P.A. Jennings. MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone. Proc Natl Acad Sci U S A 2007; 104(36): 14342-7. [60] T. Becker, R. Wagner. Mitochondrial Outer Membrane Channels: Emerging Diversity in Transport Processes. Bioessays 2018; 40(7), e1800013. [61] H. Molina, D.M. Horn, N. Tang, S. Mathivanan, A. Pandey. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 2007; 104(7): 2199-204. [62] G. Han, M. Ye, H. Zhou, X. Jiang, S. Feng, X. Jiang, R. Tian, D. Wan, H. Zou, J. Gu. Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography. Proteomics 2008; 8(7): 1346-61. [63] M. Carrascal, D. Ovelleiro, V. Casas, M. Gay, J. Abian. Phosphorylation analysis of primary human T lymphocytes using sequential IMAC and titanium oxide enrichment. J Proteome Res 2008; 7(12): 5167-76. 31

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[64] J.V. Olsen, B. Blagoev, F. Gnad, B. Macek, C. Kumar, P. Mortensen, M. Mann. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006; 127(3): 635-48. [65] B. Haas, P. Mayer, K. Jennissen, D. Scholz, M. Berriel Diaz, W. Bloch, S. Herzig, R. Fassler, A. Pfeifer. Protein kinase G controls brown fat cell differentiation and mitochondrial biogenesis. Sci Signal 2009; 2(99): ra78. [66] K. Miyashita, H. Itoh, H. Tsujimoto, N. Tamura, Y. Fukunaga, M. Sone, K. Yamahara, D. Taura, M. Inuzuka, T. Sonoyama, K. Nakao. Natriuretic peptides/cGMP/cGMP-dependent protein kinase cascades promote muscle mitochondrial biogenesis and prevent obesity. Diabetes 2009; 58(12): 2880-92. [67] E. Nisoli, E. Clementi, C. Paolucci, V. Cozzi, C. Tonello, C. Sciorati, R. Bracale, A. Valerio, M. Francolini, S. Moncada, M.O. Carruba. Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science 2003; 299(5608): 896-9. [68] A.D. Costa, K.D. Garlid, I.C. West, T.M. Lincoln, J.M. Downey, M.V. Cohen, S.D. Critz. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res 2005; 97(4): 329-36. [69] A.D. Costa, R. Jakob, C.L. Costa, K. Andrukhiv, I.C. West, K.D. Garlid. The mechanism by which the mitochondrial ATP-sensitive K+ channel opening and H2O2 inhibit the mitochondrial permeability transition. J Biol Chem 2006; 281(30): 20801-8. [70] J.Y. Yang, H.Y. Yeh, K. Lin, P.H. Wang. Insulin stimulates Akt translocation to mitochondria: implications on dysregulation of mitochondrial oxidative phosphorylation in diabetic myocardium. J Mol Cell Cardiol 2009; 46(6): 919-26. [71] K. Hojlund, K. Wrzesinski, P.M. Larsen, S.J. Fey, P. Roepstorff, A. Handberg, F. Dela, J. Vinten, J.G. McCormack, C. Reynet, H. Beck-Nielsen. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes. J Biol Chem 2003; 278(12): 10436-42. [72] D.E. Kelley, L.J. Mandarino. Fuel selection in human skeletal muscle in insulin resistance: a reexamination. Diabetes 2000; 49(5): 677-83. [73] S. Papa, D. De Rasmo, S. Scacco, A. Signorile, Z. Technikova-Dobrova, G. Palmisano, A.M. Sardanelli, F. Papa, D. Panelli, R. Scaringi, A. Santeramo. Mammalian complex I: a regulable and vulnerable pacemaker in mitochondrial respiratory function. Biochimica et biophysica acta 2008; 1777(7-8): 719-28. [74] J.R. Friedman, A. Mourier, J. Yamada, J.M. McCaffery, J. Nunnari. MICOS coordinates with respiratory complexes and lipids to establish mitochondrial inner membrane architecture. Elife 2015; 4. doi: 10.7554/eLife.07739. [75] V. Guarani, E.M. McNeill, J.A. Paulo, E.L. Huttlin, F. Frohlich, S.P. Gygi, D. Van Vactor, J.W. Harper. QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology. Elife 2015; 4. doi: 10.7554/eLife.06265. [76] S. Akabane, M. Uno, N. Tani, S. Shimazaki, N. Ebara, H. Kato, H. Kosako, T. Oka. PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60. Mol Cell 2016; 62(3): 371-384. [77] Y.T. Peng, P. Chen, R.Y. Ouyang, L. Song. Multifaceted role of prohibitin in cell survival and apoptosis. Apoptosis 2015; 20(9): 1135-49. [78] S. Rao, O. Schmidt, A.B. Harbauer, B. Schonfisch, B. Guiard, N. Pfanner, C. Meisinger. Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import. Mol Biol Cell 2012; 23(9): 1618-27. [79] E.S. Boja, D. Phillips, S.A. French, R.A. Harris, R.S. Balaban. Quantitative mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with high energy collision dissociation. J Proteome Res 2009; 8(10): 4665-75. 32

Journal Pre-proof Table 1. Details of the muscle samples included in the study (NDP: No diagnostic pathology)

Control

(i) NDP (n=3) (biceps, quadriceps)

Age (y) 15-40

Duration of illness (y) 1-10

15:5 5:5 7:3 3:7

20-35 17-40 23-40 7-67

2-18 2-10 <1

lP

re

-p

ro

Dysfy DMRV PM

(ii) Paraspinal (n=20) 10 10 10

Gender (m:f) 1:2

of

No. of samples

na

2 3 4

Group

Jo ur

Sl. No. 1

33

Journal Pre-proof Table 2. List of phosphorylated mitochondrial proteins in human skeletal muscle healthy control). The Uniprot accession id, protein symbol, description and phosphosite are indicated. The sites in bold are previously reported in muscle. “n” in superscript indicates novel site identified. The number of sites in each complex/ pathway is indicated in parenthesis.

NDUFA7

Q16795

NDUFA9

O43677

NDUFC1

P28331

NDUFS1

O43181

NDUFS4

P49821

NDUFV1

P19404

NDUFV2

P56181

NDUFV3

P31040

SDHA

P21912

SDHB

P14927

UQCRB

P31930

UQCRC1

P22695

UQCRC2

P47985

UQCRFS1

of

O95182

ro

NDUFA3

-p

O95167

re

NDUFA2

Jo ur

O43678

Protein description Phosphorylation site Oxidative Phosphorylation Complex I (n=14) NADH dehydrogenase [ubiquinone] 1 alpha S59ⁿ, S96ⁿ subcomplex subunit 2 isoform 1 NADH dehydrogenase [ubiquinone] 1 alpha Y41 subcomplex subunit 3 NADH dehydrogenase [ubiquinone] 1 alpha S72ⁿ subcomplex subunit 7 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial S246ⁿ precursor NADH dehydrogenase [ubiquinone] 1 Y69ⁿ subunit C1, mitochondrial precursor NADH-ubiquinone oxidoreductase 75 kDa T613ⁿ, S664ⁿ subunit, mitochondrial isoform 5 NADH dehydrogenase [ubiquinone] ironsulfur protein 4, mitochondrial isoform 1 S136ⁿ precursor NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial isoform 1 S31ⁿ precursor NADH dehydrogenase [ubiquinone] S145ⁿ, T163ⁿ flavoprotein 2, mitochondrial precursor NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial isoform a S98ⁿ, S470ⁿ precursor Complex II (n=5) Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial isoform S177, T256ⁿ, S321, 1 S530 Succinate dehydrogenase [ubiquinone] ironS173ⁿ sulfur subunit, mitochondrial precursor Complex III (n=17) Cytochrome b-c1 complex subunit 7 isoform S8 3 Cytochrome b-c1 complex subunit 1, S107, S178ⁿ, T384ⁿ, mitochondrial precursor Y420ⁿ, S439ⁿ Cytochrome b-c1 complex subunit 2, T91, Y195ⁿ, S247ⁿ, T369 mitochondrial precursor Cytochrome b-c1 complex subunit Rieske, S99, T100, S157

lP

SYM

na

Uniprot accession id

34

Journal Pre-proof mitochondrial

COX5A

P10606 P14854 P09669

COX5B COX6B1 COX6C

P48771

COX7A2

P15954

COX7C

P25705

ATP5A1

P06576

ATP5B

P36542

ATP5C1

P56381

ATP5E

P24539

ATP5F1

O75947 P56385

ATP5H ATP5I

P18859 O75964

ATP5J ATP5L

P48047

ATP5O

Q16134

ETFDH

P24752

ACAT1

of

P20674

ro

COX4I1

-p

P13073

re

CYC1

lP

P08574

Cytochrome b-c1 complex subunit 8 Y78ⁿ Cytochrome b-c1 complex subunit 9 isoform S8ⁿ a Cytochrome c1, heme protein, mitochondrial precursor Y140, S182 Complex IV (n=13) Cytochrome c oxidase subunit 4 isoform 1, S26, S72, S158 mitochondrial isoform 1 precursor Cytochrome c oxidase subunit 5A, S104ⁿ mitochondrial precursor Cytochrome c oxidase subunit 5B, S 82ⁿ mitochondrial precursor Cytochrome c oxidase subunit 6B1 S19ⁿ, T27ⁿ, T81 Cytochrome c oxidase subunit 6C Y53, S73 Cytochrome c oxidase subunit 7A2, Y87ⁿ mitochondrial precursor Cytochrome c oxidase subunit 7C, Y19ⁿ, S30ⁿ mitochondrial precursor Complex V (n=35) S53, T64, S76, S198, ATP synthase subunit alpha, mitochondrial S236ⁿ, S254,S346ⁿ, isoform a precursor S419, T432, S536ⁿ ATP synthase subunit beta, mitochondrial T60ⁿ, T116ⁿ, S128, precursor S327, S415, S529 ATP synthase subunit gamma, mitochondrial isoform H (heart) precursor S116, S265 ATP synthase subunit epsilon, S11ⁿ, S16ⁿ mitochondrial ATP synthase F(0) complex subunit B1, S142, S155ⁿ, S228 mitochondrial precursor ATP synthase subunit d, mitochondrial T39 isoform a ATP synthase subunit e, mitochondrial Y32, S66 ATP synthase-coupling factor 6, Y75ⁿ mitochondrial isoform b precursor ATP synthase subunit g, mitochondrial S59, S64 ATP synthase subunit O, mitochondrial S47, S77, S91, S155, precursor S166, S180 Oxidation-reduction process (n=1) Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial isoform 1 S551 precursor Fatty acid oxidation (n=17) Acetyl-CoA acetyltransferase, mitochondrial T185ⁿ precursor

na

UQCRQ UQCR10

Jo ur

O14949 Q9UDW1

35

Journal Pre-proof

ECH1

P33121

ACSL1

P09622

DLD

P08559

PDHA1

P11177

PDHB

P40925

MDH1

P40926

MDH2

P10515

DLAT

P36957

DLST

P48735

IDH2

P50213

IDH3A

Q02218

OGDH

Q9P2R7 O75390

SUCLA2 CS

Q13423

NNT

P43304

GPD2

of

Q13011

ro

ACADVL

-p

P49748

re

HADHB CPT1B

lP

P55084 Q92523

Trifunctional enzyme subunit alpha, mitochondrial precursor Trifunctional enzyme subunit beta, T40ⁿ,S126ⁿ, S237ⁿ mitochondrial isoform 1 precursor Carnitine O-palmitoyltransferase 1, muscle S330, S401 Isoform a Very long-chain specific acyl-CoA dehydrogenase, mitochondrial isoform 3 S512ⁿ Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial precursor S108, S274ⁿ Long-chain-fatty-acid--CoA ligase 1 isoform a S295ⁿ Tricarboxylic acid cycle (n=24) Dihydrolipoyl dehydrogenase, S297, S502 mitochondrial isoform 1 precursor Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial S331, S270, S277ⁿ isoform 2 precursor Pyruvate dehydrogenase E1 component subunit beta, mitochondrial isoform 1 S180ⁿ precursor Malate dehydrogenase, peroxisomal isoform Y119 MDH1x Malate dehydrogenase, mitochondrial T235 isoform 1 precursor Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial T154, S484ⁿ precursor Dihydrolipoyllysine-residue succinyltransferase component of 2oxoglutarate dehydrogenase complex, S81, T348ⁿ mitochondrial isoform 1 precursor Isocitrate dehydrogenase [NADP], S100, T350ⁿ, S423 mitochondrial isoform 1 precursor Isocitrate dehydrogenase [NAD] subunit S176ⁿ alpha, mitochondrial precursor 2-Oxoglutarate dehydrogenase, S344ⁿ mitochondrial isoform 1 precursor Succinyl-CoA ligase [ADP-forming] subunit S279, S423ⁿ beta, mitochondrial precursor Citrate synthase, mitochondrial precursor Y185ⁿ S122, S385ⁿ, T440, NAD(P) transhydrogenase, mitochondrial S769 Gluconeogenesis (n=14) Glycerol-3-phosphate dehydrogenase, S415ⁿ, S623 mitochondrial precursor

na

HADHA

Jo ur

P40939

S59ⁿ,S120, S316 , T393, S524ⁿ, S669, Y724

36

Journal Pre-proof

SLC25A12

P12694

BCKDHA

P21964

COMT

P27338

MAOB

P17540

CKMT2

O14874

BCKDK

P07237

P4HB

O14880

MGST3

P51648

ALDH3A2

P00387 P02794

CYB5R3 FTH1

O95831 Q9H7Z7

AIFM1 PTGES2

Q99623

PHB2

P35232 P17152

PHB TMEM11

O60313

OPA1

Q9UJZ1

STOML2

P53985

SLC16A1

of

O75746

ro

GOT2 SLC25A11

-p

P00505 Q02978

re

SLC25A1

lP

P53007

Triosephosphate isomerase isoform 2 Tricarboxylate transport protein, S163ⁿ mitochondrial isoform b Aspartate aminotransferase, mitochondrial S360 isoform 1 precursor Mitochondrial 2-oxoglutarate/malate carrier S52, T95ⁿ, Y202 protein isoform 1 Calcium-binding mitochondrial carrier protein Aralar1 Y47ⁿ, S664, S662 Glyoxylate metabolic process (n=1) 2-Oxoisovalerate dehydrogenase subunit S337 alpha, mitochondrial isoform 1 precursor Tyrosine metabolism (n=1) Catechol O-methyltransferase isoform MBS267 COMT Aminoacid metabolism (n=6) Amine oxidase [flavin-containing] B S131 Creatine kinase S-type, mitochondrial Y159, T214, Y255, precursor T361 [3-Methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial isoform a S31 precursor Cell redox homeostasis (n=2) Protein disulfide-isomerase precursor S180ⁿ, S266 Glutathione peroxidase (n=1) Microsomal glutathione S-transferase 3 S146 Oxidation-reduction process (n=5) Fatty aldehyde dehydrogenase isoform 1 S14 NADH-cytochrome b5 reductase 3 isoform S179ⁿ 3 Ferritin heavy chain S179 Apoptosis-inducing factor 1, mitochondrial S116 isoform AIF precursor Prostaglandin E synthase 2 isoform 1 S95 Mitochondrion organization (n=17) T94ⁿ, S151, S261ⁿ, Prohibitin-2 isoform 1 T266ⁿ T80ⁿ, S101, S109, T141ⁿ, S151, T180ⁿ, Prohibitin isoform 1 S213, S218, Y249 Transmembrane protein 11, mitochondrial S13, S17 Dynamin-like 120 kDa protein, S866ⁿ mitochondrial isoform 8 Stomatin-like protein 2, mitochondrial T327 isoform a Plasma membrane lactate transport (n=3) Monocarboxylate transporter 1 S467

na

TPI1

Jo ur

P60174

S58, T215, S241, S249

37

Journal Pre-proof

HK1

P45880

VDAC2

P21796

VDAC1

Q9Y277

VDAC3

Q9BXK5

BCL2L13

Q9NQG6

MIEF1

Q12983 Q9Y3D6

BNIP3 FIS1

Q9H3K2

GHITM

Q9Y512

SAMM50

Q9NX63 Q9BRQ6

CHCHD3 CHCHD6

Q16891 Q6UXV4 Q9BUR5

IMMT APOOL APOO

P49207

RPL34

Q00325 Q9Y3D3

SLC25A3 MRPS16

Q9NVI7

ATAD3A

S451ⁿ, S777ⁿ, S796ⁿ

T83ⁿ, S130ⁿ, S260ⁿ S43, S46, S57, Voltage-dependent anion-selective channel T107,T165, S167, protein 1 S234ⁿ, S240 Voltage-dependent anion-selective channel S55ⁿ, S165 protein 3 isoform 1 Activation of caspase and apoptosis (n=3) Bcl-2-like protein 13 isoform b S444, S450ⁿ, S468ⁿ Mitochondrial fusion (n=5) Mitochondrial dynamics protein MID51 S55 isoform 2 BCL2/adenovirus E1B 19 kDa proteinT137, S151, S160 interacting protein 3 Mitochondrial fission 1 protein S29ⁿ Cristae formation (n=2) Growth hormone-inducible transmembrane S70 protein Sorting and assembly machinery component S239ⁿ 50 homolog MICOS complex (n=20) S42ⁿ, S44ⁿ, Y168ⁿ, MICOS complex subunit MIC19 isoform 1 Y182ⁿ MICOS complex subunit MIC25 isoform 1 S126ⁿ S34, S103, S106, S115, S350ⁿ, S447, T481, T491ⁿ, S514, MICOS complex subunit MIC60 isoform 1 S555, S594 MICOS complex subunit MIC27 precursor T199ⁿ, S203, S228ⁿ MICOS complex subunit MIC26 precursor S65 Translation (n=6) 60S ribosomal protein L34 S12 Phosphate carrier protein, mitochondrial T199ⁿ, Y246, S297 isoform a precursor 28S ribosomal protein S16, mitochondrial T130 ATPase family AAA domain-containing S632 protein 3A isoform 1 Glycolysis / Gluconeogenesis (n=4)

of

P19367

Y473, S546ⁿ

ro

LACTB

-p

P83111

T426

re

PUS1

S219, S448

lP

Q9Y606

Monocarboxylate transporter 2 Regulation of nuclear receptor activity (n=1) tRNA pseudouridine synthase A, mitochondrial isoform 1 Hydrolase activity (n=2) Sine beta-lactamase-like protein LACTB, mitochondrial isoform a precursor Glycolytic process (n=3) Hexokinase-1 isoform HKI-ta/tb Anion transport (n=13) Voltage-dependent anion-selective channel protein 2 isoform 1

na

SLC16A7

Jo ur

O60669

38

Journal Pre-proof

P27105 O75431 P35180 O94826 P38646

Q9NZ45

Q9NX40 G3V556 Q5VST9

of

ro

Q9Y5L4

-p

O60220

re

P48163

lP

Q9BWH2

na

Q53H12

Jo ur

P04406

Glyceraldehyde-3-phosphate dehydrogenase S25, S192, S266, GAPDH S333 isoform 1 Long-chain fatty-acyl-CoA biosynthetic process (n=1) Acylglycerol kinase, mitochondrial AGK S350 precursor Mitophagy (n=1) FUNDC2 FUN14 domain-containing protein 2 S151 Pyruvate metabolism (n=2) ME1 NADP-dependent malic enzyme T91ⁿ, S92 Chaperone-mediated protein transport (n=2) Mitochondrial import inner membrane TIMM8A S96 translocase subunit Tim8 A isoform 1 Mitochondrial import inner membrane TIMM13 S61ⁿ translocase subunit Tim13 Protein targeting (n=4) EryTocyte band 7 integral membrane STOM S10 protein isoform a MTX2 Metaxin-2 isoform 1 S100 Mitochondrial import receptor subunit TOMM20 S138 TOM20 homolog Mitochondrial import receptor subunit TOMM70 S91 TOM70 Chaperone (n=1) HSPA9 Stress-70 protein, mitochondrial precursor S627 Metal ion binding (n=1) CDGSH iron-sulfur domain-containing CISD1 T94ⁿ protein 1 Unknown Function (n=6) OCIA domain-containing protein 1 isoform OCIAD1 S128, 4 C14orf2 6.8 kDa mitochondrial proteolipid isoform 1 S4ⁿ, S51ⁿ OBSCN Obscurin isoform IC S 7730 Obscurin isoform a S6345 LOC102724023 ES1 protein homolog, mitochondrial S102 ⁿ isoform X1

39

Journal Pre-proof Table 3. List of ambiguous phosphosites in mitochondrial proteins in human skeletal muscle (healthy control). The Uniprot accession id, protein symbol, description and the phosphosite are indicated. The sites in bold are previously reported in muscle tissue. “n” in superscript indicates novel site identified in this study. The number of phosphosites in each complex/ pathway is indicated in parenthesis.

Q9Y6M9 P28331

Oxidative Phosphorylation Complex I (n=4) NDUFA10 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial precursor NDUFB9 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 isoform 1 NDUFS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial isoform 5 NDUFV3 NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial isoform a precursor Complex III (n=2) UQCRC1 Cytochrome b-c1 complex subunit 1, mitochondrial precursor UQCRFS1 Cytochrome b-c1 complex subunit Rieske, mitochondrial Complex V (n=3) ATP5B ATP synthase subunit beta, mitochondrial precursor ATP5C1 ATP synthase subunit gamma, mitochondrial isoform H (heart) precursor ATP5H ATP synthase subunit d, mitochondrial isoform a Glycolytic process (n=1) HK1 Hexokinase-1 isoform HKI-ta/tb Anion transport (n=2) VDAC2 Voltage-dependent anion-selective channel protein 2 isoform 1 VDAC3 Voltage-dependent anion-selective channel protein 3 isoform 1 Mitochondrial fusion (n=2) MIEF1 Mitochondrial dynamics protein MID51 isoform 2 BNIP3 BCL2/adenovirus E1B 19 kDa proteininteracting protein 3 MICOS complex (n=4) IMMT MICOS complex subunit MIC60 isoform 1

P36542 O75947

P19367 P45880 Q9Y277

Q9NQG6 Q12983

Q16891

lP

P06576

na

P47985

Jo ur

P31930

re

-p

P56181

Protein description

of

O95299

SYM

ro

Uniprot accession id

Phosphorylation site

Y339ⁿ /S340ⁿ T89/ S90ⁿ S492ⁿ /S493ⁿ S159ⁿ /160ⁿ

T406ⁿ /T407ⁿ S 16/T 18ⁿ / S19ⁿ T337ⁿ /T 341ⁿ S 97ⁿ /S98ⁿ S29ⁿ /S30

T340/S341ⁿ T69ⁿ /S70ⁿ Y67ⁿ /T70

T58/S59 T41/S45 41

S38ⁿ /S39ⁿ /T42ⁿ, S191ⁿ /S192ⁿ, Y626/ S627ⁿ, 40

Journal Pre-proof APOO

GAPDH

P55084

HADHB

Q13011

ECH1

P99999 Q8NE86

CYCS MCU

P10620

MGST1

Glycolysis / Gluconeogenesis (n=2) Glyceraldehyde-3-phosphate dehydrogenase isoform 1 Fatty acid oxidation (n=2) Trifunctional enzyme subunit beta, mitochondrial isoform 1 precursor Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial precursor Others (n=3) cytochrome c calcium uniporter protein, mitochondrial isoform 1 precursor microsomal glutathione S-transferase 1 isoform a

Y43/S44

T153/T154 S210/T211

S67ⁿ /T69ⁿ /S70ⁿ T314ⁿ /T315ⁿ

Y47/S48 Y128ⁿ /S129ⁿ Y5ⁿ /S18ⁿ /Y19ⁿ /T21ⁿ

Jo ur

na

lP

re

-p

ro

P04406

MICOS complex subunit MIC26 precursor

of

Q9BUR5

41

Journal Pre-proof Table 4. List of mitochondrial proteins that are hyperphosphorylated in human skeletal muscle from different muscle pathologies (compared to healthy control). The Uniprot accession id, protein symbol, description and the phosphorylation site are indicated. “n” in superscript indicates novel site identified in this study. The number of phosphosites in each complex/ pathway is indicated in parenthesis. Uniprot SYM Protein description Phospho site Disease Fold change accession Vs control Id

P06576

ATP5A1

of

ATP synthase subunit beta, mitochondrial precursor

T116ⁿ, S128, S529

ATP synthase subunit alpha, T64, S346ⁿ, S419 mitochondrial isoform a precursor Tricarboxylic acid cycle (n=3) Isocitrate dehydrogenas e [NADP], mitochondrial S100, S423 isoform 1 precursor Malate dehydrogenase, mitochondrial T235 isoform 1 precursor Glyoxylate metabolic process (n=1) 2-Oxoisovalerate S337 dehydrogenase subunit alpha, mitochondrial isoform 1 precursor Mitophagy (n=1)

Jo ur

na

P25705

ATP5B

Cytochrome c S73 oxidase subunit 6C Complex V (n=6)

ro

COX6C

-p

P09669

re

COX7C

lP

P15954

Oxidative Phosphorylation Complex IV (n=2) Cytochrome c oxidase subunit 7C, mitochondrial Y19ⁿ precursor

P48735

IDH2

P40926

MDH2

P12694

BCKDHA

Q9BWH 2

FUNDC2

FUN14 domainS151 containing protein 2 MICOS complex (n=1)

Dysfy

1.5

Dysfy

1.7

PM

T116=1.8, S128= 1.9 S529=1.9

DMRV(S346 S236=1.9 ), PM(T64 T64=2.8 & S419) S419=1.6

PM(S100), Dysfy,DM R V & PM (S423)

S100=2.7 S423≥1.7

PM

1.8

Dysfy, DMRV and PM

>3.0

Dysfy, DMRV and PM

>1.7

42

Journal Pre-proof

Q13011

ECH1

VDAC1

P45880

VDAC2

3.4

PM

2

PM

S108-1.8 & S274-1.7

Voltagedependent anionselective channel protein 1 Voltagedependent anionselective channel protein 2 isoform 1

S234ⁿ

DMRV

4.4

S130ⁿ

PM

1.6

PM

>1.8

PM

2.4

na

P21796

DMRV

of

HADHA

≥2.0

ro

P40939

Dysfy, DMRV and PM

-p

ACSL1

re

P33121

lP

Q6UXV4 APOOL

MICOS complex subunit S203 MIC27 precursor Fatty acid oxidation (n=4) Long-chain-fatty-acid-CoA ligase 1 S295ⁿ isoform a Trifunctional enzyme subunit alpha, mitochondrial S316 precursor Delta(3,5)Delta(2,4)- dienoylCoA isomerase, mitochondrial S108, S274ⁿ precursor Anion transport (n=2)

Jo ur

Activation of caspase and apoptosis (n=2)

Q9BXK5 BCL2L13 P02794

FTH1

O95831

AIFM1

Q9H7Z7

PTGES2

P00505

GOT2

Bcl-2-like protein S444, S450ⁿ 13 isoform b Oxidation-reduction process (n=3) Ferritin heavy chain S179 Apoptosis-inducing factor 1, mitochondrial isoform S116 AIF precursor Prostaglandin E S95 synthase 2 isoform 1 Gluconeogenesis (n=1) Aspartate aminotransferase, mitochondrial isoform 1 precursor

S360

Dysf and PM 2

Dysf and PM 1.8 & 1.6

PM

2.4 43

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TMEM11

Q9UJZ1

STOML2

BCKDK

PM

2.2

PM

3.2

PM

2.9

PM

3

PM

2

Dysf and PM

>1.7

ro

O14874

[3-Methyl-2oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial isoform S31 a precursor Protein targeting (n=1)

of

P17152

Mitochondrion organization (n=3) Transmembran e protein 11, S13, S17 mitochondrial Stomatin-like protein 2, T327 mitochondrial isoform a Aminoacid metabolism (n=1)

SLC16A1

28S ribosomal protein T130 S16, mitochondrial Plasma membrane lactate transport (n=1) Monocarboxylate transporter 1

S467

Jo ur

P53985

-p

MRPS16

re

Q9Y3D3

lP

TOMM20

na

P35180

Mitochondrial import receptor subunit S138 TOM20 homolog Translation (n=1)

44

Journal Pre-proof Table 5. List of mitochondrial proteins that are hypophosphorylated in human skeletal muscle from different muscle pathologies (compared to healthy control). The Uniprot accession id, protein symbol, description and the phosphorylation site are indicated. “n” in superscript indicates novel site identified in this study. Uniprot accession SYM id

of

ro

Fold change vs. control

0.6

0.4

0.6

Q16891

Q02978

Jo ur

P36957

na

lP

P09669

-p

P47985

Oxidative Phosphorylation Complex I (n=1) NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial isoform 1 NDUFS4 S136ⁿ DMRV & PM precursor Complex III (n=1) Cytochrome b-c1 complex UQCRFS1 subunit Rieske, mitochondrial T100ⁿ PM Complex IV (n=1) Cytochrome c oxidase subunit COX6C S73 PM 6C Tricarboxylic acid cycle (n=1) Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial isoform 1 DLST S81 Dysfy precursor MICOS complex (n=2) Dysfy (S350) MICOS complex subunit S350ⁿ, &DMRV, PM IMMT MIC60 isoform 1 T481ⁿ (S350 & T481) Gluconeogenesis (n=1) Mitochondrial 2oxoglutarate/malate carrier SLC25A11 protein isoform 1 Y202 Dysfy Glycolysis / Gluconeogenesis (n=1)

re

O43181

Phospho Disease site

Protein description

P04406

GAPDH

P27338

MAOB

Q99623

PHB2

Glyceraldehyde-3-phosphate S333 Dysfy dehydrogenase isoform 1 Aminoacid metabolism (n=1) Amine oxidase [flavinS131 Dysfy containing] B Mitochondrion organization (n=1) Prohibitin-2 isoform 1 S151 Protein targeting (n=1)

Dysfy

0.6 S350-0.3 & T4810.2

0.5

0.4

0.5 0.6

45

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Dysfy & PM

0.6 & 0.5

Dysfy & DMRV

0.4 & 0.5

-p

ro

of

0.5

re

O75746

PM

lP

P00387

0.5

na

P51648

DMRV

Jo ur

P35180

Mitochondrial import receptor TOMM20 subunit TOM20 homolog S138 Oxidation-reduction process (n=2) Fatty aldehyde dehydrogenase ALDH3A2 isoform 1 S14 NADH-cytochrome b5 CYB5R3 S179ⁿ reductase 3 isoform 3 Gluconeogenesis (n=1) Calcium-binding mitochondrial carrier protein SLC25A12 Aralar1 S662

46

Journal Pre-proof LEGEND TO FIGURES Figure 1. Mitochondrial phosphoproteomics in human muscle samples from control and muscle pathologies. Schematic representation of the work flow in the proteomics experiment is shown. Muscle tissue from control (C) (n=10), dysfy (n=10), DMRV (n=10) and PM (n=10) cases were selected for the study, followed by isolation of total mitochondria, tryptic digestion, TMT labeling, fractionation and MS analysis. The

of

corresponding TMT labels for individual groups (C=127, dysfy=128, DMRV=129, PM=130) are shown. While one aliquot of the labelled peptide mixture (10% of sample)

ro

was utilized for total proteome analysis, the remaining sample (90 %) was considered for

re

-p

phosphoproteomics experiment.

lP

Figure 2. Analysis of the phosphoproteomics data from control muscle tissue. A, The number of phosphoproteins, phosphopeptides and phosphosites identified in total

na

and mitochondrial vs. non-mitochondrial proteins are indicated. The number of single, double and triple phosphorylated

peptides along with

the number

of phosphoserine

Jo ur

(p-ser), phosphothreonine (p-thr) and phosphotyrosine (p-tyr) residues are indicated among mitochondrial and non-mitochondrial proteins. B, Pie chart shows the distribution of phosphorylated mitochondrial proteins in different compartments. The functional pathwayspredominantly

associated

with

mitochondrial

phosphoproteins

are

also

indicated. C, Venn diagram comparing the mitochondrial phosphoproteins detected in the current study compare to the previous study (Zhao et al, 2011). D, Comparison of the number of phosphosites

reported in literature vs. novel sites identified in the current

study.

Figure 3. Gene network of all the mitochondrial phosphoproteins obtained 47

Journal Pre-proof following string-db analysis in control human skeletal muscle.

Figure 4. Gene network analysis of all the mitochondrial phosphoproteins involved in (A) metabolic pathways and (B) mitochondrial organization in control human skeletal muscle obtained following string-db analysis.

of

Figure 5. Phosphoproteins involved in respiratory complexes and TCA cycle. A, Schematic representation of the inner mitochondrial membrane and the embedded

ro

respiratory complexes I-V along with the distribution of phosphorylated subunits in

-p

different complexes. B, Quantitative comparison of different Protein Kinases

lP

re

contributing to the mitochondrial phosphoproteome in control skeletal muscle.

Figure 6. Differences in the mitochondrial phosphoproteome in muscle pathologies

na

compared to control. A, The number of phosphorylated, hypophoshorylated proteins, peptided and residues (phosphosites) among total and mitochondrial proteins are shown.

Jo ur

S- curve analysis of differentially phosphorylated mitochondrial proteins in dysfy (B), DMRV (C) and PM (D) is shown (Red= downregulated, Blue= Upregulated, Grey= Unchanged) along with the phosphorylated residue in each protein in parenthesis. E, Venn diagram showing the hyperphosphorylated and hypophosphorylated mitochondrial proteins that are common/ unique proteins among dysfy, DMRV and PM. F, Gene network analysis of differentially phosphorylated mitochondrial proteins in all three pathologies. G¸ Quantitative comparison of different Protein Kinases contributing to the mitochondrial phosphoproteome in skeletal muscle from different pathologies.

Figure 7. Modeling of the predicted structural changes induced by phosphorylation

48

Journal Pre-proof in Voltage-Dependent Anion Channel (VDAC1) and ATP Synthase from healthy muscle. A, The structure of human VDAC1 (PDB Id: 5XDO) showing the position of the phosphorylated residues recorded in the phosphoproteomics experiment. The close-up view shows that the distance between residues S234 (B), S240 (C) and pS167 (D) and the neighboring amino acids are altered, following phosphorylation indicating perturbation of local structure. E, The structure of bovine ATP Synthase (PDB Id: 5FIK) showing the position of the phosphorylated residues (in red) recorded in the phospho proteomics

of

experiment. The ATP5B1 subunit is shown in blue while the other subunits are in

ro

orange. The close-up view shows that the distance between residues T60 (F), T116 (G),

-p

S327 (H) and S415 (I) and the neighboring amino acids are altered, following

re

phosphorylation indicating perturbation of local structure.

lP

Figure 8. Phosphorylation of the human complex I subunits in a healthy muscle. A,

na

The structure of human complex I (PDB Id: 5XTD and 5XTB) showing the position of the phosphorylated residues (in violet/cyan) recorded in the phosphoproteomics

Jo ur

experiment. The three subunits NDUFV1, NDUFV2 and NDUFS1 at the distal end of peripheral arm are highlighted (red box). B, The close-up view of the three subunits NDUFV1, NDUFV2 and NDUFS1 along with the phosphorylated residues (violet: defined residues; cyan: ambiguous residues), co-factors (FMN) and iron-sulfur clusters (N1a, N3, N1b, N4 and N5).

Figure 9. Molecular dynamics simulation of CI containing NDUFV1, NDUFV2 and NDUFS1 subunits. A, RMSF values of the three subunits is shown. The individual phosphorylated/ unphosphorylated is shown. RMSD values of all three chains (B), NDUFV1 (C), NDUFS1 (D), NDUFV2 (E) in the phosphorylated form (blue) compared to the control (yellow) are shown. Distance between FMN and Fe-S cluster N1a (F), FMN 49

Journal Pre-proof and Fe-S cluster N3 (G) and Fe-S clusters N3 and N1b (H) in the phosphorylated form

Jo ur

na

lP

re

-p

ro

of

(blue) compared to the control (yellow) are shown.

50

Journal Pre-proof Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle

B. Sunitha, Manish Kumar, Niya Gowthami, Sruthi Unni, Narayanappa Gayathri, T.S. Keshava Prasad, Atchayaram Nalini, Kiran Polavarapu, Seena Vengalil, Veeramani Preethish-Kumar, B. Padmanabhan, M. M. Srinivas Bharath

SIGNIFICANCE OF THE STUDY

of

Most human muscle pathologies are associated with significant mortality and

ro

morbidity. While diagnostics of these diseases has significantly advanced, their therapy has met with little success. This is because the underlying pathomechanisms are unclear.

-p

Emerging evidences indicate that mitochondrial dysfunction is crucial in muscle diseases,

re

involving altered biochemistry, proteome and physiology. The current study highlights that

lP

healthy human muscle contains widespread phosphorylation of mitochondrial proteins with structural implications and potential regulatory role in mitochondrial processes. Altered

na

mitochondrial protein phosphorylation in 3 muscle pathologies (vs. control) with varied

Jo ur

pattern, clearly demonstrates that phosphorylation could regulate mitochondrial protein function in the skeletal muscle during physiological and pathological processes.

51

Journal Pre-proof HIGHLIGHTS (Sunitha et al)  Mitochondrial dysfunction in the skeletal muscle contributes to muscle pathologies  Altered

proteome

and

post-translational

modifications

underlie

mitochondrial

dysfunction  While healthy human muscle mitochondrial proteins displayed 253 phosphorylation sites, muscle pathologies displayed 33 hyper and 14 hypophorphorylated sites.

 Altered

protein

phosphorylation

regulates

of

 Site-specific phosphorylation altered local structure of mitochondrial proteins. structure-function

relationship

of

ro

mitochondrial proteins in the skeletal muscle during physiological and pathological

Jo ur

na

lP

re

-p

processes.

52

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9