1 PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER

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PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER SEGMENT DISKS: COMMONALITIES AND DIFFERENCES Maurizio Bruschi, Andrea Petretto, Federico Caicci, Martina Bartolucci, Daniela Calzia, Laura Santucci, Lucia Manni, Luca A. Ramenghi, Gian Marco Ghiggeri, Carlo E. Traverso, Giovanni Candiano, and Isabella Panfoli J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00741 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Journal of Proteome Research

PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER SEGMENT DISKS: COMMONALITIES AND DIFFERENCES.

Maurizio Bruschi1†, Andrea Petretto2*†, Federico Caicci3, Martina Bartolucci2, Daniela Calzia4, Laura Santucci1, Lucia Manni3, Luca A. Ramenghi5, GianMarco Ghiggeri1, Carlo E. Traverso6, Giovanni Candiano1and Isabella Panfoli4* 1

Laboratory of Molecular Nephrology, Istituto Giannina Gaslini, Genoa, Italy; 2Laboratory of Mass

Spectrometry-Core Facilities, Istituto Giannina Gaslini, Genova, Italy; 3Department of Biology, Università di Padova, Padova, Italy; 4Dipartimento di Farmacia-DIFAR, Università di Genova, Genoa, Italy; 5Neonatal Intensive Care Unit, , Istituto Giannina Gaslini, Genoa, Italy; 6 Clinica Oculistica, (Di.N.O.G.M.I.) Università Department of Intensive Care di Genova, IRCCS Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy. †

Co-first authors for equal contributions.

*

Corresponding Author: dr. Isabella Panfoli Università di Genova, Dipartimento di Farmacia-DIFAR V.le Benedetto XV,3 16132 Genova, Italy phone: ++39 010 353.7397 fax: ++39 010 353.8153 e-mail: [email protected] *

Corresponding Author for proteomic analysis: Dr. Andrea Petretto Laboratory of Mass Spectrometry-Core Facilities, Istituto Giannina Gaslini, Via Gerolamo Gaslini, 5 16148 Genova, Italy e-mail: [email protected]

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ABSTRACT

The retinal rod outer segment (OS) is a stack of disks surrounded by the plasma membrane, housing proteins related to phototransduction, as well as mitochondrial proteins involved in oxidative phosphorylation (OxPhos). This prompted us to compare the proteome of bovine OS disks and mitochondria, to assess the significant top gene signatures of each sample. The two proteomes, obtained by LTQ-Orbitrap Velos mass spectrometry, were compared by statistical analyses. 4139 proteins were identified, 2045 of which overlapping in the two sets. Non-hierarchical Spearman’s correlogram revealed that the groups were clearly discriminated. Partial Least Square Discriminant plus Support Vector Machine analysis identified the major discriminative proteins, implied in photo-transduction and lipid metabolism, respectively. Gene ontology analysis identified top gene signatures of the disk proteome, enriched in vesiculation, glycolysis, and OxPhos proteins. The Tricarboxylic Acid Cycle and the electron transport proteins were similarly enriched in the two samples, but the latter was up regulated in disks. Data suggest that the mitochondrial OxPhos proteins may represent a true OS proteome component, outside the mitochondrion. This knowledge may help the scientific community in the further studies of retinal physiology and pathology.

Keywords:ATP synthase; disks; electron transfer chain; mass spectrometry; mitochondria; oxidative phosphorylation; Rod Outer Segment; transmission electron microscopy. Abbreviations: BSA, bovine serum albumin; COX IV, cytochrome c oxidase subunit IV; ETC, electron transport chain; HPR, horseradish peroxidase; MDS, Multidimensional scaling; NADH, Reduced β-nicotinamide adenine dinucleotide; PBS, Phosphate Buffer Saline; OxPhos, oxidative phosphorylation; PLS-DA Partial Least Square Discriminat Analysis; SDS-PAGE, Sodium Dodecyl Sulphate–Poly Acrylamide Gel Electrophoresis; SVM, Support Vector Machine; WB, western blot.


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INTRODUCTION

The mammalian retinal rod outer segment (OS) consists of a stack of floating lamellar membranes, the disks, enclosed by the plasma membrane, containing 85% rhodopsin (Rh). The players in photoreception-specific functions

1,2

are expressed in the OS disks. These are constantly renewed,

being progressively displaced toward the rod tip, while changing their lipid composition 3. A separation of rod OS proteins was previously conducted in a 2D-PAGE study of the bovine retina, and three proteins were identified by mass spectrometry (MS) 4. A comprehensive MS analysis of the rod disk proteome was also conducted 5. A proteomic and immuno-cytochemical study focusing on the OS trafficking and membrane fusion was also performed, that showed that these contain Rab proteins, in particular 6. The timing of the photoresponse, set at milliseconds 1,2, and the chemical energy requirement (about 9 nmol ATP/min/mg of protein7) for the photoreception would suggest that a separate OS production capability is required. In fact, the rod OS is devoid of mitochondria and biochemical measurements found glycolysis insufficient to supply ATP for photo-transduction8, even though recently the contribution of glycolysis to rod anabolism was pointed out 9. By contrast, our previous analysis of the disk proteome found that the protein complement of the OS disks comprises all of the proteins of the whole glucose oxidation to CO2 and H2O OS

11–13

10

. The mitochondrial OxPhos components are active in the

, consistently with reports of the ectopic expression of the mitochondrial F1Fo-ATP synthase

(ATP synthase) and of the electron Transfer Chain (ETC) proteins in several cellular membranes

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such as plasma membrane of cancer cells15–19, human umbilical vein endothelial cells (HUVEC)

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, ,

hepatocytes21, isolated myelin22–24, exosomes25 and lipid rafts14. The extra-mitochondrial respiring membranes are able to produce ATP consuming oxygen (O2), both in the presence of conventional respiring substrates, such as pyruvate/malate or succinate, and in the presence NADH or Tricarboxylic Acid (TCA) Cycle intermediates 22,26.


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The study presented here was set up to compare rod OS disk and mitochondria proteome by discovery approach with last generation mass spectrometry, in order to assess the proteins that characterize each sample.

EXPERIMENTAL SECTION Sample preparations Preparation of intact bovine OS Intact OS were isolated under dim red light at 4°C, from 25 bovine eyes (obtained from a local slaughterhouse) 27. Briefly, 5 retinas were placed in 5 ml of Mammalian Ringer (MR: 157 mM NaCl, 5 mM KCl, 7 mM Na2HPO4, 8 mM NaH2PO4, 0.5 mM MgCl2, 2 mM CaCl2) pH 6.9, with 35% sucrose (w/v), protease inhibitor cocktail (Sigma-Aldrich, S. Louis, MO, USA) and Ampicillin (100 µg/ml). Vortexing (for 15 s for 2 times at 300 rpm) was employed, to cause minimal disruption of retinal cell types other than rods. Suspension was centrifuged at 6,000g at 4°C (Sorvall Instruments Div., Newtown, CT). The OS suspensions were filtered through cheesecloth, pooled and carefully diluted to 10% sucrose (w/v) with MR, then centrifuged in the same rotor at 4,000g for 45 min. Rod OS pellets were retained. The procedure was repeated twice. Osmotically intact disk preparation. Osmotically intact disks were obtained as swollen vesicles from rod OS following the procedure of Smith and Litman28, minimizing contaminant IS proteins. Disks do not burst by hypotonic shock, due to their phospholipid composition29. Briefly, OS were allowed to burst for 3 h in distilled water plus 70 µg/ml leupeptin and 100 µg/ml Ampicillin, then the suspension was added of 2 ml cold water on the top of the 5% (w/v) Ficoll (Sigma-Aldrich, S. Louis, MO, USA) solution, and centrifuged for 2 h at 25,000 rpm in a Beckman FW-28 rotor, 100,000g). Disks were collected as a pink layer at the interface between Ficoll and water. Isolation of bovine liver mitochondria ACS Paragon Plus Environment

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Mitochondria were isolated by a protocol carefully chosen, able of providing their best functionality. Mitochondria-enriched fractions were purified from bovine liver obtained from a local slaughterhouse. Briefly, liver (10 g) washed in cold PBS was homogenized in BufferA, containing 0.25 M sucrose, 0,15 M KCl, 10 mM TRIS HCl pH 7.4, 1 mM EDTA, and 0,5% BSA and centrifuged at 800 g for 10 min. Supernatant was filtered and centrifuged at 12,000 g for 15 min. Pellet was resuspended in 0.25 M sucrose, 75 mM mannitol, 10 mM TRIS HCl pH 7.4, 1 mM EDTA (Buffer B), and centrifuged at 1,000 g for 10 min. The final supernatant was centrifuged again at 12,000 g for 15 min and the mitochondrial pellet was resuspended in Buffer B 30. Transmission Electron microscopy of bovine retinas. Samples were processed as described 31. Briefly, the eye cups were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS buffer solution. Then, retinas were removed from the eye capsule, cut into small pieces, dehydrated, embedded in LR White Resin 32, and polymerized at 58°C. Ultrathin sections (80 nm) were placed on Formvar-coated nickel grids and processed according a post embedding immuno-gold method, using a mouse monoclonal anti-rhodopsin (Ab) (1:100) (Sigma Aldrich, St. Louis, MO, USA) and rabbit polyclonal anti-ND1 subunit of ETC IAb (diluted 1:50) (Abcam, Cambridge, UK) overnight at 4°C. Ab binding was detected using secondary goat anti-mouse IgG (British BioCell International) (diluted 1:100) coupled to gold particles (5 nm) and goat anti-rabbit IgG (British BioCell International) (diluted 1:100) coupled to gold particles (25) nm. Sections were analyzed at a FEI Tecnai G12 transmission electron microscope operating at 100KV. Negative controls were performed by omitting primary Ab; absence of cross reactivity resulted (data not shown). Images were acquired with Tietz TVIPS F114 and OSIS Veleta cameras, collected and typeset in Corel Draw X3. Protein identification by MS analysis A preliminary set of experiments was devoted to the selection of the appropriate mitochondrial sample, and it was fond that the most suitable benchmark for the comparison were liver mitochondria. For the proteomics analyses, it was decided to use the same samples used for the biochemical analyses ACS Paragon Plus Environment

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i.e. purified disks and retinal mitochondria-enriched fractions. The latter were obtained following the protocol that maintains their OxPhos activity. Sample Preparation The disk pellets were lysed, solubilized, denatured and reduced using a solution of 6M Gdm Cl, 10 mM TCEP, 40 mM CAA, 100 mM Tris pH 8.5. Afterwards, samples were loaded into a 30 kDa filtration devices and mixtures of sequencing grade of Lys C and Trypsin were added at a ratio of 1:50 and 1:100 (µg enzyme: µg protein) respectively. After an overnight digestion at 37°C, peptides were collected with one wash of 50% CH3OH, 45% H2O, and 5% TFA. The samples were subsequently acidified and desalted on C18 StageTips before injection into Mass Spectrometer. LC-MS/MS Analysis and Data Processing Peptide mixtures were separated by reversed-phase chromatography on a 25-cm EASY-Spray column (inner diameter 75 µm, PepMap C18, 2 µm particles), using an ultra-nanoflow highperformance liquid chromatography system (ULTIMATE 3000 RSLC, Thermo Fisher Scientific) which were connected online to a Orbitrap Velos Pro instrument (Thermo Fisher Scientific) through an EASYSpray™ Ion Sources (Thermo Fisher Scientific). The peptides are separated with increasing organic solvent at a flow rate of 250 nl/min using with a nonlinear gradient of 5-45 % solution B (80% CAN and 20% H2O, 5% DMSO, 0.1% FA) in 100 min. The LTQ-Orbitrap Velos Pro instrument was operated in the data-dependent acquisition (DDA) mode to automatically switch between full-scan MS and tandem mass spectrometry (MS/MS) acquisition. Single MS survey scans were performed in the Orbitrap (mass range 350 - 1650 m/z, resolution 60000, automatic gain control 1000000 ions and maximal ion injection time of 250 ms). As an internal mass, allowing the recalibration of the spectra in the Orbitrap, was chosen the poly-dimethyl cyclosiloxane background ions (protonated DMSO cluster; m/z 401.922718). A maximum of 10 MS/MS experiments were triggered per MS scan [2]. The minimum MS signal for triggering MS/MS was set to 3000 ions using an isolation window of 2 Da. The m/z values of signals already selected for MS/MS were put on an exclusion list for 60 s using an exclusion window size of ±5 p.p.m. In all cases, one micro-scan was ACS Paragon Plus Environment

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recorded. CID was done with a target value of 5,000 ions in the linear ion trap, a maximal ion injection time of 90 ms, normalized collision energy of 35%, a Q-value of 0.25 and an activation time of 10 ms. Raw MS files were processed with MaxQuant software version 1.5.3.30, using the integrated Andromeda search engine with an FDR

1 PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER

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