Voltage-Dependent Anion Selective Channel: Comparison
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Please note this is a comparison between Version 1 by Rosaria Saletti and Version 4 by Camila Xu.

Le proteine VDAC (voltage-decanale selettivo anionico voltaggio-dipendent anion selective channel) proteins, also known as mitoche), note anche come suine mitocondrial porins, are the most abundant proteins of the outeri, sono le proteine più abbondanti della membrana mitochondrial membranee esterna (OMM) where they play a vital rol, dove svolgono un ruolo vitale in various processi cellular processes, in the regulation ofi, nella regolazione del metabolism, and in survival pathwayso e nelle vie di sopravvivenza.

  • voltage dependent anion channel
  • cysteine overoxidation
  • deamidation

1. Introductzione

1.1. VDAC Isoforms: A Family of Hub Proteins

1.1. Isoforme VDAC: una famiglia di proteine hub

Le proteine VDAC (voltage-decanale selettivo anionico voltaggio-dipendent anion selective channel) proteins, also known as mitoche), note anche come suirine mitocondrial porins, are the most abundant proteins of the outeri, sono le proteine più abbondanti della membrana mitochondrial membranee esterna (OMM) where they play a vital roldove svolgono un ruolo vitale in various processi cellular processes, in the regulation ofi, nella regolazione del metabolism, and in survival pathways. They mediate the ions ando e nelle vie di sopravvivenza. Mediano lo scambio di ioni e metabolites exchange betweeni tra i mitochondria and the rest of the cell, ensuring good functondri e il resto della cellula, garantendo una buona funzionality ofà dei complessi mitochondrial complexes andi e la produzione di energy productionia [1].
In hNegligher eukaryotes, there are three VDAC eucarioti superiori, ci sono tre isoformse VDAC (VDAC1, VDAC2, VDAC3) encoded bycodificate da geni separate genes located on different chi situati su cromosomesi diversi [2]. ThQuese pore-forming proteins have a similarte proteine che formano i pori hanno un peso molecular weightolare simile (30 kDa) and highlye sequenze altamente conserved sequences of aboutate di circa 280 amino acids with the exception ofmminoacidi ad eccezione del VDAC2 which has thche ha la frazione N-terminal moiety ofe di 11 residues longer than the otheri più lunga delle altre isoformse.
TheL'analisi evolutionary analysis va indicates VDAC3 as the oldest come l'isoform, whila più antica, mentre VDAC1 isè considered the youngestata la più giovane porina mitochondriale porin [3][4].
ThLe exstrutture sperimental 3D structures of mouse and humani 3D dell'isoforma VDAC1 isoform have been di topo e umana sono state determined using X-ray cryate utilizzando la cristallography andafia a raggi X e la NMR [5][6][7]. ThQueste analyses revealed isi hanno rivelato una structure constituted by 19 β-strands arranged to form ttura costituita da 19 β-filamenti disposti a formare una trans-membranea β-barrel and by ile e da una region containing α-helix at thee contenente α-elica al N-terminus of thedella protein. The barrel isa. La canna è organized as a regularzzata come una normale schiera antiparallel array of β-strands with the exception of strands 1 and 19 that runa di β fili ad eccezione dei fili 1 e 19 che corrono in parallel. The amphipathic α-helix tail is located inside the pore. However, the exact position and local structure of thiso. La coda anfipatica a α elica si trova all'interno del poro. Tuttavia, la posizione esatta e la struttura locale di questo segment are still elusive since these features are not perfectly overlapping in the available X-ray and NMR structureso sono ancora sfuggenti poiché queste caratteristiche non si sovrappongono perfettamente nelle strutture a raggi X e NMR disponibili [5][6][7].
Recentlyemente, the structure ofla struttura di zebrafish VDAC2 was solved at a high resolutionè stata risolta ad alta risoluzione, confirming the samermando la stessa disposizione β-barrel arrangement as di VDAC1 [8]. Zebrafish VDAC2 has one cysteine residue in theun residuo di cisteina nella sequenza e manca della sequence and lacks theza N-terminale più lunga di 11 amino acid longer N-terminal sequence present in mammalian VDACsminoacidi presente nelle VDAC dei mammiferi.
TheLa VDAC3 structure has not yet beenstruttura VDAC3 non è stata ancora determined. Severalata. Diverse previsioni bioinformatic predictions, based on the largehe, basate sulla somiglianza di grandi sequence similarity,ze, hanno proposed a barrel core such as the otherto un nucleo di botte come le altre isoforme VDAC isoforms [9]. DeNonospite the hightante l'elevata somiglianza di sequence similarity and structural homology, VDAC za e l'omologia strutturale, le isoforms display different functional properties within the celle VDAC mostrano diverse proprietà funzionali all'interno della cellula.
AL'analysis of the exisi dei livelli di espression levels of humane delle isoforme VDAC isoforms in HeLa cellsumane nelle cellule HeLa, determined by real-time ati mediante REAL-TIME PCR, suggests thatrisce che VDAC1 is the most abundant isoform, ten times more abundant compared to è l'isoforma più abbondante, dieci volte più abbondante rispetto a VDAC2 and hundred times more abundant compared to e cento volte più abbondante rispetto a VDAC3, the least characterized of thla meno caratterizzata delle isoforms. In addition, the overexe. Inoltre, la sovraespression of each single VDACe di ogni singola isoform affects the mRNA levels of the other two a VDAC influisce sui livelli di mRNA delle altre due isoformse, suggesting that the ratios betweenerendo che i rapporti tra le isoforme VDAC isoforms are subjected to asono sottoposti a un controllo reciprocal control that avoids an imbalance among theso che evita uno squilibrio tra queste proteinse [10].
ASebbene lthough the threee tre isoforms show a common involvement in cellulare mostrino un coinvolgimento comune nel mantenimento della bioenergetics maintenanca cellulare, VDAC1 ande VDAC2 havenno funzioni specialized functions inzzate nella morte cellulare programmed cell death. Forata. Per l'isoforma VDAC3 isoform,, studi recent studiesi indicate ano un ruolo central role in ROSle nel metabolism and in mitochondrialo dei ROS e nel controllo di quality controlà mitocondriale [11].
ThLe functions of VDACs are several-fold and some of these depend on, or are affected by, zioni dei VDC sono parecchie e alcune di queste dipendono o sono influenzate dall'interaction with other cyzione con altre proteine citosolic andhe e mitochondrial proteins. Due to theiri. A causa della loro localization at the OMM, VDACs are zzazione presso l'OMM, le VDAC sono considered to be hub ate proteins,e hub, che interacting with overgiscono con oltre 200 proteins in order to e al fine di integrate mitochre le funzioni mitocondrial functions with the rest of thei con il resto delle attività cellular activitiesi [12][13]. ThusPertanto, VDACle isoforms appears to be a junction for e VDAC sembrano essere una giunzione per una variety of signalsà di segnali associated with different pathways related to cell survival ori a diversi percorsi legati alla sopravvivenza cellulare o alla morte programmed death. Furthermore, the function of VDACs and theirata. Inoltre, la funzione dei VDAC e le loro interactions with otherzioni con altre proteins are affected bye sono influenzate da modificazioni post-translational modifications (PTMsduzionali (PTM) [14]. UnSfortunately, PTMs of VDAC amente, i PTM delle proteins ree VDAC rappresent a little explored field, mainly because discovery and characterization ofano un campo poco esplorato, principalmente perché la scoperta e la caratterizzazione del PTM in thesqueste proteins is very challenging, due to their poore è molto impegnativa, a causa della loro scarsa solubility andà e impossibility toà di isolatre singole isoforms. Only in recent years has the increasing number of tools aimed ate. Solo negli ultimi anni, il crescente numero di strumenti volti a identifying andicare e quantifying PTMs increased, improving the knowledge in this field and in the mechanisms that regulate functions andicare i PTM ha migliorato le conoscenze in questo campo e nei meccanismi che regolano le funzioni e le interactions of mitochzioni delle suipine mitocondrial porinsi. In particular, the development of nano-reversed phase ultra-high-performanceolare, lo sviluppo di metodi di cromatografia liquid chromatographya ad altissime prestazioni (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometrye di spettrometria di massa ad alta risoluzione (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such metha fase nano-invertita ha svolto un ruolo chiave in questo campo. I risultati ottenuti sui PTM VDAC utilizzando tali metodologies, which have permitted an in-depth characterization of these very hydrophobic, che hanno permesso una caratterizzazione approfondita di queste proteine dei pori trans-membrane pore proteins, are summarized in this reviewa molto idrofobiche, sono riassunti in questa recensione.

1.2. VDACs as Main Players in Mediating and Regulating Mitochondrial Functions with Cellular Activities

1.2. Le VDAC come attori principali nella mediazione e nella regolazione delle funzioni mitocondriali con attività cellulari

TheLa location in the OMM allows thposizione nell'OMM consente alle proteine VDAC proteins to act as anchor points fo di agire come punti di ancoraggio per diverse sets ofi insiemi di molecules thatole che interact withgiscono con i mitochondria. In this way, VDACs are able toondri. In questo modo, i VDC sono in grado di mediate and regulate the re e regolare l'integration ofzione delle funzioni mitochondrial functions withi con le attività cellular activi.
L'inties.
Theerattoma VDAC interactome includesomprende proteins locatede situate in OMM, innermembrana mitochondrial membranee interna (IMM), ispazio intermembrane spacea (IMS), cyitosol, ereticolo endoplasmic reticulum,atico, membrana plasma membrane andtica e nucleus that are involved ino che sono coinvolti nel metabolismo, apoptosis, signal transduction, trasduzione del segnale, protection againstzione contro ROS, binding tolegame a RNA or DNA, and more DNA e altro ancora.
MLa mobility ofà della regione VDAC N-terminal α-helix region is ie α-elica è important for channee per il gating but also fordei canali, ma anche per le interactions with bothzioni con proteine pro-apoptotic andhe e anti-apoptotic proteins such as he come Bax, Bak, and e Bcl-xL [15][16][17]. Il VDAC1 is involved in the release ofè coinvolto nel rilascio di fattori apoptotic factors located in the i localizzati nello spazio intermembrane space due to its ability ofa grazie alla sua capacità di oligomerizingzarsi in dimers, hexamers, and higher-order structures, to form a large pore that allows the passage of cytochrome c and apoptosis inducing factori, esameri e strutture di ordine superiore, per formare un grande poro che consente il passaggio del citocromo c e del fattore di induzione dell'apoptosi (AIF) to the cyal citosol ande di consequently the activation ofguenza l'attivazione della morte cellulare programmed cell death. Insteadata. Invece, VDAC2 functions asziona come fattore anti-apoptotic factor and it is upregulated in severalco ed è sovraregolato in diverse malattie debilitating diseases including nti tra cui l'Alzheimer’s and cancer e il cancro [18]. ThiQues property ista proprietà è probably due to the unique ability ofilmente dovuta alla capacità unica di VDAC2 todi sequester therare la proteina pro-apoptotic protein Bak in the OMM and maintain it in the inactivea Bak nell'OMM e mantenerla nello stato inattivo state [11].
VDAC1 dimosplays binding sites, located in its cyttra siti di legame, situati nella sua porzione citosolic moiety, for manya, per molti enzimi metabolic enzymes, such as glyi, come la gliceraldehyde 3-phosphate dehyide-3-fosfato deidrogenase, i, la creatine kinase, glycerol kinase, glucokinase, c-Raf kinase, and hexokinaschinasi, la glicerolo chinasi, la glucochinasi, la chinasi c-Raf e le isoforms (I ande e l'esochinasi (I e II), which need che necessitano di un accesso preferential access to mitochziale all'ATP mitocondriale ATP [19].
HL'exokinasesochinasi interacts through its hydrophobicgisce attraverso la sua sequenza idrofobica N-terminal sequence with e con Glu73 ofdi VDAC1, a binding situn sito di legame localized on one side of the barrel wall, buried in the hydrophobic environment ofzato su un lato della parete della canna, sepolto nell'ambiente idrofobo di OMM [20].
IÈ st has been demonstrated that treatment ofato dimostrato che il trattamento dei mitochondria with dicyclohexyondri con dicicloesilcarbodiimide (DCCD) inhibits hexokinase–VDAC ibisce l'interaction due to selective chemicalzione esochinasi-VDAC a causa della modification of G chimica selettiva della glu73 [21].
GIlu73 residueo is alsodi glu73 è tanche binding site for il sito di legame per le ceramides, tumor sui, lipidi oncosoppressor lipids able to act directly oni in grado di agire direttamente sui mitochondria to trigger apoptotic cell death. It is interesting that both ondri per innescare la morte delle cellule apoptotiche. È interessante notare che sia VDAC1 and 2 own, in a similar position, a cysteineche 2 possiedono, in una posizione simile, un residueo di cisteina (Cys127 in human el VDAC1 andumano e Cys138 in human VDAC2) in the form of suel VDAC2 umano) sotto forma di acido solfonic acid with a strongo con una forte carica negative charge resembling that of the a simile a quella del residuo acido glutamatemato acid residue [22]. Instvecead, VDAC3 , l'isoform does not show anya VDAC3 non mostra alcun residue ho omologous to a Cys127/138 or Glu73 incorporato nembedded in the hydrophobic moiety of the lla porzione idrofobica dell'OMM.
VDAC1 presenta una tas a chca legante il colesterol binding pocket formed, in human o formata, in isoform, bya umana, da residui di Ile123, ,Leu144, ,Tyr146, ,Ala151,e and Val171 r[ 23].
Le siduesuine [23].
Mmitochondrial porins formi formano complexes with otherssi con altre proteins, such as the adeninee, come il nucleotide translocase dell'adenina (ANT), the tranla proteina traslocator proteinrice (TSPO), also known as the peripheral-typnota anche come recettore delle benzodiazepine receptordi tipo periferico (PBR), mitochHSP70 mitocondrial HSP70, and several cytoskeletale e diverse proteins such ase citoscheletriche come tubulina, actin, dynein light chain, anda, catena leggera della dineina e gelsolina [11].
ThLa prote tranina traslocator proteinrice interacts directly with all VDAC gisce direttamente con tutte le isoformse VDAC. In particular, olare, l'interaction betweenzione tra TSPO ande VDAC1 contributes to regulate the eisce a regolare l'efficiency of mitochondrialza dei meccanismi di controllo della quality control mechanisms and inhibits mitophagyà mitocondriale e inibisce la mitofagia, preventing endo l'ubiquitination ofzione delle proteins throughe attraverso la downregulation of thedella via PINK1/Parkin pathway [24].. Il Themotivo GxxxG motifsi presents both a sia in VDAC andche in TSPO, and ised è necessary for thisio per questa interactionzione [25]. MInoreoverltre, VDAC1 ande TSPO, in association withzione con StAR (steroidogenic acute regulatory protein), form the transduceosome, aano il trasdusoma, un complesso multi-protein complex involved in chco coinvolto nel trasporto del colesterol transport. In a former hypothesiso. In una precedente ipotesi, VDAC1 ande TSPO in OMM, ANT in IMM, and e Cyclophilin D in thnella matrice mitochondrial matrix weree erano candidates to constitute the permeabilityi a costituire il poro di transition porezione di permeabilità (PTP), a high un poro ad alta conductance and non-ttanza e non specific pore that allowso che consente il gonfiore mitochondrial swelling and release ofe e il rilascio di proteine apoptogenic proteins. More recently, it washe. Più recentemente, è stato proposed that PTP could bto che il PTP potesse essere formed by dimers of theato da dimeri del complesso ATP synthaseintasi complex [26].
Recenti studies have focused attention on the role of VDAC proteins in hanno focalizzato l'attenzione sul ruolo delle proteine VDAC nella disfunzione mitochondrial dysfunction typical of many pathe tipica di molte condizioni patological conditions including strokehe tra cui ictus, cancer, mitochro, encefalomiopatie mitocondrial encephalomyopathies, and aging, as well as i e invecchiamento, nonché disturbi neurodegenerative disordersi [27][28].
ML'anass speclisi della spettrometry analysis revealed the ia di massa ha rivelato l'association between VDACs and the zione tra VDAC e l'ubiquitina ligasei Parkin. In presence of damagedza di mitochondria, as inondri danneggiati, come nel morbo di Parkinson’s disease,, La Parkin is phosphorylated bya viene fosforilata da PINK1 ande di consequentlyguenza ubiquitinates le proteins that reside on the OMM, targeting thee che risiedono sull'OMM, prendendo di mira i mitochondria for ondri per la degradationzione. Parkin is a cy è una proteina citosolic protein but translocates to thea ma trasloca nei mitocondri per partecipare ai meccanismi di controllo della qualità mitocondriale. Le proteine VDAC rappresentano un sito di attracco di Parkin sui mitochondria toondri difettosi [29].
Moreover, VDAC1 reparticipate in esents the main docking site at the mitochondrial quality control mechanisms. VDAClevel for misfolded and aggregated proteins represent a docking site of, a common feature of neurodegenerative disorders known as proteinopathies, such as Alzheimer’s disease (AD), Parkin on defective mitochondriason’s disease (PD), Creutzfeldt–Jacob disease (CJD), dementia with Lewy bodies (DLB), Huntington disease (HD), and amyotrophic lateral sclerosis (ALS) [29].
Moreover, VDAC1 represents the main docking site at the mitochondrial level for misfolded and aggregated proteins, a common feature of neurodegenerative disorders known as proteinopathies, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Creutzfeldt–Jacob disease (CJD), dementia with Lewy bodies (DLB), Huntington disease (HD), and amyotrophic lateral sclerosis (ALS) [30].
.
For example, in AD post-mortem brains, in neuroblastoma cells and in an AD mouse model, a direct association was demonstrated between VDAC1, specifically its N-terminal region, and hyper-phosphorylated Tau but also with amyloid beta (Aβ), both in its monomeric and oligomeric forms [31]. These interactions can have a dramatic effect on mitochondrial functions in AD neuron because they block the PTP formation, disrupt the transport of mitochondrial proteins and metabolites, and impair gating, conductance, and physiological interactome of VDACs [32].
In Parkinson’s disease, α-synuclein directly interacts with mitochondria, blocks VDAC1, and impairs metabolite fluxes leading, consequently, to an energetic crisis able to compromise cell viability [33].
In ALS, several SOD1 mutants are able to bind VDAC1 [34]. This interaction impairs ATP/ADP exchange, VDAC1 conductance and mitochondrial membrane potential. Recently, the competition between SOD1G93A and HK1 was demonstrated in binding VDAC1, in NSC34 motor-neuron cell lines [35].
In literature, the role of VDAC1 in neurodegeneration is rather well known; however, the involvement of the other two isoforms in these pathways remains poorly defined. This is likely associated with the relative abundance of VDAC1 compared to other isoforms which are more difficult to isolate in pure form.
Recent studies demonstrated that Cytoskeleton-associated protein 4 (CKAP4), a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER), regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites [36].
VDAC2 binds inositol trisphosphate receptors (IP3R) and regulates the release of Ca2+ from the ER. In addition, several other interaction partners have been reported for VDAC2 isoform, which imply its effect in multiple cellular functions. Specifically, VDAC2 has been linked to many cellular proteins, including apoptotic factors as Bak and Bax, StAR, Metaxin2, eNOS (nitric oxide synthesize), GSK3β, tubulin, and Mcl1 [19]. In addition, VDAC2 and RACK1 (receptor of activated protein kinase C1) function as receptors for lymphocystis disease virus (LCDV) and for bursal disease virus in host cells [37].
VDAC2 together with VDAC3 binds Erastin, the activator of ferroptosis, a new pathway that regulates cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Interaction between VDAC2/3 and Erastin results in degradation of the channels following activation of ubiquitin protein ligase Nedd4 [38].
Finally, the VDAC3 isoform is associated with cytosolic proteins as tubulins and cytoskeletal proteins, stress sensors, chaperones, and proteasome components, redox-mediating enzymes such as protein disulfide isomerase [39].

2. Proteomics of VDAC Isoforms

2.1. Sample Preparation

Sample preparation has a profound effect on the final results of a proteomic workflow. Protein extraction methods and protein separation techniques should provide an unbiased and reliable map representative of all proteins present in a specific sample. The different extraction and fractionation approaches are based on proteins physicochemical and structural characteristics, such as molecular weight, solubility, hydrophobicity, and isoelectric point. A specific protocol has to be optimized for each particular sample, to maximize protein recovery and minimize the possible proteolysis and amino acid modifications. For these reasons, there is no universal extraction protocol and not a unique buffer composition. Regarding the extraction method, the different strategies available need to be compatible with both the amount of the processed material and the subsequent analytical approach (i.e., separation or MS).
The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, and the impossibility to separate them from other mitochondrial proteins of similar hydrophobicity and to easily isolate each single isoform. In fact, isolation of VDACs has been possible exclusively for plant VDAC isoforms by chromatofocusing, thanks to the absence of phosphorylation sites in their structure [40]. Consequently, it is necessary to analyze them as components of a relatively complex mixture.
A bottom-up proteomic approach was used to investigate the VDAC3 from rat liver mitochondria (rVDAC3) [41]. According with a standard procedure [42], mitochondria were extracted and lysed with a buffer containing 3% Triton X-100 at pH 7.0. The VDAC proteins were partially purified by hydroxyapatite (HTP) chromatography, which allows to obtain a VDACs enriched fraction which comprises also other mitochondria hydrophobic proteins. After precipitation with cold acetone, the protein pellet was solubilized in SDS buffer and loaded on a 17% polyacrylamide gel (1D-SDS-PAGE). The bands in the range 30–35 kDa were manually excised from the gel, cut in small pieces, and subjected to reduction with DTT and alkylation by addition of IAA. Finally, the reduced and carboxyamidomethylated proteins were in gel-digested using trypsin and chymotrypsin, and the resulting peptide mixtures were analyzed by nUHPLC/HRMS [41]. MS data showed that rVDAC3 was found in the whole range 30–35 kDa, together with other proteins, mainly VDAC1, VDAC2, and several other mitochondrial proteins. The reason for VDAC3 electrophoretic heterogeneity probably stems from (i) the different pattern of cysteine oxidations that can modify the protein mobility; (ii) the different amount and quality of cysteine oxidations in various molecules (“redox isomers”).
The gel-digestion procedure shows some disadvantages: (i) larger peptides can get trapped between the gel meshes and lost during the extraction phase of the peptides from the gel; (ii) the electrophoretic procedure itself could damage the samples and alter the redox state of the sulfur amino acids (due to possible over heating generated by the applied voltage and to the presence of residual quantities of the catalysts used for the polyacrylamide polymerization). Furthermore, electrophoresis requires a relatively high amount of sample and the utilization of dyes and detergents. These last molecules could interfere with subsequent MS analyses because these compounds are difficult to eliminate from the sample.
2-DE could potentially represent a useful alternative to 1-DE to improve the separation of VDAC isoforms, but its utilization presents other problems. Actually, this kind of proteins has been under-represented in 2-DE gels due to difficulties in extracting and solubilizing them in the isoelectric focusing sample buffer. In fact, the most effective solubilizing agent for highly hydrophobic membrane proteins is SDS, but this detergent is incompatible with 2-DE. In addition to the difficulties in entering IPG (immobilized pH gradient) gels, membrane proteins tend to precipitate at their isoelectric point during IEF. Furthermore, their tendency to absorb the IPG matrix prevents their migration into the SDS-PAGE gel.
An improvement in the rVDAC3 mass spectrometric analysis was obtained following the introduction of a gel-free shotgun proteomic approach [41]. According to this procedure, to avoid any possible artefact due to air exposure and manipulations, reduction/alkylation was carried out before VDACs purification from the mitochondria. Afterwards, all the proteins present in the HTP eluate, without previous electrophoretic separation, were purified from non-protein contaminating molecules with the PlusOne 2-D Clean-Up kit, and the desalted protein pellet was then re-dissolved in ammonium bicarbonate containing RapiGest SF to improve the solubility. In fact, this surfactant makes the proteins more susceptible to enzymatic cleavage without modifying the sample or inhibiting endoprotease activity. Furthermore, the RapiGest SF is compatible with enzymes such as trypsin or chymotrypsin and does not influence subsequent MS analysis because it can be easily removed in acidic conditions.
Separate aliquots of reduced and alkylated proteins were then subjected to digestion with modified porcine trypsin and chymotrypsin. In this experiment, every protein in the HTP eluate was digested, producing a very complex peptide mixture, which was finally analyzed by nUHPLC/HR nESI-MS/MS.
The new “in solution-digestion” protocol associated with nUHPLC/HR ESI-MS/MS allowed to extend the coverage of the rat and human VDACs sequences with respect to that obtained with the previous procedure [43], so that it was possible to completely cover the rat and human VDAC1 sequences and almost completely the rat and human VDAC2 and VDAC3 sequences [22][41][44]. It should be noted that the short regions not identified in VDAC2 and VDAC3 correspond to small tryptic or chymotryptic peptides or even to single amino acids, which cannot be detected in LC/MS analysis.
Moreover, by means of this new procedure a detailed characterization of PTMs of the three VDACs was obtained (see next paragraphs).

2.2. Mass Spectrometry Analysis of Post-Translational Modifications

The mammalian proteome is vastly more complex than the related genome. The reasons for this difference reside both in the molecular mechanisms that allow a single gene to encode for multiple proteins (genomic recombination, transcription initiation at alternative promoters, differential transcription termination, and alternative splicing of the transcript) and in the post-translational modifications (PTMs) which represent a wide range of chemical changes that proteins can undergo after synthesis. They include the specific cleavage of protein precursors, the covalent addition or removal of low-molecular weight groups (i.e., acetylation, glycosylation, hydroxylation, phosphorylation, ubiquitination) and the formation of disulfide bonds or other redox modifications [45][46][47].
PTMs play crucial roles in cell biology since they can change protein physical or chemical properties, activity, localization, and/or stability. Traditionally, PTMs have been identified by Edman degradation, amino acid analysis, isotopic labeling, or immunochemistry. Within recent years, MS has proven to be extremely useful in PTM discovery. Post-translationally modified amino acids always have a different molecular mass than the original, unmodified residues and this mass increment or deficit is usually the basis for the detection and characterization of PTM by MS (commonly by LC-ESI-MS/MS).
MS has several advantages for characterization of PTMs, including (i) very high sensitivity; (ii) discovery of novel PTMs; (iii) ability to identify PTMs and the modified sites, even in complex protein mixtures; and (iv) ability to quantify the relative changes in PTM occupancy at distinct sites. None of the other techniques provide all these features, so the greater majority of the known PTMs have been described by MS [48].
To improve sensibility, several methods have been developed to enrich the samples in proteins or peptides with specific PTMs prior to MS/MS analysis, such as anti-pY antibodies, IMAC (immobilized metal affinity chromatography) and TiO2 for phosphorylation [49][50], affinity capture with lectins for glycosylated proteins [51], and resin coupled with anti-acetyl-lysine for acetylated proteins [52]. Although, as previously described, isolation of single isoforms of VDACs cannot be obtained, application of combined HPLC and high-resolution ESI-MSMS analysis has resulted in the identification of several PTM in these proteins. In the following, a summary of the MS-based PTMs characterized in VDACs is reported and the respective biological significance discussed. These results are resumed in Table 1 and Figure 1.
Ijms 22 12833 g001 550
Figure 1. Post-translational modifications of human (upper panel) and rattus (lower panel) VDAC isoforms. The image shows only the modified amino acids and their positions with respect to the cytosol, the outer mitochondrial membrane (OMM), and the intermembrane space (IMS). In the rVDAC1 Cys232 faces the aqueous inside of the pore; in the rVDAC3 Cys8 is located inside of the pore.
Table 1. Post-translational modifications in VDAC isoforms obtained using mass spectrometry. PTM type, mass shift (Da), source of the sample, modified residue, MS method and relative reference are reported. Studies are described by listing first author + year.
ISOFORM PTM Type ΔMass (Da) Source Residue Method Study
VDAC1 Protein N-terminal acetylation 42.0106 Rat liver Ala 2 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2018
HAP1 cells Ala 2 Pittalà et al., 2020
Acetylation 42.0106 Mouse liver Lys 33, 41, 74, 234 nHPLC MS/MS in an LTQ MS Kim et al., 2006
Lys 41, 122, 132 nHPLC MS/MS in an LTQ 2D

ion-trap MS		 Schwer et al., 2009
Mouse liver and heart Lys 237 UPLC Velos-FT MS Yang et al., 2011
Human liver Lys 28 LC/LC-MS/MS in an FTICR/MS Zhao et al., 2010
Oxidation 15.9949 Rat liver Met 155 LC/LC-MS/MS in an FTICR/MS Guan et al., 2003
nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2018
HAP1 cells Met 129, 155 Pittalà et al., 2020
Trioxidation 47.9847 Rat liver Cys 127, 232 Saletti et al., 2018
HAP1 cells Cys 127 Pittalà et al., 2020
Phosphorylation 79.9663 Rat liver Ser 12, 136 HPLC MS/MS in an LTQ MS Distler et al., 2007
Mouse liver Ser 117 nHPLC MS/MS in an LTQ MS Lee et al., 2007
HeLa cells Ser 101, 102, 104,

Thr 107		 nHPLC MS/MS in an

LTQ-Orbitrap MS		 Olsen et al., 2006
Mouse brain Tyr 80, 208 LC-MS/MS in an LTQ FT MS Ballif et al., 2008
VDAC2 Protein N-terminal acetylation 42.0106 Rat liver Ala 2 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2018
HAP1 cells Ala 2 Pittalà et al., 2020
Acetylation 42.0106 Mouse liver Lys 32, 75 nHPLC MS/MS in an LTQ MS Kim et al., 2006
Lys 121 nHPLC MS/MS in an LTQ 2D

ion-trap MS		 Schwer et al., 2009
Oxidation 15.9949 Rat liver Met 167 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2018
HAP1 cells Met 12, 166 Pittalà et al., 2020
Trioxidation 47.9847 Rat liver Cys 48, 77, 104, 211 Saletti et al., 2018
HAP1 cells Cys 47, 76, 103, 138, 210 Pittalà et al., 2020
Succination 116.0110 Mouse brain Cys 48, 77 LC-nESI-MS/MS in an

LTQ-Orbitrap MS		 Piroli et al., 2016
Rat liver Cys 48 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2018
Phosphorylation 79.9663 HeLa cells Ser 115, Thr 118 nHPLC MS/MS in an

LTQ-Orbitrap MS		 Olsen et al., 2006
Rat liver Thr 109 SCX-RP-MS/MS in an

LTQ-Orbitrap MS		 Deng et al., 2010
Rat liver Tyr 237 HPLC MS/MS in an LTQ MS Distler et al., 2007
Mouse brain Tyr 207 LC-MS/MS in an LTQ FT MS Ballif et al., 2008
VDAC3 Protein N-terminal acetylation 42.0106 Rat liver Cys 2 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2016
HAP1 cells Cys 2 Pittalà et al., 2020
Acetylation 42.0106 Mouse liver Lys 20, 61, 226 nHPLC MS/MS in an LTQ MS Kim et al., 2006
Lys 63, 109 nHPLC MS/MS in an LTQ 2D

ion-trap MS		 Schwer et al., 2009
Human liver Lys 28 LC/LC-MS/MS in an FTICR-MS Zhao et al., 2010
Oxidation 15.9949 Rat liver Met 26, 155 nUHPLC/high resolution

nESI-MS/MS in a Q-QT-qIT MS		 Saletti et al., 2016
HAP1 cells Met 26, 155, 226 Pittalà et al., 2020
Trioxidation 47.9847 Rat liver Cys 36, 65, 165, 229 Saletti et al., 2016
HAP1 cells Cys 36, 65 Pittalà et al., 2020
Succination 116.0110 Rat liver Cys 8, 36, 229 Saletti et al., 2018
Phosphorylation 79.9663 Rat liver Ser 241, Thr 33 HPLC MS/MS in an LTQ MS Distler et al., 2007
Mouse brain Tyr 49 LC-MS/MS in an LTQ FT MS Ballif et al., 2008

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