Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Combined robotic inguinal and iliac-obturator lymphadenectomy for stage III skin cancers: Surgical technique and preliminary results.

BACKGROUND: Ilio-inguinal lymphadenectomy for stage III melanoma and skin cancers still represents the best therapeutic option for a subset of patients, although the incidence of post-operative complications is dramatically high. Only a paucity of papers on robotic approach have been published, reporting experiences on isolated pelvic or inguinal lymphadenectomy, and no series on combined dissections have been described yet. We present the preliminary results achieved with combined robotic approach, with special emphasis on lymph nodal mapping, dissection technique and postoperative complications linked with the lymphatic system. METHODS: Between September 2019 and September 2021, 10 patients were submitted to robotic inguinal and iliac-obturator lymphadenectomy. RESULTS: Post-operative course was characterised by early mobilisation and minimal post-operative pain. Only one lymphoedema occurred and lymph nodal harvesting was more than satisfactory. CONCLUSIONS: Robotic surgery provides meticulous lymph nodal dissections, with promising functional and oncologic outcomes. Further series are advocated to confirm these preliminary results.

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

A multicenter retrospective study on 4480 implanted PICC-ports: A GAVeCeLT project.

BACKGROUND: PICC-ports may be defined as totally implantable central venous devices inserted in the upper limb using the current state-of-the-art techniques of PICC insertion (ultrasound-guided venipuncture of deep veins of the arm, micro-puncture kits, proper location of the tip preferably by intracavitary ECG), with placement of the reservoir at the middle third of the arm. A previous report on breast cancer patients demonstrated the safety and efficacy of these devices, with a very low failure rate. METHODS: This retrospective multicenter cohort study-developed by GAVeCeLT (the Italian Group of Long-Term Venous Access Devices)-investigated the outcomes of PICC-ports in a large cohort of unselected patients. The study included 4480 adult patients who underwent PICC-port insertion in five Italian centers, during a period of 60 months. The primary outcome was device failure, defined as any serious adverse event (SAE) requiring removal. The secondary outcome was the incidence of temporary adverse events (TAE) not requiring removal. RESULTS: The median follow-up was 15.5 months. Device failure occurred in 52 cases (1.2%), the main causes being local infection (n = 7; 0.16%) and CRBSI (n = 19; 0.42%). Symptomatic catheter-related thrombosis occurred in 93 cases (2.1%), but removal was required only in one case (0.02%). Early/immediate and late TAE occurred in 904 cases (20.2%) and in 176 cases (3.9%), respectively. CONCLUSIONS: PICC-ports are safe venous access devices that should be considered as an alternative option to traditional arm-ports and chest-ports when planning chemotherapy or other long-term intermittent intravenous treatments.

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function.

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

Effects of magnesium supplementation on post-thyroidectomy hypocalcemia: a prospective single-center study.

BACKGROUND: Severe and/or symptomatic hypocalcemia due to hypoparathyroidism is the main contraindication for discharge in patients who have undergone thyroid surgery. Hypomagnesemia may contribute to the onset of hypoparathyroidism and is frequently observed after thyroid surgery in hypocalcemic patients. The impact of prophylactic and postoperative Magnesium supplementation on postoperative hypocalcemia and hypomagnesemia was prospectively evaluated by comparing patients undergoing prophylactic supplementation to a control group of patients who had only received Magnesium after evidence of postoperative hypoMg. METHODS: One hundred and twenty patients who underwent a total thyroidectomy participated in the study. Seventy-three patients were included in the study group, 47 in the control group. Prior to surgery, patients in the study group were given Magnesium orally for 5 days; postoperatively, Calcium and Magnesium was administered to all patients who displayed hypocalcemia and hypomagnesemia. RESULTS: Postoperative biochemical hypocalcemia (serum Calcium<8.5 mg/dL, regardless of its clinical severity) was found in 60 patients (50%) on D1 and in 58 patients (48.4%) on D2. Among hypocalcemic patients, hypomagnesemia was recorded in 29 at D1 (48%), and in 46 at D2 (79%). A significant positive correlation was found between Magnesium, Calcium, and parathyroid hormone in the first two postoperative days, while a significant inverse correlation occurred for these same parameters and length of hospital stay (p<0.001). One hundred and five patients (87.5%) were discharged as expected on the second postoperative day (Study group = 65, Control group = 40, p = 0.724), whereas 15 patients (12.5%) required prolonged hospitalization (Study group = 8, Control group = 7, p = 0.721). The Study group only showed significantly higher Magnesium levels on the first postoperative day (p=0.03). CONCLUSIONS: Although Magnesium and Calcium levels showed the same trend after thyroidectomy, neither Magnesium prophylaxis nor Magnesium treatment influenced the clinical course of postoperative hypocalcemia.

Voltage-Dependent Anion Selective Channel 3: Unraveling Structural and Functional Features of the Least Known Porin Isoform.

Voltage-dependent anion-selective channels (VDAC) are pore-forming proteins located in the outer mitochondrial membrane. Three isoforms are encoded by separate genes in mammals (VDAC1-3). These proteins play a crucial role in the cell, forming the primary interface between mitochondrial and cellular metabolisms. Research on the role of VDACs in the cell is a rapidly growing field, but the function of VDAC3 remains elusive. The high-sequence similarity between isoforms suggests a similar pore-forming structure. Electrophysiological analyzes revealed that VDAC3 works as a channel; however, its gating and regulation remain debated. A comparison between VDAC3 and VDAC1-2 underlines the presence of a higher number of cysteines in both isoforms 2 and 3. Recent mass spectrometry data demonstrated that the redox state of VDAC3 cysteines is evolutionarily conserved. Accordingly, these residues were always detected as totally reduced or partially oxidized, thus susceptible to disulfide exchange. The deletion of selected cysteines significantly influences the function of the channel. Some cysteine mutants of VDAC3 exhibited distinct kinetic behavior, conductance values and voltage dependence, suggesting that channel activity can be modulated by cysteine reduction/oxidation. These properties point to VDAC3 as a possible marker of redox signaling in the mitochondrial intermembrane space. Here, we summarize our current knowledge about VDAC3 predicted structure, physiological role and regulation, and possible future directions in this research field.

Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation.

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier .

High-Resolution Respirometry Reveals MPP+ Mitochondrial Toxicity Mechanism in a Cellular Model of Parkinson's Disease.

MPP+ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson's disease models. Within the cells, MPP+ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP+ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP+ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP+, relevant for the standardization of studies that employ this neurotoxin as a disease model.

Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.

A High Resolution Mass Spectrometry Study Reveals the Potential of Disulfide Formation in Human Mitochondrial Voltage-Dependent Anion Selective Channel Isoforms (hVDACs).

The voltage-dependent anion-selective channels (VDACs), which are also known as eukaryotic porins, are pore-forming proteins, which allow for the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions that regulate organelle and cellular metabolism. We have recently reported the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing, for the first time, the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed for us to extend the investigation to the human VDACs. The over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as also certained in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs that are found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell. Data are available via ProteomeXchange with identifier PXD017482.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics.

Possible role of low magnesium levels in the onset of postoperative hypoparathyroidism following thyroidectomy.

BACKGROUND: A positive correlation between calcium and magnesium serum levels is well known and depends upon various factors. This study aims at verifying the existence of this association in a retrospective series of patients who underwent thyroid surgery. METHODS: Two hundred and eighty-five consecutive patients (202 female, 83 male, mean age 57 years) who underwent at least total thyroidectomy (TT) and had a complete clinical and biochemical pre- and postoperative evaluation were included in the study. Patients were evaluated with regard to: sex, age, indications for surgery, operative time, number of accidentally removed parathyroids, extent and time of surgery, thyroiditis, final histology, pre- and postoperative levels of calcium, magnesium, vitamin D, and creatinine, presence of symptoms of hypocalcemia. Statistical analysis was performed using the R software. RESULTS: In the postoperative period, biochemical hypocalcemia (<8.5 mg/dL) was observed in 126 patients (44%) and severe hypocalcemia (<7.5 mg/dL, a level indicating the need for longer hospitalization) was seen in 40 (14%). When analyzing patients with and without postoperative hypocalcemia, the factors affecting postoperative biochemical hypocalcemia were: older age (P=0.019), longer operative time (P=0.039), and a highly significant correlation between postoperative calcium and magnesium levels (r=0.432; P<0.001). CONCLUSIONS: The only factor among the ones we analyzed in this retrospective study that would appear to be linked to the onset of clinically relevant hypocalcemia is low magnesium levels in the postoperative period. A prospective randomized study with a group of patients undergoing magnesium replacement in the postoperative period can clarify the possible role of magnesium repletion on hypocalcemia.

Sentinel lymph node biopsy in patients with malignant melanoma: analysis of post-operative complications.

BACKGROUND: This study investigates the incidence of post-operative complications and risk factors of sentinel lymph node biopsy (SLNB) in melanoma patients. METHODS: A retrospective cohort study was conducted at a single cancer institution on 408 consecutive SLNBs. RESULTS: Fifty-five post-operative complications occurred in 39 (9.5%) patients and included: wound infection in 24 (5.9%), seroma and lymphorrhea in 15 (3.7%), wound dehiscence in seven (1.7%), lymphocele in six (1.5%) and others in three (0.7%). Univariate analysis failed to identify possible risk factors (i.e. gender, age, lymph node region, number of excised lymph nodes, Breslow index, pT levels, comorbidities, length of surgery and hospital stay). Metastatic sentinel nodes occurred in four of 135 (3%) patients with thin melanoma (Breslow <1 mm) and in 68 of 262 (25.9%) patients with Breslow >1 mm. CONCLUSION: For patients with thin melanoma in whom the incidence of lymph node metastasis is low, the reported post-operative morbidity of almost 10% of SLNB highlights the need for careful patient selection.

Sentinel lymph node biopsy in porocarcinoma: A case reports.

INTRODUCTION: Eccrine porocarcinoma (EPC) is a slow-growing carcinoma arising from the eccrine sweat glands. Based on its clinical presentation it can be confused with malignant and benign skin lesions, both. Histological examination is essential to formulate a correct diagnosis. Surgical excision with clear margins is the standard therapeutic approach while the role of sentinel lymph node biopsy (SNLB) remains controversial. CASE PRESENTATION: The Authors report two cases of EPC of the lower limbs occurred in two women. Patients were treated by wide surgical excision of the lesion and SNLB. 6 months follow-up was disease free for both patients. CONCLUSION: Although a rare cutaneous tumor, EPC has to be taken into account in the differential diagnosis of malignant skin lesions because of its possible loco-regional aggressiveness and related morbidity. Among the available treatment options, surgical excision is considered the standard approach whereas the role of SNLB is controversial although the Authors discuss a possible usefulness for staging and diagnosis.

Post-translational modifications of VDAC1 and VDAC2 cysteines from rat liver mitochondria.

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.

VDAC1 as Pharmacological Target in Cancer and Neurodegeneration: Focus on Its Role in Apoptosis.

Cancer and neurodegeneration are different classes of diseases that share the involvement of mitochondria in their pathogenesis. Whereas the high glycolytic rate (the so-called Warburg metabolism) and the suppression of apoptosis are key elements for the establishment and maintenance of cancer cells, mitochondrial dysfunction and increased cell death mark neurodegeneration. As a main actor in the regulation of cell metabolism and apoptosis, VDAC may represent the common point between these two broad families of pathologies. Located in the outer mitochondrial membrane, VDAC forms channels that control the flux of ions and metabolites across the mitochondrion thus mediating the organelle's cross-talk with the rest of the cell. Furthermore, the interaction with both pro-apoptotic and anti-apoptotic factors makes VDAC a gatekeeper for mitochondria-mediated cell death and survival signaling pathways. Unfortunately, the lack of an evident druggability of this protein, since it has no defined binding or active sites, makes the quest for VDAC interacting molecules a difficult tale. Pharmacologically active molecules of different classes have been proposed to hit cancer and neurodegeneration. In this work, we provide an exhaustive and detailed survey of all the molecules, peptides, and microRNAs that exploit VDAC in the treatment of the two examined classes of pathologies. The mechanism of action and the potential or effectiveness of each compound are discussed.

Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities.

The voltage-dependent anion channel 1 (VDAC1), an outer mitochondria membrane (OMM) protein, serves as a mitochondrial gatekeeper, mediating the transport of nucleotides, Ca2+ and other metabolites across the OMM. VDAC1 also plays a central role in mitochondria-mediated apoptosis by facilitating the release of apoptotic proteins and by association with both pro- and anti-apoptotic proteins. Tumor cells, which are constantly exposed to hypoxic conditions, affect the cell via the transcription factor hypoxia-inducible factor (HIF) that induces transcriptional activity. In cultured cells and in lung cancer patients, hypoxia induces VDAC1 truncation at the C-terminus (VDAC1-ΔC). However, the molecular mechanisms involved in VDAC1-ΔC formation are unknown. Here, we show that hypoxia-induced VDAC1-ΔC formation is inhibited by the Ca2+ chelator BAPTA-AM, by calpain inhibitor-1, by inhibitor of the asparagine endopeptidase (AEP) and by si-RNA targeting HIF1-α or Ca2+-activated protease calpain-1 expression but not that of calpain-2. Finally, VDAC1-ΔC expressed in bacteria and reconstituted into a planar lipid bilayer exhibited decreased channel conductance relative to the full-length protein, yet retained voltage-dependent conductance. These findings suggest that hypoxia, acting via HIF-1α expression, leads to VDAC1 cleavage involving the activation of calpain 1 and AEP.

Anti-Cancer Compounds Targeted to VDAC: Potential and Perspectives.

BACKGROUND: VDAC (Voltage-Dependent Anion selective Channel) is a small family of abundant pore-forming proteins located in the outer mitochondrial membrane. Their role range from the most intuitive, the formation of a hydrophilic conduit through the membrane thanks to its beta-barrel structure, to less understood functions that make them essential actors in the cross-talk between the bioenergetics metabolism and the cytosol components. Due to this localization, VDAC1, in particular, has been reported to be involved in apoptosis, Hexokinase and tubulin binding, and in the Warburg effect. For these reasons, an involvement of VDAC in cancer is considered consequential and a number of compounds have been proposed and used in experimental trials to demonstrate the efficacy of molecules affecting the functions of VDAC. OBJECTIVES: In this work, we thus survey the literature describing drug compounds acting on the cancerous proliferation through VDAC. Three main categories have been assigned: molecules acting on the VDAC-Hexokinase binding, molecules directly inhibiting the VDAC conductance, molecules affecting the expression levels of the VDAC gene. The application of biological peptides for this purpose is also considered. CONCLUSION: Since the knowledges about the functional properties of VDAC protein are still insufficient, VDAC as a pharmacological target in the fight against cancer is still a very open, but very promising, field.

High resolution mass spectrometry characterization of the oxidation pattern of methionine and cysteine residues in rat liver mitochondria voltage-dependent anion selective channel 3 (VDAC3).

Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.