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Interactions of VDAC with Proteins Involved in Neurodegenerative Aggregation: An Opportunity for Advancement on Therapeutic Molecules
Abstract
Background:
The Voltage Dependent Anion Channel (VDAC) proteins represent the most important pore-forming proteins of the mitochondrial outer membrane, directly involved in metabolism and apoptosis regulation. Literature has highlighted a key role of VDACs in mitochondrial dysfunction typical of many neurodegenerative disorders. In particular, the principal isoform VDAC1 represents the main mitochondrial docking site of many misfolded proteins, such as amyloid β and Tau in Alzheimer's disease, α-synuclein in Parkinson's disease and several SOD1 mutants in Amyotrophic Lateral Sclerosis. The interaction of misfolded proteins with VDAC1 has a strong impact on both cellular bioenergetics and apoptosis' pathways alteration. Therefore, VDACs represent a promising therapeutic target in neurodegeneration.
Objective:
This review summarizes the roles of VDAC isoforms, and particularly of VDAC1, in the most common neurological disorders and analyzes in detail molecules and peptides available so far, able to interact and modulate VDAC1 in any considered pathological condition.
Conclusion:
This review offers a description of the most promising therapeutic strategies acting on VDAC1, for the treatment of neurodegenerative diseases.
... The finding that αSyn localizes in a close proximity (~ 30-40 nm) to VDAC and COX IV is suggestive of a direct interaction with these two proteins, but does not provide definitive evidence in either case (Fig. 2) and does not show whether VDAC forms a pathway for αSyn translocation across MOM. As the current knowledge indicates VDAC1 as the major docking site at the MOM for misfolded proteins involved in neurodegenerative diseases [37], to further evaluate the role of VDAC in αSyn mitochondrial localization, VDAC1 expression was downregulated in differentiated SH-SY5Y cells using siRNA. Western blot in Fig. 3a, b shows 85% decrease of VDAC1 protein after siRNA treatment. ...
... According to this model [16][17][18], VDAC can serve as a gateway for αSyn translocation across the MOM into the mitochondrial intermembrane space. Interaction of αSyn with VDAC has been reported in various cell and animal models of PD (reviewed in [37]) using co-localization or co-immunoprecipitation approaches. In Olesoxime partitions into the MOM and hinders αSyn translocation through VDAC by interacting with the pore-lipid interface. ...
... This leads to the loss of mitochondrial membrane potential and finally to mitochondrial dysfunction. This model is in accord with the current trend positing VDAC as the main mitochondrial docking site for various neurodegenerative disease-related misfolded proteins [37]. In particular, similar to αSyn, some of these proteins-i.e., amyloid β associated with Alzheimer disease-could have the ability to translocate through the VDAC pore to reach and impair the ETC [37,79]. ...
An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson’s disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC β-barrel at the lipid–protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.
... The modulating effect of β-amyloid on VDAC1 conductance was also shown in in vitro experiments using BLMs [133]. The interaction of β-amyloid with VDAC1 was demonstrated in SH-SY5Y neuroblastoma cells, in which VDAC1 phosphorylation promoted release of proapoptotic molecules from the mitochondria and exacerbated the neurotoxic effect of β-amyloid [134]. Reducing VDAC1 levels in VDAC1 +/− mice protected brain cells from degenerative changes by preserving their mitochondrial function [135]. ...
... The binding of VDAC1 to misfolded proteins and accompanying disturbances in the mitochondrial homeostasis were also found in amyotrophic lateral sclerosis (ALS), a progressive disease characterized by muscle paralysis caused by degeneration of motor neurons [136,137]. The main cause in the development of hereditary ALS forms is aggregation of mutant superoxide dismutase 1 (SOD1) [134]. It is assumed that mutant SOD1, alone or together with other intracellular components, forms oligomers, which is followed by the generation of high-molecular-weight aggregates. ...
Voltage-dependent anion channels (VDAC1-3) of the outer mitochondrial membrane are a family of pore-forming β-barrel proteins that carry out controlled "filtration" of small molecules and ions between the cytoplasm and mitochondria. Due to the conformational transitions between the closed and open states and interaction with cytoplasmic and mitochondrial proteins, VDACs not only regulate the mitochondrial membrane permeability for major metabolites and ions, but also participate in the control of essential intracellular processes and pathological conditions. This review discusses novel data on the molecular structure, regulatory mechanisms, and pathophysiological role of VDAC proteins, as well as future directions in this area of research.
... Recent studies have focused attention on the role of VDAC proteins in mitochondrial dysfunction typical of many pathological conditions including stroke, cancer, mitochondrial encephalomyopathies, and aging, as well as neurodegenerative disorders [27,28]. ...
... The interaction of misfolded proteins with VDAC1 has a strong impact on both cellular bioenergetics and apoptosis' pathways alteration. Therefore, VDACs represent a promising therapeutic target in neurodegenerative diseases [28]. ...
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.
... Due to its central role in controlling MOM permeability, and consequently mitochondrial function, VDAC is emerging as a promising pharmacological target for treating a wide variety of mitochondria-associated pathologies (Shoshan- Barmatz and Ben-Hail, 2012;Camara et al., 2017;Karachitos et al., 2017;Shoshan-Barmatz et al., 2018b). In this context, proteomics and biochemical approaches along with molecular dynamics simulations have identified a large number of non-polar compounds, such as natural and synthetic steroids, smallmolecule anticancer and neuroprotective drugs, and general anesthetics, which are able to bind to VDAC (Shoshan- Barmatz and Ben-Hail, 2012;Magri and Messina, 2017;Reina and De Pinto, 2017;Leanza et al., 2019). These findings have sparked speculation that both natural steroids and synthetic hydrophobic drugs regulate mitochondrial physiology by directly affecting VDAC channel properties, despite the lack of experimental evidence in most of the cases. ...
... Considering the established unique role of VDAC in regulation of mitochondrial physiology (600 vs 9,000 usages in Google Scholar) (Colombini, 2004;Lemasters and Holmuhamedov, 2006;Shoshan-Barmatz et al., 2018a), the identification of pharmacological agents for VDAC regulation is absolutely crucial for improving outcomes of mitochondriaassociated diseases (Camara et al., 2017;Magri and Messina, 2017;Reina and De Pinto, 2017). There is an urgent need for a deeper knowledge about the molecular mechanisms of VDAC interaction with plethora of structurally and functionally unrelated endogenous proteins and steroids and synthetic drugs. ...
There is accumulating evidence that endogenous steroids and non-polar drugs are involved in the regulation of mitochondrial physiology. Many of these hydrophobic compounds interact with the Voltage Dependent Anion Channel (VDAC). This major metabolite channel in the mitochondrial outer membrane (MOM) regulates the exchange of ions and water-soluble metabolites, such as ATP and ADP, across the MOM, thus governing mitochondrial respiration. Proteomics and biochemical approaches together with molecular dynamics simulations have identified an impressively large number of non-polar compounds, including endogenous, able to bind to VDAC. These findings have sparked speculation that both natural steroids and synthetic hydrophobic drugs regulate mitochondrial physiology by directly affecting VDAC ion channel properties and modulating its metabolite permeability. Here we evaluate recent studies investigating the effect of identified VDAC-binding natural steroids and non-polar drugs on VDAC channel functioning. We argue that while many compounds are found to bind to the VDAC protein, they do not necessarily affect its channel functions in vitro. However, they may modify other aspects of VDAC physiology such as interaction with its cytosolic partner proteins or complex formation with other mitochondrial membrane proteins, thus altering mitochondrial function.
... Since the current state of knowledge dissociates the MOM channel from the mPTP, VDAC contribution to mitochondrial damages associated with CIPN might be thought from a different perspective. Lately, the interest for this channel has spanned into the neuroscience field with the successive findings of VDAC1 serving as the docking site for several neurodegenerative disease-related misfolded proteins ( Magri and Messina, 2018). As a result, VDAC is considered as a potent pharmacological target for new molecules and peptides (Magri and Messina, 2018), which potentially protects against mitochondrial dysfunctions associated with neurodegenerative disorders. ...
... Lately, the interest for this channel has spanned into the neuroscience field with the successive findings of VDAC1 serving as the docking site for several neurodegenerative disease-related misfolded proteins ( Magri and Messina, 2018). As a result, VDAC is considered as a potent pharmacological target for new molecules and peptides (Magri and Messina, 2018), which potentially protects against mitochondrial dysfunctions associated with neurodegenerative disorders. A multidisciplinary approach to further clarify the relevance of tubulin regulation of mitochondrial bioenergetics in neurons would certainly benefit to CIPN field of research. ...
Tubulin is a well-established target of microtubule-targeting agents (MTAs), a widely used class of chemotherapeutic drugs. Yet, aside from their powerful anti-cancer efficiency, MTAs induce a dose-limiting and debilitating peripheral neurotoxicity. Despite intensive efforts in the development of neuroprotective agents, there are currently no approved therapies to effectively manage chemotherapy-induced peripheral neuropathy (CIPN). Over the last decade, attempts to unravel the pathomechanisms underlying the development of CIPN led to the observation that mitochondrial dysfunctions stand as a common feature associated with axonal degeneration. Concomitantly, mitochondria emerged as crucial players in the anti-cancer efficiency of MTAs. The findings that free dimeric tubulin could be associated with mitochondrial membranes and interact directly with the voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane strongly suggested the existence of an interplay between both subcellular compartments. The biological relevance of the interaction between tubulin and VDAC came from subsequent in vitro studies, which found dimeric tubulin to be a potent modulator of VDAC and ultimately of mitochondrial membrane permeability to respiratory substrates. Therefore, one of the hypothetic mechanisms of CIPN implies that MTAs, by binding directly to the tubulin associated with VDAC, interferes with mitochondrial function in the peripheral nervous system. We review here the foundations of this hypothesis and discuss them in light of the current knowledge. A focus is set on the molecular mechanisms behind MTA interference with dimeric tubulin and VDAC interaction, the potential relevance of tubulin isotypes and availability as a free dimer in the specific context of MTA-induced CIPN. We further highlight the emerging interest for VDAC and its interacting partners as a promising therapeutic target in neurodegeneration.
... αSyn was found to be associated with both mitochondrial membranes, causing impairment of the mitochondrial respiratory complexes [20,21] [22], oxidative stress [23], and fission [24] [25]. At the MOM, monomeric αSyn interacts with VDAC (reviewed in [26]) and translocates through VDAC1 into the mitochondrial intermembrane space as was shown in neuroblastoma and HeLa cells [27,28]. ...
The Voltage Dependent Anion Channel (VDAC) is the major conduit of water-soluble metabolites and small ions into and out of the mitochondria. In mammals, VDAC exists in three isoforms, VDAC1, VDAC2, and VDAC3, each characterized by distinct tissue-dependent distribution and physiological role. VDAC2 is the most notable among the three isoforms because its knockout results in embryonic lethality and regulates the BAK/BAX-dependent apoptosis pathways. Yet, understanding of the biophysical underpinnings of VDAC2 functions remains limited. In this study, we reevaluate VDAC2's properties, utilizing recombinant human VDAC2 WT and its three mutants - cysteine-less VDAC2, VDAC2 with truncated first 11 residues, and E84A - to explore the biophysical basis that distinguishes VDAC2 from the other isoforms using single-molecule electrophysiology. We found that contrary to VDAC1 and VDAC3, which are characterized by a unique open state, VDAC2 displays dynamic switching between a few high-conductive anion-selective substates. We employed alpha-synuclein (αSyn) - a known potent cytosolic regulator of VDAC1 and VDAC3 - as a sensitive molecular probe to show that it induces characteristic blockage events in all open substates of VDAC2 but with up to ten-fold different on-rates and blockage times. A substate with higher conductance always corresponds to a higher on-rate of the αSyn-VDAC2 interaction but proportionally lower blockage times. This gives the same equilibrium constant for all substates, thus resulting in the same affinity of the αSyn-VDAC2 interaction. The pronounced difference is limited to the kinetic parameters, suggesting that once the αSyn molecule is captured, its physical state and free energy are the same for all substates. These striking results imply that the αSyn molecule senses the dynamic structural variations within the channel prior to its final capture by the pore. We propose that the discovered conformational flexibility may allow VDAC2 to recognize a larger number of binding partners, thus explaining the physiological significance of this isoform, namely, its ability to adapt to mitochondrial metabolic conditions in cells dynamically.
... The isoforms are characterized by similar molecular weights (28-32 kDa) and by approximately 70% sequence similarity. Despite the high sequence homology, VDAC isoforms display different roles in physiological and pathological conditions, expression levels, and tissue specificity [21][22][23]. Even though VDACs have been extensively investigated, only a few studies focus on VDAC proteins in male germinal tissues and sperm cells [24][25][26][27][28][29], which have suggested that VDACs might play important roles in spermatogenesis, playing a critical role in male fertility. ...
Male infertility affects nearly 50% of infertile couples, with various underlying causes, including endocrine disorders, testicular defects, and environmental factors. Spermatozoa rely on mitochondrial oxidative metabolism for motility and fertilization, with mitochondria playing a crucial role in sperm energy production, calcium regulation, and redox balance. Voltage-dependent anion channels (VDACs), located on the outer mitochondrial membrane, regulate energy and metabolite exchange, which are essential for sperm function. This review offers an updated analysis of VDACs in the male reproductive system, summarizing recent advances in understanding their expression patterns, molecular functions, and regulatory mechanisms. Although VDACs have been widely studied in other tissues, their specific roles in male reproductive physiology still remain underexplored. Special attention is given to the involvement of VDAC2/3 isoforms, which may influence mitochondrial function in sperm cells and could be implicated in male fertility disorders. This update provides a comprehensive framework for future research in reproductive biology, underscoring the significance of VDACs as a molecular link between mitochondrial function and male fertility.
... Furthermore, the dysregulated accumulation of cholesterol in mitochondrial membranes influences their physical properties, modulating mitochondrial permeability transition pore dynamics during cell death induction by Bax and death ligands 44,45 . In neurodegenerative conditions such as Alzheimer's disease, a change in mitochondrial lipid profiles is also observed 32 , including increased mitochondrial cholesterol levels 33,46,47 , which is particularly relevant given VDAC's involvement in Alzheimer pathology 48 . Therefore, our findings suggest that lipid variation could serve as an effective mechanism for regulating VDAC organization, offering flexibility, given the MOM's propensity for rapid changes in lipid composition due to lipid flux and membrane interactions. ...
Mitochondrial physiology is intricately linked to the oligomerization of voltage-dependent anion channels (VDAC), acting as gatekeepers to mitochondria. However, the molecular determinants of VDAC oligomerization remain poorly understood. Here, we used atomic force microscopy to investigate the effects of three lipids of the Mitochondrial Outer Membrane (MOM) on VDAC assemblies. We observed that VDAC forms lipid-sensitive clusters, termed honeycombs, and their compaction is regulated by cholesterol. Molecular dynamics simulations revealed VDAC’s affinity for cholesterol and phosphatidylethanolamine, both of which affect the formation of these honeycombs. Although we identified honeycomb-like assemblies akin to those in the native MOM, deviations from the physiological lipid composition resulted in varying degrees of disruption of these native-like structures. This emphasizes the profound impact of the lipid environment on VDAC organization. These findings underscore the physiological significance of lipid heterogeneity and changes within biological membranes arising from membrane contacts or pathologies in modulating VDAC behavior.
... In vivo, overexpression of VDAC1 in myofibroblasts or treatment mice with the VDAC1-derived R-Tf-D-LP4 peptide significantly alleviates transverse aortic constriction (TAC)induced cardiac fibrosis and rescues cardiac function in mice [137]. In addition to forming large pores in OMM through oligomerization, VDAC1 can also hetero-oligomerize with various proteins, such as Aβ peptide, α-synuclein (αSyn), tau, polyglutamine, and superoxide dismutase, which may result in various diseases [138,139]. For example, oligomerization of VDAC1 with Aβ peptide is associated with Alzheimer's disease and the induction of apoptosis [138]. ...
... These differences could be associated with the lower mitochondrial ROS production and slower aging rate of long-lived animals. VDAC1 is the major mitochondrial docking site for misfolded α-syn in Parkinson's disease [41]. These mitochondrial characteristics were also confirmed in our study. ...
An increase in α-synuclein (α-syn) levels and mutations in proteins associated with mitochondria contribute to the development of familial Parkinson’s disease (PD); however, the involvement of α-syn and mitochondria in idiopathic PD remains incompletely understood. The voltage-dependent anion channel I (VDAC1) protein, which serves as a crucial regulator of mitochondrial function and a gatekeeper, plays a pivotal role in governing cellular destiny through the control of ion and respiratory metabolite flux. The ability of resveratrol (RES), which is a potent phytoalexin with antioxidant and anti-inflammatory properties, to regulate VDAC1 in PD is unknown. The objective of this study was to evaluate the role of VDAC1 in the pathological process of PD and to explore the mechanism by which resveratrol protects dopaminergic neurons by regulating VDAC1 to maintain the mitochondrial permeability transition pore (mPTP) and calcium ion balance. The effects of RES on the motor and cognitive abilities of A53T mice were evaluated by using small animal behavioral tests. Various techniques, including immunofluorescence staining, transmission electron microscopy, enzyme-linked immunoadsorption, quantitative polymerase chain reaction (PCR), and Western blotting, among others, were employed to assess the therapeutic impact of RES on neuropathy associated with PD and its potential in regulating mitochondrial VDAC1. The findings showed that RES significantly improved motor and cognitive dysfunction and restored mitochondrial function, thus reducing oxidative stress levels in A53T mice. A significant positive correlation was observed between the protein expression level of VDAC1 and mitochondrial α-syn expression, as well as disease progression, whereas no such correlation was found in VDAC2 and VDAC3. Administration of RES resulted in a significant decrease in the protein expression of VDAC1 and in the protein expression of α-syn both in vivo and in vitro. In addition, we found that RES prevents excessive opening of the mPTP in dopaminergic neurons. This may prevent the abnormal aggregation of α-syn in mitochondria and the release of mitochondrial apoptosis signals. Furthermore, the activation of VDAC1 reversed the resveratrol-induced decrease in the accumulation of α-syn in the mitochondria. These findings highlight the potential of VDAC1 as a therapeutic target for PD and identify the mechanism by which resveratrol alleviates PD-related pathology by modulating mitochondrial VDAC1.
... At the same time, VDAC1 directly mediates the mitochondrial toxicity of SOD1 mutants in ALS motor neurons, being the main interactor of misfolded proteins at the organelle in the affected tissues [33]. Precisely, the interaction between VDAC1 and SOD1 G93A dramatically decreases the channel conductance and ADP accumulation within the mitochondrion and interferes with the physiological interaction of VDAC1 with HKs [33][34][35]. As a result, the downregulation of VDAC1 expression in SOD1 transgenic rats accelerates the disease onset and further reduces animals' survival [33]. ...
Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD ⁺ /NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.
... As the factors and circumstances leading to the regulation of VDAC genes are still ongoing [5,8,9], understanding whether there is an involvement or a role for their pseudogenes could provide an important key to understanding the whole regulatory network of VDAC isoforms expression. This is even more important considering that VDAC is already considered a main player in several diseases, and is becoming a prognostic factor and a possible therapeutic target [61,62]. ...
Background
Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1 , 2 and 3 , mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered.
Results
We investigated the relevance of processed pseudogenes of human VDAC genes , both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML).
Conclusions
Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.
... since 1998 [54] and is considered a consequence of the combination of different factors, including the limited availability of NADH-linked substrates due to the deposition of SOD1 mutants on the MOM [24,26,55,56]. Additionally, we found a significative reduction of Sirt3 expression in transgenic mice (Fig. 3E) that can explain the deficiency in the ET chain activity. ...
Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mouse model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinson's disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.
... Furthermore, VDAC1 acts as an anchor for many cytosolic proteins, including Hexokinases and specific members of the Bcl-2 family, participating in the regulation of mitochondrial-mediated apoptosis [17][18][19][20]. In light of these considerations, VDAC1 has rapidly become a pharmacological target in cancer and neurodegeneration [21][22][23][24]. Similar to isoform 1, VDAC2 is a key modulator of cell death and survival, exerting both pro-and antiapoptotic functions by interacting with Bak and Bax [25][26][27]. ...
Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.
... Thus, the concomitant reduction of ATP availability and E-Excess makes mitochondria more susceptible to further toxic insults.Notably, reduction of respiration coupled to complex I and II, here calculated as the specific contribution of each complex in the activation of the ET chain and analyzed in real time upon the addition of specific substrates (Fig. 3A and C), is not related to variation in the protein levels of specific complexes subunits, nor to changes in mitochondrial mass, as demonstrated by Western blot inFig. 3Band D. Certainly, inhibition of complex I in ALS is known since 1998[54] and is considered a consequence of the combination of different factors, including the limited availability of NADH-linked substrates due to the deposition of SOD1 mutants on the MOM [24,26,55,56]. ...
Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mice model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinson's disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker, Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.
... As the factors and circumstances leading to the regulation of VDAC genes are still ongoing (5,8,9), understanding whether there is an involvement or a role for their pseudogenes could provide an important key to understanding the whole regulatory network of VDAC isoforms expression. This is even more important considering that VDAC is already considered a main player in several diseases, and is becoming a prognostic factor and a possible therapeutic target (59,60) To compensate for the lack of information, in this work we investigated VDAC pseudogenes by means of an in-silico analysis integrating multi-omic data in terms of genomic features and expression profile, and also validated data in AML cell lines. ...
Background. Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered.
Results. We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML).
Conclusions. Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.
... Multipass β-Sheet Membrane Protein: The voltage-dependent anion channel 1 (VDAC-1) is an abundant β-barrel protein localized predominantly in the outer membrane of mitochondria. This protein, which has been implicated in Alzheimer's and Parkinson's disease, [54] occurs in monomeric, homodimeric, and homotrimeric forms. [55] Indeed, we observed a broad elution profile peaking at ≈100 kDa but with a pronounced shoulder on the left-hand side, indicative of oligomeric species ( Figure 7D). ...
Membrane proteins can be examined in near‐native lipid‐bilayer environments with the advent of polymer‐encapsulated nanodiscs. These nanodiscs self‐assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent‐solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral—yet water‐soluble—polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo‐DIBMA and Sulfo‐SMA(2:1). Sulfo‐DIBMA and Sulfo‐SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein–lipid interactions through lab‐on‐a‐chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.
... In addition to regulation of mitochondrial metabolism and energetic functions, VDAC also appears to be convergence point for a variety of cell survival and death signals [12]. VDAC1 also represents the main mitochondrial docking site of various disease related misfolded proteins, like amyloid β and Tau involved in Alzheimer's disease, α-synuclein involved in Parkinson's disease and several SOD1 mutants involved in Amyotrophic Lateral Sclerosis [13]. ...
Mitochondrial homeostasis regulates energy metabolism, calcium buffering, cell function and apoptosis. The present study has been conducted to investigate the implications of ubiquitin-encoding gene UBA52 in mitochondrial physiology. Transient expression of Myc-UBA52 in neurons significantly inhibited the rotenone-induced increase in reactive oxygen species generation, nitrite level and depleted glutathione level. Mass spectrometric and co-immunoprecipitation data suggested the profound interaction of UBA52 with mitochondrial outer membrane channel protein, VDAC1 in both the wild-type and Myc-alpha-synuclein overexpressed neuronal cells and in the Parkinson disease (PD)-specific substantia nigra and striatal region of the rat brain. In vitro ubiquitylation assay revealed that UBA52 participates in the ubiquitylation of VDAC1 through E3 ligase CHIP. Myc-UBA52 overexpression in neurons further improved the mitochondrial functionality and cell viability by preventing the alteration in mitochondrial membrane potential, mitochondrial complex-I activity, translocation of cytochrome-c and p-Nrf2 along with effect on intracellular calcium uptake, thus collectively inhibiting the opening of mitochondrial permeability transition pore. Additionally, Myc-UBA52 expression in neuronal cells offered protection against apoptotic and autophagic cell death. Altogether, our findings delineate functional association between UBA52 and mitochondrial homeostasis, providing new insights into the deterrence of dopaminergic cell death during acute PD pathogenesis.
... Three VDAC isoforms (VDAC1, VDAC2, and VDAC3) are found in mammalian cells, and VDAC1 is the most widely expressed, followed by VDAC2 and VDAC3 (Cesar and Wilson, 2004;Messina et al., 2012). VDAC1 is linked to the toxicity of pathogenic proteins in neurodegenerative disorders, including phosphorylated tau, Aβ, α-synuclein, and γ-secretase (Magri and Messina, 2017). Furthermore, significant changes in the VDAC1 level were found in neurodegenerative illnesses such as AD, PD, ALS, and HD (Risiglione et al., 2021). ...
Mitochondria are essential organelles for neuronal function and cell survival. Besides the well-known bioenergetics, additional mitochondrial roles in calcium signaling, lipid biogenesis, regulation of reactive oxygen species, and apoptosis are pivotal in diverse cellular processes. The mitochondrial proteome encompasses about 1,500 proteins encoded by both the nuclear DNA and the maternally inherited mitochondrial DNA. Mutations in the nuclear or mitochondrial genome, or combinations of both, can result in mitochondrial protein deficiencies and mitochondrial malfunction. Therefore, mitochondrial quality control by proteins involved in various surveillance mechanisms is critical for neuronal integrity and viability. Abnormal proteins involved in mitochondrial bioenergetics, dynamics, mitophagy, import machinery, ion channels, and mitochondrial DNA maintenance have been linked to the pathogenesis of a number of neurological diseases. The goal of this review is to give an overview of these pathways and to summarize the interconnections between mitochondrial protein dysfunction and neurological diseases.
... Furthermore, the mitochondrial fractions of HKs and therefore the HK/VDAC1 complexes were found to be significantly reduced in case of neurodegenerative disorders, thus clearing the way for the easiest docking onto VDAC1 of misfolded proteins involved in neurodegenerative diseases. 18,19 In this context, VDAC1 was identified as a key player of Schwann cell (SC) demyelination. 9 Indeed, methyl jasmonate (MJ) a phytohormone, which is able to detach HK from VDAC1, 20 induces a spontaneous peripheral nerve demyelination in vivo. ...
The voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca2+ homeostasis. Notably, the Ca2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies.
... Thus, aggregations of Lewy bodies (containing alphasynuclein) are observed in Parkinson's disease, polyglutamine repeats and excess glutamine in the synaptic clefts in Huntington's disease, and amyloid plaques are typical for pathologies associated with Alzheimer's disease (Scheltens et al., 2016;Cieri et al., 2017;Jimenez-Sanchez et al., 2017). These protein aggregates accumulate to cytotoxic levels and directly possess the neurotoxic effect on neurons or can disrupt mitochondrial proteins like cytochrome c oxidase, ATP synthase, membrane translocases, and voltage-dependent anion channels (Dong et al., 2009;Hernandez-Zimbron et al., 2012;Pinho et al., 2014;Magri and Messina, 2017). Impaired functions of these proteins lead to decreased mitochondrial membrane potential, low ATP production, and increased oxidative stress and trigger apoptosis of neurons (Zhang et al., 2010;Liang et al., 2012;Chen and Zhong, 2014). ...
Light is a natural agent consisting of a range of visible and invisible electromagnetic spectrum travels in waves. Near-infrared (NIR) light refers to wavelengths from 800 to 2,500 nm. It is an invisible spectrum to naked eyes and can penetrate through soft and hard tissues into deep structures of the human body at specific wavelengths. NIR light may carry different energy levels depending on the intensity of emitted light and therapeutic spectrum (wavelength). Stimulation with NIR light can activate intracellular cascades of biochemical reactions with local short- and long-term positive effects. These properties of NIR light are employed in photobiomodulation (PBM) therapy, have been linked to treating several brain pathologies, and are attracting more scientific attention in biomedicine. Transcranial brain stimulations with NIR light PBM in recent animal and human studies revealed a positive impact of treatment on the progression and improvement of neurodegenerative processes, management of brain energy metabolism, and regulation of chronic brain inflammation associated with various conditions, including traumatic brain injury. This scientific overview incorporates the most recent cellular and functional findings in PBM with NIR light in treating neurodegenerative diseases, presents the discussion of the proposed mechanisms of action, and describes the benefits of this treatment in neuroprotection, cell preservation/detoxification, anti-inflammatory properties, and regulation of brain energy metabolism. This review will also discuss the novel aspects and pathophysiological role of the glymphatic and brain lymphatics system in treating neurodegenerative diseases with NIR light stimulations. Scientific evidence presented in this overview will support a combined effort in the scientific community to increase attention to the understudied NIR light area of research as a natural agent in the treatment of neurodegenerative diseases to promote more research and raise awareness of PBM in the treatment of brain disorders.
... That the interplay between Aβ and VDAC1 occurs was proven by adding soluble Aβ oligomers to SH-SY5Y cell cultures: increased levels of total VDAC1 was observed, and in addition, VDAC1 phosphorylation facilitated the escape of mitochondrial proapoptotic molecules contributing to the neurotoxic effects of Aβ [103]. ...
Alzheimer's disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two 'bioenergetic' proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer's proteins. The pathological convergence of tau and amyloid β-peptide (Aβ) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aβ or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.
... The impairment of mitochondrial bioenergetics is widely considered a key event for neurodegeneration. It depends on many factors, including the accumulation of misfolded proteins on the organelle surface, the increased oxidative stress, lipid peroxidation and calcium dysregulation [31,32]. Among misfolded proteins, αSyn is responsible for a broad set of neurodegenerative diseases namely synucleinopathies. ...
α-synuclein (αSyn) is a small neuronal protein whose accumulation correlates with Parkinson's disease. αSyn A53T mutant impairs mitochondrial functions by affecting substrate import within the organelle, activity of complex I and the maximal respiratory capacity. However, the precise mechanism initiating the bioenergetic dysfunction is not clearly understood yet. By overexpressing αSyn A53T in SH-SY5Y cells, we investigated the specific changes in the mitochondrial respiratory profile using High-Resolution Respirometry. We found that αSyn A53T increases dissipative fluxes across the intermembrane mitochondrial space: this does not compromise the oxygen flows devoted to ATP production while it reduces the bioenergetic excess capacity of mitochondria, providing a possible explanation of the increased cell susceptibility observed in the presence of further stress stimuli.
... In the substantia nigra of transgenic mice, inactivation of TFAM alleles induces respiratory chain deficiency in dopaminergic neurons and aggravates PD [39]. In addition, Voltage-Dependent Anion Channels (VDACs) also serve as docking sites for misfolded or mutated proteins, associated with many neurodegenerative disorders, including PD. Interactions with these abnormal proteins alter the physiological activity of VDAC, contributing to mitochondrial dysfunction typical of these pathologies [40,41]. The VDAC proteins represent the most important pore-forming proteins of the mitochondrial outer membrane, directly involved in metabolism and apoptosis regulation [42]. ...
Hyperbaric oxygen therapy (HBOT) has been suggested as a potential adjunctive therapy for Parkinson’s disease (PD). PD is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The aim of this study was to investigate the protective mechanisms of HBOT on neurons and motor function in a 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD and 1-methyl-4-phenylpyridinium (MPP+)-mediated neurotoxicity in SH-SY5Y cells on the potential protective capability. In vivo: male C57BL/6 mice were randomly divided into three groups: MPTP group, MPTP+HBOT group, and control. The MPTP-treated mice were intraperitoneally administered MPTP (20 mg/kg) four times at 2 h intervals each day. The day after MPTP treatment, MPTP+HBOT mice were exposed to hyperbaric oxygen at 2.5 atmosphere absolute (ATA) with 100% oxygen for 1 h once daily for 7 consecutive days. In vitro: retinoic acid (RA)-differentiated SH-SY5Y cells were treated with MPP+ for 1 h followed by hyperbaric oxygen at 2.5 ATA with 100% oxygen for 1 h. The results showed that MPTP induced a significant loss in tyrosine hydroxylase (TH)-positive neurons in the SNpc of mice. HBOT treatment significantly increased the number of TH-positive neurons, with enhanced neurotrophic factor BDNF, decreased apoptotic signaling and attenuated inflammatory mediators in the midbrain of MPTP-treated mice. In addition, MPTP treatment decreased the locomotor activity and grip strength of mice, and these effects were shown to improve after HBOT treatment. Furthermore, MPTP decreased mitochondrial biogenesis signaling (SIRT-1, PGC-1α and TFAM), as well as mitochondrial marker VDAC expression, while HBOT treatment was shown to upregulate protein expression. In cell experiments, MPP+ reduced neurite length, while HBOT treatment attenuated neurite retraction. Conclusions: the effects of HBOT in MPTP-treated mice might come from promoting mitochondrial biogenesis, decreasing apoptotic signaling and attenuating inflammatory mediators in the midbrain, suggesting its potential benefits in PD treatment.
... Due to its crucial role in cellular metabolism and apoptosis, VDAC proteins are implicated in a wide range of diseases (Caterino et al., 2017;Magrì et al., 2018), including cancer (Maldonado et al., 2010;Reina and De Pinto, 2017;Magrì et al., 2018), neurodegenerative disorders, including Parkinson's disease (Rostovtseva et al., 2015), Amyotrophic Lateral Sclerosis (Magrì et al., 2016;Magri and Messina, 2017), and Alzheimer's disease (Manczak and Reddy, 2012). The knowledge about VDAC3 involvement in pathologies is very restricted. ...
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.
... One of the hallmarks of mammalian VDAC interactions is its nonspecific association with several cellular proteins that result in various diseases (Table 1). VDACs are known to heterooligomerize with the Aβ peptide, α-synuclein (αSyn), tau, polyglutamine, 172 and superoxide dismutase, 49,50 which results in protein aggregation in the membrane. For example, Alzheimer's disease is associated with mammalian VDAC1 oligomerization with Aβ peptide and the induction of Figure 13. ...
... The use of NHK1 and other interfering peptides could be a convincing strategy aimed at recovering mitochondrial dysfunction in ALS. Also, given that the interaction of misfolded proteins, such as ↵-synuclein and A peptide, with VDAC1 represents a common mechanism shared by many other neurodegenerative disorders [84][85][86][87], it is plausible to expect that NHK1 might have similar beneficial effect in other diseases. ...
Mutations in Cu/Zn Superoxide Dismutase (SOD1) gene represent one of the most common causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that specifically affects motor neurons (MNs). The dismutase-active SOD1 G93A mutant is responsible for the formation of toxic aggregates onto the mitochondrial surface, using the Voltage-Dependent Anion Channel 1 (VDAC1) as an anchor point to the organelle. VDAC1 is the master regulator of cellular bioenergetics and by binding to hexokinases (HKs) it controls apoptosis. In ALS, however, SOD1 G93A impairs VDAC1 activity and displaces HK1 from mitochondria, promoting organelle dysfunction, and cell death. Using an ALS cell model, we demonstrate that a small synthetic peptide derived from the HK1 sequence (NHK1) recovers the cell viability in a dose-response manner and the defective mitochondrial respiration profile relative to the ADP phosphorylation. This correlates with an unexpected increase of VDAC1 expression and a reduction of SOD1 mutant accumulation at the mitochondrial level. Overall, our findings provide important new insights into the development of therapeutic molecules to fight ALS and help to better define the link between altered mitochondrial metabolism and MNs death in the disease.
... It is thus reasonable to expect that if VDAC plays a crucial role in MOM permeability, there should exist mitochondrial or cytosolic endogenous VDAC regulators. Currently emerging proteomic, biochemical, and computational data identify VDAC as an interaction partner with a panoply of natural and synthetic compounds, anticancer and neuroprotective small molecule drugs, most of which are hydrophobic [25][26][27][28][29]. VDAC is also known to interact with a plethora of cytosolic proteins such as hexokinases, glycolytic enzymes, neuronal and cytoskeletal proteins [30][31][32][33][34], as well as with its neighbors in the MOM membrane, such as cholesterol transporter, TSPO [35,36], and Bcl-2-family proteins [37][38][39]. ...
The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC’s sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics—reduced metabolite flux and increased calcium flux—are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.
... In this contest, VDAC proteins (and VDAC1 in particular) play a crucial role in mediating mitochondrial dysfunction. In fact, most of the previously cited proteins are able to aggregate onto the cytosolic surface of mitochondria using VDAC as an anchor point (Magrì and Messina, 2017). Thus, the use of por1 mutant, transformed or not with plasmids carrying encoding sequences for human VDAC isoforms or mutants, represents an important opportunity to clarify the specific roles of porins in pathological contexts. ...
Mitochondrial porins, also known as voltage-dependent anion selective channels (VDACs), are pore-forming molecules of the outer mitochondrial membranes, involved in the regulation of metabolic flux between cytosol and mitochondria. Playing such an essential role, VDAC proteins are evolutionary conserved and isoforms are present in numerous species. The quest for specific function(s) related to the raise of multiple isoforms is an intriguing theme. The yeast Saccharomyces cerevisiae genome is endowed with two different VDAC genes encoding for two distinct porin isoforms, definitely less characterized in comparison to mammalian counterpart. While yVDAC1 has been extensively studied, the second isoform, yVDAC2, is much less expressed, and has a still misunderstood function. This review will recapitulate the known and poorly known information in the literature, in the light of the growing interest about the features of VDAC isoforms in the cell.
... In addition to the physiological interactors pattern, VDACs, and in particular VDAC1, act as a docking site for misfolded or mutated proteins associated with many neurodegenerative disorders, including PD. Interaction with these abnormal proteins alters the physiological activity of VDAC, contributing to the mitochondrial dysfunction typical of these pathologies (as reviewed in [21,141]). In Alzheimer's disease, β-amyloid peptide and hyperphosphorylated tau co-precipitate with VDAC1 in brains patients [142]. ...
Alpha-Synuclein (αSyn) is a protein whose function is still debated, as well as its role in modulation of mitochondrial function in both physiological and pathological conditions. Mitochondrial porins or Voltage-Dependent Anion Channel (VDAC) proteins are the main gates for ADP/ATP and various substrates towards the organelle. Furthermore, they act as a mitochondrial hub for many cytosolic proteins, including αSyn. This review analyzes the main aspects of αSyn-mitochondria interaction, focusing on the role of VDAC and its emerging involvement in the pathological processes.
... VDAC is known to interact with a quite impressive number of various genetically, structurally, and physiologically unrelated cytosolic and mitochondrial membrane proteins (see reviews by [58,72,73]). The former include glycolytic enzymes, hexokinase, dimeric tubulin, and neuronal proteins intimately involved in different neurological diseases such as α-synuclein (αSyn), Aβ peptide, tau, and SOD1 [73][74][75][76][77][78][79]. The latter include the cholesterol transporter, translocator protein 18 kDa (TSPO) [80], and Bcl-2 family proteins that ultimately link VDAC to apoptosis [52,53,81,82]. ...
Voltage-dependent anion channel (VDAC), the most abundant mitochondrial outer membrane protein, is important for a variety of mitochondrial functions including metabolite exchange, calcium transport, and apoptosis. While VDAC’s role in shuttling metabolites between the cytosol and mitochondria is well established, there is a growing interest in understanding the mechanisms of its regulation of mitochondrial calcium transport. Here we review the current literature on VDAC’s role in calcium signaling, its biophysical properties, physiological function, and pathology focusing on its importance in cardiac diseases. We discuss the specific biophysical properties of the three VDAC isoforms in mammalian cells—VDAC 1, 2, and 3—in relationship to calcium transport and their distinct roles in cell physiology and disease. Highlighting the emerging evidence that cytosolic proteins interact with and regulate VDAC calcium permeability, we advocate for continued investigation into the VDAC interactome at the contact sites between mitochondria and organelles and its role in mitochondrial calcium transport.
... The increasing occurrence of neurodegeneration in humans and the likely existence of an evolutionary pressure to balance protein stability and function with turnover highlights the urgent need to investigate biophysical basis for assembly of toxic protein aggregates. For example, the human mitochondrial VDACs co-aggregate with A, Parkin, ␣-synuclein, Tau, and other amyloidogenic proteins and accelerate the progression of neurodegeneration (29,36). Despite obvious differences in the protein source, sequence, and microenvironment, we observe similarities in the molecular mechanism of aggregation of Yersinia Ail (Fig. 7A) and human VDAC (29). ...
Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.
... Protein aggregates interact with the cytosolic surface of mitochondria and impair metabolic exchanges with the organelle. A oligomers in Alzheimer's disease [44,45], SOD1 mutants in Amyotrophic Lateral Sclerosis (ALS) [46][47][48], and ↵-syn in PD [49], all associate with the Voltage-Dependent Anion Channels (VDAC) isoform 1, reducing dramatically the mitochondria synthesized ATP availability [50]. VDACs are the most abundant mitochondrial porin, evolutionarily conserved from yeast to humans, playing a fundamental role for organelle physiology [51][52][53]. ...
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.
... There are three VDAC isoforms identified in mammals, includingVDAC1, VDAC2, and VDAC3 [10]. Increasing evidence point out that VDAC1 plays an essential role in mitochondria-mediated apoptosis, which is implicated in neurodegenerative disorders [12][13][14] as well as in cancer [10,15]. Though there are some studies concerning the connection between apoptosis and epilepsy, little is known about the role of VDAC1 in epilepsy as well as in refractory epilepsy [16]. ...
The purpose of this study was to investigate the role of voltage-dependent anion channel (VDAC) in mitochondria-mediated apoptosis of neurons in refractory epilepsy. Western blot analyses were carried out to detect the changes in cytochrome C, caspase 9, Bax, and Bcl-2. TUNEL assays were also carried out to investigate cell apoptosis under the upregulation and downregulation of VDAC1 with or without Bax or Bcl-2. VDAC1 induced Bax, Bcl-2, and caspase 9, increasing the release of cytochrome C. VDAC1 played an essential role in the apoptotic cell death of refractory epilepsy. It is concluded that VDAC1 plays an important role in refractory epilepsy and could be a possible therapeutic target of anti-epileptic drugs. The current study provides a new understanding of the possible mechanisms of refractory epilepsy.
... The association of misfolded proteins with VDAC1 has a substantial impact on both cellular bioenergetics and apoptosis pathway change. Therefore, VDACs represent a promising therapeutic target in neurodegeneration (Magri and Messina, 2018). In addition to this, CYC interacts with mitochondria in Lewy bodies to promote their aggregation, as well as activating cell death pathways. ...
Parkinson's disease (PD) is the second-most common neurodegenerative disorder, and the actual cause of this disease is still unknown. Identifying the target genes that are associated with disease plays an essential role in the treatment of PD. Various genetic studies have determined the significant target genes for disease progression, although this continues to be challenging in the field of drug designing. In this study, we proposed a network-based approach to identify target genes for PD using gene mutation, gene expression, and gene deletion analysis. The subnetwork of PD genes was constructed from human protein-protein interaction network, and the potential genes were identified using network centrality measures. Two genes, PARK1 and PARK2, were identified as target genes by integrating gene mutation and expression data into the subnetwork. Gene deletion analysis was carried out to determine the significant target, and results revealed that VDAC1 and ATP5C1 genes were crucial for the Parkinson's subnetwork. Thus, findings from the network-based approach will provide additional insight for understanding the disease mechanism of PD. Future enhancement of this study may help in predicting disease biomarkers as well as designing novel compounds in rational drug designing.
... A peptide based on the first 11 N-terminal residues of HKI (NHK1) showed the ability to hamper toxicity in ALS models [119]. NHK1 would impair the interaction between the mutant form of superoxide dismutase 1 (SOD1), SOD1 G93A and VDAC1, in turn adjusting VDAC1-conductance and boosting mitochondrial bioenergetics [119,120]. Conversely, a peptide encompassing the HKII N-terminal domain (pHK) and flanked by a penetration accelerating sequence (pHK-PAS) could trigger apoptosis in multiple cancer models [121][122][123] by dislodging HKII from mitochondria and inducing mitochondrial dysfunction. However, the precise mechanism of action of these peptides is still controversial and the direct contribution of mitochondrial Ca 2+ fluxes has not yet been ruled out. ...
Intracellular Ca2+-flux systems located at the ER-mitochondrial axis govern mitochondrial Ca2+ balance and cell fate. Multiple yet incurable pathologies are characterized by insufficient or excessive Ca2+ fluxes toward the mitochondria, in turn leading to aberrant cell life or death dynamics. The discovery and ongoing molecular characterization of the main interorganellar Ca2+ gateways have resulted in a novel class of peptide tools able to regulate relevant protein-protein interactions (PPIs) underlying this signaling scenario. Here, we review peptides, molecularly derived from Ca2+-flux systems or their accessory proteins. We discuss how they alter Ca2+-signaling protein complexes and modulate cell survival in light of their forthcoming therapeutic applications.
... The increasing occurrence of neurodegeneration in humans and the likely existence of an evolutionary pressure to balance protein stability and function with turnover highlights the urgent need to investigate biophysical basis for assembly of toxic protein aggregates. For example, the human mitochondrial voltage-dependent anion channels (VDACs) coaggregate with Aβ, Parkin, α-synuclein, Tau, and other amyloidogenic proteins and accelerate the progression of neurodegeneration (29,36). Despite obvious differences in the protein source, sequence, and microenvironment, we observe similarities in the molecular mechanism of aggregation of Yersinia Ail (Fig. 7A) and human VDAC (29). ...
Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.
... The primary function of VDAC proteins is to allow the traffic of small hydrophilic metabolites and ions through the OMM, a function 1 3 supported by its structure, solved as a transmembrane barrel made of amphipathic β-strands [3][4][5][6]. In higher eukaryotes, VDAC proteins have been involved in many other cellular processes or biochemical interactions [7][8][9][10]. Recently, it was robustly asserted that, in yeast, VDAC1 is also a coupling factor for protein translocation into the inner mitochondrial membrane (IMM), in particular for the group of inner membrane metabolite carriers [11]. ...
The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ⁰ cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a “branched” fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.
... The β-barrel outer mitochondrial membrane porin, VDAC1, is regulated by a diverse number of ligands and proteins [1,34,35], and its roles in mitochondrial metabolism and apoptosis make it a potential therapeutic target in cancer and neurodegenerative disorders [9,36]. Proteome-wide photolabeling using neurosteroid or cholesterol re-agents in rat brain [16] or mammalian cell lines [15] have found VDAC1 to be one of the most efficiently photolabeled targets. ...
Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.
Voltage-dependent anion channels of the outer membrane of mitochondria are a family of pore-forming β-barrel proteins (VDAC1-3), which carry out controlled “filtration” of small molecules and ions between the cytoplasm and mitochondria. The possibility of temporary conformational transitions between the closed and open states of VDAC proteins, as well as their interaction with a number of cytoplasmic and mitochondrial proteins, allows these channels not only to regulate membrane permeability for major metabolites and ions, but also to participate in the control of vital intracellular processes and pathological conditions. This work is devoted to the analysis of novel data obtained on the putative molecular structure, regulatory mechanisms, and pathophysiological role of VDAC family proteins, as well as possible future directions in this area of research.
Mitochondria, essential organelles responsible for cellular energy production, emerge as a key factor in the pathogenesis of neurodegenerative disorders. This review explores advancements in mitochondrial biology studies that highlight the pivotal connection between mitochondrial dysfunctions and neurological conditions such as Alzheimer’s, Parkinson’s, Huntington’s, ischemic stroke, and vascular dementia. Mitochondrial DNA mutations, impaired dynamics, and disruptions in the ETC contribute to compromised energy production and heightened oxidative stress. These factors, in turn, lead to neuronal damage and cell death. Recent research has unveiled potential therapeutic strategies targeting mitochondrial dysfunction, including mitochondria targeted therapies and antioxidants. Furthermore, the identification of reliable biomarkers for assessing mitochondrial dysfunction opens new avenues for early diagnosis and monitoring of disease progression. By delving into these advancements, this review underscores the significance of understanding mitochondrial biology in unraveling the mechanisms underlying neurodegenerative disorders. It lays the groundwork for developing targeted treatments to combat these devastating neurological conditions.
Mitochondrial homeostasis regulates energy metabolism, calcium buffering, cell function, and apoptosis. The present study has been conducted to investigate the implications of the ubiquitin-encoding gene UBA52 in mitochondrial physiology. Transient expression of Myc-UBA52 in neurons significantly inhibited the rotenone-induced increase in reactive oxygen species generation, nitrite level, and depleted glutathione level. Mass spectrometric and coimmunoprecipitation data suggested the profound interaction of UBA52 with mitochondrial outer membrane channel protein, VDAC1 in both the wild-type and Myc-α-synuclein overexpressed neuronal cells and in the Parkinson's disease (PD)-specific substantia nigra and striatal region of the rat brain. In vitro ubiquitylation assay revealed that UBA52 participates in the ubiquitylation of VDAC1 through E3 ligase CHIP. Myc-UBA52 overexpression in neurons further improved the mitochondrial functionality and cell viability by preventing the alteration in mitochondrial membrane potential, mitochondrial complex I activity, and translocation of cytochrome c and p-Nrf2 along with the effect on intracellular calcium uptake, thus collectively inhibiting the opening of mitochondrial permeability transition pore. Additionally, Myc-UBA52 expression in neuronal cells offered protection against apoptotic and autophagic cell death. Altogether, our findings delineate a functional association between UBA52 and mitochondrial homeostasis, providing new insights into the deterrence of dopaminergic cell death during acute PD pathogenesis.
Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.
Mitochondria are critical for tumor growth and metastasis. A number of traditional antitumor drugs have poor water solubility and must penetrate multiple cellular barriers to reach the mitochondria. Because mitochondria have a unique transmembrane potential and an inner membrane with a low permeability, it is difficult for most drugs to enter mitochondria. In recent years, mitochondria-targeted delivery systems that use functional peptides to modify drugs have received increasing attention. Introducing functional peptides can change the original physicochemical properties of drugs and actively target mitochondria. Functional peptide-drug conjugates (PDCs, peptide-drug conjugates) can decompose and release drugs over time or due to certain stimuli in tumors. This preserves the biological activity of the drug while increasing intratumor uptake through the enhanced permeability and retention effect (EPR, the enhanced permeability and retention effect). In this review, we focus on the direction of cancer therapy and review the application of different functional peptides in the mitochondria-targeted tumor treatments reported in recent years.
Mitochondria dysfunction is an important factor involved in PD pathogenesis. We reported neuroprotective actions of vitamin D (VD3) on a PD model, and now we investigated the VD3 effects on the brain mitochondrial function. We focused on oxygen consumption, respiratory control ratio (RCR), ADP/O ratio, mitochondria swelling, H2O2 production, and SOD activity. Additionally, immunohistochemistry assays for the dopamine system markers (TH and DAT) and mitochondrial markers (VDAC1 and Hsp60) were also carried out in the striata. Young adult male Wistar rats (250 g, 2.5 months age) were anesthetized and subjected to stereotaxic surgery and injection of saline (SO group) or 6-OHDA, into the right striatum. Brain mitochondria were isolated from the groups: sham-operated (SO), 6-OHDA, 6-OHDA pretreated with VD3 for 7, days before the 6-OHDA lesion (6-OHDA+VD3, pre-) or treated with VD3 for 14 days, after the 6-OHDA lesion (6-OHDA+VD3, post-). VD3 prevented decreases in oxygen consumption, RCR, and ADP/O ratio observed after 6-OHDA injury. Noteworthy, a very low (oxygen consumption and RCR) or no improvement (ADP/O) were observed in the 6-OHDA+VD3 post- group. VD3 also prevented the increased mitochondria swelling and H2O2 production and a decrease in SOD activity, respectively, in the 6-OHDA injured mitochondria. Also, VD3 supplementation protected the hemiparkinsonian brain from decreases in TH and DAT expressions and decreased the upregulation of mitochondrial markers, as VDAC 1 and Hsp60. In conclusion, VD3 showed neuroprotective actions on brain mitochondria injured by 6-OHDA and should stimulate translational studies focusing on its use as a therapeutic strategy for the treatment of neurodegenerative diseases as PD.
Regulation of VDAC by α-Synuclein (αSyn) is a rich and instructive example of protein-protein interactions catalyzed by a lipid membrane surface. αSyn, a peripheral membrane protein involved in Parkinson's disease pathology, is known to bind to membranes in a transient manner. αSyn's negatively charged C-terminal domain is then available to be electromechanically trapped by the VDAC β-barrel, a process that is observed in vitro as the reversible reduction of ion flow through a single voltage-biased VDAC nanopore. Binding of αSyn to the lipid bilayer is a prerequisite of the channel-protein interaction; surprisingly, however, we find that the strength of αSyn binding to the membrane does not correlate in any simple way with its efficiency of blocking VDAC, suggesting that the lipid-dependent conformations of the membrane-bound αSyn control the interaction. Quantitative models of the free energy landscape governing the capture and release processes allow us to discriminate between several αSyn (sub-) conformations on the membrane surface. These results, combined with known structural features of αSyn on anionic lipid membranes, point to a model in which the lipid composition determines the fraction of αSyn molecules for which the charged C terminal domain is constrained to be close, but not tightly bound, to the membrane surface and thus readily captured by VDAC nanopore. We speculate that changes in the mitochondrial membrane lipid composition may be key regulators of the αSyn-VDAC interaction and consequently of VDAC-facilitated transport of ions and metabolites in and out of mitochondria and, therefore, mitochondrial metabolism.
Tau is a neuronal microtubule-associated protein (MAP) that is involved in the regulation of axonal microtubule assembly. However, as a protein with intrinsically disordered regions (IDRs), tau also interacts with many other partners in addition to microtubules. Phosphorylation at selected sites modulates tau’s various intracellular interactions and regulates the properties of IDRs. In Alzheimer’s disease (AD) and other tauopathies, tau exhibits pathologically increased phosphorylation (hyperphosphorylation) at selected sites and aggregates into neurofibrillary tangles (NFTs). By bioinformatics means, we tested the hypothesis that the sequence of tau has changed during the vertebrate evolution in a way that novel interactions developed and also the phosphorylation pattern was affected, which made tau prone to the development of tauopathies. We report that distinct regions of tau show functional specialization in their molecular interactions. We found that tau’s amino-terminal region, which is involved in biological processes related to “membrane organization” and “regulation of apoptosis,” exhibited a strong evolutionary increase in protein disorder providing the basis for the development of novel interactions. We observed that the predicted phosphorylation sites have changed during evolution in a region-specific manner, and in some cases the overall number of phosphorylation sites increased owing to the formation of clusters of phosphorylatable residues. In contrast, disease-specific hyperphosphorylated sites remained highly conserved. The data indicate that novel, non-microtubule related tau interactions developed during evolution and suggest that the biological processes, which are mediated by these interactions, are of pathological relevance. Furthermore, the data indicate that predicted phosphorylation sites in some regions of tau, including a cluster of phosphorylatable residues in the alternatively spliced exon 2, have changed during evolution. In view of the “antagonistic pleiotropy hypothesis” it may be worth to take disease-associated phosphosites with low evolutionary conservation as relevant biomarkers into consideration.
Mitochondria-targeting peptides represent an emergent approach for cancer inhibition. Here supramolecular assemblies of novel amphiphilic cell-penetrating peptides for targeting cancer cells mitochondria are reported. The employed strategy aims at amplifying the apoptotic stimuli by weakening the mitochondrial VDAC1 (voltage-dependent anion channel-1)–hexokinase-II (HK-II) interaction. Peptide engineering is performed with the N-terminus of the HK-II protein, which binds to VDAC1. First, a designed positively-charged segment (pKV) is anchored to the specific 15 aminoacid sequence (MIASHLLAYFFTELN) to yield a cell-penetrating peptide (pHK-pKV). Second, a lipid chain (Pal) is conjugated to the N-terminus of pHK-pKV in order to enhance the intracellular delivery of the HK-II scaffold. The self-assembly properties of these two synthetic peptides are investigated by synchrotron small-angle X-ray scattering (BioSAXS) and cryogenic transmission electron (cryo-TEM) imaging, which evidence the formation of nanoassemblies of ellipsoid-like shapes. Circular dichroism (CD) spectroscopy demonstrates the induction of partial in the amphiphilic peptides. Confocal microscopy reveals the specific mitochondrial location of Pal-pHK-pKV assemblies in human non-small cell lung cancer (NSCLC) A549 cells.The cytotoxicity and apoptotic studies indicate the enhanced bioactivity of Pal-pHK-pKV self-assembled reservoirs, which cause massive A549 cell death with regard to pHK-pKV. Of significance, Pal-pHK-pKV treatment of non-cancerous NCM460 cells resulted in substantially lower cytotoxicity. The results demonstrate the potential of self-assembled lipo-peptide (HK-II-derived) conjugates as a promising strategy in cancer therapy.
VDAC3 is the least known isoform of the mammalian voltage-dependent anion selective channels of the outer mitochondrial membrane. It has been recently shown that cysteine residues of VDAC3 are found over-oxidized. The VDAC3 cysteine over-oxidation was associated with the oxidizing environment and the abundance of reactive oxygen species (ROS) in the intermembrane space. In this work, we have examined the role of VDAC3 in general pathogenic mechanisms at the basis of mitochondrial dysfunction and involving the mitochondrial quality control. Many of the diseases reported here, including cancer and viral infections, are often associated with significant changes in the intracellular redox state. In this sense, VDAC3 bearing oxidative modifications could become marker of the oxidative load in the mitochondria and part of the ROS signaling pathway.
It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington's disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application.
Apoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e., Parkinson's and Alzheimer's diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondria-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondria dysfunction, restoring dissipated mitochondria membrane potential, and thus cell energy and metabolism, decreasing ROS production, preventing detachment of hexokinase bound to mitochondria and preventing disruption of intracellular Ca2+ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis and mitochondria dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and points to VDAC1 as a promising target for therapeutic intervention.
Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies.
Approximately 20 % of familial Amyotrophic Lateral Sclerosis (ALS) is caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several conformation-restricted antibodies have been generated that specifically recognize misfolded SOD1 protein, and have been used as therapeutics in pre-clinical models. Misfolded SOD1 selectively associates with spinal cord mitochondria in SOD1 rodent models. Using the SOD1G93A rat model, we find that SOD1 conformational specific antibodies AMF7-63 and DSE2-3H1 labeled a fibrillar network concentrated in the anterior horn; while A5C3, B8H10, C4F6 and D3H5 labeled motor neurons as well as puncta in the neuropil. There is a time-dependent accumulation of misfolded SOD1 at the surface of spinal cord mitochondria with AMF7-63-labeled mitochondria having increased volume in contrast to a mitochondrial subset labeled with B8H10. In spinal cord homogenates and isolated mitochondria, AMF7-63, DSE2-3H1 and B8H10 detect misfolded SOD1 aggregates. SOD1 that lacks its metal cofactors has an increased affinity for naïve mitochondria and misfolded SOD1 antibodies B8H10 and DSE2-3H1 readily detect demetalated mutant and wild-type SOD1. Together, these data suggest that multiple non-native species of misfolded SOD1 may exist, some of which are associated with mitochondrial damage. Conformational antibodies are invaluable tools to identify and characterize the variation in misfolded SOD1 species with regards to biochemical characteristics and toxicity. This information is highly relevant to the further development of these reagents as therapeutics.
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In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.
The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is over-expressed in post-mortem brains of Alzheimer's disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid beta (Aβ). In this study, we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction.
The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.
Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Though recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point towards the long-sought-after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.
Background/Aims: Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has not been assigned to the VDAC3 isoform. Methods : Electrophysiological characterization of the recombinant protein purified and refolded was obtained after incorporation into planar lipid bilayers. Results: Here we report for the first time that recombinant hVDAC3, upon expression in E.coli and purification-refolding, shows a channel activity with a very small conductance (90 pS in 1 M KCl) with respect to the conductance of hVDAC1 (>3500 pS in 1 M KCl). Purified hVDAC3 allowed the passage of both chloride and gluconate anions and did not distinguish between potassium, sodium and calcium used as cations. In contrast to VDAC1, the channel was active also at transmembrane voltages higher than +/-40 mV and displayed a relatively high open probability even at +/-80 mV. hVDAC3 was only slightly voltage-dependent, displaying a tendency to adopt lower-conductance states at positive voltages applied to the cis chamber. In accordance with the small conductance of the pore, expression of hVDAC3 in a porin-less yeast strain allowed only partial recovery of the growth under non-permissive conditions. Conclusion: The observed electrophysiological properties of hVDAC3 are surprisingly different from the other isoforms and are discussed in relation to the proposed physiological role of the protein in mammalian cells. © 2014 S. Karger AG, Basel.
In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli.
In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and 3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the 2 and 3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish (zf) VDAC2. Further, we resolved the structure of zfVDAC2 at 2.8Å resolution revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ~20% in LDAO detergent micelles and ~40% in lipidic bicelles with the presence of some higher order oligomers in the latter. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s) of sporadic ALS, which accounts for ~90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1) protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a "toxic conformation" that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.
Voltage-Dependent Anion selective Channel maintains the permeability of the outer mitochondrial membrane and is relevant in bioenergetic metabolism and apoptosis. The structure of the protein was shown to be a β-barrel formed by 19 strands. The topology or sideness of the pore has been predicted with various approaches but a general consensus was never reached. This is an important issue since VDAC is considered receptor of Hexokinase and Bcl-2. We fused at VDAC1 C-terminus two tags separated by a caspase cleavage site. Activation in cellulo of caspases was used to eventually separate the two reporters. This experiment did not require the isolation of mitochondria and limited the possibility of outer membrane rupture due to similar procedures. Our results show that the C-terminus end of VDAC faces the mitochondrial inter-membrane space.
Neurodegenerative diseases are multifactorial debilitating disorders of the nervous system that affect approximately 30 millionindividuals worldwide. Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis diseases are the consequence of misfolding and dysfunctional trafficking of proteins. Beside that, mitochondrial dysfunction, oxidative stress, and/or environmental factors strongly associated with age have also been implicated in causing neurodegeneration. After years of intensive research, considerable evidence has accumulated that demonstrates an important role of these factors in the etiology of common neurodegenerative diseases. Despite the extensive efforts that have attempted to define the molecular mechanisms underlying neurodegeneration, many aspects of these pathologies remain elusive. However, in order to explore the therapeutic interventions directed towards treatment of neurodegenerative diseases, neuroscientists are now fully exploiting the data obtained from studies of these basic mechanisms that have gone awry. The novelty of these mechanisms represents a challenge to the identification of viable drug targets and biomarkers for early diagnosis of the diseases. In this paper, we are reviewing various aspects associated with the disease and the recent trends that may have an application for the treatment of the neurodegenerative disorders.
Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.
Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide pre-clinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies. © 2013 The Authors Aging Cell © 2013 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer. Found at the outer mitochondrial membrane, VDAC1 assumes a crucial position in the cell, controlling the metabolic cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC1 to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. As a metabolite transporter, VDAC1 contributes to the metabolic phenotype of cancer cells. This is reflected by VDAC1 over-expression in many cancer types, and by inhibition of tumor development upon silencing VDAC1 expression. Along with regulating cellular energy production and metabolism, VDAC1 is also a key protein in mitochondria-mediated apoptosis, participating in the release of apoptotic proteins and interacting with anti-apoptotic proteins. The involvement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space is discussed, as is VDAC1 oligomerization as an important step in apoptosis induction. VDAC also serves as an anchor point for mitochondria-interacting proteins, some of which are also highly expressed in many cancers, such as hexokinase (HK), Bcl2, and Bcl-xL. By binding to VDAC, HK provides both metabolic benefit and apoptosis-suppressive capacity that offers the cell a proliferative advantage and increases its resistance to chemotherapy. VDAC1-based peptides that bind specifically to HK, Bcl2, or Bcl-xL abolished the cell's abilities to bypass the apoptotic pathway. Moreover, these peptides promote cell death in a panel of genetically characterized cell lines derived from different human cancers. These and other functions point to VDAC1 as a rational target for the development of a new generation of therapeutics.
Background:
Parkin is recruited to defective mitochondria to promote degradation by an autophagy mechanism (mitophagy).
Results:
VDACs specifically interact with Parkin on defective mitochondria and are required for efficient targeting of Parkin to mitochondria and subsequent mitophagy.
Conclusion:
VDACs recruit Parkin to defective mitochondria.
Significance:
A novel mechanistic aspect of Parkin-dependent mitophagy is proposed that may be relevant to Parkinson disease. Mutations in the ubiquitin ligase Parkin and the serine/threonine kinase PINK1 can cause Parkinson disease. Both proteins function in the elimination of defective mitochondria by autophagy. In this process, activation of PINK1 mediates translocation of Parkin from the cytosol to mitochondria by an unknown mechanism. To better understand how Parkin is targeted to defective mitochondria, we purified affinity-tagged Parkin from mitochondria and identified Parkin-associated proteins by mass spectrometry. The three most abundant interacting proteins were the voltage-dependent anion channels 1, 2, and 3 (VDACs 1, 2, and 3), pore-forming proteins in the outer mitochondrial membrane. We demonstrate that Parkin specifically interacts with VDACs when the function of mitochondria is disrupted by treating cells with the proton uncoupler carbonyl cyanide p-chlorophenylhydrazone. In the absence of all three VDACs, the recruitment of Parkin to defective mitochondria and subsequent mitophagy are impaired. Each VDAC is sufficient to support Parkin recruitment and mitophagy, suggesting that VDACs can function redundantly. We hypothesize that VDACs serve as mitochondrial docking sites to recruit Parkin from the cytosol to defective mitochondria.
An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson's disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP)-mainly macroautophagy and chaperone-mediated autophagy-have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies.