Biology subjects

Rostovtseva, T. K.

Publications and source records attributed to Rostovtseva, T. K..

8 recordsLinked to original sources

bioRxiv2026

Towards VDAC pharmacology: Developing small-peptide channel inhibitors inspired by nature

The Voltage-Dependent Anion Channel (VDAC) forms a unique pathway across the mitochondrial outer membrane for small ions and water-soluble metabolites, such as ATP and ADP. Despite VDAC's involvement in both normal regulation of respiration and in a wide variety of mitochondria-associated pathologies, there are, surprisingly, no known FDA-approved pharmacological agents targeting VDAC. Earlier, we have shown that VDAC can be regulated through its interactions with well-known cytosolic proteins--dimeric tubulin and -synuclein (Syn)--that are able to reversibly block this channel at nanomolar concentrations. We hypothesized that the architecture of a prospective VDAC inhibitor should fulfill two main requirements: possess a negatively charged disordered polypeptide C- or N-terminus as a pore blocking domain and feature a mitochondrial membrane-binding domain. Using Syn as a template, we engineered three peptides and demonstrated that they all characteristically block VDAC reconstituted into planar lipid membranes in a controllable manner, thereby mimicking the actions of tubulin and Syn. The VDAC blockage times induced by the synthetic peptides are comparable to Syn-induced blockages, though the half-effect solution concentration for synthetic blockers is a hundred times higher than that of Syn. Our work can be considered a proof-of-principle study that informs future development of mitochondria-targeting pharmacological tools.

biophysics↗

bioRxiv2026

So similar, yet so different: α-synuclein recognition of the isoform-specific VDAC protein-lipid interface

In mammals, VDAC exists as three isoforms--VDAC1, VDAC2, and VDAC3--each characterized by a distinct tissue-dependent distribution and physiologic role. Isoform-specific knockout studies in cells and mice demonstrate distinct physiological roles for each VDAC isoform. The isoforms share about 70% sequence homology and a conserved {beta}-barrel structure and, when reconstituted into planar lipid membranes, form similar highly conductive, anion-selective, voltage-gated channels. A natural question, then, is how relatively minor differences in isoform sequences lead to striking differences in their interactions with protein partners and, consequently, in their physiological functions. To address this question, we used a small, intrinsically disordered cytosolic protein -synuclein (Syn) as a sensitive molecular probe. A comparative study with recombinant VDACs reconstituted into planar lipid membranes shows that, depending on the isoform, the Syn-induced blockage exhibits up to 100-fold differences in on-rates and equilibrium constants. The isoforms also display distinct translocation regimes for Syn, as evidenced by altered dependences of the off-rates on the applied voltage. Deletion of cysteine residues has a significant effect on Syn binding. Combined with corresponding data obtained for VDAC2 substates--which show an order of magnitude variation in on-rates while maintaining the conserved equilibrium constants--and the results of molecular dynamics simulations, these findings point to a previously unrecognized step in VDAC inhibition by Syn: an isoform-specific Syn interaction with the VDAC protein-lipid interface. This finding opens a new paradigm for understanding VDAC isoform functions and developing isoform-specific therapeutics.

biophysics↗

bioRxiv2025

A Membrane-Disruptive Action of VBIT-4 Challenges Its Role as a Widely Used VDAC Oligomerization Inhibitor

Voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane and a key regulator of metabolite exchange and mitochondrial physiology. Its oligomerization has been proposed to control processes such as mitochondrial DNA release and membrane remodeling, yet the underlying mechanisms remain poorly defined. VBIT-4 has been widely used over the last decade as a putative inhibitor of VDAC1 oligomerization, despite limited mechanistic validation. Here, using high-speed atomic force microscopy (AFM), we directly visualized VDAC1 assemblies in lipid membranes and examined the effect of VBIT-4. Unexpectedly, VBIT-4 induced membrane defects and permeabilization at micromolar concentrations, independently of VDAC1. Quantitative AFM analysis further shows that VBIT-4 does not alter VDAC1 cluster organization. Complementary electrophysiology, microscale thermophoresis, and coarse-grained molecular dynamics demonstrate that VBIT-4 partitions into lipid bilayers, increases membrane permeability, and destabilizes membrane structure, without detectable effects on VDAC1 channel properties or assemblies. Consistent with this mechanism, VBIT-4 induces VDAC1-independent cytotoxicity in HeLa cells at concentrations above 10 {micro}M. Together, these results demonstrate that VBIT-4 does not act as a specific inhibitor of VDAC1 oligomerization but instead functions as a membrane-active compound. This work provides a revised framework for interpreting studies using VBIT-4 and highlights the importance of systematically assessing drug-membrane interactions when targeting membrane proteins.

biochemistry↗

bioRxiv2025

VDAC isoforms play distinct roles in modulating metabolism and maintaining mitochondrial function

The Voltage Dependent Anion Channel (VDAC) is the most ubiquitous protein in the mitochondrial outer membrane. This channel facilitates the flux of water-soluble metabolites and ions like calcium across the mitochondrial outer membrane. Beyond this canonical role, VDAC has been implicated, through interactions with protein partners, in several cellular processes such as apoptosis, calcium signaling, and lipid metabolism. There are three VDAC isoforms in mammalian cells, VDAC 1, 2, and 3, with varying tissue-specific expression profiles. From a biophysical standpoint, all three isoforms can conduct metabolites and ions with similar efficiency. However, isoform knockouts (KOs) in mice lead to distinct phenotypes, which may be due to differences in VDAC isoform interactions with partner proteins. To understand the functional role of each VDAC isoform within a single cell type, we created functional KOs of each isoform in HeLa cells and performed a comparative study of their metabolic activity and proteomics. We found that each isoform KO alters the proteome differently, with VDAC3 KO dramatically downregulating key members of the electron transport chain (ETC) while shifting the mitochondria into a glutamine-dependent state. Importantly, this unexpected dependence of mitochondrial function on the VDAC3 isoform is not compensated by the more ubiquitously expressed VDAC1 and VDAC2 isoforms. In contrast, VDAC2 KO did not affect respiration but upregulated ETC components and decreased key enzymes in the glutamine metabolic pathway. VDAC1 KO specifically reduced glycolytic activity linked to decreased hexokinase localization to mitochondria. These results reveal non-redundant roles of VDAC isoforms in cancer cell metabolic adaptability.

cell biology↗

bioRxiv2024

Conformational plasticity of mitochondrial VDAC2 controls the kinetics of its interaction with cytosolic proteins

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 VDAC2s 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 -synuclein (Syn) - 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 Syn-VDAC2 interaction but proportionally lower blockage times. This gives the same equilibrium constant for all substates, thus resulting in the same affinity of the Syn-VDAC2 interaction. The pronounced difference is limited to the kinetic parameters, suggesting that once the Syn molecule is captured, its physical state and free energy are the same for all substates. These striking results imply that the Syn 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.

biophysics↗

bioRxiv2022

Restricting α-Synuclein Transport into Mitochondria by Inhibition of α-Synuclein-VDAC Complexation as a Potential Therapeutic Target for Parkinson's Disease Treatment

Involvement of alpha-synuclein (Syn) in Parkinsons disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently we established that Syn can regulate mitochondrial function by voltage-activated complexation with the Voltage-Dependent Anion Channel (VDAC) of the outer mitochondrial membrane. When complexed with Syn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, Syn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of Syn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on the Syn membrane binding, and hence on Syn complex formation with VDAC and translocation through it. In electrophysiology experiments, addition of HK2p at micromolar concentrations to the same side of the membrane as Syn results in dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of Syn from lipid membranes. Experiments with live HeLa cells using proximity ligation assay confirmed that HK2p impedes Syn entry into mitochondria. Our results demonstrate that it is possible to regulate Syn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate Syn mitochondrial toxicity in PD and other synucleinopathies.

biophysics↗

bioRxiv2021

MspA Porin as a Local Nanopore Probe for Membrane-bound Proteins

Nanopore sensing is based on detection and analysis of nanopore transient conductance changes induced by analyte capture. We have recently shown that -Synuclein (Syn), an intrinsically disordered, membrane-active, neuronal protein implicated in Parkinson disease, can be reversibly captured by the VDAC nanopore. The capture process is a highly voltage dependent complexation of the two proteins where transmembrane potential drives the polyanionic C-terminal domain of Syn into VDAC--exactly the mechanism by which generic nanopore-based interrogation of proteins and polynucleotides proceeds. The complex formation, and the motion of Syn in the nanopore, thus may be expected to be only indirectly dependent on the pore identity. Here, we confirm this prediction by demonstrating that when VDAC is replaced with a different transmembrane pore, the engineered mycobacterial porin M2MspA, all the qualitative features of the Syn/nanopore interaction are preserved. The rate of Syn capture by M2MspA rises exponentially with the applied field, while the residence time displays a crossover behavior, indicating that at voltages >50 mV M2MspA-bound Syn largely undergoes translocation to the other side of the membrane. The translocation is directly confirmed using the selectivity tag method, in which the polyanionic C-terminal and neutral N-terminal regions of Syn alter the selectivity of the M2MspA channel differently, allowing direct discrimination of translocation vs retraction for single Syn molecules. We thus prove that the physical model of the motion of disordered protein chains in the nanopore confinement and the selectivity tag technique are not limited to VDAC but are broadly applicable to nanopore-based protein detection, analysis, and separation technologies.

biophysics↗

bioRxiv2020

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport

When the Parkinsons disease (PD) related neuronal protein, alpha-synuclein (Syn), is added to the reconstituted mitochondrial voltage-dependent anion channel (VDAC), it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, Syn reverses the channels selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by Syn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. These findings reveal a new regulatory role of Syn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

biophysics↗