Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities.

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels.

Retrieving functional pathways of biomolecules from single-particle snapshots.

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important.

Structure of the cell-binding component of the binary toxin reveals a di-heptamer macromolecular assembly.

Targeting infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent strains often have a binary toxin termed the toxin, in addition to the enterotoxins TsdA and TsdB. The toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb.

Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown.

Preserving Insulin Secretion in Diabetes by Inhibiting VDAC1 Overexpression and Surface Translocation in β Cells.

Type 2 diabetes (T2D) develops after years of prediabetes during which high glucose (glucotoxicity) impairs insulin secretion. We report that the ATP-conducting mitochondrial outer membrane voltage-dependent anion channel-1 (VDAC1) is upregulated in islets from T2D and non-diabetic organ donors under glucotoxic conditions. This is caused by a glucotoxicity-induced transcriptional program, triggered during years of prediabetes with suboptimal blood glucose control.

Novel Compounds Targeting the Mitochondrial Protein VDAC1 Inhibit Apoptosis and Protect against Mitochondrial Dysfunction.

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.

VDAC1-interacting anion transport inhibitors inhibit VDAC1 oligomerization and apoptosis.

Mitochondria-mediated apoptosis involves pro-apoptotic protein release from the mitochondria to the cytosol, triggering apoptosis. However, the mechanisms by which apoptotic initiators cross the outer mitochondrial membrane (OMM) remain unclear. The voltage-dependent anion channel 1 (VDAC1), an OMM protein, is central to mitochondria-mediated apoptosis.

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

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 overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ).

A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells.

The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release.

The mitochondrial voltage-dependent anion channel 1 in tumor cells.

VDAC1 is found at the crossroads of metabolic and survival pathways. VDAC1 controls metabolic cross-talk between mitochondria and the rest of the cell by allowing the influx and efflux of metabolites, ions, nucleotides, Ca2+ and more. The location of VDAC1 at the outer mitochondrial membrane also enables its interaction with proteins that mediate and regulate the integration of mitochondrial functions with cellular activities.

Ca(2+)-mediated regulation of VDAC1 expression levels is associated with cell death induction.

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release.

Assays of mitochondrial Ca2+ transport and Ca2+ efflux via the MPTP.

Studying Ca(2+) transport in mitochondria in connection with energy production, as well as cell death, is of great importance. Ca(2+) activates several key enzymes in the mitochondrial matrix to enhance ATP production. This provides an important mechanism for synchronizing energy production with the energy demands of Ca(2+)-activated processes, such as contraction, allowing important feedback effects to help shape cytosolic Ca(2+) signals.

Assay of Ca2+ transport by VDAC1 reconstituted into liposomes.

Ca(2+) permeability mediated by voltage-dependent anion-selective channel protein 1 (VDAC1) can be tested by reconstitution of purified VDAC1 into liposomes. Here, we describe a setup for this membranal system, which has been used to study the transport activity of various transporters, including VDAC1, and allows detection of the passage of molecules across the lipid bilayer. Despite the disadvantage of needing radiolabeled molecules, this system is highly desirable when the transport properties of noncharged molecules and/or active transporters are studied.

Reconstitution of purified VDAC1 into a lipid bilayer and recording of channel conductance.

The functional properties of purified voltage-dependent anion-selective channel protein 1 (VDAC1) have been examined in reconstituted systems based on artificially prepared phospholipid bilayers. The most widespread method for the characterization of the pore-forming activity of the mitochondrial VDAC1 protein requires reconstitution of the channel activity into a planar lipid bilayer (PLB) that separates two aqueous compartments. This system is able to produce a refined and large set of information on channel activity.

Purification of VDAC1 from rat liver mitochondria.

To make biophysical measurements of functions such as the pore-forming activity of mitochondrial voltage-dependent anion-selective channel protein 1 (VDAC1), it is first necessary to obtain a source of purified VDAC protein. In this protocol, we present a method for obtaining rat liver mitochondria as a source of VDAC1 and then describe two methods, one using a nonionic detergent and the other an ionic detergent, for purifying VDAC1 from the isolated mitochondria. This produces a source of VDAC1 proteins that are suitable for subsequent incorporation into artificially prepared phospholipid bilayers.

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells.

The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis.

The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca(2+), and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release.

New fluorescent reagents specific for Ca(2+)-binding proteins.

Ca(2+) carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca(2+)-dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru.

Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein.

The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity.

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins.

Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed.

Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae.

VDAC, a multi-functional mitochondrial protein as a pharmacological target.

Regulation of mitochondrial physiology requires an efficient exchange of molecules between mitochondria and the cytoplasm via the outer mitochondrial membrane (OMM). The voltage-dependent anion channel (VDAC) lies in the OMM and forms a common pathway for the exchange of metabolites between the mitochondria and the cytosol, thus playing a crucial role in the regulation of metabolic and energetic functions of mitochondria. VDAC is also recognized to function in mitochondria-mediated apoptosis and in apoptosis regulation via interaction with anti-apoptotic proteins, namely members of Bcl-2 family, and the pro-survival protein, hexokinase, overexpressed in many cancer types.