A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC

Abstract

The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes, but they do not predominate in detergent-solubilized VDAC samples. In this study, a cholesterol analog, cholesteryl-hemisuccinate (CHS), was shown to induce the formation of detergent-soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation, and size-exclusion chromatography small angle x-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (K d of 27 5 6 mM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.

Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5′ Terminal Regions

“…To determine the binding affinity of a previously uncharacterized interaction system containing DDX3X 132–607 and 5′ TR of JEV, we employed microscale thermophoresis (MST) as performed previously [ 71 , 72 ]. DDX3X 132–607 was titrated against the constant concentration of the fluorescent ncRNAs (non-coding RNAs) to determine their dissociation constant (K d ).…”

A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro

“…Structural predictions for VDACs from multiple sources (Bay et al, 2012) support this model. A comparison of the VDAC isolated in decyl-maltoside (DM)solubilized mitochondrial membranes of Neurospora crassa and the equivalent protein expressed in E. coli and folded in the same detergent support the equivalence of the native and recombinant forms (Ferens et al, 2019).…”

“…The expression, folding, and analysis of WT-VDAC and VDAC-C were carried out as described (Ferens et al, 2019) (see Supplementary File 1). Circular dichroism (CD), size exclusion chromatography (SEC), and AUC experiments were carried out as performed in previous studies (Ferens et al, 2019).…”

“…The full-length N. crassa VDAC oligomerizes in sterolcontaining detergents (Ferens et al, 2019). To explore the ability of the VDAC-C β-barrels to interact with each other, the protein was over-expressed in E. coli.…”

“…The self-association of detergent-solubilized VDAC has been observed in chemically cross-linked samples (Zalk et al, 2005;Malia and Wagner, 2007). In the absence of a cross-linker, N. crassa VDAC, expressed in E. coli and folded in DM, assembles into higher order structures only in the presence of sterol; models of dimers and of hexamers that resembled those observed by (Mannella et al, 1986) were obtained from a combination of SEC, alone or in combination with small-angle scattering (SEC-SAX) and analytical ultracentrifugation (AUC) (Ferens et al, 2019).…”