Biology subjects

Holthuis, J. C. M.

Publications and source records attributed to Holthuis, J. C. M..

11 recordsLinked to original sources

bioRxiv2026

Ceramides disrupt hexokinase HKI-VDAC complex assembly and modulate VDAC tilting

Ceramides bind voltage-dependent anion channels (VDACs) to promote mitochondrial apoptosis. However, the underlying mechanism is unclear. Ceramide binding relies on a charged, bilayer-facing glutamate that also controls complex formation between VDACs and pro-survival hexokinase HKI, suggesting that ceramides may exert their pro-apoptotic activity by displacing HKI from VDACs. Here, we challenged this model using coarse-grain molecular dynamics simulations. We show that ceramides impair VDAC-HKI complex assembly, but not via direct competition with HKI for binding to the crucial glutamate. Instead, we find that ceramides disrupt the membrane thinning capacity of polar channel residues adjacent to the glutamate, thereby congesting a low-energy passageway used by HKI for efficient complex formation. We also demonstrate that VDACs are tilted in the membrane and that ceramides act as modulators of channel tilt by neutralizing the hydrophobic mismatch imposed by the polar channel residues. We postulate that this ceramide action further modulates the oligomerization propensity of VDACs. Altogether, our data reveal a mechanistic basis for how ceramides may trigger apoptotic cell death.

biophysics↗

bioRxiv2025

Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

Ceramides are essential but potentially toxic intermediates of sphingolipid metabolism that can act directly on mitochondria to trigger apoptotic cell death, but the underlying mechanism is unclear. While one model postulates that ceramides form stable channels in the outer mitochondrial membrane that induce cell death through direct release of cytochrome c, an alternative view is that ceramides self-assemble into microdomains that facilitate membrane insertion and oligomerization of the pro-apoptotic Bcl-2 protein Bax into cytochrome c-conducting pores. To challenge these models, we here analyzed the influence of ceramides in combination with recombinant Bax on the leakiness of model membranes. We show that ceramides on their own are unable to support membrane passage of even the smallest fluorescence markers. Moreover, we find that ceramides cannot substitute for cardiolipin in facilitating membrane recruitment of Bax and its subsequent assembly into functional pores. Our data argue against a direct role of ceramides in apoptotic pore formation and indicate that the mechanism by which ceramides initiate permeabilization of the outer mitochondrial membrane is independent of ceramide channels or ceramide acting autonomously as translocation platform for Bax.

biochemistry↗

bioRxiv2025

Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) regulates the metabolic fate of glucose and promotes cell survival in hyperglycolytic tumors. Computer simulations indicated that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged bilayer-facing glutamate on the outer wall of VDACs. Protonation of this residue blocks complex formation in silico, explaining the release of HKI from mitochondria observed upon cytosolic acidification. To validate these findings, we here developed an in vitro assay for interrogating HKI binding to VDAC1 reconstituted in vesicles captured on a functionalized surface. TIRF-based quantitative interaction studies with a fluorescent peptide comprising the N-terminal -helix of HKI revealed a crucial role of the bilayer-facing glutamate in complex assembly and recapitulated an exquisite sensitivity of HKI-VDAC binding to fluctuations in pH. Our assay opens up important opportunities to investigate the impact of membrane environment on HKI-VDAC complex assembly and may benefit the development of therapeutics that target pathogenic imbalances in this process.

biophysics↗

bioRxiv2025

VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair

Perturbations in lysosome integrity are tightly linked to neurological disorders and ageing, but the underlying pathogenic mechanisms are incompletely understood. Using an unbiased proteomic approach, we here identified the bridge-like lipid transport protein VPS13C/PARK23 as a key component of a global early response pathway to lysosome damage. VPS13C readily binds lysosomes under mechanical or osmotic tension in anticipation of membrane lesions. The latter trigger a conformational change in the proteins C-terminus, involving its ATG2C domain acting as sensor of damage-induced lipid packing defects. We show that ER-lysosome contacts formed by VPS13C provide critical binding platforms for OSBP/ORPs to enable efficient ER wrapping of damaged lysosomes. A chemical approach to assess directional ER-to-lysosome lipid transport revealed that VPS13C is essential for large-scale lipid delivery to acutely damaged lysosomes to facilitate their repair. Our findings offer new mechanistic insights into how loss-of-function mutations in VPS13C may enhance the risk of Parkinsons disease.

cell biology↗

bioRxiv2024

Optical control of sphingolipid biosynthesis using photoswitchable sphingosines

Sphingolipid metabolism comprises a complex interconnected web of enzymes, metabolites and modes of regulation that influence a wide range of cellular and physiological processes. Deciphering the biological relevance of this network is challenging as numerous intermediates of sphingolipid metabolism are short-lived molecules with often opposing biological activities. Here, we introduce clickable, azobenzene-containing sphingosines, termed caSphs, as light-sensitive substrates for sphingolipid biosynthesis. Photo-isomerization of the azobenzene moiety enables reversible switching between a straight trans- and curved cis-form of the lipids hydrocarbon tail. Combining in vitro enzyme assays with metabolic labeling studies, we demonstrate that trans-to-cis isomerization of caSphs profoundly stimulates their metabolic conversion by ceramide synthases and downstream sphingomyelin synthases. These light-induced changes in sphingolipid production rates are acute, reversible, and can be implemented with great efficiency in living cells. Our findings establish caSphs as versatile tools with unprecedented opportunities to manipulate sphingolipid biosynthesis and function with the spatiotemporal precision of light.

biochemistry↗

bioRxiv2024

Hexokinase-I directly binds to a charged membrane-buried glutamate of mitochondrial VDAC1 and VDAC2

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) has far-reaching physiological implications. However, the structural basis of this interaction is unclear. Combining computer simulations with experiments in cells, we here show that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged membrane-buried glutamate on the outer wall of VDAC1 and VDAC2. Protonation of this residue blocks complex formation in silico while acidification of the cytosol causes a reversable release of HKI from mitochondria. Membrane insertion of HKI occurs adjacent to the bilayer-facing glutamate where a pair of polar channel residues mediates a marked thinning of the cytoplasmic leaflet. Disrupting the membrane thinning capacity of VDAC1 dramatically impairs its ability to bind HKI in silico and in cells. Our data reveal key topological and mechanistic insights into HKI-VDAC complex assembly that may benefit the development of therapeutics to counter pathogenic imbalances in this process.

biochemistry↗

bioRxiv2023

Systematic analysis of the sphingomyelin synthase family in C. elegans

Sphingomyelin (SM) is a major component of mammalian cell membranes and particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is catalyzed by SM synthases SMS1 and SMS2, which interconvert phosphatidylcholine and ceramide to diacylglycerol and SM in the Golgi and at the plasma membrane, respectively. As the lipids participating in this reaction fulfill both structural and signaling functions, SMS enzymes have considerable potential to influence diverse important cellular processes. The nematode Caenorhabditis elegans is an attractive model for studying both animal development and human disease. The organism contains five SMS homologues but none of these have been characterized in any detail. Here, we carried out the first systematic analysis of SMS family members in C. elegans. Using heterologous expression systems, genetic ablation, metabolic labeling and lipidome analyses, we show that C. elegans harbors at least three distinct SM synthases and one ceramide phosphoethanolamine (CPE) synthase. Moreover, C. elegans SMS family members have partially overlapping but also unique subcellular distributions and together occupy all principal compartments of the secretory pathway. Our findings shed light on crucial aspects of sphingolipid metabolism in a valuable animal model and opens avenues for exploring the role of SM and its metabolic intermediates in organismal development.

molecular biology↗

bioRxiv2023

Complex Sphingolipid Profiling and Identification of an Inositol Phosphorylceramide Synthase in Dictyostelium discoideum

Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries.

cell biology↗

bioRxiv2022

Mitochondrial phospholipid import mediated by VDAC, a dimeric beta barrel scramblase

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identified the voltage-dependent ion channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a new class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act by an unprecedented mechanism to import lipids into mitochondria.

biochemistry↗

bioRxiv2022

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

cell biology↗

bioRxiv2021

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

cell biology↗