Biology subjects

van der Goot, F. G.

Publications and source records attributed to van der Goot, F. G..

17 recordsLinked to original sources

bioRxiv2026

The luminal and cytosolic domains of CLIMP-63 cooperate to set ER sheet width

The endoplasmic reticulum (ER) synthesized and folds secreted and transmembrane protein in regions composed of flattened sheets with a defined luminal width of [~]50 nm. CLIMP-63 has been proposed to be the principal determinant of this spacing, but the molecular mechanism by which it controls luminal width remains unclear. The luminal domain of CLIMP-63 has been proposed to act as a fixed-length spacer through antiparallel coiled-coil dimerization between molecules on opposing membranes. Here we revise this model. Using systematic cysteine trapping across the luminal domain, we demonstrate that CLIMP-63 assembles into parallel trimers. An AlphaFold-guided screen of CLIMP-63 orthologs combined with cryo-electron microscopy reveals that the luminal domain forms an elongated trimeric rod with intrinsic conformational flexibility at hinge regions. This flexibility is functionally required: replacing the human luminal domain with a more rigid ortholog causes complete ER vacuolation. Higher-order assembly of CLIMP-63 trimers beyond the trimer depends on the cytosolic tail rather than on luminal trans interactions, and the tails role in higher order assembly is separable from its other architectural functions. Together, these findings show that luminal trimeric extension and cytosolic tail-mediated clustering cooperate to determine ER sheet width.

cell biology↗

bioRxiv2026

An overexpression platform reveals the functional diversity of human KRAB-Zinc Finger Proteins in maintaining cellular homeostasis

Kruppel associated box zinc finger proteins (KZFPs) form the largest family of transcriptional regulators in mammals, yet most remain uncharacterized. Here we established a scalable framework to probe KZFP function. An arrayed inducible overexpression screen of 366 human KZFPs in K562 cells identified factors that alter cellular proliferation, enabling functional prioritization. Integrative transcriptomic, chromatin and proteomic analyses revealed diverse mechanisms, including transposable element-linked repression (ZNF43), promoter proximal regulation (ZNF257), and SCAN domain dependent transcriptional activation (ZNF498/ZSCAN25 and ZNF18). These results highlight the functional diversity of KZFPs and provide a strategy for their annotation.

genetics↗

bioRxiv2026

Dynamic S-acylation controls CMG2 maturation, extracellular matrix regulation, and anthrax toxin entry

CMG2/ANTXR2 functions as a Collagen VI receptor and the primary portal for anthrax toxin entry. We find that CMG2 is regulated by ordered cycles of S-acylation and deacylation throughout its life cycle. Following synthesis in the endoplasmic reticulum, acylation by ZDHHC7 on two juxtamembrane cysteines protects folding intermediates from ER-associated degradation, resulting in a 5-fold increase in CMG2 biogenesis. The cytosolic thioesterase APT2 can remove these acyl chains, thereby controlling CMG2 levels. In the Golgi, CMG2 acylation by ZDHHC3 on a third cysteine to permit Arf6-dependent transport to the plasma membrane. At the cell surface, S-acylated CMG2 recruits APT2 in response to ligand binding, enabling release from the actin cytoskeleton and endocytosis. Accordingly, blocking APT2 suppresses the intracellular delivery of anthrax toxin, and inhibits CMG2-dependent Collagen VI degradation. These results define S-acylation-deacylation cycles as key regulators of CMG2 biogenesis and function, and highlight APT2 inhibition as a strategy to modulate CMG2 levels or prevent anthrax intoxication.

cell biology↗

bioRxiv2025

Antxr2-mediated fine-tuning of Collagen VI ensures skeletal muscle function

Tissue function relies on the extracellular matrix (ECM) that surrounds cells, providing structural and biochemical support. The complex ECM composition depends on an adequately tuned balance between the deposition and degradation of each of its components. Disequilibrium may cause disease, as observed for Collagen VI (COL6), for which mutations lead to muscular dystrophy. Here, we investigated the role of Anthrax Toxin Receptor 2 (ANTXR2/CMG2), a receptor that controls the turnover of COL6, in skeletal muscle. We show that ANTXR2 is mostly expressed by fibro-adipogenic precursors and that its deficiency in ANTXR2 null (Antxr2-/-) mice leads to a premature and irregular COL6 accumulation in intramuscular connective tissue. This results in tissue stiffening and gradual, non-functional muscle hypertrophy, marked by impaired locomotion and myopathic signs. Our findings further indicate that COL6 accretion drives these alterations, as revealed by Antxr2-/-::Col6a1-/- double knockout mice, highlighting the essential role of ANTXR2-mediated COL6 remodeling in maintaining ECM homeostasis and muscle functionality. SIGNIFICANCE STATEMENTRemodelling of the extracellular matrix (ECM) was long thought to rely almost exclusively on extracellular proteases. Increasing evidence, however, indicates that some ECM components may undergo intracellular degradation following receptor-mediated endocytosis, as we have found for Collagen VI (COL6). Here, we identify the COL6 receptor ANTXR2 as a critical regulator of ECM turnover in skeletal muscle.. When ANTXR2 is absent, COL6 builds up, followed by the accumulation of fibrillar collagens, without changes in gene expression. These alterations in muscle ECM lead to increased stiffness, myofiber defects and impaired locomotor activity. Our findings establish ANTXR2 as a key regulator in ECM remodelling, offering new insights into potential treatments for conditions associated with defective ECM remodeling, such as aging and congenital muscular dystrophies.

cell biology↗

bioRxiv2025

Anthrax toxin receptor 2 is the Receptor for Clostridium perfringens NetF: Structural Insights into Toxin Binding and Pore Formation

Hemolysin {beta}-pore-forming toxins ({beta}PFTs) are key virulence factors of Clostridium perfringens, associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium {beta}PFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 - as does the natural ANTXR2 ligand collagen type VI - NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin {beta}PFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses.

microbiology↗

bioRxiv2025

Molecular Regulation and Physiological Role of 1 GOLPH3-mediated Golgi retention

The Golgi complex serves as the central hub of the biosynthetic pathway, where anterograde and retrograde trafficking converge. How cargo and Golgi-resident proteins traverse this organelle has long been debated. Recent studies have identified a molecular machinery that sorts resident proteins into retrograde-directed COPI vesicles during cisternal maturation. Golgi phosphoprotein 3 (GOLPH3) is a key component of this system; however, its physiological relevance and regulatory mechanisms remain poorly defined. Here, we show that GOLPH3 depletion in mice disrupts both protein and lipid glycosylation, causes partially penetrant embryonic lethality, and severely impairs growth and bone mineralization. At the molecular level, we find that GOLPH3 is regulated by functionally antagonistic S-acylation events that control the topology of its membrane association. To mediate retrograde trafficking of Golgi-resident glycosyltransferases, GOLPH3 must bind their cytosolic tails. This occurs via a negatively charged surface region, which is correctly oriented only in one of the S-acylated GOLPH3 conformations. Together, these findings reveal a lipid-mediated regulatory mechanism for intra-Golgi trafficking and establish the critical role of GOLPH3 in vertebrate development.

cell biology↗

bioRxiv2025

Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

Membrane phospholipids are vulnerable to oxidative radicals, and uncontrolled lipid peroxidation affects cell viability. Cells have evolved quality control and defense mechanisms, of which the genetic regulation is not fully understood. Here, we identify what we have coined the Lipid Oxygen Radical Defense (LORD) pathway. It is epigenetically repressed by a complex comprising the KRAB-zinc finger protein ZNF354A, the scaffold protein KAP1/TRIM28, the histone methyltransferase SETDB1, and the transcriptional activator ATF2. Upon lipid peroxide accumulation, p38- and JNK-dependent phosphorylation of ATF2, KAP1/TRIM28, and ZNF354A leads to disassembly of the repressive complex, releasing ZNF354A from specific DNA loci and activating a network of protective genes, including NRF2 targets. The pathway affects the cellular sensitivity to oxidative stress and ferroptosis, revealing a novel layer of epigenetic control in lipid quality control and damage repair. This positions the LORD pathway as a promising therapeutic target for diseases linked to chronic inflammation, neurodegeneration and cancer.

cell biology↗

bioRxiv2025

Intestinal stem cell renewal controlled by capillary morphogenesis gene 2 following injury

Patients with the rare genetic disorder Hyaline Fibromatosis Syndrome (HFS) often succumb before 18 months of age due to severe diarrhea and protein-losing enteropathy. As HFS is caused by loss-of-function mutations in the gene encoding capillary morphogenesis gene 2 (CMG2), also known as Anthrax Toxin Receptor 2, these symptoms highlight a critical yet unclear role for CMG2 in the gut. Here, we demonstrate that CMG2 knockout mice exhibit normal colon morphology and no signs of inflammation until the chemical induction of colitis. In these conditions, the colons of knockout mice do not regenerate despite previously experiencing similarly severe colitis, due to an inability to replenish their intestinal stem cell pool. Specifically, CMG2 knockout impairs the transition from fetal-like to Lgr5+ adult stem cells, which is associated with a defect in {beta}-catenin nuclear translocation. Our findings suggest that CMG2 functions as a context-specific modulator of Wnt signaling, essential for replenishing the pool of intestinal stem cells following injury. This study provides new insights into the molecular mechanisms underlying protein-losing enteropathy in HFS and offers a broader understanding of fetal-like regenerative responses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/631493v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1a38f2corg.highwire.dtl.DTLVardef@10ca815org.highwire.dtl.DTLVardef@1ef3bddorg.highwire.dtl.DTLVardef@1433671_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

SARS-CoV-2 shifts transcription of host gene to increase Spike acylation and boost infectivity

SARS-CoV-2 infection requires Spike protein mediating fusion between the viral and cellular membranes. The fusogenic activity of Spike requires its post-translational lipid modification by host S-acyltransferases, predominantly ZDHHC20. Previous observations indicate that SARS-CoV-2 infection augments the S-acylation of Spike when compared to transfection. Here, we find that SARS-CoV-2 infection triggers a change in the transcriptional start site of the zddhc20 gene, both in cells and in an in vivo infection model, resulting in a 67-amino-acid-long N-terminally extended protein with 37-times higher Spike acylating activity, leading to enhanced viral infectivity. Furthermore, we observed the same induced transcriptional change in response to other challenges, such as chemically induced colitis, indicating that SARS-CoV-2 hijacks an existing cell damage response pathway to generate more infectious viruses.

cell biology↗

bioRxiv2022

Anthrax intoxication reveals that ER-Golgi membrane contact sites control the formation of plasma membrane lipid nanodomains

To promote infections, pathogens exploit host cell machineries including structural elements of the plasma membrane. Studying these interactions and identifying involved molecular players is an ideal way to gain insights into the fundamental biology of the host cell. Here, using the anthrax toxin, we screened a 1500-gene library of regulatory, cell surface, and membrane trafficking genes for their involvement in the intoxication process. We found that the ER-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the cell surface, an essential step for anthrax intoxication that depends on localization to cholesterol-rich lipid nanodomains. Further biochemical, morphological and mechanistic analyses showed that TMED2 and TMED10 are essential components of a multiprotein supercomplex that operates exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodelling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

cell biology↗

bioRxiv2021

S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S-acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

cell biology↗

bioRxiv2021

S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 is required for TCR assembly and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel had reduced mobility in lipids, accumulated in cholesterol-poor domains, formed tiny clusters, failed to reach the IS and unexpectedly also prevented TCR recruitment to the IS. Our results establish S-acylation as a critical regulator of ORAI1 channel assembly and function at the IS and reveal that local Ca2+ fluxes are required for TCR recruitment to the synapse.

biochemistry↗

bioRxiv2020

Molecular mode of action of an Acyl Protein thioesterase

Many biochemical reactions occur at the membrane interfaces. The proper control of these reactions requires spatially and temporally controlled recruitment of protein complexes. These assemblies are largely regulated by post-translational modifications and a frequent one is S-acylation, which consists of the addition of medium length acyl chains. Reversibility of this modification is ensured by acyl protein thioesterases (APTs), which are poorly understood enzymes. Using a combination of computational, structural, biochemical, and cellular approaches, we dissect the mode of action of a major cellular thioesterase, APT2 (LYPLA2). We show that for APT2 to encounter its targets, it must interact with membranes by two consecutive steps, the insertion of a hydrophobic loop and subsequent S-acylation by the ZDHHC3 or ZDHHC7 palmitoyltransferases. Once bound, APT2 deforms the lipid bilayer to extract the acyl chain bound to its substrate, capturing it in a hydrophobic pocket and allowing hydrolysis. Deacylation releases APT2, allowing it to bind to other membranes, but also renders it vulnerable to ubiquitination and proteasomal degradation. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated depalmitoylation throughout the endomembrane system.

cell biology↗

bioRxiv2019

Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20

Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton-channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by acyl-RAC and confirmed by 3H-palmitate labelling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co- localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the hemagglutinin-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs.

microbiology↗

bioRxiv2019

A toxic palmitoylation on Cdc42 drives a severe autoinflammatory syndrome

BackgroundAutoinflammatory diseases (AID) result from dysregulation of the first lines of innate immune responses. Recently, development of high throughput genome sequencing technology led to the rapid emergence of important knowledge in the genetic field. About 20 genes have been identified so far in monogenic forms of distinct AID. However, 70-90 % of patients with AID remain without genetic diagnosis.\n\nObjectiveWe report the identification and characterization of a mutation in the C-terminal region of the Rho GTPase Cdc42 in a patient presenting a severe autoinflammatory phenotype.\n\nMethodsWe have analyzed the consequences of the mutation on the subcellular localization of the Cdc42 protein using imaging techniques. Molecular studies were performed using proteomic and biochemical experiments to provide mechanistic bases of the observed defects. Functional assays were also conducted using flow cytometry and cytokine production measurements.\n\nResultsWe show that mutant Cdc42 is trapped in the Golgi apparatus due to the aberrant addition of a palmitate that both enhances the interaction of mutant Cdc42 with Golgi membranes and inhibit its extraction by GDP dissociation inhibitor (GDI), thus impairing its cytosol/membrane shuttling. At the functional level, mutant Cdc42 fails to sustain actin filaments polymerization and induces an exacerbated profile of pro-inflammatory cytokine production due to increased NF-{kappa}B activation.\n\nConclusionsOur study now provides a molecular explanation for mutations that have been identified recently in our AID patient and others in the C-terminal part of Cdc42. Mutations located in this region of Cdc42 impair the intracellular localization of Cdc42, preventing its interaction with the plasma membrane. Thus, our results definitively link mutations in the CDC42 gene to a complex immune-hemato-autoinflammatory phenotype in humans.

genetics↗

bioRxiv2018

The architecture of the endoplasmic reticulum is regulated by the reversible lipid modification of the shaping protein CLIMP-63

The endoplasmic reticulum (ER) has a complex morphology generated and maintained by membrane-shaping proteins and membrane energy minimization, though not much is known about how it is regulated. The architecture of this intracellular organelle is balanced between large, thin sheets that are densely packed in the perinuclear region and a connected network of branched, elongated tubules that extend throughout the cytoplasm. Sheet formation is known to involve the cytoskeleton-linking membrane protein 63 (CLIMP-63), though its regulation and the depth of its involvement remain unknown. Here we show that the post-translational modification of CLIMP-63 by the palmitoyltransferase ZDHHC6 controls the relative distribution of CLIMP-63 between the ER and the plasma membrane. By combining data-driven mathematical modeling, predictions, and experimental validation, we found that the attachment of a medium chain fatty acid, so-called S-palmitoylation, to the unique CLIMP-63 cytoplasmic cysteine residue drastically reduces its turnover rate, and thereby controls its abundance. Light microscopy and focused ion beam electron microcopy further revealed that enhanced CLIMP-63 palmitoylation leads to strong ER-sheet proliferation. Altogether, we show that ZDHHC6-mediated S-palmitoylation regulates the cellular localization of CLIMP-63, the morphology of the ER, and the interconversion of ER structural elements in mammalian cells through its action on the CLIMP-63 protein.\n\nSignificance StatementEukaryotic cells subcompartmentalize their various functions into organelles, the shape of each being specific and necessary for its proper role. However, how these shapes are generated and controlled is poorly understood. The endoplasmic reticulum is the largest membrane-bound intracellular compartment, accounting for more than 50% of all cellular membranes. We found that the shape and quantity of its sheet-like structures are controlled by a specific protein, cytoskeleton-linking membrane protein 63, through the acquisition of a lipid chain attached by an enzyme called ZDHHC6. Thus, by modifying the ZDHHC6 amounts, a cell can control the shape of its ER. The modeling and prediction technique used herein also provides a method for studying the interconnected function of other post-translational modifications in organelles.

cell biology↗

bioRxiv2017

Identification and dynamics of the DHHC16-DHHC6 palmitoylation cascade

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC family of palmitoyltransferases is very limited. Here we show that mammalian DHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, DHHC16, revealing the first palmitoylation cascade. Combination of site specific mutagenesis of the three DHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the 8 differentially palmitoylated DHHC6 species. We found that species rapidly interconvert through the action of DHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its DHHC6 activity.

cell biology↗