Biology subjects

Bruegger, B.

Publications and source records attributed to Bruegger, B..

13 recordsLinked to original sources

bioRxiv2025

Secretion and signaling properties of Wnt11 and Wnt3 are determined by their N-terminus

Wnt proteins are evolutionarily conserved signaling molecules that control cell-cell communication in health and disease. While different Wnt proteins share a requirement for palmitoleoylation and the cargo receptor Evi/Wls for their secretion, they interact with diverse signaling receptors and co-receptors to elicit distinct outcomes in signal-receiving cells. The molecular mechanisms underlying this contrast between conserved post-translational modification and secretion and diverse receptor interactions remain poorly understood. Here, we demonstrate that Wnt11 maintains partial secretion capability in the absence of Evi/Wls. Unexpectedly, the secreted protein is also non-palmitoleoylated, yet retains its ability to activate downstream signaling in receiving cells through the Frizzled 6 receptor. We identify the N-terminus as the mediator of this unusual behavior, as Wnt3, which strictly requires Evi/Wls, becomes Evi/Wls-independent when its N-terminus is replaced with that of Wnt11. In different cell lines and in Xenopus laevis embryos, the chimeric Wnt11Nterm-Wnt3 protein recapitulates known Wnt11 phenotypes, demonstrating that the N-terminal portion of Wnt11 is sufficient to determine signaling specificity. Our findings reveal novel determinants of Wnt protein specificity and provide mechanistic insights into the molecular basis of Wnt secretion and downstream signaling.

cell biology↗

bioRxiv2024

T cell interaction partners of DHHC20

Reversible palmitoylation of proteins at cysteine residues represents a post-translational modification that can alter the cellular localization of proteins, change their distribution within lipid membranes or modulate their conformation and molecular interaction patterns. DHHC enzymes catalyze protein palmitoylation, while thioesterases or hydrolases can rapidly remove the acyl-chain. In human T cells, DHHC proteins have been shown to modify proteins that are involved in major signaling pathways such as Ca2+-signaling or kinase-dependent activation of transcription factors for cytokines. For DHHC20, a role in the palmitoylation of the Orai1 Ca2+-channel has been demonstrated, but otherwise its T cell interaction partners are largely unknown. Here, we show that recombinantly expressed DHHC20 robustly interacts with 28 proteins from Jurkat T cells, as shown by affinity enrichment combined with mass spectrometric analysis. We find a robust interaction between DHHC20 and the {beta}-subunit of the trimeric G protein Gs{beta}1{gamma}2, while the typically palmitoylated Ga subunit is not identified. Cross-linking mass spectrometry with purified DHHC20 and Gs{beta}1{gamma}2 then confirms a direct interaction between the {beta}1{gamma}2 domains and the enzyme, while rigid docking offers structural poses that are in agreement with the observed intermolecular cross-linking constraints. Thus, we suggest a model where the {beta}1{gamma}2 subunits of a trimeric G protein serve as a stable interaction partner of a DHHC enzyme, presumably acting as a landing platform for the G subunit that is subsequently palmitoylated by the enzyme.

biochemistry↗

bioRxiv2022

Simple, rapid, and sensitive quantification of dolichyl phosphates using phosphate methylation and reverse-phase liquid chromatography-high resolution mass spectrometry

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is crucial for all glycosylation processes, as these lipids serve as membrane-anchored building blocks that various types of glycosyltransferases use to generate complex post-translational modifications of proteins and lipids. Analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) has remained challenging due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane (TMSD)-dependent phosphate methylation and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa and human skin fibroblasts from steroid 5--reductase 3-congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to further our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

biochemistry↗

bioRxiv2022

IFITM3 blocks viral entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically impacts virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

molecular biology↗

bioRxiv2022

The Ebola virus VP40 matrix undergoes endosomal disassembly essential for membrane fusion

Ebola viruses (EBOVs) are filamentous particles, whose shape and stability are determined by the VP40 matrix. Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

microbiology↗

bioRxiv2022

The glycolipid GlcCer is recruited into the viral envelope to promote phenuivirus binding to host cells

Virus-receptor interactions largely contribute to the tropism and outcome of an infection. Here, we found that the glycolipid glucosylceramide (GlcCer) is a major component of Uukuniemi phenuivirus and allows viral binding to host cells. A lipidomic analysis with mass spectrometry revealed the lipidome of UUKV particles and indicated that GlcCer was enriched in both infected cells and viral particles. In addition, the infectivity of UUKV depended on the conversion of ceramide (Cer) into GlcCer in the Golgi network of producer cells. In contrast, depletion of GlcCer in virions profoundly impaired the attachment of UUKV and other related viruses to target cells. Furthermore, competing GlcCer ligands prevented virus binding to various cell types. Altogether, our results demonstrate that glycolipids are essential structural determinants of the virions necessary for virus attachment to host cells and have strong implications for future work on the identification of virus receptors.

microbiology↗

bioRxiv2022

The intra-mitochondrial contact site formed by Cqd1 and the Por1-Om14 complex modulates architecture and morphology of mitochondria

Mitochondria are essential organelles of eukaryotic cells that are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study we identified a novel mitochondrial contact site that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to the mitochondrial porin, Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain containing kinases). It was recently shown that Cqd1 in cooperation with Cqd2 controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 in addition is involved in the homeostasis of phospholipids and contributes to the maintenance of mitochondrial morphology and architecture. Summary statementHere, we show that the conserved mitochondrial inner membrane protein Cqd1 interacts with the outer membrane proteins Por1 and Om14. Additionally, we provide evidence that Cqd1 is important for maintaining mitochondrial homeostasis.

biochemistry↗

bioRxiv2021

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/ER modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here we identified a short amphipathic -helix (AH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AH is disrupted show NPC biogenesis and NE integrity defects, without impacting upon Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic -helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE during NPC biogenesis.

cell biology↗

bioRxiv2021

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Control of endoplasmic reticulum membrane biogenesis by regulators of lipid metabolism

Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the regulatory mechanisms that govern ER membrane biogenesis are largely unclear. Here, we conduct a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, which is called lipin in metazoa. Ice2 inhibits the conserved Nem1-Spo7 complex, which dephosphorylates and thus activates Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain ER homeostasis during ER stress. These findings establish inhibition of the lipin phosphatase complex as an important mechanism for controlling ER membrane biogenesis.

cell biology↗

bioRxiv2020

Oncogenic memory underlying minimal residual disease in breast cancer

Tumor relapse is responsible for most breast cancer related deaths1,2. The disease recurrence stems from treatment refractory cancer cells that persist as minimal residual disease (MRD) for years following initial therapy3. Yet, the molecular characteristics defining the malignancy of MRD remain elusive due to difficulties in observing these rare cells in patients or in model organisms. Here, we use a tractable organoid system and multi-omics analysis to show that the dormant MRD cells retain metabolic peculiarities reminiscent of the tumor state. While the MRD cells were distinct from both normal and tumor cells at a global transcriptomic level, their metabolomic and lipidomic profile markedly resembled that of the tumor state. The MRD cells particularly exhibited a de-regulated urea cycle and elevated glycolysis. We find the latter being crucial for their survival and could be selectively targeted using a small molecule inhibitor of glycolytic activity. We validated these metabolic peculiarities of the MRD cells in corresponding tissues obtained from the mouse model as well as in transcriptomic data from patients following neo-adjuvant therapy. Together, our results show that the treatment surviving MRD cells retain features of the tumor state over an extended period suggestive of an oncogenic memory. In accord, we found striking similarity in DNA methylation profiles between the tumor and the MRD cells. The distinction of MRD from normal breast cells comes as a surprise, considering their phenotypic similarity with regards to proliferation and polarized epithelial organization. The metabolic aberrances of the MRD cells offer a therapeutic opportunity towards tackling emergence of breast tumor recurrence in post-treatment care.

cancer biology↗

bioRxiv2019

Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.

biochemistry↗

bioRxiv2019

PINK1 Regulates Dopamine and Lipids at Mitochondria to Maintain Synapses and Neuronal Function

Mitochondrial dysfunction contributes to the pathogenesis of Parkinsons disease but it is not clear why inherent mitochondrial defects lead specifically to the death of dopaminergic neurons of the mid brain. PINK1 is mitochondrial kinase and PINK1 mutations cause early onset Parkinsons disease.\n\nWe found that in neuronal progenitors, PINK1 regulates mitochondrial morphology, mitochondrial contact to the endoplasmic reticulum (ER) and the phosphorylation of Miro1. A compensatory metabolic shift towards lipid synthesis provides mitochondria with the components needed for membrane renewal and oxidative phosphorylation, maintaining the mitochondrial network once mature.\n\nCholesterol is increased by loss of PINK1, promoting overall membrane rigidity. This alters the distribution of phosphorylated DAT at synapses and impairs dopamine uptake. PINK1 is required for the phosphorylation of tyrosine hydroxylase at Ser19, dopamine and calcium homeostasis and dopaminergic pacemaking.\n\nWe suggest a novel mechanism for PINK1 pathogenicity in Parkinsons disease in addition to but not exclusive of mitophagy. We also provide a basis for potential therapeutics by showing that low doses of the cholesterol depleting drug {beta}-cyclodextrin reverse PINK1-specific phenotypes.

neuroscience↗