Glucosylceramide in bunyavirus particles is essential for virus binding to host cells.

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase.

Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and nonsterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.

Metabolic switch from fatty acid oxidation to glycolysis in knock-in mouse model of Barth syndrome.

Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease-specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid-driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell-derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V . Treatment of mutant cells with AMPK activator reestablishes fatty acid-driven OXPHOS and protects mice against cardiac dysfunction.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

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

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). 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.

The UbiB family member Cqd1 forms a novel membrane contact site in mitochondria.

Mitochondria are essential organelles of eukaryotic cells and 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 in Saccharomyces cerevisiae that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to what is found for 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 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes.

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

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but 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 and SARS-CoV-2 replication and proper DMV formation. 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 and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper 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. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

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

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (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 α-helix (AαH) 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 AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting 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 AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.

Metabolic memory underlying minimal residual disease in breast cancer.

Tumor relapse from treatment-resistant cells (minimal residual disease, MRD) underlies most breast cancer-related deaths. Yet, the molecular characteristics defining their malignancy have largely remained elusive. Here, we integrated multi-omics data from a tractable organoid system with a metabolic modeling approach to uncover the metabolic and regulatory idiosyncrasies of the MRD. We find that the resistant cells, despite their non-proliferative phenotype and the absence of oncogenic signaling, feature increased glycolysis and activity of certain urea cycle enzyme reminiscent of the tumor. This metabolic distinctiveness was also evident in a mouse model and in transcriptomic data from patients following neo-adjuvant therapy. We further identified a marked similarity in DNA methylation profiles between tumor and residual cells. Taken together, our data reveal a metabolic and epigenetic memory of the treatment-resistant cells. We further demonstrate that the memorized elevated glycolysis in MRD is crucial for their survival and can be targeted using a small-molecule inhibitor without impacting normal cells. The metabolic aberrances of MRD thus offer new therapeutic opportunities for post-treatment care to prevent breast tumor recurrence.

Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex.

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 underlying regulatory mechanisms are largely unclear. Here, we describe 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, called lipin in metazoa. Specifically, Ice2 inhibits the conserved Nem1-Spo7 complex and thus suppresses the dephosphorylation and activation of Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain cell homeostasis during ER stress. These findings establish the control of the lipin phosphatase complex as an important mechanism for regulating ER membrane biogenesis.

Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins.

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites.

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.

TMEM147 interacts with lamin B receptor, regulates its localization and levels, and affects cholesterol homeostasis.

The structurally and functionally complex endoplasmic reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147, and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of TMEM147 drastically reduces the level of lamin B receptor (LBR) at the inner nuclear membrane and results in mistargeting of LBR to the ER. LBR possesses a modular structure and corresponding bifunctionality, acting in heterochromatin organization via its N-terminus and in cholesterol biosynthesis via its sterol-reductase C-terminal domain. We show that TMEM147 physically interacts with LBR, and that the C-terminus of LBR is essential for their functional interaction. We find that TMEM147 also physically interacts with the key sterol reductase DHCR7, which is involved in cholesterol biosynthesis. Similar to what was seen for LBR, TMEM147 downregulation results in a sharp decline of DHCR protein levels and co-ordinate transcriptional decreases of LBR and DHCR7 expression. Consistent with this, lipidomic analysis upon TMEM147 silencing identified changes in cellular cholesterol levels, cholesteryl ester levels and profile, and in cellular cholesterol uptake, raising the possibility that TMEM147 is an important new regulator of cholesterol homeostasis in cells.This article has an associated First Person interview with the first author of the paper.

Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

A novel neurodegenerative spectrum disorder in patients with MLKL deficiency.

Mixed lineage kinase domain-like (MLKL) is the main executor of necroptosis, an inflammatory form of programmed cell death. Necroptosis is implicated in combating infections, but also in contributing to numerous other clinical conditions, including cardiovascular diseases and neurodegenerative disorders. Inhibition of necroptosis is therefore of therapeutic interest. Here we report two siblings both of whom over the course of 35 years developed a similar progressive, neurodegenerative spectrum disorder characterized by paresis, ataxia and dysarthria. Magnetic resonance imaging of their central nervous system (CNS) revealed severe global cerebral volume loss and atrophy of the cerebellum and brainstem. These brothers are homozygous for a rare haplotype identified by whole genome sequencing carrying a frameshift variant in MLKL, as well as an in-frame deletion of one amino acid in the adjacent fatty acid 2-hydroxylase (FA2H) gene. Functional studies of patient-derived primary cells demonstrated that the variant in MLKL leads to a deficiency of MLKL protein resulting in impairment of necroptosis. Conversely, shotgun lipidomic analysis of the variant in FA2H shows no impact on either the abundance or the enzymatic activity of the encoded hydroxylase. To our knowledge, this is the first report of complete necroptosis deficiency in humans. The findings may suggest that impaired necroptosis is a novel mechanism of neurodegeneration, promoting a disorder that shares some clinical features with primary progressive multiple sclerosis (PPMS) and other neurodegenerative diseases. Importantly, the necroptotic deficiency does not cause symptoms outside the nervous system, nor does it confer susceptibility to infections. Given the current interest in pharmacological inhibition of necroptosis by targeting MLKL and its associated pathways, this strategy should be developed with caution, with careful consideration of the possible development of adverse neurological effects.

Lipid Profiles of Five Essential Phospholipid Preparations for the Treatment of Nonalcoholic Fatty Liver Disease: A Comparative Study.

Nonalcoholic fatty liver disease (NAFLD) is associated with an imbalance in fatty acid composition and can progress from simple steatosis to steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. Essential phospholipids (EPL), which contain high levels of 1,2-dilinoleoylphosphatidylcholine, can be used to treat NAFLD. Polyenylphosphatidylcholine (PPC) preparations are external, commercially available EPL products. The lipid composition of five commercially available PPC preparations, including Essentiale Forte, Fortifikat, Hepatoprotect Regenerator, Fortifikat Forte, and Esentin Forte were compared, the outcome of which may impact physician choice in the treatment of NAFLD. Following lipid extraction, a comparative analysis of key lipid content was performed using a QTRAP6500+ triple quadruple ion trap hybrid mass spectrometer (Sciex) in nanoelectrospray ionization mode. The glycerophospholipid composition of each PPC was determined, including levels of phosphatidylcholine (PtdCho), and phosphatidylethanolamine (PtdEtn) species, as well as PtdCho:PtdEtn ratio. Of the five preparations analyzed, Essentiale Forte contained the highest PtdCho levels (61.9 mol%) and lowest PtdEtn levels (4.9 mol%). PtdCho 36:4 levels, a polyunsaturated species of PtdCho, were highest in Esentin Forte (39.3 mol%) and Essentiale Forte (38.3 mol%) compared with other PPCs (28.7-35.8 mol%). Levels of lysophosphatidylcholine, phosphatidylinositol, phosphatidic acid, and phosphatidylglycerol were low in all five preparations. Lipid composition was consistent between the preparations. The high PtdCho:PtdEtn ratio composition of Essentiale Forte compared with the other PPC analyzed, as well as the presence of polyunsaturated fatty acids, suggest it could be the most clinically beneficial commercially available hepatoprotective product in the treatment of NAFLD.

Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes.

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

In vitro quantification of botulinum neurotoxin type A1 using immobilized nerve cell-mimicking nanoreactors in a microfluidic platform.

The bacterial toxin botulinum neurotoxin A (BoNT/A) is not only an extremely toxic substance but also a potent pharmaceutical compound that is used in a wide spectrum of neurological disorders and cosmetic applications. The quantification of the toxin is extremely challenging due to its extraordinary high physiological potency and is further complicated by the toxin's three key functionalities that are necessary for its activity: receptor binding, internalization-translocation, and catalytic activity. So far, the industrial standard to measure the active toxin has been the mouse bioassay (MBA) that is considered today as outdated due to ethical issues. Therefore, recent introductions of cell-based assays were highly anticipated; their impact however remains limited due to their labor-intensive implementation. This report describes a new in vitro approach that combines a nanosensor based on the use of nerve cell-mimicking nanoreactors (NMN) with microfluidic technology. The nanosensor was able to measure all three key functionalities, and therefore suitable to quantify the amount of physiologically active BoNT/A. The integration of such a sensor in a microfluidic device allowed the detection and quantification of BoNT/A amounts in a much shorter time than the MBA (<10 h vs. 2-4 days). Lastly, the system was also able to reliably quantify physiologically active BoNT/A within a simple final pharmaceutical formulation. This complete in vitro testing system and its unique combination of a highly sensitive nanosensor and microfluidic technology represent a significant ethical advancement over in vivo measures and a possible alternative to cell-based in vitro detection methods.