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1.
EMBO J ; 42(11): e113578, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37082863

ABSTRACT

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.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Humans , Hemorrhagic Fever, Ebola/metabolism , Membrane Fusion , Viral Core Proteins/metabolism , Endosomes/metabolism , Viral Matrix Proteins
2.
J Biol Chem ; 300(2): 105644, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38218226

ABSTRACT

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.


Subject(s)
Farnesyl-Diphosphate Farnesyltransferase , Mevalonic Acid , Aspartic Acid Endopeptidases , Cholesterol/metabolism , Farnesyl-Diphosphate Farnesyltransferase/genetics , Farnesyl-Diphosphate Farnesyltransferase/metabolism , Mevalonic Acid/metabolism , Terpenes , HEK293 Cells , Humans
3.
EMBO J ; 40(22): e107958, 2021 11 15.
Article in English | MEDLINE | ID: mdl-34617598

ABSTRACT

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.


Subject(s)
Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Phosphatidate Phosphatase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum Stress , Gene Expression Regulation, Fungal , Lipid Metabolism , Membrane Proteins/genetics , Multiprotein Complexes/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Organic Chemicals/metabolism , Phosphatidate Phosphatase/genetics , Phosphorylation , Repressor Proteins/genetics , Repressor Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Unfolded Protein Response
4.
J Cell Sci ; 136(10)2023 05 15.
Article in English | MEDLINE | ID: mdl-37073556

ABSTRACT

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.


Subject(s)
Mitochondria , Saccharomyces cerevisiae Proteins , Humans , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Endoplasmic Reticulum/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism
5.
Cell Mol Life Sci ; 81(1): 71, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38300320

ABSTRACT

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.


Subject(s)
Orthobunyavirus , Glucosylceramides , Virus Attachment , Lipidomics , Mass Spectrometry
6.
Mol Genet Metab ; 139(3): 107610, 2023 07.
Article in English | MEDLINE | ID: mdl-37245379

ABSTRACT

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.


Subject(s)
Congenital Disorders of Glycosylation , Phosphotransferases (Phosphomutases) , Humans , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/therapy , Congenital Disorders of Glycosylation/metabolism , Glycosylation , Phosphotransferases (Phosphomutases)/genetics , Amino Acids/metabolism , Lipids
7.
J Cell Sci ; 133(16)2020 08 21.
Article in English | MEDLINE | ID: mdl-32694168

ABSTRACT

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.


Subject(s)
Nuclear Envelope , Receptors, Cytoplasmic and Nuclear , Cholesterol , HeLa Cells , Homeostasis , Humans , Membrane Proteins , Nerve Tissue Proteins , Nuclear Envelope/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Lamin B Receptor
8.
Mol Syst Biol ; 17(10): e10141, 2021 10.
Article in English | MEDLINE | ID: mdl-34694069

ABSTRACT

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.


Subject(s)
Breast Neoplasms , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Female , Humans , Mice , Neoplasm Recurrence, Local , Neoplasm, Residual/genetics
9.
Proc Natl Acad Sci U S A ; 115(15): E3446-E3453, 2018 04 10.
Article in English | MEDLINE | ID: mdl-29581260

ABSTRACT

Huntington's disease is caused by the expansion of a polyglutamine (polyQ) tract in the N-terminal exon of huntingtin (HttEx1), but the cellular mechanisms leading to neurodegeneration remain poorly understood. Here we present in situ structural studies by cryo-electron tomography of an established yeast model system of polyQ toxicity. We find that expression of polyQ-expanded HttEx1 results in the formation of unstructured inclusion bodies and in some cases fibrillar aggregates. This contrasts with recent findings in mammalian cells, where polyQ inclusions were exclusively fibrillar. In yeast, polyQ toxicity correlates with alterations in mitochondrial and lipid droplet morphology, which do not arise from physical interactions with inclusions or fibrils. Quantitative proteomic analysis shows that polyQ aggregates sequester numerous cellular proteins and cause a major change in proteome composition, most significantly in proteins related to energy metabolism. Thus, our data point to a multifaceted toxic gain-of-function of polyQ aggregates, driven by sequestration of endogenous proteins and mitochondrial and lipid droplet dysfunction.


Subject(s)
Peptides/metabolism , Saccharomyces cerevisiae/metabolism , Humans , Huntington Disease/genetics , Huntington Disease/metabolism , Inclusion Bodies/chemistry , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Lipid Droplets/chemistry , Lipid Droplets/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Peptides/chemistry , Peptides/toxicity , Proteomics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
10.
PLoS Pathog ; 14(10): e1007348, 2018 10.
Article in English | MEDLINE | ID: mdl-30321240

ABSTRACT

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cell Wall/metabolism , Group II Phospholipases A2/immunology , Immunity, Innate/drug effects , Polysaccharides, Bacterial/pharmacology , Streptococcal Infections/microbiology , Streptococcus/immunology , Blood Bactericidal Activity , Group II Phospholipases A2/blood , Group II Phospholipases A2/genetics , Host-Pathogen Interactions , Humans , Streptococcal Infections/blood , Streptococcal Infections/enzymology , Streptococcus/pathogenicity
11.
J Inherit Metab Dis ; 43(5): 1046-1055, 2020 09.
Article in English | MEDLINE | ID: mdl-32441337

ABSTRACT

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.


Subject(s)
Arrhythmias, Cardiac/drug therapy , Chondrodysplasia Punctata, Rhizomelic/drug therapy , Glyceryl Ethers/pharmacology , Plasmalogens/biosynthesis , Administration, Oral , Animals , Arrhythmias, Cardiac/etiology , Chondrodysplasia Punctata, Rhizomelic/physiopathology , Dietary Supplements , Disease Models, Animal , Electrocardiography , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phospholipid Ethers/pharmacology
12.
Analyst ; 144(19): 5755-5765, 2019 Sep 23.
Article in English | MEDLINE | ID: mdl-31433410

ABSTRACT

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.


Subject(s)
Biomimetic Materials , Botulinum Toxins, Type A/analysis , Cells, Immobilized , Lab-On-A-Chip Devices , Nanostructures , Neurons , Animals , Biosensing Techniques , Drugs, Chinese Herbal/chemistry , In Vitro Techniques/methods , Liposomes/chemistry , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Protein Binding , Serum Albumin, Human/chemistry , Surface Plasmon Resonance , Swine
13.
J Biol Chem ; 292(33): 13702-13713, 2017 08 18.
Article in English | MEDLINE | ID: mdl-28659343

ABSTRACT

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.


Subject(s)
HIV-1/pathogenicity , Lipid Metabolism , Membrane Proteins/metabolism , Virion/pathogenicity , nef Gene Products, Human Immunodeficiency Virus/metabolism , Antigens, Surface/genetics , Antigens, Surface/metabolism , Binding, Competitive , Cell Line, Tumor , Gene Deletion , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HIV-1/chemistry , HIV-1/physiology , Humans , Kinetics , Liposomes , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Milk Proteins/genetics , Milk Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphatidylserines/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sphingosine/metabolism , Surface Properties , Virion/chemistry , Virion/physiology , Virus Assembly , nef Gene Products, Human Immunodeficiency Virus/genetics
14.
Mol Genet Metab ; 123(3): 364-374, 2018 03.
Article in English | MEDLINE | ID: mdl-29396028

ABSTRACT

Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. >100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T>G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H+-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the ß-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.


Subject(s)
Congenital Disorders of Glycosylation/genetics , Cutis Laxa/genetics , Exocrine Pancreatic Insufficiency/genetics , Metabolome/genetics , Vacuolar Proton-Translocating ATPases/genetics , Acyl-CoA Oxidase/metabolism , Catalase/metabolism , Congenital Disorders of Glycosylation/diagnosis , Congenital Disorders of Glycosylation/metabolism , Cutis Laxa/diagnosis , Cutis Laxa/metabolism , Exocrine Pancreatic Insufficiency/diagnosis , Exocrine Pancreatic Insufficiency/metabolism , Fatty Acids/metabolism , Genes, X-Linked/genetics , Humans , Infant , Male , Metabolomics , Oxidation-Reduction , Vacuolar Proton-Translocating ATPases/deficiency , Exome Sequencing
16.
Nat Commun ; 15(1): 6914, 2024 Aug 12.
Article in English | MEDLINE | ID: mdl-39134548

ABSTRACT

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.


Subject(s)
Cyclooxygenase 1 , Electron Transport Complex IV , Inflammation , Liver , Oxidative Phosphorylation , Reactive Oxygen Species , Animals , Female , Humans , Male , Mice , DEAD Box Protein 58 , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Electron Transport Complex IV/metabolism , Electron Transport Complex IV/genetics , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Liver/metabolism , Liver/pathology , Membrane Proteins , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Mutation , Protein Biosynthesis , Reactive Oxygen Species/metabolism , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolism
17.
J Cell Biol ; 222(6)2023 06 05.
Article in English | MEDLINE | ID: mdl-37154843

ABSTRACT

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.


Subject(s)
Nuclear Envelope , Nuclear Pore Complex Proteins , Saccharomyces cerevisiae Proteins , Cell Membrane/metabolism , Nuclear Envelope/genetics , Nuclear Envelope/metabolism , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/chemistry
18.
EMBO Mol Med ; 15(9): e17399, 2023 09 11.
Article in English | MEDLINE | ID: mdl-37533404

ABSTRACT

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.


Subject(s)
Barth Syndrome , Mice , Animals , Barth Syndrome/metabolism , Barth Syndrome/pathology , Cardiolipins/metabolism , AMP-Activated Protein Kinases/metabolism , Glycolysis , Fatty Acids/metabolism , Adenosine Triphosphate
19.
Open Biol ; 11(11): 210250, 2021 11.
Article in English | MEDLINE | ID: mdl-34814743

ABSTRACT

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.


Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Membrane Proteins/genetics , Mutation , Nuclear Envelope/metabolism , Nuclear Pore/metabolism , Phosphatidic Acids/metabolism , Protein Conformation, alpha-Helical , Protein Domains , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
20.
Nat Commun ; 12(1): 2673, 2021 05 11.
Article in English | MEDLINE | ID: mdl-33976123

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Inositol Phosphates/metabolism , Membrane Lipids/metabolism , Animals , Biological Transport , COS Cells , Chlorocebus aethiops , Cholesterol/metabolism , Endocytosis , HEK293 Cells , HeLa Cells , Humans , Inositol Polyphosphate 5-Phosphatases/genetics , Inositol Polyphosphate 5-Phosphatases/metabolism , Microscopy, Confocal , Phosphatidylinositol Phosphates/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Trihexosylceramides/metabolism
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