RESUMO
Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.
Assuntos
Lisossomos , Transdução de Sinais , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes , Fenômenos Fisiológicos CelularesRESUMO
Ectopic lipid deposition and altered mitochondrial dynamics contribute to the development of obesity and insulin resistance. However, the mechanistic link between these processes remained unclear. Here we demonstrate that the C16:0 sphingolipid synthesizing ceramide synthases, CerS5 and CerS6, affect distinct sphingolipid pools and that abrogation of CerS6 but not of CerS5 protects from obesity and insulin resistance. We identify proteins that specifically interact with C16:0 sphingolipids derived from CerS5 or CerS6. Here, only CerS6-derived C16:0 sphingolipids bind the mitochondrial fission factor (Mff). CerS6 and Mff deficiency protect from fatty acid-induced mitochondrial fragmentation in vitro, and the two proteins genetically interact in vivo in obesity-induced mitochondrial fragmentation and development of insulin resistance. Our experiments reveal an unprecedented specificity of sphingolipid signaling depending on specific synthesizing enzymes, provide a mechanistic link between hepatic lipid deposition and mitochondrial fragmentation in obesity, and define the CerS6-derived sphingolipid/Mff interaction as a therapeutic target for metabolic diseases.
Assuntos
Proteínas de Membrana/metabolismo , Obesidade/metabolismo , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Animais , Apoptose , Linhagem Celular , Células HeLa , Humanos , Resistência à Insulina/fisiologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Obesidade/fisiopatologia , Esfingolipídeos/fisiologia , Esfingosina N-Aciltransferase/fisiologiaRESUMO
Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems. Lipidomics has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Here, I review state-of-the-art electrospray ionization tandem mass spectrometric approaches and direct infusion to quantitatively assess lipid compositions of cells and subcellular fractions. Finally, I discuss a few examples of the power of mass spectrometry-based lipidomics in addressing cell biological questions.
Assuntos
Lipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cardiolipinas/química , Físico-Química , Biologia Computacional , Humanos , Mitocôndrias/metabolismo , Organelas/química , Solventes/química , Frações Subcelulares/químicaRESUMO
Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina C-Palmitoiltransferase/genética , Animais , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Feminino , Humanos , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais/imunologia , Esfingolipídeos/biossínteseRESUMO
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.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Doença pelo Vírus Ebola/metabolismo , Fusão de Membrana , Proteínas do Core Viral/metabolismo , Endossomos/metabolismo , Proteínas da Matriz ViralRESUMO
Adaptive immune responses comprise the activation of T cells by peptide antigens that are presented by proteins of the Major Histocompatibility Complex (MHC) on the surface of an antigen-presenting cell. As a consequence of the T cell receptor interacting productively with a certain peptide-MHC complex, a specialized cell-cell junction known as the immunological synapse forms and is accompanied by changes in the spatiotemporal patterning and function of intracellular signaling molecules. Key modifications occurring at the cytoplasmic leaflet of the plasma and internal membranes in activated T cells comprise lipid switches that affect the binding and distribution of proteins within or near the lipid bilayer. Here, we describe two major classes of lipid switches that act at this critical water/membrane interface. Phosphoinositides are derived from phosphatidylinositol, an amphiphilic molecule that contains two fatty acid chains and a phosphate group that bridges the glycerol backbone to the carbohydrate inositol. The inositol ring can be variably (de-)phosphorylated by dedicated kinases and phosphatases, thereby creating phosphoinositide signatures that define the composition and properties of signaling molecules, molecular complexes, or whole organelles. Palmitoylation refers to the reversible attachment of the fatty acid palmitate to a substrate protein's cysteine residue. DHHC enzymes, named after the four conserved amino acids in their active site, catalyze this post-translational modification and thereby change the distribution of proteins at, between, and within membranes. T cells utilize these two types of molecular switches to adjust their properties to an activation process that requires changes in motility, transport, secretion, and gene expression.
Assuntos
Sinapses Imunológicas , Linfócitos T , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/imunologia , Humanos , Animais , Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/citologia , Fosfatidilinositóis/metabolismo , LipoilaçãoRESUMO
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.
Assuntos
Farnesil-Difosfato Farnesiltransferase , Ácido Mevalônico , Ácido Aspártico Endopeptidases , Colesterol/metabolismo , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácido Mevalônico/metabolismo , Terpenos , Células HEK293 , HumanosRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidato Fosfatase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático , Regulação Fúngica da Expressão Gênica , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos Orgânicos/metabolismo , Fosfatidato Fosfatase/genética , Fosforilação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não DobradasRESUMO
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.
Assuntos
Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
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.
Assuntos
Orthobunyavirus , Glucosilceramidas , Ligação Viral , Lipidômica , Espectrometria de MassasRESUMO
S-acylation is a reversible posttranslational protein modification consisting of attachment of a fatty acid to a cysteine via a thioester bond. Research over the last few years has shown that a variety of different fatty acids, such as palmitic acid (C16:0), stearate (C18:0), or oleate (C18:1), are used in cells to S-acylate proteins. We recently showed that GNAI proteins can be acylated on a single residue, Cys3, with either C16:0 or C18:1, and that the relative proportion of acylation with these fatty acids depends on the level of the respective fatty acid in the cell's environment. This has functional consequences for GNAI proteins, with the identity of the acylating fatty acid affecting the subcellular localization of GNAIs. Unclear is whether this competitive acylation is specific to GNAI proteins or a more general phenomenon in the proteome. We perform here a proteome screen to identify proteins acylated with different fatty acids. We identify 218 proteins acylated with C16:0 and 308 proteins acylated with C18-lipids, thereby uncovering novel targets of acylation. We find that most proteins that can be acylated by C16:0 can also be acylated with C18-fatty acids. For proteins with more than one acylation site, we find that this competitive acylation occurs on each individual cysteine residue. This raises the possibility that the function of many different proteins can be regulated by the lipid environment via differential S-acylation.
Assuntos
Cisteína , Ácido Palmítico , Proteoma , Ácidos Esteáricos , Acilação , Cisteína/metabolismo , Ácido Palmítico/metabolismo , Proteoma/metabolismo , Células HEK293 , Células HeLa , Humanos , Ácidos Esteáricos/metabolismoRESUMO
Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge 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-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-α-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 expand 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.
Assuntos
Cromatografia de Fase Reversa , Fosfatos , Humanos , Metilação , Fosfatos/metabolismo , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Fosfatos de Dolicol/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Defeitos Congênitos da Glicosilação , Fosfotransferases (Fosfomutases) , Humanos , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/terapia , Defeitos Congênitos da Glicosilação/metabolismo , Glicosilação , Fosfotransferases (Fosfomutases)/genética , Aminoácidos/metabolismo , LipídeosRESUMO
Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.
Assuntos
Proteínas de Transporte/metabolismo , Cavéolas/metabolismo , Ácidos Graxos/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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.
Assuntos
Membrana Nuclear , Receptores Citoplasmáticos e Nucleares , Colesterol , Células HeLa , Homeostase , Humanos , Proteínas de Membrana , Proteínas do Tecido Nervoso , Membrana Nuclear/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptor de Lamina BRESUMO
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.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Camundongos , Recidiva Local de Neoplasia , Neoplasia Residual/genéticaRESUMO
Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics studies require high-quality spectral libraries for reliable metabolite identification. We have constructed EMBL-MCF (European Molecular Biology Laboratory-Metabolomics Core Facility), an open LC-MS/MS spectral library that currently contains over 1600 fragmentation spectra from 435 authentic standards of endogenous metabolites and lipids. The unique features of the library include the presence of chromatographic profiles acquired with different LC-MS methods and coverage of different adduct ions. The library covers many biologically important metabolites with some unique metabolites and lipids as compared with other public libraries. The EMBL-MCF spectral library is created and shared using an in-house-developed web application at https://curatr.mcf.embl.de/. The library is freely available online and also integrated with other mass spectral repositories.
Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Biblioteca Gênica , ÍonsRESUMO
Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.
Assuntos
Espectrometria de Massas/métodos , Metaloendopeptidases/química , Proteínas/análise , Proteômica/métodos , Estreptavidina/química , Tripsina/química , Arginina/análogos & derivados , Arginina/química , Biotinilação/métodos , Imunoprecipitação da Cromatina/métodos , Células HeLa , Humanos , Lisina/análogos & derivados , Lisina/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteólise , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Peptídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Corpos de Inclusão/química , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Peptídeos/química , Peptídeos/toxicidade , Proteômica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.