RESUMEN
Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.
Asunto(s)
Membrana Celular/metabolismo , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/fisiología , Acilación/fisiología , Células HeLa , Humanos , Lipoilación/fisiología , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/fisiología , Proteínas/metabolismo , Especificidad por Sustrato , Tioléster Hidrolasas/genéticaRESUMEN
Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.
Asunto(s)
Venenos de Cnidarios/toxicidad , Células Eucariotas/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Potasio/metabolismo , Anémonas de Mar/patogenicidad , Animales , Células Cultivadas , Cricetinae , Células Eucariotas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.
Asunto(s)
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pliegue de Proteína , Multimerización de Proteína/fisiología , Precursores de Proteínas/metabolismo , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Humanos , Chaperonas Moleculares/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Precursores de Proteínas/genética , Estructura Terciaria de ProteínaRESUMEN
The complex architecture of the endoplasmic reticulum (ER) comprises distinct dynamic features, many at the nanoscale, that enable the coexistence of the nuclear envelope, regions of dense sheets and a branched tubular network that spans the cytoplasm. A key player in the formation of ER sheets is cytoskeleton-linking membrane protein 63 (CLIMP-63). The mechanisms by which CLIMP-63 coordinates ER structure remain elusive. Here, we address the impact of S-acylation, a reversible post-translational lipid modification, on CLIMP-63 cellular distribution and function. Combining native mass-spectrometry, with kinetic analysis of acylation and deacylation, and data-driven mathematical modelling, we obtain in-depth understanding of the CLIMP-63 life cycle. In the ER, it assembles into trimeric units. These occasionally exit the ER to reach the plasma membrane. However, the majority undergoes S-acylation by ZDHHC6 in the ER where they further assemble into highly stable super-complexes. Using super-resolution microscopy and focused ion beam electron microscopy, we show that CLIMP-63 acylation-deacylation controls the abundance and fenestration of ER sheets. Overall, this study uncovers a dynamic lipid post-translational regulation of ER architecture.
Asunto(s)
Retículo Endoplásmico , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Cinética , Retículo Endoplásmico/metabolismo , Acilación , LípidosRESUMEN
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.
Asunto(s)
Ceramidas , Esfingolípidos , Humanos , Ceramidas/metabolismo , Homeostasis , Mutación , Esfingolípidos/genética , Esfingolípidos/metabolismoRESUMEN
Pore-forming toxins (PFTs) are secreted proteins that contribute to the virulence of a great variety of bacterial pathogens. They inflict one of the more disastrous damages a target cell can be exposed to: disruption of plasma membrane integrity. Since this is an ancient form of attack, which bears similarities to mechanical membrane damage, cells have evolved response pathways to these perturbations. Here, it is reported that PFTs trigger very diverse yet specific response pathways. Many are triggered by the decrease in cytoplasmic potassium, which thus emerges as a central regulator. Upon plasma membrane damage, cells activate signalling pathways aimed at restoring plasma membrane integrity and ion homeostasis. Interestingly these pathways do not require protein synthesis. Cells also trigger signalling cascades that allow them to enter a quiescent-like state, where minimal energy is consumed while waiting for plasma membrane damage to be repaired. More specifically, protein synthesis is arrested, cytosolic constituents are recycled by autophagy and energy is stored in lipid droplets.
Asunto(s)
Bacterias/patogenicidad , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Estrés Fisiológico , Autofagia , Línea Celular , Membrana Celular/metabolismo , Humanos , Metabolismo de los Lípidos , Potasio/metabolismo , Biosíntesis de Proteínas , Transducción de SeñalRESUMEN
Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.
Asunto(s)
Fibroblastos , Piel , Esfingolípidos , Fibroblastos/química , Fibroblastos/clasificación , Fibroblastos/metabolismo , Humanos , Lipidómica/métodos , Redes y Vías Metabólicas , Piel/química , Piel/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Esfingolípidos/análisis , Esfingolípidos/metabolismo , TranscriptomaRESUMEN
The amyloid cascade model hypothesizes that neurotoxic oligomers or aggregates formed by the Alzheimer amyloid peptide (Abeta) cause disease pathology in Alzheimer's disease. Attempted treatment strategies for Alzheimer's disease have involved either inhibiting Abeta oligomerization or aggregation, or dissolving existing aggregates. Blocking such downhill processes, however, has proved daunting. We have used a different approach that targets Abeta before the oligomerization cascade begins. We predicted that an amphipathic helix could convert Abeta into a native-like protein and inhibit initiation of oligomerization and aggregation. This idea was tested with a designed library and genetic screen. We exhaustively screened a library of semi-randomized amphipathic helical sequences, each expressed as a fusion protein with an Abeta42-yellow fluorescent protein sequence serving as a reporter for folding and solubilization. This yielded an amphipathic helix capable of initiating native-like folding in Abeta42 and preventing aggregation. This amphipathic helix has direct application to Alzheimer's disease therapy development.
Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Amiloide/química , Amiloide/ultraestructura , Péptidos beta-Amiloides/genética , Secuencia de Bases , Cartilla de ADN/genética , Escherichia coli/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Biblioteca de Péptidos , Péptidos/química , Péptidos/genética , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's beta-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Superóxido Dismutasa/química , Zinc/metabolismo , Esclerosis Amiotrófica Lateral/etiología , Cobre/metabolismo , Dimerización , Guanidina/farmacología , Humanos , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , Desnaturalización Proteica , Superóxido Dismutasa/metabolismo , Termodinámica , Triptófano/genética , Triptófano/metabolismoRESUMEN
Can unique protein structures arise from a limited set of amino acids present on the prebiotic earth? To address this question, we have determined the stability and structure of KIA7, a 20-residue polypeptide containing chiefly Lys, Ile, and Ala. NMR methods reveal that KIA7 tetramerizes and folds on the millisecond time scale to adopt a four-helix X-bundle structure with a tightly and specifically packed core. Denaturation studies and hydrogen exchange measurements of KIA7 and several variants demonstrate that ridges-into-grooves packing of Ala and Ile side chains and the packing of a C-terminal aromatic group into the hydrophobic core are sufficient to give rise to a rather stable, well folded protein structure, with no favorable electrostatic interactions or tertiary or quaternary hydrogen bonds. Both modern proteins and RNAs can adopt specific structures, but RNAs do so with a limited "alphabet" of residues and types of stabilizing interactions. The results reported here show that specific, well folded protein structures can also arise from a highly reduced set of stabilizing interactions and amino acids that are thought to have been present on the prebiotic earth.
Asunto(s)
Origen de la Vida , Péptidos/química , Proteínas/química , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Evolución Molecular , Enlace de Hidrógeno , Isoleucina/química , Isoleucina/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/síntesis química , Conformación Proteica , Pliegue de Proteína , Solventes/químicaRESUMEN
Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.
Asunto(s)
Apoptosis/fisiología , Trastornos de Estrés por Calor/metabolismo , Proteínas de Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Sustitución de Aminoácidos , Caspasa 3 , Inhibidores de Caspasas , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Citoplasma/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Luciferasas/química , Luciferasas/metabolismo , Membrana Nuclear/metabolismo , Pliegue de Proteína , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , TransfecciónRESUMEN
A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.