RESUMO
Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMCs were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stoichiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome-scale modeling and 13C-fluxomics. Our results reveal that contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing to significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli.IMPORTANCEThe properties of bacterial microcompartments make them an ideal tool for building orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modeling, and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in Escherichia coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.
Assuntos
Proteínas de Escherichia coli , Etanolamina , Etanolamina/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Óperon/genética , Redes e Vias Metabólicas/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteômica/métodosRESUMO
Bacterial micro-compartments (BMC) are complex macromolecular assemblies that participate in varied metabolic processes in about 20% of bacterial species. Most of these organisms carry BMC genetic information organized in operons that often include several paralog genes coding for components of the compartment shell. BMC shell constituents can be classified depending on their oligomerization state as hexamers (BMC-H), pentamers (BMC-P) or trimers (BMC-T). Formation of hetero-oligomers combining different protein homologs is theoretically feasible, something that could ultimately modify BMC shell rigidity or permeability, for instance. Despite that, it remains largely unknown whether hetero-oligomerization is a widespread phenomenon. Here, we demonstrated that the tripartite GFP (tGFP) reporter technology is an appropriate tool that might be exploited for such purposes. Thus, after optimizing parameters such as the size of linkers connecting investigated proteins to GFP10 or GFP11 peptides, the type and strength of promoters, or the impact of placing coding cassettes in the same or different plasmids, homo-oligomerization processes could be successfully monitored for any of the three BMC shell classes. Moreover, the screen perfectly reproduced published data on hetero-association between couples of CcmK homologues from Syn. sp. PCC6803, which were obtained following a different approach. This study paves the way for mid/high throughput screens to characterize the extent of hetero-oligomerization occurrence in BMC-possessing bacteria, and most especially in organisms endowed with several BMC types and carrying numerous shell paralogs. On the other hand, our study also unveiled technology limitations deriving from the low solubility of one of the components of this modified split-GFP approach, the GFP1-9.
Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Bactérias/genética , Genes BacterianosRESUMO
A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.
Assuntos
Nanoestruturas , Proteínas , Difração de Raios X , Espalhamento a Baixo Ângulo , Proteínas/química , Temperatura , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Conformação de Ácido NucleicoRESUMO
Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.
Assuntos
Plasmócitos , Proteínas SNARE , Camundongos , Animais , Plasmócitos/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Retículo Endoplasmático/metabolismo , Transporte BiológicoRESUMO
The twisted structures of the chitin-based cuticle of beetles confer specific optical characteristics on them. Intrigued by the observation of Bragg gratings with a depth-dependent periodicity in the cuticle of Chrysina beetles, we determine the experimental conditions leading to their transcription into cholesteric liquid-crystal oligomers. We correlate the optical properties of reflectors thus produced with their internal morphology, as observed by transmission electron microscopy. With the use of a single parameter, thermal annealing time, the reflection color is made time-tunable. Different spectral bands and reflection colors from golden yellow to NIR are available, and the irreversibility of the final color is reached at the end. On the basis of the design concept and these properties, these hybrid chiral-achiral materials inspire the fabrication of smart reflective labels. When encapsulated in the package of a product to be kept under cold conditions, the label records the history of the product conservation. Two kinds of information based on color changes are recorded as follows: qualitative information reporting that the product was kept outside of the specified storage temperature and quantitative information giving an indication of the time elapsed since the temperature exceeded the storage temperature of the product.
RESUMO
Mycobacterium tuberculosis (Mtb) regulates the macrophage metabolic state to thrive in the host, yet the responsible mechanisms remain elusive. Macrophage activation toward the microbicidal (M1) program depends on the HIF-1α-mediated metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Here, we ask whether a tuberculosis (TB) microenvironment changes the M1 macrophage metabolic state. We expose M1 macrophages to the acellular fraction of tuberculous pleural effusions (TB-PEs) and find lower glycolytic activity, accompanied by elevated levels of OXPHOS and bacillary load, compared to controls. The eicosanoid fraction of TB-PE drives these metabolic alterations. HIF-1α stabilization reverts the effect of TB-PE by restoring M1 metabolism. Furthermore, Mtb-infected mice with stabilized HIF-1α display lower bacillary loads and a pronounced M1-like metabolic profile in alveolar macrophages (AMs). Collectively, we demonstrate that lipids from a TB-associated microenvironment alter the M1 macrophage metabolic reprogramming by hampering HIF-1α functions, thereby impairing control of Mtb infection.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose Pleural/metabolismo , Animais , Carga Bacteriana , Eicosanoides/farmacologia , Feminino , Glicólise/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Derrame Pleural , Tuberculose Pleural/microbiologiaRESUMO
Replicating biological patterns is promising for designing materials with multifaceted properties. Twisted cholesteric liquid crystal patterns are found in the iridescent tessellated cuticles of many insects and a few fruits. Their accurate replication is extremely difficult since discontinuous patterns and colors must coexist in a single layer without discontinuity of the structures. Here, a solution is demonstrated by addressing striped insect cuticles with a complex twisted organization. Geometric constraints are met by controlling the thermal diffusion in a cholesteric oligomer bilayer subjected to local changes in the molecular anchoring conditions. A multicriterion comparison reveals a very high level of biomimicry. Proof-of-concept prototypes of anti-counterfeiting tags are presented. The present design involves an economy of resources and a high versatility of chiral patterns unreached by the current manufacturing techniques such as metallic layer vacuum deposition, template embossing and various forms of lithography which are limited and often prohibitively expensive.
Assuntos
Biomimética/métodos , Biofísica/métodos , Cristais Líquidos/química , Óptica e Fotônica/métodos , Animais , InsetosRESUMO
Determining the path of single ribonucleoprotein (RNP) particles through the 100 nm-wide nuclear pore complex (NPC) by fluorescence microscopy remains challenging due to resolution limitation and RNP labeling constraints. By using high-pressure freezing and electron tomography, here we captured snapshots of the translocation of native RNP particles through NPCs in yeast and analyzed their trajectory at nanometer-scale resolution. Morphological and functional analyses indicate that these particles mostly correspond to pre-ribosomes. They are detected in 5-6% of the NPCs, with no apparent bias for NPCs adjacent to the nucleolus. Their path closely follows the central axis of the NPC through the nuclear and inner rings, but diverges at the cytoplasmic ring, suggesting interactions with the cytoplasmic nucleoporins. By applying a probabilistic queueing model to our data, we estimated that the dwell time of pre-ribosomes in the yeast NPC is ~90 ms. These data reveal distinct steps of pre-ribosome translocation through the NPC.
Assuntos
Tomografia com Microscopia Eletrônica , Poro Nuclear/metabolismo , Ribossomos/ultraestrutura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Microscopia de Fluorescência , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestruturaRESUMO
The outermost part of insect cuticles is very often covered with wax, which prevents desiccation and serves for chemical communication in many species. Earlier studies on cuticular waxes have mainly focused on their chemical composition revealing complex mixtures of lipids. In the absence of information on their physical organization, cuticular waxes have been considered isotropic. Here we report the presence of parallel stripes in the wax layer of the carapace of the scarab beetle, Chrysina gloriosa, with a textural periodicity of ca. 28 nm, as revealed by electron microscopy of transverse sections. Observations at oblique incidence argue for a layered organization of the wax, which might be related to a layer-by-layer deposition of excreted wax. Our findings may lay the foundation for further studies on the internal structure of cuticular waxes for other insects.
Assuntos
Exoesqueleto/ultraestrutura , Besouros/ultraestrutura , Exoesqueleto/química , Animais , Besouros/química , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ceras/químicaRESUMO
In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.
Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Macrófagos/fisiologia , Microscopia de Força Atômica/métodos , Podossomos/fisiologia , Animais , HumanosRESUMO
Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.
Assuntos
Reabsorção Óssea/etiologia , Infecções por HIV/complicações , HIV-1/fisiologia , Osteoclastos/virologia , Actinas/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Osso e Ossos/metabolismo , Adesão Celular , Feminino , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of monogenic genodermatoses that encompasses non-syndromic disorders of keratinization. The pathophysiology of ARCI has been linked to a disturbance in epidermal lipid metabolism that impaired the stratum corneum function, leading to permeability barrier defects. Functional characterization of some genes involved in ARCI contributed to the identification of molecular actors involved in epidermal lipid synthesis, transport or processing. Recently, PNPLA1 has been identified as a gene causing ARCI. While other members of PNPLA family are key elements in lipid metabolism, the function of PNPLA1 remained unclear. We identified 5 novel PNPLA1 mutations in ARCI patients, mainly localized in the putative active enzymatic domain of PNPLA1. To investigate Pnpla1 biological role, we analysed Pnpla1-deficient mice. KO mice died soon after birth from severe epidermal permeability defects. Pnpla1-deficient skin presented an important impairment in the composition and organization of the epidermal lipids. Quantification of epidermal ceramide species highlighted a blockade in the production of ω-O-acylceramides with a concomitant accumulation of their precursors in the KO. The virtually loss of ω-O-acylceramides in the stratum corneum was linked to a defective lipid coverage of the resistant pericellular shell encapsulating corneocytes, the so-called cornified envelope, and most probably disorganized the extracellular lipid matrix. Finally, these defects in ω-O-acylceramides synthesis and cornified envelope formation were also evidenced in the stratum corneum from PNPLA1-mutated patients. Overall, our data support that PNPLA1/Pnpla1 is a key player in the formation of ω-O-acylceramide, a crucial process for the epidermal permeability barrier function.
Assuntos
Ictiose Lamelar/genética , Lipase/genética , Lipase/metabolismo , Idoso , Animais , Ceramidas/metabolismo , Criança , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Feminino , Genes Recessivos , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Permeabilidade , Pele/metabolismoRESUMO
Malignant progression results from a dynamic cross-talk between stromal and cancer cells. Recent evidence suggests that this cross-talk is mediated to a significant extent by exosomes, nanovesicles secreted by most cell types and which allow the transfer of proteins, lipids, and nucleic acids between cells. Adipocytes are a major component of several tumor microenvironments, including that of invasive melanoma, where cells have migrated to the adipocyte-rich hypodermic layer of the skin. We show that adipocytes secrete exosomes in abundance, which are then taken up by tumor cells, leading to increased migration and invasion. Using mass spectrometry, we analyzed the proteome of adipocyte exosomes. Interestingly, these vesicles carry proteins implicated in fatty acid oxidation (FAO), a feature highly specific to adipocyte exosomes. We further show that, in the presence of adipocyte exosomes, FAO is increased in melanoma cells. Inhibition of this metabolic pathway completely abrogates the exosome-mediated increase in migration. Moreover, in obese mice and humans, both the number of exosomes secreted by adipocytes as well as their effect on FAO-dependent cell migration are amplified. These observations might in part explain why obese melanoma patients have a poorer prognosis than their nonobese counterparts. Cancer Res; 76(14); 4051-7. ©2016 AACR.
Assuntos
Adipócitos/fisiologia , Exossomos/fisiologia , Ácidos Graxos/metabolismo , Melanoma/patologia , Obesidade/complicações , Células 3T3 , Animais , Movimento Celular , Humanos , Masculino , Melanoma/etiologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.