RESUMEN
Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.
Asunto(s)
Linaje de la Célula , Rastreo Celular/métodos , Metástasis de la Neoplasia/patología , Células Madre Neoplásicas/patología , Tejido Parenquimatoso/patología , Coloración y Etiquetado/métodos , Nicho de Células Madre , Microambiente Tumoral , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diferenciación Celular , Técnicas de Cocultivo , Células Epiteliales/patología , Femenino , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Metástasis de la Neoplasia/inmunología , Neutrófilos/patología , Organoides/patología , Nicho de Células Madre/inmunología , Microambiente Tumoral/inmunología , Proteína Fluorescente RojaRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Histona Demetilasas con Dominio de Jumonji , Proteínas de Ciclo Celular/metabolismo , Humanos , Hidroxilación , Proteínas Intrínsecamente Desordenadas/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Dominios Proteicos , Factores de Transcripción/metabolismoRESUMEN
In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/metabolismo , Fosfolipasa C gamma/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Animales , Autorrenovación de las Células , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Células Madre Neoplásicas/metabolismo , Proteínas de Fusión Oncogénica/genética , Fosfolipasa C gamma/genética , Proteoma , Proteína 1 Compañera de Translocación de RUNX1/genética , Transcriptoma , Translocación GenéticaRESUMEN
Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.
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Leucemia Mieloide Aguda , Proteómica , Humanos , Ratones , Animales , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción/genética , Mutación , Expresión GénicaRESUMEN
Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para-ortholog, Smed-exoc3, abrogates in vivo tissue homeostasis and regeneration-processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2-deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin (Vim)-a known inducer of LD formation. Smed-exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl-L-carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2-deficient ESCs and organ maintenance in Smed-exoc3-depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance.
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Metabolismo de los Lípidos , Planarias , Animales , Diferenciación Celular , Homeostasis , Metabolismo de los Lípidos/genética , Ratones , Planarias/genética , Interferencia de ARNRESUMEN
Astrocytes are increasingly being recognized as contributors to physiological brain function and behavior. Astrocytes engage in glia-synaptic interactions through peripheral astrocyte processes, thus modulating synaptic signaling, for example, by handling glutamate removal from the synaptic cleft and (re)provision to axonal terminals. Peripheral astrocyte processes are ultrafine membrane protrusions rich in the membrane-to-actin cytoskeleton linker Ezrin, an essential component of in vitro filopodia formation and in vivo peripheral astrocyte process motility. Consequently, it has been postulated that Ezrin significantly contributes to neurodevelopment as well as astrocyte functions within the adult brain. However, while Ezrin has been studied in vitro within cultured primary astrocytes, in vivo studies on the role of Ezrin in astrocytes remain to be conducted and consequences of its depletion to be studied. Here, we investigated consequences of Ezrin deletion in the mouse brain starting from early neuronal specification. While Ezrin knockout did not impact prenatal cerebral cortex development, behavioral phenotyping depicted reduced exploratory behavior. Starting with postnatal appearance of glia cells, Ezrin was verified to remain predominantly expressed in astrocytes. Proteome analysis of Ezrin deficient astrocytes revealed alterations in glutamate and ion homeostasis, metabolism and cell morphology - important processes for synaptic signal transmission. Notably, Ezrin deletion in astrocytes provoked (GFAP) glial fibrillary acidic protein upregulation - a marker of astrocyte activation and reactive astrogliosis. However, this spontaneous, reactive astrogliosis exhibited proteome changes distinct from ischemic-induced reactive astrogliosis. Moreover, in experimental ischemic stroke, Ezrin knockout mice displayed reduced infarct volume, indicating a protective effect of the Ezrin deletion-induced changes and astrogliosis.
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Astrocitos , Gliosis , Animales , Astrocitos/metabolismo , Proteínas del Citoesqueleto , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/metabolismo , Ácido Glutámico/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Embarazo , Proteoma/metabolismo , Regulación hacia ArribaRESUMEN
Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetismâwith optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffersâall confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applicationsâall while retaining the speed and compatibility of SP3.
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Proteoma , Proteómica , Fenómenos Magnéticos , Agregado de Proteínas , Proteoma/análisis , Reproducibilidad de los Resultados , SolventesRESUMEN
Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented due to the production of several carotenoids and their derivatives. Two of these pigment molecules, salinixanthin and retinal, are reported to be essential cofactors of the xanthorhodopsin, a light-driven proton pump unique to this bacterium. Here, we isolate and characterize an outer membrane porin-like protein that retains salinixanthin. The characterization by mass spectrometry identified an unknown protein whose structure, predicted by AlphaFold, consists of a 8 strands beta-barrel transmembrane organization typical of porins. The protein is found to be part of a functional network clearly involved in the outer membrane trafficking. Cryo-EM micrographs showed the shape and dimensions of a particle comparable with the ones of the predicted structure. Functional implications, with respect to the high representativity of this protein in the outer membrane fraction, are discussed considering its possible role in primary functions such as the nutrients uptake and the homeostatic balance. Finally, also a possible involvement in balancing the charge perturbation associated with the xanthorhodopsin and ATP synthase activities is considered.
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Bacteroidetes , Porinas , Porinas/metabolismo , Bacteroidetes/química , Bacteroidetes/metabolismo , Carotenoides/química , Carotenoides/metabolismoRESUMEN
In the extremophile bacterium Deinococcus radiodurans, the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent n-dodecyl-ß-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.
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Proteínas Bacterianas/química , Deinococcus/química , Glicoproteínas de Membrana/química , Complejos Multiproteicos/química , Proteínas Bacterianas/genética , Carotenoides/química , Membrana Celular/química , Pared Celular/química , Deinococcus/genética , Complejos Multiproteicos/genética , Porinas/química , Unión Proteica/genéticaRESUMEN
A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.
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Envejecimiento/metabolismo , Encéfalo/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Ribosomas/metabolismo , Envejecimiento/genética , Animales , Fenómenos Biofísicos , Ciprinodontiformes/genética , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Factores de Riesgo , Transcriptoma/genéticaRESUMEN
The interpatient variability of tumor proteomes has been investigated on a large scale but many tumors display also intratumoral heterogeneity regarding morphological and genetic features. It remains largely unknown to what extent the local proteome of tumors intrinsically differs. Here, we used hepatocellular carcinoma as a model system to quantify both inter- and intratumor heterogeneity across human patient specimens with spatial resolution. We defined proteomic features that distinguish neoplastic from the directly adjacent nonneoplastic tissue, such as decreased abundance of NADH dehydrogenase complex I. We then demonstrated the existence of intratumoral variations in protein abundance that re-occur across different patient samples, and affect clinically relevant proteins, even in the absence of obvious morphological differences or genetic alterations. Our work demonstrates the suitability and the benefits of using mass spectrometry-based proteomics to analyze diagnostic tumor specimens with spatial resolution. Data are available via ProteomeXchange with identifier PXD007052.
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Hígado/metabolismo , Masculino , Espectrometría de Masas , Ratones , Persona de Mediana Edad , ProteómicaRESUMEN
Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid-protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo-cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann-Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.
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Diglicéridos/metabolismo , Lípidos/análisis , Proteoma/metabolismo , Proteómica/métodos , Esfingosina/química , Señalización del Calcio , Diglicéridos/química , Fibroblastos/metabolismo , Células HeLa , Humanos , Metabolismo de los Lípidos , Lípidos/química , Microscopía Confocal , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Unión Proteica , Transporte de Proteínas , Proteoma/química , Esfingosina/metabolismo , Imagen de Lapso de Tiempo/métodosRESUMEN
Label-free quantification (LFQ) and isobaric labeling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT SPS/MS3 10-plex workflow to a label free single shot data-independent acquisition (DIA) workflow on a controlled sample set. The sample set consisted of ten samples derived from 10 biological replicates of mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. For a fair comparison, we matched the instrument time for the two workflows. The LC-MS data were acquired at two facilities to assess interlaboratory reproducibility. Both methods resulted in a high proteome coverage (>5000 proteins) with low missing values on protein level (<2%). The TMT workflow led to 15-20% more identified proteins and a slightly better quantitative precision, whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA showed a similar performance. The quantitative performance of the DIA data stayed in a similar range when searching the spectra against a fasta database directly, instead of using a project-specific library. Our experiments also demonstrated that both workflows are readily transferrable between facilities.
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Cerebelo/metabolismo , Proteoma/genética , Proteómica/métodos , Animales , Cromatografía Liquida , Ratones , Proteómica/normas , Coloración y Etiquetado , Espectrometría de Masas en Tándem , Flujo de TrabajoRESUMEN
The monomeric photosystem I-light-harvesting antenna complex I (PSI-LHCI) supercomplex from the extremophilic red alga Cyanidioschyzon merolae represents an intermediate evolutionary link between the cyanobacterial PSI reaction center and its green algal/higher plant counterpart. We show that the C. merolae PSI-LHCI supercomplex is characterized by robustness in various extreme conditions. By a combination of biochemical, spectroscopic, mass spectrometry, and electron microscopy/single particle analyses, we dissected three molecular mechanisms underlying the inherent robustness of the C. merolae PSI-LHCI supercomplex: (1) the accumulation of photoprotective zeaxanthin in the LHCI antenna and the PSI reaction center; (2) structural remodeling of the LHCI antenna and adjustment of the effective absorption cross section; and (3) dynamic readjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK core subunit. We show that the largest low light-treated C. merolae PSI-LHCI supercomplex can bind up to eight Lhcr antenna subunits, which are organized as two rows on the PsaF/PsaJ side of the core complex. Under our experimental conditions, we found no evidence of functional coupling of the phycobilisomes with the PSI-LHCI supercomplex purified from various light conditions, suggesting that the putative association of this antenna with the PSI supercomplex is absent or may be lost during the purification procedure.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Rhodophyta/fisiología , Adaptación Biológica , Clorofila/metabolismo , Dicroismo Circular , Cianobacterias/química , Cianobacterias/fisiología , Evolución Molecular , Concentración de Iones de Hidrógeno , Luz , Rhodophyta/química , Espectrometría de Fluorescencia , Temperatura , Zeaxantinas/metabolismoRESUMEN
BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.
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Envejecimiento , Hígado/metabolismo , Longevidad , Ratas Topo/fisiología , Proteoma , Adulto , Anciano , Anciano de 80 o más Años , Animales , Caenorhabditis elegans/fisiología , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Especificidad de la EspecieRESUMEN
We have isolated and analysed the cell envelope of the thermophilic bacterium Thermus thermophilus HB8. Isolated cell walls, characterized by the dominance of the S-layer protein SlpA, are found to be constituted by several protein complexes of high molecular weights. Further isolation steps, starting from the cell wall samples, led to the selective release of the S-layer protein SlpA in solution as confirmed by mass spectrometry. Blue Native gel electrophoresis on these samples showed that SlpA is organized into a specific hierarchical order of oligomeric states that are consistent with the complexes at high molecular weight identified on the total cell wall fraction. The analysis showed that SlpA bases this peculiar organization on monomers and exceptionally stable dimers, leading to the formation of tetramers, heptamers, and decamers. Furthermore, the two elementary units of SlpA, monomers and dimers, are regulated by the presence of calcium not only for the assembling of monomers into dimers, but also for the splitting of dimers into monomers. Finally, the SlpA protein was found to be subjected to specific proteolysis leading to characteristic degradation products. Findings are discussed in terms of S-layer assembling properties as bases for understanding its structure, turn-over and organization.
Asunto(s)
Proteínas Bacterianas/metabolismo , Thermus thermophilus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Calcio/química , Calcio/metabolismo , Pared Celular/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Espectrometría de MasasRESUMEN
The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.
Asunto(s)
Fragaria/enzimología , Fragaria/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Empalme Alternativo/fisiología , Secuencia de Bases , ADN de Plantas/genética , Frutas/enzimología , Frutas/metabolismo , Redes y Vías Metabólicas/fisiología , Modelos Moleculares , Oxidorreductasas/genética , Proteínas de Plantas/genética , Conformación Proteica , Isoformas de ProteínasRESUMEN
We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single-step staining-destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed.
Asunto(s)
Electroforesis en Gel de Poliacrilamida Nativa/métodos , Proteínas del Complejo del Centro de Reacción Fotosintética/análisis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Colorantes de Rosanilina/química , Coloración y Etiquetado/métodos , Tilacoides/química , Nicotiana/química , Nicotiana/citologíaRESUMEN
The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C(CORT), a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C).