RESUMO
In 1939, directly after the worst earthquake in the country's history, the Chilean state began implementing an electrification program. This plan shaped energy goals for years to come and defined the interconnected grid that dominates the country's energy infrastructure today. Based on extensive archival work, this article describes the birth of energopolitics in the country, using technology sociologist Michel Callon's notion of "interessement" to describe the strategies of a group of engineers who acted as system builders. Their four main strategies were embracing technological futurisms, forging heterogeneous networks, articulating and mobilizing knowledge, and using crises as windows of opportunity for change. The article shows not only the historical impact of past energy choices on today's world but also that current challenges to energy transitions are not without precedent. Using a sociological framework to tell this story allows us to highlight the mechanisms through which energy systems can change.
Assuntos
Terremotos , Chile , História do Século XX , Terremotos/história , Humanos , Desastres/história , PolíticaRESUMO
This paper presents an integrative case study of Chile's national strategy of research, development, and innovation (R&D+i) for disaster resilience and progress towards developing the institutional conditions necessary for its successful implementation. The paper covers the period between 2016 and 2021, concentrating on the work of the Chilean Commission of R&D+i for Resilience to Disasters of Natural Origin (CREDEN). Through an analysis of the official records of the initiative at all of its stages, and 29 semi-structured interviews with CREDEN members and stakeholders, we aim to present a successful example of strengthening the role of science and technology in disaster risk reduction. Chile's experience is particularly interesting because its strategy focused on R&D+i and proposed developing an industry of scientific-based technological solutions for disaster resilience. The study also illustrates how strategic interaction between academia, state, and industry can be a key factor in aligning knowledge production to tackle current socio-technical challenges.
Assuntos
Comportamento de Redução do Risco , Tecnologia , Humanos , ChileRESUMO
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.
Assuntos
Fenômenos Biológicos , Mycobacterium tuberculosis , Proteínas de Bactérias/genética , Humanos , Hipóxia , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/genética , Proteoma , ProteômicaRESUMO
The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.
Assuntos
Proteínas de Bactérias/metabolismo , Dictyostelium/metabolismo , Proteoma/metabolismo , Salmonella typhimurium/metabolismo , Vacúolos/metabolismo , Amoeba/metabolismo , Animais , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Mutação , Proteômica , Proteínas de Protozoários/metabolismo , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genéticaRESUMO
The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.
Assuntos
Proteínas de Bactérias/genética , Quimiotaxia/genética , Quimiotaxia/imunologia , Flagelos/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas aeruginosa/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Membrana Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Mutação , Fenótipo , Diester Fosfórico Hidrolases/genética , Proteoma , Proteômica/métodosRESUMO
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.
Assuntos
Calgranulina B/imunologia , Exossomos/patologia , Leucemia Linfocítica Crônica de Células B/patologia , NF-kappa B/imunologia , Basigina/análise , Basigina/imunologia , Calgranulina B/análise , Progressão da Doença , Exossomos/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , NF-kappa B/análise , Proteoma/análise , Proteoma/imunologiaRESUMO
The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.
Assuntos
Equinococose/veterinária , Echinococcus/química , Proteínas de Helminto/química , Lipoproteínas/química , Doenças dos Suínos/parasitologia , Animais , Equinococose/parasitologia , Echinococcus/genética , Echinococcus/imunologia , Echinococcus/isolamento & purificação , Eletroforese em Gel Bidimensional , Genótipo , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Espectrometria de Massas , Proteômica , SuínosRESUMO
The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Dicroísmo Circular , Humanos , Macrófagos/imunologia , Espectrometria de Massas , Mutação , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.
Assuntos
Eritrócitos/enzimologia , Proteínas de Homeodomínio/metabolismo , Nitrogênio/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/metabolismo , Animais , Domínio Catalítico , Echinococcus granulosus/enzimologia , Ativação Enzimática/fisiologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tirosina/metabolismoRESUMO
PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Rubredoxinas/metabolismo , Alcenos/química , Alcenos/metabolismo , Domínio Catalítico/fisiologia , Dicroísmo Circular , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Mutagênese Sítio-Dirigida , Nitrocompostos/química , Nitrocompostos/metabolismo , Rubredoxinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cystathionine beta-synthase (CBS) is a homocysteine metabolizing enzyme that contains pyridoxal phosphate (PLP) and a six-coordinate heme cofactor of unknown function. CBS was inactivated by peroxynitrite, the product of nitric oxide and superoxide radicals. The IC(50) was approximately 150microM for 5microM ferric CBS. Stopped-flow kinetics and competition experiments showed a direct reaction with a second-order rate constant of (2.4-5.0)x10(4)M(-1)s(-1) (pH 7.4, 37 degrees C). The radicals derived from peroxynitrite, nitrogen dioxide and carbonate radical, also inactivated CBS. Exposure to peroxynitrite did not modify bound PLP but led to nitration of Trp208, Trp43 and Tyr223 and alterations in the heme environment including loss of thiolate coordination, conversion to high-spin and bleaching, with no detectable formation of oxo-ferryl compounds nor promotion of one-electron processes. This study demonstrates the susceptibility of CBS to reactive oxygen/nitrogen species, with potential relevance to hyperhomocysteinemia, a risk factor for cardiovascular diseases.