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How does one help a struggling trainee, in or out of their own lab?
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Can we do better when it comes to the "other-race effect"?
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Academias e Institutos , Biología/educación , Educación Continua , Docentes , Viaje , Recursos HumanosRESUMEN
Research in the life sciences is an inherently wasteful endeavor. Could we all do better to reduce waste by our laboratories?
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Administración de Residuos , LaboratoriosRESUMEN
As COVID wanes and scientific conferences come back, some advice on how to deal with, organise and enjoy sharing science at meetings.
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COVID-19 , COVID-19/epidemiología , HumanosRESUMEN
PIs do not need to stay away from bench work; in fact, they are often overall the best and most experienced experimentalists in the lab.
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There are many paths into and through academic science. Heran Darwin describes how she eventually got hooked on research.
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Investigación , Femenino , Humanos , CienciaRESUMEN
Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera. To understand the function of this domain, we deleted discrete sequences from the Dop loop and assessed pupylation in mutant strains of Mycobacterium tuberculosis. We determined that various Dop loop mutations resulted in altered pupylome profiles, in particular when mutant dop alleles were overexpressed. Taken together, our data suggest these conserved amino acids play a role in substrate selectivity for Dop.
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Proteínas Bacterianas , Mycobacterium tuberculosis , Complejo de la Endopetidasa Proteasomal , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinas/metabolismo , Secuencia Conservada/genéticaRESUMEN
Mycobacterium tuberculosis is a fascinating object of study: it is one of the deadliest pathogens of humankind, able to fend off persistent attacks by the immune system or drugs.
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Miel , Mustelidae , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculosis , Animales , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiologíaRESUMEN
2020 has been one of the craziest and strangest years we have lived through. Now that it's over, it's an opportunity to show gratitude for all the good things.
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There is no perfect recipe to balance work and life in academic research. Everyone has to find their own optimal balance to derive fulfilment from life and work.
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Equilibrio entre Vida Personal y LaboralRESUMEN
Good manners make a difference-in science and elsewhere. This includes our social media etiquette as researchers.
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One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.
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Aminohidrolasas/metabolismo , Citocininas/biosíntesis , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Aldehídos/metabolismo , Aminohidrolasas/genética , Animales , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Citocininas/metabolismo , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Óxido Nítrico/farmacología , Supresión GenéticaRESUMEN
Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1-6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique ß-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis-driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its ß-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.
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Adenosina Trifosfatasas/metabolismo , Mycobacterium tuberculosis/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/química , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
COVID-19 has caused a "Hunger Games" like run for emergency funding that risks detracting away from other diseases that ravage humanity.
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Betacoronavirus , Infecciones por Coronavirus , Pandemias , Neumonía Viral , COVID-19 , Humanos , Hambre , SARS-CoV-2RESUMEN
The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.
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Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Chaperonina 60/genética , Proteínas de Choque Térmico/genética , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Compuestos de Amonio/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Nitrógeno/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/patologíaRESUMEN
Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.
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Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tirfostinos/farmacología , Ubiquitinas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Relación Estructura-Actividad , Tirfostinos/síntesis química , Tirfostinos/química , Ubiquitinas/metabolismoRESUMEN
Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology.
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Proteínas Bacterianas/metabolismo , Mycobacterium/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Archaea/metabolismo , Proteínas Bacterianas/química , Células Eucariotas/metabolismo , Mycobacterium tuberculosis/metabolismo , Complejo de la Endopetidasa Proteasomal/química , ProteolisisRESUMEN
The cell envelope of Mycobacterium tuberculosis is a key target for antibiotics, yet its assembly and maintenance remain incompletely understood. Here we report that Rv2700, a previously uncharacterized M. tuberculosis gene, contributes to envelope integrity. Specifically, an Rv2700 mutant strain had a decreased growth rate, increased sensitivity to antibiotics that target peptidoglycan crosslinking, and increased cell envelope permeability. We propose that Rv2700 be named a "cell envelope integrity" gene (cei). Importantly, a cei mutant had attenuated virulence in mice. Cei shares predicted structural homology with another M. tuberculosis protein, VirR (Rv0431), and we found that a virR mutant had growth rate, antibiotic sensitivity, and envelope permeability phenotypes similar to those of the cei mutant. Both Cei and VirR are predicted to consist of a transmembrane helix and an extracellular LytR_C domain. LytR_C domains have no known function, but they are also found in a family of proteins, the LytR-Cps2A-Psr (LCP) enzymes, that perform important cell envelope functions in a range of bacteria. In mycobacteria, LCP enzymes attach arabinogalactan to peptidoglycan, and mycobacterial LCP enzyme mutants have phenotypes similar to those of virR- and cei-deficient strains. Collectively, our results suggest that LytR_C domain proteins may contribute to the cell envelope functions performed by LCP proteins. This study provides a framework for further mechanistic investigations of LytR_C proteins and, more broadly, for advancing our understanding of the cell envelopes of mycobacteria and other medically and economically important genera.IMPORTANCEMycobacterium tuberculosis causes about 1.5 million deaths per year. The unique composition of the Mycobacterium tuberculosis cell envelope is required for this bacterium to cause disease and is the target for several critical antibiotics. By better understanding the mechanisms by which mycobacteria assemble and maintain their cell envelope, we might uncover new therapeutic targets. In this work, we show that a previously uncharacterized protein, Rv2700, is important for cell envelope integrity in Mycobacterium tuberculosis and that loss of Rv2700 attenuates virulence in mice. This family of proteins is found in a broad group of bacterial species, so our work provides a first insight into their potential functions in many species important to the environment, industry, and human health.