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1.
J Biol Chem ; 298(10): 102478, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36100038

RESUMO

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.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Complexo de Endopeptidases do Proteassoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismo , Sequência Conservada/genética
2.
Mol Ecol ; 26(8): 2331-2347, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28141898

RESUMO

Chromosomal rearrangements between sympatric species often contain multiple loci contributing to assortative mating, local adaptation and hybrid sterility. When and how these associations arise during the process of speciation remains a subject of debate. Here, we address the relative roles of local adaptation and assortative mating on the dynamics of rearrangement evolution by studying how a rearrangement covaries with sexual and ecological trait divergence within a species. Previously, a chromosomal rearrangement that suppresses recombination on the Z (sex) chromosome was identified in European corn borer moths (Ostrinia nubilalis). We further characterize this recombination suppressor and explore its association with variation in sex pheromone communication and seasonal ecological adaptation in pairs of populations that are divergent in one or both of these characteristics. Direct estimates of recombination suppression in pedigree mapping families indicated that more than 39% of the Z chromosome (encompassing up to ~10 megabases and ~300 genes) resides within a nonrecombining unit, including pheromone olfactory receptor genes and a major quantitative trait locus that contributes to ecotype differences (Pdd). Combining direct and indirect estimates of recombination suppression, we found that the rearrangement was occasionally present between sexually isolated strains (E vs. Z) and between divergent ecotypes (univoltine vs. bivoltine). However, it was only consistently present when populations differed in both sexual and ecological traits. Our results suggest that independent of the forces that drove the initial establishment of the rearrangement, a combination of sexual and ecological divergence is required for rearrangement spread during speciation.


Assuntos
Rearranjo Gênico , Especiação Genética , Mariposas/genética , Simpatria , Adaptação Fisiológica , Animais , Mapeamento Cromossômico , Ecótipo , Feminino , Genética Populacional , Masculino , América do Norte , Linhagem , Locos de Características Quantitativas , Recombinação Genética , Estações do Ano , Atrativos Sexuais/genética
3.
bioRxiv ; 2024 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-38585835

RESUMO

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pup∼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup∼PanB in response to pantothenate levels. One Sentence Summary: A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .

4.
Curr Opin Struct Biol ; 67: 120-126, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33221704

RESUMO

Bacteria use gated proteolytic machines for routine protein quality control and regulated responses to environmental conditions. This review discusses recent advances in understanding the structure and regulation of ClpP proteases, nanomachines widely distributed across bacteria, and the bacterial proteasome, a protease found in relatively few species. For both machines, activators confer substrate specificity. We highlight new data from organisms encoding two ClpP isoforms and the central role of activators as platforms for integrating regulatory signals. Because proteolytic systems contribute to survival and virulence of many bacterial pathogens, understanding their forms and functions enables new approaches to design targeted therapeutics.


Assuntos
Endopeptidase Clp , Complexo de Endopeptidases do Proteassoma , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteólise , Especificidade por Substrato
5.
J Med Chem ; 64(9): 6262-6272, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33949190

RESUMO

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.


Assuntos
Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Desenho de Fármacos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/química , Relação Estrutura-Atividade
6.
Curr Biol ; 29(20): 3501-3509.e5, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31607536

RESUMO

Synchronizing the annual timing of physiological, morphological, and behavioral transitions with seasons enables survival in temperate environments [1]. The capacity to adjust life history timing and track local seasonal cycles can facilitate geographic expansion [2], adaptation [3], and tolerance [4-6] during rapid environmental change. Understanding the proximate causes of variation in seasonal timing improves prediction of future response and persistence [7, 8]. However, relatively little is known about the molecular basis generating this diversity [9], particularly in Lepidoptera, a group with many species in decline [10, 11]. In insects, the stress-tolerant physiological state of diapause enables coping with seasonal challenges [1, 12-15]. Seasonal changes in photoperiod and temperature are used to synchronize diapause with winter, and timing of diapause transitions varies widely within and among species [9, 16]. Changes in spring diapause termination in the European corn borer moth (Ostrinia nubilalis) have allowed populations to respond to shorter winters and emerge ∼3 weeks earlier in the year [17]. Multiple whole-genome approaches suggest two circadian clock genes, period (per) and pigment-dispersing factor receptor (Pdfr), underlie this polymorphism. Per and Pdfr are within interacting quantitative trait loci (QTL) and differ in allele frequency among individuals that end diapause early or late, with alleles maintained in high linkage disequilibrium. Our results provide testable hypotheses about the physiological role of circadian clock genes in the circannual timer. We predict these gene candidates will be targets of selection for future adaptation under continued global climate change [18].


Assuntos
Genoma de Inseto/fisiologia , Mariposas/genética , Animais , Genômica , Ritmo Infradiano/genética , Fatores de Tempo
7.
ISME J ; 11(12): 2718-2728, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28742070

RESUMO

Vibrio cholerae is a water-borne bacterial pathogen and causative agent of cholera. Although V. cholerae is a halophile, it can survive in fresh water, and this has a major role in cholera epidemics through consumption of contaminated water and subsequent fecal-oral spread. After dissemination from humans back into fresh water, V. cholerae encounters limited nutrient availability and an abrupt drop in conductivity but little is known about how V. cholerae adapts to, and survives in this environment. In this work, by abolishing or altering the expression of V. cholerae genes in a high-throughput manner, we observed that many osmotic shock tolerant mutants exhibited slowed or arrested growth, and/or generated a higher proportion of persister cells. In addition, we show that growth-arrested V. cholerae, including a persister subpopulation, are generated during infection of the intestinal tract and together allow for the successful dissemination to fresh water. Our results suggest that growth-arrested and persister subpopulations enable survival of V. cholerae upon shedding to the aquatic environment.


Assuntos
Vibrio cholerae/crescimento & desenvolvimento , Cólera/microbiologia , Água Doce/microbiologia , Humanos , Pressão Osmótica , Vibrio cholerae/química , Vibrio cholerae/genética
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