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1.
Nat Commun ; 11(1): 3394, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636383

RESUMO

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.


Assuntos
Ritmo Circadiano , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Relógios Circadianos , Criptocromos/genética , Feminino , Genes Reporter , Teste de Complementação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Oscilometria , Transdução de Sinais , Núcleo Supraquiasmático/citologia
2.
PLoS One ; 15(7): e0235853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701967

RESUMO

PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Teste de Complementação Genética/métodos , Fases de Leitura Aberta , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Escherichia coli , Biblioteca Gênica , Mycobacterium tuberculosis , beta-Lactamases/metabolismo
3.
PLoS Biol ; 18(5): e3000627, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421706

RESUMO

Despite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. These duplicated genes may have been free to diverge in function since their expansion, and it is unclear how or at what rate ancestral functions are retained or partitioned among co-orthologs between species and within gene families. Thus, in order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (1 yeast gene to 2 human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replaceability across different ortholog classes, with an obvious trend toward differential replaceability inside gene families, and rarely observe replaceability by all members of a family. We quantify the ability of various properties of the orthologs to predict replaceability, showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and tissue-specific expression of the human co-orthologs, i.e., the human proteins that are less diverged from their yeast counterpart and more ubiquitously expressed across human tissues more often replace their single yeast ortholog. These trends were consistent with in silico simulations demonstrating that when only one ortholog can replace its corresponding yeast equivalent, it tends to be the least diverged of the pair. Replaceability of yeast genes having more than 2 human co-orthologs was marked by retention of orthologous interactions in functional or protein networks as well as by more ancestral subcellular localization. Overall, we performed >400 human gene replaceability assays, revealing 50 new human-yeast complementation pairs, thus opening up avenues to further functionally characterize these human genes in a simplified organismal context.


Assuntos
Evolução Molecular , Genes Duplicados , Genes Fúngicos , Família Multigênica , Saccharomycetales/genética , Expressão Gênica , Teste de Complementação Genética , Humanos , Homologia de Sequência do Ácido Nucleico
4.
PLoS Genet ; 16(4): e1008330, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324744

RESUMO

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.


Assuntos
Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alelos , Anticódon , Citosol/enzimologia , Teste de Complementação Genética , Humanos , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Alinhamento de Sequência , Especificidade por Substrato
5.
PLoS One ; 15(4): e0230981, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251488

RESUMO

Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/fisiologia , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/fisiologia , Aminoácidos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Bortezomib/farmacologia , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Genes Fúngicos , Teste de Complementação Genética , Humanos , Técnicas In Vitro , Mutação , Nitrogênio/metabolismo , Estresse Oxidativo/genética , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional , Vacúolos/metabolismo
6.
Hum Genet ; 139(10): 1247-1259, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32306098

RESUMO

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.


Assuntos
Complexo 1 de Proteínas Adaptadoras/genética , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Surdez/genética , Diarreia/genética , Ictiose/genética , Deficiência Intelectual/genética , Ceratodermia Palmar e Plantar/genética , Mutação de Sentido Incorreto , Complexo 1 de Proteínas Adaptadoras/deficiência , Subunidades sigma do Complexo de Proteínas Adaptadoras/deficiência , Sequência de Bases , Células CACO-2 , Claudina-3/genética , Claudina-3/metabolismo , Consanguinidade , Surdez/diagnóstico , Surdez/metabolismo , Surdez/patologia , Diarreia/diagnóstico , Diarreia/metabolismo , Diarreia/patologia , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Ictiose/diagnóstico , Ictiose/metabolismo , Ictiose/patologia , Lactente , Recém-Nascido , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/metabolismo , Ceratodermia Palmar e Plantar/patologia , Linhagem , Permeabilidade , Sequenciamento Completo do Exoma , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
7.
Plant Mol Biol ; 103(1-2): 197-210, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32130643

RESUMO

DEEPER ROOTING 1 (DRO1) contributes to the downward gravitropic growth trajectory of roots upstream of lateral auxin transport in monocots and dicots. Loss of DRO1 function leads to horizontally oriented lateral roots and altered gravitropic set point angle, while loss of all three DRO family members results in upward, vertical root growth. Here, we attempt to dissect the roles of AtDRO1 by analyzing expression, protein localization, auxin gradient formation, and auxin responsiveness in the atdro1 mutant. Current evidence suggests AtDRO1 is predominantly a membrane-localized protein. Here we show that VENUS-tagged AtDRO1 driven by the native AtDRO1 promoter complemented an atdro1 Arabidopsis mutant and the protein was localized in root tips and detectable in nuclei. atdro1 primary and lateral roots showed impairment in establishing an auxin gradient upon gravistimulation as visualized with DII-VENUS, a sensor for auxin signaling and proxy for relative auxin distribution. Additionally, PIN3 domain localization was not significantly altered upon gravistimulation in atdro1 primary and lateral roots. RNA-sequencing revealed differential expression of known root development-related genes in atdro1 mutants. atdro1 lateral roots were able to respond to exogenous auxin and AtDRO1 gene expression levels in root tips were unaffected by the addition of auxin. Collectively, the data suggest that nuclear localization may be important for AtDRO1 function and suggests a more nuanced role for DRO1 in regulating auxin-mediated changes in lateral branch angle. KEY MESSAGE: DEEPER ROOTING 1 (DRO1) when expressed from its native promoter is predominately localized in Arabidopsis root tips, detectable in nuclei, and impacts auxin gradient formation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares/fisiologia , Raízes de Plantas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Núcleo Celular/metabolismo , Teste de Complementação Genética , Gravitação , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
8.
Nat Commun ; 11(1): 870, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054839

RESUMO

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.


Assuntos
Aciltransferases/metabolismo , Embriófitas/metabolismo , Liases Intramoleculares/metabolismo , Proteínas de Plantas/metabolismo , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Biocatálise , Vias Biossintéticas/genética , Chalconas/biossíntese , Embriófitas/genética , Evolução Molecular , Flavonoides/biossíntese , Genes de Plantas , Teste de Complementação Genética , Liases Intramoleculares/genética , Cinética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Policetídeos/metabolismo , Especificidade por Substrato
9.
Chem Commun (Camb) ; 56(19): 2925-2928, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32039410

RESUMO

Cytochalasans are highly complex fungal metabolites which exhibit diverse biological activities. Little is known of the chemical steps involved in the construction of the tricyclic core, which consists of an octahydro-isoindole skeleton fused to a macrocyclic ring. Here, using a directed gene knockout and complementation strategy, we show that PyiF is implicated as the proposed intramolecular [4+2] Diels-Alderase required for construction of the tricyclic core of pyrichalasin H 1.


Assuntos
Reação de Cicloadição , Catálise , Fungos/genética , Fungos/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética
10.
Appl Microbiol Biotechnol ; 104(5): 2179-2191, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31900562

RESUMO

O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O-acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production.


Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Acetilação , Alginatos/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Complexos Multiproteicos/genética , Periplasma/metabolismo , Polimerização , Ligação Proteica , Estabilidade Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade
11.
mBio ; 11(1)2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992617

RESUMO

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design.IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.


Assuntos
Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Farmacorresistência Bacteriana Múltipla , Interações Hospedeiro-Patógeno , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/virologia , Mutação , Bacteriófagos/ultraestrutura , Evolução Molecular , Teste de Complementação Genética , Interações Hospedeiro-Patógeno/genética , Humanos , Testes de Sensibilidade Microbiana , Porinas/genética , Porinas/metabolismo , Ligação Viral
12.
Plant Mol Biol ; 102(4-5): 501-515, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31919641

RESUMO

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.


Assuntos
Oryza/fisiologia , Fosfatidilserinas/biossíntese , Folhas de Planta/fisiologia , Proteínas de Plantas/fisiologia , Clorofila/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Malondialdeído/metabolismo , Mutação , Oryza/genética , Estresse Oxidativo , Fenótipo , Fotossíntese , Pigmentação , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
13.
Plant Mol Biol ; 102(4-5): 517-535, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927660

RESUMO

KEY MESSAGE: We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids. Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.


Assuntos
Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/metabolismo , Flores/enzimologia , Lavandula/enzimologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Catálise , Dimetilaliltranstransferase/genética , Difosfatos , Diterpenos , Escherichia coli/genética , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Lavandula/genética , Monoterpenos/metabolismo , Fases de Leitura Aberta , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Terpenos/metabolismo , Tabaco/metabolismo
14.
Nucleic Acids Res ; 48(1): 486-499, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31745563

RESUMO

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.


Assuntos
Adenina/biossíntese , Vias Biossintéticas/genética , Carboxiliases/genética , Cromossomos Artificiais Humanos/química , Peptídeo Sintases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Carboxiliases/metabolismo , Cromossomos Artificiais Humanos/metabolismo , Teste de Complementação Genética , Engenharia Genética/métodos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
15.
Nucleic Acids Res ; 48(1): 332-348, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777930

RESUMO

Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metaloproteínas/genética , RNA Ribossômico 16S/genética , Sinorhizobium meliloti/genética , Zinco/metabolismo , Pareamento de Bases , Cátions Bivalentes , Ensaios Enzimáticos , Escherichia coli/metabolismo , Expressão Gênica , Teste de Complementação Genética , Isoenzimas/deficiência , Isoenzimas/genética , Metaloproteínas/deficiência , Mutação , Conformação de Ácido Nucleico , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico 16S/metabolismo , Sinorhizobium meliloti/metabolismo
16.
Nat Chem Biol ; 16(1): 42-49, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31636431

RESUMO

Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines, and present evidence for an unprecedented biosynthetic model that invokes intermodule substrate activation and tailoring, module skipping and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate driven. Expanding bidirectional intermodule domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and challenges our understanding of the potential elasticity of multimodular megaenzymes.


Assuntos
Família Multigênica , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/biossíntese , Catálise , Cromatografia Líquida , Clonagem Molecular , Elasticidade , Deleção de Genes , Teste de Complementação Genética , Espectrometria de Massas , Mutação , Policetídeo Sintases/metabolismo , Domínios Proteicos , Proteobactérias/enzimologia , Especificidade por Substrato
17.
Chemosphere ; 240: 124914, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31557642

RESUMO

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.


Assuntos
Arsênico/toxicidade , Glutationa/biossíntese , Hebeloma/efeitos dos fármacos , Micorrizas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Antioxidantes/metabolismo , Arsênico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Hebeloma/genética , Hebeloma/metabolismo , Inativação Metabólica , Mutação , Micorrizas/genética , Micorrizas/metabolismo , Saccharomyces cerevisiae/metabolismo , Poluentes do Solo/metabolismo
18.
Appl Environ Microbiol ; 86(5)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31862728

RESUMO

The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in citric acid hyperproduction. The ΔlaeA strain exhibited a significant reduction in citric acid production. Cap analysis gene expression (CAGE) revealed that laeA is required for the expression of a putative citrate exporter-encoding cexA gene, which is critical for citric acid production. Deficient citric acid production by a ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of a cexA-overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachii IMPORTANCE A. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation tests, and ChIP-qPCR, it was found that laeA is required for citric acid production through the regulation of cexA in A. kawachii The epigenetic regulation of citric acid production elucidated in this study will be useful for controlling the fermentation processes of shochu.


Assuntos
Aspergillus/genética , Ácido Cítrico/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Sequência de Aminoácidos , Aspergillus/metabolismo , Imunoprecipitação da Cromatina , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Metiltransferases/química , Metiltransferases/metabolismo , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência
19.
BMC Genomics ; 20(1): 942, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31810444

RESUMO

BACKGROUND: Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The PrrAB two-component genetic regulatory system is essential in M. tuberculosis and represents an attractive therapeutic target. In this study, transcriptomic analysis (RNA-seq) of an M. smegmatis ΔprrAB mutant was used to define the PrrAB regulon and provide insights into the essential nature of PrrAB in M. tuberculosis. RESULTS: RNA-seq differential expression analysis of M. smegmatis wild-type (WT), ΔprrAB mutant, and complementation strains revealed that during in vitro exponential growth, PrrAB regulates 167 genes (q < 0.05), 57% of which are induced in the WT background. Gene ontology and cluster of orthologous groups analyses showed that PrrAB regulates genes participating in ion homeostasis, redox balance, metabolism, and energy production. PrrAB induced transcription of dosR (devR), a response regulator gene that promotes latent infection in M. tuberculosis and 21 of the 25 M. smegmatis DosRS regulon homologues. Compared to the WT and complementation strains, the ΔprrAB mutant exhibited an exaggerated delayed growth phenotype upon exposure to potassium cyanide and respiratory inhibition. Gene expression profiling correlated with these growth deficiency results, revealing that PrrAB induces transcription of the high-affinity cytochrome bd oxidase genes under both aerobic and hypoxic conditions. ATP synthesis was ~ 64% lower in the ΔprrAB mutant relative to the WT strain, further demonstrating that PrrAB regulates energy production. CONCLUSIONS: The M. smegmatis PrrAB two-component system regulates respiratory and oxidative phosphorylation pathways, potentially to provide tolerance against the dynamic environmental conditions experienced in its natural ecological niche. PrrAB positively regulates ATP levels during exponential growth, presumably through transcriptional activation of both terminal respiratory branches (cytochrome c bc1-aa3 and cytochrome bd oxidases), despite transcriptional repression of ATP synthase genes. Additionally, PrrAB positively regulates expression of the dormancy-associated dosR response regulator genes in an oxygen-independent manner, which may serve to fine-tune sensory perception of environmental stimuli associated with metabolic repression.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Mutação , Mycobacterium smegmatis/fisiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Teste de Complementação Genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Cianeto de Potássio/farmacologia , Regulon , Análise de Sequência de RNA/métodos
20.
Cell Rep ; 29(13): 4620-4631.e4, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875565

RESUMO

Patients with pathogenic mutations in NGLY1 cannot make tears and have global developmental delay and liver dysfunction. Traditionally, NGLY1 cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins before proteasomal degradation. We demonstrate that Ngly1-null mouse embryonic fibroblasts, NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient mouse embryonic fibroblasts swell slower and have reduced aquaporin1 mRNA and protein expression. Ngly1 knockdown and overexpression confirms that Ngly1 regulates aquaporin1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 knockout cells show reduced aquaporin11 mRNA, supporting NGLY1 as regulating expression of multiple aquaporins across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restores normal hypotonic lysis and aquaporin1 protein. We show that transcription factors Atf1/Creb1 regulate aquaporin1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient mouse embryonic fibroblasts. These results identify a non-enzymatic, regulatory function of NGLY1 in aquaporin transcription, possibly related to alacrima and neurological symptoms.


Assuntos
Aquaporina 1/genética , Aquaporinas/genética , Defeitos Congênitos da Glicosilação/genética , Oftalmopatias Hereditárias/genética , Doenças do Aparelho Lacrimal/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Transcrição Genética , Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/metabolismo , Animais , Aquaporina 1/metabolismo , Aquaporinas/metabolismo , Linhagem Celular , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Teste de Complementação Genética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Doenças do Aparelho Lacrimal/metabolismo , Doenças do Aparelho Lacrimal/patologia , Camundongos , Camundongos Knockout , Osmose , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/antagonistas & inibidores , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
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