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1.
Biochemistry ; 60(39): 2925-2931, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34506130

RESUMO

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.


Assuntos
Antivirais/metabolismo , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Isoxazóis/metabolismo , Fenilalanina/análogos & derivados , Pirrolidinonas/metabolismo , SARS-CoV-2/enzimologia , Valina/análogos & derivados , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Enterovirus Humano D/enzimologia , Ligação de Hidrogênio , Isoxazóis/química , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Pirrolidinonas/química , Eletricidade Estática , Valina/química , Valina/metabolismo
2.
Science ; 373(6555): 662-673, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353949

RESUMO

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias/genética , RNA Longo não Codificante/genética , Acetilgalactosamina , Animais , Biopterina/análogos & derivados , Biopterina/metabolismo , Biopterina/uso terapêutico , Dieta , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Fenilalanina/administração & dosagem , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêutico
3.
Biophys J ; 120(16): 3382-3391, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34237287

RESUMO

FG nucleoporins (FG Nups) are intrinsically disordered proteins and are the putative regulators of nucleocytoplasmic transport. They allow fast, yet selective, transport of molecules through the nuclear pore complex, but the underlying mechanism of nucleocytoplasmic transport is not yet fully discovered. As a result, FG Nups have been the subject of extensive research in the past two decades. Although most studies have been focused on analyzing the conformation and function of FG Nups from a biophysical standpoint, some recent studies have investigated the sequence-function relationship of FG Nups, with a few investigating amino acid sequences of a large number of FG Nups to understand common characteristics that might enable their function. Previously, we identified an evolutionarily conserved feature in FG Nup sequences, which are extended subsequences with low charge density, containing only positive charges, and located toward the N-terminus of FG Nups. We named these patterns longest positive like charge regions (lpLCRs). These patterns are specific to positively charged residues, and negatively charged residues do not demonstrate such a pattern. In this study, we compare FG Nups with other disordered proteins obtained from the DisProt and UniProt database in terms of presence of lpLCRs. Our results show that the lpLCRs are virtually exclusive to FG Nups and are not observed in other disordered proteins. Also, lpLCRs are what differentiate FG Nups from DisProt proteins in terms of charge distribution, meaning that excluding lpLCRs from the sequences of FG Nups make them similar to DisProt proteins in terms of charge distribution. We also previously showed the biophysical effect of lpLCRs in conformation of FG Nups. The results of this study are in line with our previous findings and imply that lpLCRs are virtually exclusive and functionally significant characteristics of FG Nups and nucleocytoplasmic transport.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Fenilalanina , Transporte Ativo do Núcleo Celular , Glicina/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/metabolismo
4.
Nat Commun ; 12(1): 4322, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262048

RESUMO

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.


Assuntos
Meiose , Oócitos/citologia , Securina/metabolismo , Motivos de Aminoácidos , Animais , Segregação de Cromossomos , Camundongos , Mutação , Oócitos/metabolismo , Fenilalanina/genética , Fenilalanina/metabolismo , Prometáfase , Ligação Proteica , Securina/química , Securina/genética , Separase/metabolismo
5.
J Virol ; 95(16): e0061721, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34105996

RESUMO

The current pandemic of COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, we first analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human angiotensin-converting enzyme 2 (ACE2) receptor. Among 32,123 genomes of SARS-CoV-2 isolates (December 2019 through March 2020), 302 nonsynonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F+D614G), which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. IMPORTANCE A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious has been the research hot spot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human angiotensin-converting enzyme 2 (ACE2) receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , COVID-19/transmissão , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Cinética , Simulação de Dinâmica Molecular , Fenilalanina/química , Fenilalanina/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/classificação , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Termodinâmica , Valina/química , Valina/metabolismo , Virulência , Ligação Viral
6.
Nat Commun ; 12(1): 2805, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990606

RESUMO

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Terapia Biológica/métodos , Microbioma Gastrointestinal , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Fenilcetonúrias/terapia , Animais , Células CACO-2 , Simulação por Computador , Escherichia coli/metabolismo , Engenharia Genética , Células HT29 , Humanos , Técnicas In Vitro , Microfluídica , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/microbiologia , Primatas , Biologia Sintética
7.
Food Funct ; 12(9): 4092-4104, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977979

RESUMO

Baicalin shows excellent protective effects against Mycoplasma gallisepticum (MG) induced inflammatory injury as discussed in our previous studies. However, the physiological effects of baicalin are notable in contrast to its low bioavailability, and the critical mechanism for the protective effects of baicalin against MG infection is still unclear. The main objective of this study was to investigate whether baicalin alleviates MG-induced lung inflammatory injury through regulating gut microbiota. Using an MG infection model, results showed that baicalin treatment significantly reduced MG colonization and ameliorated the abnormal pathological changes in the lung. Baicalin treatment also reduced the level of proinflammatory cytokines and suppressed proinflammatory protein expression. Notably, MG infection changed the gut microbiota composition, however, the abnormal gut microbiota composition was partially alleviated by baicalin treatment. Baicalin significantly enriched the commensal bacterium Bacteroides fragilis, and gavaged with Bacteroides fragilis alleviating MG infection-induced inflammatory injury in the lung. In addition, baicalin reversed peripheral accumulation of phenylalanine induced by MG infection. Importantly, increased phenylalanine induced excessive necroptosis through the modulation of gga-miR-190a-3p-Fas-associated protein with death domain (FADD) axis in HD11 macrophages. Together, our findings highlighted the role of gut microbiota and phenylalanine metabolism in MG infection and confirmed that baicalin could effectively inhibit MG-induced inflammatory injury in the lung by remodeling the gut microbiota and phenylalanine metabolism.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Galinhas , Flavonoides/uso terapêutico , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum , Fenilalanina/metabolismo , Animais , Bacteroides fragilis/crescimento & desenvolvimento , Citocinas/metabolismo , Disbiose/tratamento farmacológico , Disbiose/veterinária , Inflamação/veterinária , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/metabolismo , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/patologia , Mycoplasma gallisepticum/crescimento & desenvolvimento , Necroptose , Necrose , Fenilalanina/farmacologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia
8.
Plant Cell ; 33(3): 671-696, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33955484

RESUMO

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.


Assuntos
Plântula/metabolismo , Triptofano/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Arabidopsis/metabolismo , Cicloexenos/metabolismo , Fenilalanina/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
9.
World J Microbiol Biotechnol ; 37(5): 84, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855641

RESUMO

L-phenylalanine is an important amino acid that is widely used in the fields of food flavors and pharmaceuticals. Apart from L-phenylalanine itself, various commercially valuable chemical compounds can also be generated via the L-phenylalanine biosynthesis pathway. Compared with direct extraction from plants or synthesis by chemical reaction, microbial production of L-phenylalanine -derived compounds can overcome the drawbacks of environmental pollution, low yield, and mixtures of stereoisomeric products. Accordingly, increasing intracellular levels of precursors, deregulating feedback inhibition and transcription repression, engineering global regulators and other effective strategies have been implemented to produce different L-phenylalanine -derived compounds in the excellent chassis host Escherichia coli. Finally, this review highlights principal strategies for improving the production of L-phenylalanine and/or its derivatives in E. coli, and discusses the future outlook for further enhancing the titer and yields of these compounds.


Assuntos
Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Fenilalanina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrocarbonetos Aromáticos/metabolismo , Microbiologia Industrial , Engenharia Metabólica
10.
Biomolecules ; 11(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808760

RESUMO

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Quinolonas/química , Quinolonas/farmacologia , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Fluorometria , Células HEK293 , Humanos , Doenças Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Ressonância de Plasmônio de Superfície , Tripsina
11.
Viruses ; 13(3)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804121

RESUMO

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 µM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.


Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/metabolismo , HIV-1/efeitos dos fármacos , Indóis/metabolismo , Fenilalanina/análogos & derivados , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fármacos Anti-HIV/isolamento & purificação , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Desenho de Fármacos , Células HEK293 , Humanos , Indóis/farmacologia , Fígado/efeitos dos fármacos , Camundongos , Microssomos/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Replicação Viral/efeitos dos fármacos
12.
FEBS Lett ; 595(11): 1525-1541, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792910

RESUMO

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Transporte/química , Cloroplastos/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/genética , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismo
13.
Nat Commun ; 12(1): 2073, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824313

RESUMO

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.


Assuntos
Amiloide/metabolismo , Fígado/enzimologia , Mutação/genética , Estresse Oxidativo , Fenilalanina Hidroxilase/genética , Agregados Proteicos , Animais , Autofagia , Biomarcadores/metabolismo , Peso Corporal , Cruzamento , Feminino , Regulação da Expressão Gênica , Genótipo , Metabolismo dos Lipídeos , Fígado/patologia , Masculino , Metaboloma , Camundongos , Proteínas Mutantes/metabolismo , Neurotransmissores/metabolismo , Estresse Oxidativo/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Pterinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Respiração , Ubiquitina/metabolismo , Ubiquitinação
14.
Molecules ; 26(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806510

RESUMO

It has been reported that monoamine neurotransmitters can be produced by gut microbiota, and that several related metabolites of amino acids in these pathways are associated with nervous system (NVS) diseases. Herein, we focused on three pathways, namely, phenylalanine (Phe), tryptophan (Trp), and glutamic acid (Glu), and established an underivatized liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of nineteen monoamine neurotransmitters and related metabolites in the gut microbiota. The neurotransmitters and related metabolites included Phe, tyrosine (Tyr), l-dopa (Dopa), dopamine (DA), 3-methoxytyramine, Trp, hydroxytryptophan, 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), kynurenine (KN), kynurenic acid (KYNA), melatonin, tryptamine (TA), indole-3-lactic acid (ILA), indole-3-acetic acid (IAA), indolyl-3-propionic acid (IPA), Glu, gamma-aminobutyric acid (GABA), and acetylcholine (Ach). A fluoro-phenyl bonded column was used for separation, and the mobile phase consisted of methanol:acetonitrile (1:1) and water, with 0.2% formic acid in both phases. The compounds exhibited symmetric peak shapes and sufficient sensitivity under a total analysis time of 8.5 min. The method was fully validated with acceptable linearity, accuracy, precision, matrix effect, extraction recovery, and stability. The results showed that neurotransmitters, such as Dopa, DA, 5-HT, GABA, and Ach, were present in the gut microbiota. The metabolic pathway of Trp was disordered under depression, with lower levels of 5-HT, 5-HIAA, KN, KYNA, TA, ILA, IAA, IPA, and Glu, and a higher ratio of KYNA/KN. In addition, some first-line NVS drugs, such as sertraline, imipramine, and chlorpromazine, showed regulatory potential on these pathways in the gut microbiota.


Assuntos
Monoaminas Biogênicas/análise , Microbioma Gastrointestinal , Ácido Glutâmico/metabolismo , Neurotransmissores/análise , Fenilalanina/metabolismo , Triptofano/metabolismo , Animais , Masculino , Redes e Vias Metabólicas , Ratos , Ratos Sprague-Dawley
15.
Molecules ; 26(8)2021 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-33920347

RESUMO

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.


Assuntos
Metaboloma , Metabolômica/métodos , Neoplasias da Bexiga Urinária/urina , Idoso , Ácido Benzoico/urina , Estudos de Casos e Controles , Cromatografia Líquida , Ácidos Cumáricos/urina , Feminino , Glicerofosfolipídeos/urina , Hipuratos/urina , Histidina/urina , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Fenilalanina/metabolismo , Microextração em Fase Sólida/métodos , Espectrometria de Massas em Tandem , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , beta-Alanina/urina
16.
Nucleic Acids Res ; 49(9): 5351-5368, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33885823

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.


Assuntos
Mycobacterium tuberculosis/enzimologia , Fenilalanina-tRNA Ligase/química , RNA de Transferência de Fenilalanina/química , Aminoacilação de RNA de Transferência , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mycobacterium tuberculosis/genética , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina-tRNA Ligase/metabolismo , Ligação Proteica , RNA de Transferência de Fenilalanina/metabolismo , Thermus thermophilus/enzimologia
17.
Biomolecules ; 11(2)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670496

RESUMO

Obesity is one of the most incident and concerning disease worldwide. Definite strategies to prevent obesity and related complications remain elusive. Among the risk factors of the onset of obesity, gut microbiota might play an important role in the pathogenesis of the disease, and it has received extensive attention because it affects the host metabolism. In this study, we aimed to define a metabolic profile of the segregated obesity-associated gut dysbiosis risk factor. The study of the metabolome, in an obesity-associated gut dysbiosis model, provides a relevant way for the discrimination on the different biomarkers in the obesity onset. Thus, we developed a model of this obesity risk factors through the transference of gut microbiota from obese to non-obese male Wistar rats and performed a subsequent metabolic analysis in the receptor rats. Our results showed alterations in the lipid metabolism in plasma and in the phenylalanine metabolism in urine. In consequence, we have identified metabolic changes characterized by: (1) an increase in DG:34:2 in plasma, a decrease in hippurate, (2) an increase in 3-HPPA, and (3) an increase in o-coumaric acid. Hereby, we propose these metabolites as a metabolic profile associated to a segregated dysbiosis state related to obesity disease.


Assuntos
Disbiose/metabolismo , Disbiose/microbiologia , Microbioma Gastrointestinal/fisiologia , Obesidade/metabolismo , Obesidade/microbiologia , Animais , Ácidos Cumáricos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Masculino , Metabolômica/métodos , Fenilalanina/metabolismo , Projetos Piloto , Ratos , Ratos Wistar
18.
J Gerontol A Biol Sci Med Sci ; 76(6): 996-999, 2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-33539523

RESUMO

Older adults' skeletal muscle has shown to be less responsive to anabolic stimuli as compared to young both in vitro, in short and controlled in vivo settings and in long-term training studies. However, to translate controlled mechanistic findings to long-term adaptations intermediate measures allowing daily life routines with regard to activity and diet would be useful to evaluate physiological interventions. The purpose of this study was to investigate the exercise effect in young and older adults with 2 independent methods to measure muscle protein synthesis rate. Healthy young and old men were recruited to the study protocol where myofibrillar fractional synthesis rate was measured during 2 days allowing normal activities of daily living with D2O-labeled alanine and during 4 hours in the overnight fasted state with [13C6]phenylalanine infusion. During this period 1 leg completed an exercise session every day (exercise leg) while the contralateral leg was kept inactive (normal leg). Both legs were used for activities of daily living. Two-day myofibrillar fractional synthesis rate was significantly higher in the exercise leg in both young and old as compared to normal leg with no age difference. The 4-hour overnight fasted myofibrillar fractional synthesis rate showed that only young exercise leg was significantly higher than normal leg. The present findings support the notion that anabolic resistance exists in the skeletal muscle of healthy older men when evaluated in controlled settings. However, this response is not as clear when measured during daily life where variance is greater, which calls for further investigations in larger cohorts.


Assuntos
Exercício Físico/fisiologia , Proteínas Musculares/biossíntese , Atividades Cotidianas , Adolescente , Adulto , Fatores Etários , Idoso , Alanina/metabolismo , Humanos , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Fenilalanina/metabolismo , Adulto Jovem
19.
Int J Sport Nutr Exerc Metab ; 31(3): 217-226, 2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33588378

RESUMO

Protein ingestion and exercise stimulate myofibrillar protein synthesis rates. When combined, exercise further increases the postprandial rise in myofibrillar protein synthesis rates. It remains unclear whether protein ingestion with or without exercise also stimulates muscle connective tissue protein synthesis rates. The authors assessed the impact of presleep protein ingestion on overnight muscle connective tissue protein synthesis rates at rest and during recovery from resistance-type exercise in older men. Thirty-six healthy, older men were randomly assigned to ingest 40 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine-labeled casein protein (PRO, n = 12) or a nonprotein placebo (PLA, n = 12) before going to sleep. A third group performed a single bout of resistance-type exercise in the evening before ingesting 40 g intrinsically-labeled casein protein prior to sleep (EX+PRO, n = 12). Continuous intravenous infusions of L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine were applied with blood and muscle tissue samples collected throughout overnight sleep. Presleep protein ingestion did not increase muscle connective tissue protein synthesis rates (0.049 ± 0.013 vs. 0.060 ± 0.024%/hr in PLA and PRO, respectively; p = .73). Exercise plus protein ingestion resulted in greater overnight muscle connective tissue protein synthesis rates (0.095 ± 0.022%/hr) when compared with PLA and PRO (p < .01). Exercise increased the incorporation of dietary protein-derived amino acids into muscle connective tissue protein (0.036 ± 0.013 vs. 0.054 ± 0.009 mole percent excess in PRO vs. EX+PRO, respectively; p < .01). In conclusion, resistance-type exercise plus presleep protein ingestion increases overnight muscle connective tissue protein synthesis rates in older men. Exercise enhances the utilization of dietary protein-derived amino acids as precursors for de novo muscle connective tissue protein synthesis during overnight sleep.


Assuntos
Tecido Conjuntivo/metabolismo , Proteínas na Dieta/administração & dosagem , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Treinamento de Força , Sono/fisiologia , Idoso , Glicemia/análise , Proteínas Sanguíneas/análise , Caseínas/administração & dosagem , Caseínas/sangue , Caseínas/metabolismo , Proteínas na Dieta/metabolismo , Método Duplo-Cego , Fenômenos Fisiológicos da Nutrição do Idoso , Humanos , Insulina/sangue , Leucina/administração & dosagem , Leucina/sangue , Leucina/metabolismo , Masculino , Miofibrilas/metabolismo , Fenilalanina/administração & dosagem , Fenilalanina/sangue , Fenilalanina/metabolismo , Período Pós-Prandial/fisiologia
20.
Biomolecules ; 11(2)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562549

RESUMO

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.


Assuntos
Antifúngicos/farmacologia , Arabidopsis/efeitos dos fármacos , Botrytis/patogenicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Espermidina/farmacologia , Espermina/farmacologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/imunologia , Asparagina/imunologia , Asparagina/metabolismo , Botrytis/imunologia , Catalase/genética , Catalase/imunologia , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Frutose/imunologia , Frutose/metabolismo , Glucose/imunologia , Glucose/metabolismo , Peróxido de Hidrogênio , Nitrato Redutase/genética , Nitrato Redutase/imunologia , Peroxidase/genética , Peroxidase/imunologia , Fenilalanina/imunologia , Fenilalanina/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/prevenção & controle , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Sacarose/imunologia , Sacarose/metabolismo , Ácido gama-Aminobutírico/imunologia , Ácido gama-Aminobutírico/metabolismo
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