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1.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3268-3275, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622634

RESUMO

Polyethylene terephthalate (PET) is a synthetic polymer consisting of ester bond-linked terephthalate and ethylene glycol. Tremendous amounts of PET have been produced and majority of them enters terrestrial and marine environment as wastes, posing serious threats to the global ecosystems. In 2016, a PET hydrolase from a PET-assimilating bacterium Ideonalla sakaiensis was reported and termed as IsPETase. This enzyme outperforms other PET-hydrolyzing enzymes in terms of its PET hydrolytic activity at ambient temperature, thus holds a great promise for PET biodegradation. In order to improve IsPETase activity, we conducted structure-based engineering to modify the putative substrate-binding tunnel. Among the several variants to the N233 residue of IsPETase, we discovered that the substitution of N233 with alanine increases its PET hydrolytic activity, which can be further enhanced when combined with a R280A mutation. We also determined the X-ray crystal structure of the IsPETase N233A variant, which shares nearly identical fold to the WT protein, except for an open end of subsite Ⅱ. We hypothesized that the smaller side chain of N233A variant might lead to an extended subsite Ⅱ for PET binding, which subsequently increases the enzymatic activity. Thus, this study provides new clues for further structure-based engineering of PETase.


Assuntos
Burkholderiales , Hidrolases , Polietilenotereftalatos/metabolismo , Burkholderiales/enzimologia , Hidrolases/genética , Engenharia de Proteínas
2.
Nanoscale ; 13(31): 13401-13409, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477745

RESUMO

Enzymes are the most efficient catalysts in nature that possess an impressive range of catalytic activities, albeit limited by stability in adverse conditions. Functional peptides have emerged as alternative robust biocatalysts to mimic complex enzymes. Here, a rational design of minimalistic amyloid-inspired peptides 1-2 is demonstrated, which leads to pathway-driven self-assembly triggered by heat, light and chemical cues to render 1D and 2D nanostructures by the interplay of hydrogen bonding, host-guest interaction and reversible photodimerization. Such in situ transformable peptide nanostructures by means of external cues are envisaged as a catalytic amyloid for the first time to mimic the hydrolase enzyme activity. Michaelis Menten's enzyme kinetic parameters for the hydrolysis rate correlate the external cue-mediated structure-function augmentation with the twisted bundles, 1TB being the most efficient biocatalyst among all the dimensionally diverse nanostructures. Unlike the natural enzyme, the peptide nanostructures exhibited the robust nature of the hydrolase activity over a broad range of temperature and pH. Finally, the peptide nanostructures are explored as efficient heterogeneous flow catalysts to improve the turnover number for the hydrolase activity.


Assuntos
Hidrolases , Nanoestruturas , Catálise , Ligação de Hidrogênio , Peptídeos
3.
Enzyme Microb Technol ; 150: 109868, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489027

RESUMO

Poly(ethylene terephthalate) (PET) is a class of polyester plastic composed of terephthalic acid (TPA) and ethylene glycol (EG). The accumulation of large amount of PET waste has resulted in severe environmental and health problems. Microbial polyester hydrolases with the ability to degrade PET provide an economy- and environment-friendly approach for the treatment of PET waste. In recent years, many PET hydrolases have been discovered and characterized from various microorganisms and engineered for better performance under practical application conditions. Here, recent progress in the discovery, characterization, and enzymatic mechanism elucidation of PET hydrolases is firstly reviewed. Then, structure-guided protein engineering of PET hydrolases with increased enzymatic activities, expanded substrate specificity, as well as improved protein stability is summarized. In addition, strategies for efficient expression of recombinant PET hydrolases, including secretory expression and cell-surface display, are briefly introduced. This review is concluded with future perspectives in biodegradation and subsequent biotransformation of PET wastes to produce value-added compounds.


Assuntos
Ácidos Ftálicos , Polietilenotereftalatos , Etilenos , Hidrolases/genética
4.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548397

RESUMO

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.


Assuntos
Cisteína/química , Hidrolases/metabolismo , Lactobacillus plantarum/enzimologia , Níquel/metabolismo , Nucleotídeos/biossíntese , Piridinas/química , Racemases e Epimerases/metabolismo , Carboxiliases , Catálise , Cristalografia por Raios X , Hidrolases/química , Hidrólise , Modelos Moleculares , Conformação Proteica , Racemases e Epimerases/química , Especificidade por Substrato
5.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502074

RESUMO

Amyloids are supramolecular assemblies composed of polypeptides stabilized by an intermolecular beta-sheet core. These misfolded conformations have been traditionally associated with pathological conditions such as Alzheimer's and Parkinson´s diseases. However, this classical paradigm has changed in the last decade since the discovery that the amyloid state represents a universal alternative fold accessible to virtually any polypeptide chain. Moreover, recent findings have demonstrated that the amyloid fold can serve as catalytic scaffolds, creating new opportunities for the design of novel active bionanomaterials. Here, we review the latest advances in this area, with particular emphasis on the design and development of catalytic amyloids that exhibit hydrolytic activities. To date, three different types of activities have been demonstrated: esterase, phosphoesterase and di-phosphohydrolase. These artificial hydrolases emerge upon the self-assembly of small peptides into amyloids, giving rise to catalytically active surfaces. The highly stable nature of the amyloid fold can provide an attractive alternative for the design of future synthetic hydrolases with diverse applications in the industry, such as the in situ decontamination of xenobiotics.


Assuntos
Amiloide/química , Hidrolases/química , Amiloide/síntese química , Amiloide/metabolismo , Animais , Domínio Catalítico , Humanos , Hidrolases/síntese química , Hidrolases/metabolismo
6.
J Hazard Mater ; 416: 126075, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492896

RESUMO

The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.


Assuntos
Burkholderiales , Polietilenotereftalatos , Hidrolases
7.
Molecules ; 26(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34443596

RESUMO

FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.


Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Humanos , Hidrolases/química , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica
8.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361727

RESUMO

Enzymatic pretreatment of seeds is a novel approach that enhances the health benefits of the extracted oil. The study investigated the influence of the enzymatic pretreatment of seeds on the quality of oil from different pomegranate cultivars. The quality of the ultrasound-assisted (and ethanol-extracted) oil was studied, with respect to the refractive index (RI), yellowness index (YI), conjugated dienes (K232), peroxide value (PV) ρ-anisidine value (AV), total oxidation value (TOTOX), total carotenoid content (TCC), total phenolic compounds (TPC), fatty acid composition, phytosterol composition, ferric reducing antioxidant power (FRAP), and 2.2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging capacity. The seeds of three different pomegranate cultivars ('Wonderful', 'Herskawitz', and 'Acco') were digested with an equal mixture of Pectinex Ultra SPL, Flavourzyme 100 L, and cellulase crude enzymes, at a concentration, pH, temperature, and time of 1.7%, 4.5, 40 °C, and 5 h, respectively. Enzymatic pretreatment of PS increased oil yield, PV, TPC, TCC, and DPPH radical scavenging capacity, but decreased the YI. The levels of K232, AV and TOTOX, fatty acids, phytosterols, RI, and FRAP, were not significantly affected by enzymatic pretreatment of PS. Principal component analysis (PCA) established that oil extracted from the 'Acco' seed after enzymatic pretreatment had higher yield, TPC, TCC, and DPPH radical scavenging capacity. Therefore, enzyme-pretreated 'Acco' pomegranate fruit seed is a source of quality seed oil with excellent antioxidant properties.


Assuntos
Antioxidantes/isolamento & purificação , Hidrolases/química , Extração Líquido-Líquido/métodos , Óleos Vegetais/isolamento & purificação , Romã (Fruta)/química , Sementes/química , Antioxidantes/química , Antioxidantes/farmacologia , Compostos de Bifenilo/antagonistas & inibidores , Carotenoides/química , Carotenoides/isolamento & purificação , Carotenoides/farmacologia , Etanol/química , Ácidos Graxos/química , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/farmacologia , Frutas/química , Alimento Funcional/provisão & distribuição , Humanos , Oxirredução , Fenóis/química , Fenóis/isolamento & purificação , Fenóis/farmacologia , Fitosteróis/química , Fitosteróis/isolamento & purificação , Fitosteróis/farmacologia , Picratos/antagonistas & inibidores , Óleos Vegetais/química , Análise de Componente Principal , Solventes/química , Sonicação/métodos
9.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
10.
Nutrients ; 13(8)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34444642

RESUMO

Fatty acids play a significant role in maintaining cellular and DNA protection and we previously found an inverse relationship between blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and DNA damage. The aim of this study was to explore differences in proteomic profiles, for 117 pro-inflammatory proteins, in two previously defined groups of individuals with different DNA damage and EPA and DHA levels. Healthy children and adolescents (n = 140) aged 9 to 13 years old in an urban area of Brazil were divided by k-means cluster test into two clusters of DNA damage (tail intensity) using the comet assay (cluster 1 = 5.9% ± 1.2 and cluster 2 = 13.8% ± 3.1) in our previous study. The cluster with higher DNA damage and lower levels of DHA (6.2 ± 1.6 mg/dL; 5.4 ± 1.3 mg/dL, p = 0.003) and EPA (0.6 ± 0.2 mg/dL; 0.5 ± 0.1 mg/dL, p < 0.001) presented increased expression of the proteins CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB, which are involved in pro-inflammatory pathways. Our findings support the hypothesis that low levels of n-3 long-chain PUFA may have a less protective role against DNA damage through expression of pro-inflammatory proteins, such as CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB.


Assuntos
Dano ao DNA , Ácidos Docosa-Hexaenoicos/sangue , Ácido Eicosapentaenoico/sangue , Ácidos Graxos Ômega-3/sangue , Adolescente , Brasil , Criança , Classe I de Fosfatidilinositol 3-Quinases/sangue , Classe Ia de Fosfatidilinositol 3-Quinase/sangue , Estudos Transversais , Ciclina C/sangue , Quinase 8 Dependente de Ciclina/sangue , Feminino , Humanos , Hidrolases/sangue , Inflamação/metabolismo , Masculino , Proteína Quinase C beta/sangue , Proteômica
11.
Enzyme Microb Technol ; 149: 109832, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311877

RESUMO

Haloalkane dehalogenase DhaA catalyzes the hydrolytic cleavage of carbon-halogen bonds and produces alcohol, a proton and a halide. However, DhaA suffers from the poor environmental stability, such as sensitivity to high temperature, low pH, hypersaline and organic solvent. In order to improve the environmental stability of DhaA, DhaA was covalently conjugated with inulin, a hydrophilic polysaccharide in the present study. Each DhaA was averagely conjugated with 7∼8 inulin molecules. The conjugated inulin could form a hydration layer around DhaA, which increased the conformational rigidity and decreased the entropy of the enzyme. Conjugation of inulin maintained 75.5 % of the enzymatic activity of DhaA and slightly altered the structure of DhaA. As compared with DhaA, the conjugate (inu-DhaA) showed slightly different kinetic parameters (Km of 2.9 µmol/L and Kcat of 1.0 s-1). Inulin conjugation could delay the structural unfolding and/or slow the protonation process of DhaA under undesirable environment, including the long-term storage, low pH, hypersaline and organic solvent stability. As a result, the environmental stability of DhaA was markedly increased upon conjugation with inulin. Thus, inulin conjugation was an effective approach to enhance the environmental stability of DhaA.


Assuntos
Inulina , Rhodococcus , Hidrolases/genética , Hidrólise
12.
J Med Chem ; 64(14): 9759-9785, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34213320

RESUMO

Much of the experimental evidence in the literature has linked altered lipid metabolism to severe diseases such as cancer, obesity, cardiovascular pathologies, diabetes, and neurodegenerative diseases. Therefore, targeting key effectors of the dysregulated lipid metabolism may represent an effective strategy to counteract these pathological conditions. In this context, α/ß-hydrolase domain (ABHD) enzymes represent an important and diversified family of proteins, which are involved in the complex environment of lipid signaling, metabolism, and regulation. Moreover, some members of the ABHD family play an important role in the endocannabinoid system, being designated to terminate the signaling of the key endocannabinoid regulator 2-arachidonoylglycerol. This Perspective summarizes the research progress in the development of ABHD inhibitors and modulators: design strategies, structure-activity relationships, action mechanisms, and biological studies of the main ABHD ligands will be highlighted.


Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Transtornos do Metabolismo dos Lipídeos/tratamento farmacológico , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Hidrolases/metabolismo , Transtornos do Metabolismo dos Lipídeos/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
13.
Appl Environ Microbiol ; 87(18): e0002021, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34260304

RESUMO

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo, we have developed a gene disruption system in I. sakaiensis. The pT18mobsacB-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (pyrF) from the genome of the wild-type strain, producing the ΔpyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the ΔpyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δpetase and Δmhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δpetase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis, which was then applied for constructing Δpetase and Δmhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis, while PETase and MHETase play essential roles in its PET assimilation.


Assuntos
Proteínas de Bactérias/genética , Burkholderiales/genética , Burkholderiales/metabolismo , Hidrolases/genética , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/metabolismo , Etilenoglicol/metabolismo , Genes Bacterianos , Hidrolases/metabolismo , Hidrólise , Engenharia Metabólica , Ácidos Ftálicos/metabolismo , Reciclagem
14.
Nat Plants ; 7(7): 923-931, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34226693

RESUMO

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals2. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.


Assuntos
Catálise , Glucosídeos/biossíntese , Hidrolases/metabolismo , Pirimidinonas/metabolismo , Sementes/metabolismo , Vicia faba/genética , Vicia faba/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Dinamarca , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosídeos/genética , Hidrolases/genética , Sementes/genética
15.
Molecules ; 26(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202153

RESUMO

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6-N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.


Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Catálise , Domínio Catalítico , Ácido Úrico/análogos & derivados , Ácido Úrico/química
16.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
17.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(4): 619-623, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34323040

RESUMO

Objective: To prepare and characterize D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) modified arginine deiminase (ADI) sulfobutyl-ß-Cyclodextrin liposome nanoparticles (ATCL), and to investigate the pharmacokinetic characteristics of ATCL in animals. Methods: The reverse evaporation method was used to prepare ATCL, and the particle size and Zeta potential of ATCL were measured. Thiosemicarbazone-diacetylmonooxime colorimetric method was used to measure the activity of ADI. After intravenous administration, blood was drawn at set intervals of time and the enzyme activity in the plasma was measured. Enzyme activity-time curve was drawn subsequently and Debris Assessment Software (DAS) 2.1.1 was used to analyze the pharmacokinetic characteristics. Results: The particle size and the potential of ATCL were (216.1±13.6) nm and (-19.4±2.1) mV, respectively. The optimal temperature and optimal pH for the catalytic reaction of ADI and ATCL were the same, both being 37 ℃ and pH6.5. Results of the analysis showed that the AUC (0-168 h), MRT (0-168 h), C max, T max, and t 1/2 of ATCL were 3.99, 2.56, 1.58, 3.2, and 9.88 times those of free ADI, respectively. Compared with ADI, the bioavailability of ATCL increased by 298.54%. Conclusion: ATCL prepared in the study can effectively improve the enzyme activity and bioavailability of ADI in Sprague-Dawley rats.


Assuntos
Hidrolases , Nanopartículas , Animais , Arginina , Lipídeos , Polietilenoglicóis , Ratos , Ratos Sprague-Dawley
18.
Oncoimmunology ; 10(1): 1943253, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34290906

RESUMO

Background: Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods: A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results: Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1-3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions: The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Hidrolases , Neoplasias/tratamento farmacológico , Polietilenoglicóis , Microambiente Tumoral
19.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299249

RESUMO

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.


Assuntos
Hidrolases/farmacologia , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Arginina/deficiência , Arginina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Hidrolases/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
20.
Fish Physiol Biochem ; 47(4): 1211-1227, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34173183

RESUMO

Cichlasoma dimerus is a neotropical cichlid that has been used as a biological model for neuroendocrinology studies. However, its culture is problematic in terms of larval feeding to allow having enough fry quantity and quality. Larviculture requires full knowledge about the digestive system and nutrition; therefore, this study was intended to assess the digestive enzymes' changes at different ages during the early ontogeny. Acid protease activity was detectable from the first day after hatching (dah), increasing to its maximum peaks on 9 dah. In contrast, alkaline proteases had low activity in the first days of life but reached their maximum activity on 17 dah. Chymotrypsin, L-aminopeptidase, and carboxypeptidase A activities increased at 6 dah, while trypsin activity was first detected on 13 dah and reached its maximum activity on 17 dah. Lipase and α-amylase activity were detectable at low levels in the first days of life, but the activity fluctuated and reaching its maximum activity at 21 dah. Alkaline phosphatase continued to oscillate and had two maximum activity peaks, the first at 6 dah and the second at 19 dah. Zymograms of alkaline proteases on day 6 dah six revealed four activity bands with molecular weights from 16.1 to 77.7 kDa. On 13 dah, two more activity bands of 24.4 and 121.9 kDa were detected, having a total of six proteases. The enzymatic activity analyzes indicate the digestive system shows the low activity of some enzymes in the first days after hatching, registering significant increases on 6 dah and the maximum peaks of activities around at 17 dah. Therefore, we recommend replacing live food with dry feed and only providing dry feed after day 17 dah.


Assuntos
Ciclídeos/crescimento & desenvolvimento , Ciclídeos/metabolismo , Hidrolases/metabolismo , Animais , Digestão , Larva/crescimento & desenvolvimento , Larva/metabolismo
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