Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.408
Filtrar
1.
Biochemistry (Mosc) ; 89(8): 1519-1530, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39245459

RESUMO

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.


Assuntos
Aldeído Liases , Sistema Enzimático do Citocromo P-450 , Daucus carota , Proteínas de Plantas , Daucus carota/enzimologia , Daucus carota/genética , Daucus carota/metabolismo , Aldeído Liases/metabolismo , Aldeído Liases/genética , Aldeído Liases/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Peróxidos Lipídicos/metabolismo , Especificidade por Substrato , Sequência de Aminoácidos , Ácidos Linoleicos
2.
J Enzyme Inhib Med Chem ; 39(1): 2388207, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39140692

RESUMO

The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis (MtFolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of MtFolB. These compounds exhibited IC50 values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting MtFolB, an attractive molecular target for TB drug development.


Assuntos
Aldeído Liases , Antituberculosos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Antituberculosos/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Relação Estrutura-Atividade , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/metabolismo , Aldeído Liases/química , Células Vero , Estrutura Molecular , Cristalografia por Raios X , Chlorocebus aethiops , Animais , Guanina/farmacologia , Guanina/química , Guanina/análogos & derivados , Guanina/síntese química , Simulação de Acoplamento Molecular , Células Hep G2 , Modelos Moleculares
3.
Extremophiles ; 28(3): 42, 2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39215799

RESUMO

Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4­(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints.


Assuntos
Proteínas Arqueais , Estabilidade Enzimática , Methanocaldococcus , Methanocaldococcus/enzimologia , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Mathanococcus/enzimologia , Termotolerância , Aldeído Liases/metabolismo , Aldeído Liases/genética , Aldeído Liases/química , Temperatura Alta , Simulação de Dinâmica Molecular
4.
Int J Biol Macromol ; 278(Pt 4): 134994, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39181367

RESUMO

l-threonine aldolase (LTA) catalyzes the synthesis of ß-hydroxy-α-amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T5060 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains.


Assuntos
Estabilidade Enzimática , Simulação de Dinâmica Molecular , Mutação , Temperatura , Bacillus/enzimologia , Bacillus/genética , Ligação de Hidrogênio , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Domínio Catalítico , Cinética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Engenharia de Proteínas/métodos
5.
Appl Environ Microbiol ; 90(8): e0104724, 2024 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-39028195

RESUMO

Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn2+-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP+-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO2 and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.IMPORTANCEPhloroglucinol is an important intermediate in the bacterial degradation of polyphenols, a highly abundant class of plant natural products. Recent research has identified key enzymes of the phloroglucinol degradation pathway in butyrate-producing anaerobic bacteria, which involves cleavage of a linear triketide intermediate by a beta ketoacid cleavage enzyme, requiring acetyl-CoA as a co-substrate. This paper reports a variant of the pathway in the lactic acid bacterium Collinsella sp. zg1085, which involves cleavage of the triketide intermediate by a homolog of deoxyribose-5-phosphate aldolase, highlighting the variety of mechanisms for phloroglucinol degradation by different anaerobic bacterial taxa.


Assuntos
Redes e Vias Metabólicas , Floroglucinol , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Redes e Vias Metabólicas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Aldeído Liases/metabolismo , Aldeído Liases/genética , Animais
6.
Nat Commun ; 15(1): 5969, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39013920

RESUMO

The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.


Assuntos
Metanol , Methylobacterium , Methylobacterium/metabolismo , Methylobacterium/genética , Methylobacterium/enzimologia , Methylobacterium/crescimento & desenvolvimento , Metanol/metabolismo , Simbiose , Mutação , Aldeído Liases/metabolismo , Aldeído Liases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Folhas de Planta/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Methylobacterium extorquens/crescimento & desenvolvimento , Methylobacterium extorquens/enzimologia , Desenvolvimento Vegetal , Microbiota/genética , Biomassa
7.
Nat Commun ; 15(1): 6326, 2024 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-39068153

RESUMO

The sensitivity and responsiveness of living cells to environmental changes are enabled by dynamic protein structures, inspiring efforts to construct artificial supramolecular protein assemblies. However, despite their sophisticated structures, designed protein assemblies have yet to be incorporated into macroscale devices for real-life applications. We report a 2D crystalline protein assembly of C98/E57/E66L-rhamnulose-1-phosphate aldolase (CEERhuA) that selectively blocks or passes molecular species when exposed to a chemical trigger. CEERhuA crystals are engineered via cobalt(II) coordination bonds to undergo a coherent conformational change from a closed state (pore dimensions <1 nm) to an ajar state (pore dimensions ~4 nm) when exposed to an HCN(g) trigger. When layered onto a mesoporous silicon (pSi) photonic crystal optical sensor configured to detect HCN(g), the 2D CEERhuA crystal layer effectively blocks interferents that would otherwise result in a false positive signal. The 2D CEERhuA crystal layer opens in selective response to low-ppm levels of HCN(g), allowing analyte penetration into the pSi sensor layer for detection. These findings illustrate that designed protein assemblies can function as dynamic components of solid-state devices in non-aqueous environments.


Assuntos
Aldeído Liases , Aldeído Liases/metabolismo , Aldeído Liases/química , Cristalização , Cobalto/química , Conformação Proteica , Silício/química , Engenharia de Proteínas , Modelos Moleculares
8.
J Lipid Res ; 65(8): 100587, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38950680

RESUMO

Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-ßH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA.


Assuntos
Células Secretoras de Insulina , Gotículas Lipídicas , Lisofosfolipídeos , Esfingosina , Células Secretoras de Insulina/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Ratos , Animais , Gotículas Lipídicas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Aldeído Liases/metabolismo , Aldeído Liases/genética , Metabolismo dos Lipídeos , Humanos , Linhagem Celular , Estresse Oxidativo , Espaço Intracelular/metabolismo
9.
Neurobiol Dis ; 199: 106585, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38955289

RESUMO

Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.


Assuntos
Aldeído Liases , Lisofosfolipídeos , Esfingosina , Aldeído Liases/metabolismo , Aldeído Liases/antagonistas & inibidores , Humanos , Animais , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico
10.
Biomolecules ; 14(7)2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-39062466

RESUMO

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.


Assuntos
Aldeído Liases , Escherichia coli , Frutose-Bifosfato Aldolase , Simulação de Acoplamento Molecular , Nucleosídeos de Pirimidina , Thermotoga maritima , Animais , Escherichia coli/enzimologia , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/síntese química , Aldeído Liases/metabolismo , Aldeído Liases/química , Coelhos , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Thermotoga maritima/enzimologia , Fosfato de Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/química , Estereoisomerismo
11.
BMC Plant Biol ; 24(1): 590, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38902595

RESUMO

BACKGROUND: The Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China. RESULTS: To identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants. CONCLUSIONS: This work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.


Assuntos
Amigdalina , Prunus , Sementes , Amigdalina/metabolismo , Prunus/genética , Prunus/metabolismo , Prunus/enzimologia , Sementes/metabolismo , Sementes/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Óleos de Plantas/metabolismo , Aldeído Liases/metabolismo , Aldeído Liases/genética , Regulação da Expressão Gênica de Plantas
12.
Nutrients ; 16(11)2024 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-38892556

RESUMO

Sphingosine-1-phosphate lyase (SPL) resides at the endpoint of the sphingolipid metabolic pathway, catalyzing the irreversible breakdown of sphingosine-1-phosphate. Depletion of SPL precipitates compromised muscle morphology and function; nevertheless, the precise mechanistic underpinnings remain elusive. Here, we elucidate a model of SPL functional deficiency in Caenorhabditis elegans using spl-1 RNA interference. Within these SPL-deficient nematodes, we observed diminished motility and perturbed muscle fiber organization, correlated with the accumulation of sphingoid bases, their phosphorylated forms, and ceramides (collectively referred to as the "sphingolipid rheostat"). The disturbance in mitochondrial morphology was also notable, as SPL functional loss resulted in heightened levels of reactive oxygen species. Remarkably, the administration of the antioxidant N-acetylcysteine (NAC) ameliorates locomotor impairment and rectifies muscle fiber disarray, underscoring its therapeutic promise for ceramide-accumulation-related muscle disorders. Our findings emphasize the pivotal role of SPL in preserving muscle integrity and advocate for exploring antioxidant interventions, such as NAC supplementation, as prospective therapeutic strategies for addressing muscle function decline associated with sphingolipid/ceramide metabolism disruption.


Assuntos
Acetilcisteína , Aldeído Liases , Caenorhabditis elegans , Ceramidas , Esfingolipídeos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Acetilcisteína/farmacologia , Ceramidas/metabolismo , Aldeído Liases/metabolismo , Esfingolipídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , Interferência de RNA , Esfingosina/análogos & derivados , Esfingosina/metabolismo
13.
Phytochemistry ; 224: 114151, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38768880

RESUMO

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.


Assuntos
Aldeído Liases , Cucumis sativus , Sistema Enzimático do Citocromo P-450 , Oxilipinas , Cucumis sativus/metabolismo , Cucumis sativus/enzimologia , Aldeído Liases/metabolismo , Aldeído Liases/química , Oxilipinas/metabolismo , Oxilipinas/química , Oxilipinas/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Estrutura Molecular
14.
Biomed Pharmacother ; 174: 116575, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38599060

RESUMO

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice.


Assuntos
Aldeído Liases , Camundongos Endogâmicos C57BL , Sepse , Receptores de Esfingosina-1-Fosfato , Animais , Sepse/tratamento farmacológico , Sepse/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidores , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/metabolismo , Camundongos , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/metabolismo , Masculino , Modelos Animais de Doenças , Linhagem Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Citocinas/metabolismo , Camundongos Knockout
15.
Chembiochem ; 25(11): e202400118, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38526556

RESUMO

Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0 Šresolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.


Assuntos
Aldeído Liases , Mutagênese Sítio-Dirigida , Aldeído Liases/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Animais , Benzaldeídos/metabolismo , Benzaldeídos/química , Acetonitrilas/química , Acetonitrilas/metabolismo , Modelos Moleculares , Cristalografia por Raios X , Nitrilas/metabolismo , Nitrilas/química , Estereoisomerismo
16.
ACS Synth Biol ; 13(3): 888-900, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-38359048

RESUMO

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.


Assuntos
Células Artificiais , Metanol , Metanol/metabolismo , NAD/metabolismo , Frutose-Bifosfato Aldolase , Aldeído Liases/metabolismo
17.
Int J Mol Sci ; 25(4)2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38396648

RESUMO

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Dióxido de Silício/química , Aldeído Liases/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Biocatálise
18.
J Pathol ; 263(1): 22-31, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38332723

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Dependovirus , Fibrose Pulmonar Idiopática , Lisofosfolipídeos , Esfingosina/análogos & derivados , Humanos , Camundongos , Animais , Dependovirus/genética , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/terapia , Fibrose Pulmonar Idiopática/metabolismo , Bleomicina , Modelos Animais , Terapia Genética , Aldeído Liases/genética , Aldeído Liases/metabolismo
19.
Int J Mol Sci ; 24(21)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37958544

RESUMO

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1, the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic "CAG" promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.


Assuntos
Terapia Genética , Parvovirinae , Esfingosina , Animais , Humanos , Camundongos , Aldeído Liases/genética , Aldeído Liases/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos Knockout , Parvovirinae/metabolismo , Fosfatos , Esfingosina/metabolismo
20.
J Immunother Cancer ; 11(10)2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37857526

RESUMO

BACKGROUND: Post-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered immune responses. One such modification is carbamylation (homocitrullination) that results in the formation of homocitrulline (Hcit) residues in a non-enzymatic reaction of cyanate with the lysine residues in the polypeptide chain. Homocitrullination occurs in the tumor microenvironment and CD4-mediated immune responses to Hcit epitopes can target stressed tumor cells and provide a potent antitumor response in mouse models. METHODS: Homocitrullinated peptides were identified and assessed in vitro for HLA-A2 binding and in vivo in human leukocyte antigen (HLA) transgenic mouse models for immunogenicity. CD8 responses were assessed in vitro for cytotoxicity and in vivo tumor therapy. Human tumor samples were analyzed by targeted mass spectrometry for presence of homocitrullinated peptides. RESULTS: Homocitrullinated peptides from aldolase and cytokeratin were identified, that stimulated CD8-mediated responses in vivo. Modified peptides showed enhanced binding to HLA-A2 compared with the native sequences and immunization of HLA-A2 transgenic mice generated high avidity modification specific CD8 responses that killed peptide expressing target cells. Importantly, in vivo the homocitrullinated aldolase specific response was associated with efficient CD8 dependent antitumor therapy of the aggressive murine B16 tumor model indicating that this epitope is naturally presented in the tumor. In addition, the homocitrullinated aldolase epitope was also detected in human tumor samples. CONCLUSION: This is the first evidence that homocitrullinated peptides can be processed and presented via MHC-I and targeted for tumor therapy. Thus, Hcit-specific CD8 T-cell responses have potential in the development of future anticancer therapy.


Assuntos
Linfócitos T CD8-Positivos , Antígeno HLA-A2 , Camundongos , Humanos , Animais , Antígenos de Histocompatibilidade Classe II/metabolismo , Vacinação , Camundongos Transgênicos , Peptídeos , Antígenos de Histocompatibilidade Classe I , Epitopos , Processamento de Proteína Pós-Traducional , Aldeído Liases/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA