RESUMEN
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.
Asunto(s)
Aldehído-Liasas , Antituberculosos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Antituberculosos/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Relación Estructura-Actividad , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Células Vero , Estructura Molecular , Cristalografía por Rayos X , Chlorocebus aethiops , Animales , Guanina/farmacología , Guanina/química , Guanina/análogos & derivados , Guanina/síntesis química , Simulación del Acoplamiento Molecular , Células Hep G2 , Modelos MolecularesRESUMEN
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.
Asunto(s)
Aldehído-Liasas , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Cristalización , Cobalto/química , Conformación Proteica , Silicio/química , Ingeniería de Proteínas , Modelos MolecularesRESUMEN
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.
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Aldehído-Liasas , Lisofosfolípidos , Esfingosina , Aldehído-Liasas/metabolismo , Aldehído-Liasas/antagonistas & inhibidores , Humanos , Animales , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéuticoRESUMEN
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.
Asunto(s)
Aldehído-Liasas , Escherichia coli , Fructosa-Bifosfato Aldolasa , Simulación del Acoplamiento Molecular , Nucleósidos de Pirimidina , Thermotoga maritima , Animales , Escherichia coli/enzimología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/síntesis química , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Conejos , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Thermotoga maritima/enzimología , Dihidroxiacetona Fosfato/metabolismo , Dihidroxiacetona Fosfato/química , EstereoisomerismoRESUMEN
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.
Asunto(s)
Metanol , Methylobacterium , Methylobacterium/metabolismo , Methylobacterium/genética , Methylobacterium/enzimología , Methylobacterium/crecimiento & desarrollo , Metanol/metabolismo , Simbiosis , Mutación , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hojas de la Planta/microbiología , Hojas de la Planta/crecimiento & desarrollo , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Methylobacterium extorquens/crecimiento & desarrollo , Methylobacterium extorquens/enzimología , Desarrollo de la Planta , Microbiota/genética , BiomasaRESUMEN
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.
Asunto(s)
Amigdalina , Prunus , Semillas , Amigdalina/metabolismo , Prunus/genética , Prunus/metabolismo , Prunus/enzimología , Semillas/metabolismo , Semillas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Aceites de Plantas/metabolismo , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
Asunto(s)
Acetilcisteína , Aldehído-Liasas , Caenorhabditis elegans , Ceramidas , Esfingolípidos , Animales , Caenorhabditis elegans/efectos de los fármacos , Acetilcisteína/farmacología , Ceramidas/metabolismo , Aldehído-Liasas/metabolismo , Esfingolípidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Músculos/efectos de los fármacos , Músculos/metabolismo , Interferencia de ARN , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
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.
Asunto(s)
Aldehído-Liasas , Cucumis sativus , Sistema Enzimático del Citocromo P-450 , Oxilipinas , Cucumis sativus/metabolismo , Cucumis sativus/enzimología , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Oxilipinas/metabolismo , Oxilipinas/química , Oxilipinas/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Estructura MolecularRESUMEN
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.
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Aldehído-Liasas , Ratones Endogámicos C57BL , Sepsis , Receptores de Esfingosina-1-Fosfato , Animales , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/metabolismo , Ratones , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/metabolismo , Masculino , Modelos Animales de Enfermedad , Línea Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Citocinas/metabolismo , Ratones NoqueadosRESUMEN
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 2.0â Å resolutions, 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.
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Aldehído-Liasas , Mutagénesis Sitio-Dirigida , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Aldehído-Liasas/genética , Animales , Benzaldehídos/metabolismo , Benzaldehídos/química , Acetonitrilos/química , Acetonitrilos/metabolismo , Modelos Moleculares , Cristalografía por Rayos X , Nitrilos/metabolismo , Nitrilos/química , EstereoisomerismoRESUMEN
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.
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Dependovirus , Fibrosis Pulmonar Idiopática , Lisofosfolípidos , Esfingosina/análogos & derivados , Humanos , Ratones , Animales , Dependovirus/genética , Pulmón/metabolismo , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/terapia , Fibrosis Pulmonar Idiopática/metabolismo , Bleomicina , Modelos Animales , Terapia Genética , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismoRESUMEN
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.
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Células Artificiales , Metanol , Metanol/metabolismo , NAD/metabolismo , Fructosa-Bifosfato Aldolasa , Aldehído-Liasas/metabolismoRESUMEN
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.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Dióxido de Silicio/química , Aldehído-Liasas/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , BiocatálisisRESUMEN
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.
Asunto(s)
Terapia Genética , Parvovirinae , Esfingosina , Animales , Humanos , Ratones , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Lisofosfolípidos/metabolismo , Ratones Noqueados , Parvovirinae/metabolismo , Fosfatos , Esfingosina/metabolismoRESUMEN
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.
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Linfocitos T CD8-positivos , Antígeno HLA-A2 , Ratones , Humanos , Animales , Antígenos de Histocompatibilidad Clase II/metabolismo , Vacunación , Ratones Transgénicos , Péptidos , Antígenos de Histocompatibilidad Clase I , Epítopos , Procesamiento Proteico-Postraduccional , Aldehído-Liasas/metabolismoRESUMEN
NAcetylDneuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-D-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.
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Aldehído-Liasas , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Aldehído-Liasas/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Ácido Pirúvico/metabolismo , Biotransformación , Ácido N-Acetilneuramínico/metabolismoRESUMEN
The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.
Asunto(s)
Aldehído-Liasas , Carbono , Acetilcoenzima A , Carbono/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Glucosa/metabolismo , Ingeniería MetabólicaRESUMEN
Background: Sphingosine phosphate lyase insufficiency syndrome (SPLIS) is associated with biallelic variants in SGPL1, comprising a multisystemic disease characterized by steroid resistant nephrotic syndrome, primary adrenal insufficiency, neurological problems, skin abnormalities and immunodeficiency in described cases. Signal transducer and activator of transcription 1 (STAT1) plays an important role in orchestrating an appropriate immune response through JAK-STAT pathway. Biallelic STAT1 loss of function (LOF) variants lead to STAT1 deficiency with a severe phenotype of immunodeficiency with increased frequency of infections and poor outcome if untreated. Case presentation: We report novel homozygous SGPL1 and STAT1 variants in a newborn of Gambian ethnicity with clinical features of SPLIS and severe combined immunodeficiency. The patient presented early in life with nephrotic syndrome, severe respiratory infection requiring ventilation, ichthyosis, and hearing loss, with T-cell lymphopenia. The combination of these two conditions led to severe combined immunodeficiency with inability to clear respiratory tract infections of viral, fungal, and bacterial nature, as well as severe nephrotic syndrome. The child sadly died at 6 weeks of age despite targeted treatments. Conclusion: We report the finding of two novel, homozygous variants in SGPL1 and STAT1 in a patient with a severe clinical phenotype and fatal outcome early in life. This case highlights the importance of completing the primary immunodeficiency genetic panel in full to avoid missing a second diagnosis in other patients presenting with similar severe clinical phenotype early in life. For SPLIS no curative treatment is available and more research is needed to investigate different treatment modalities. Hematopoietic stem cell transplantation (HSCT) shows promising results in patients with autosomal recessive STAT1 deficiency. For this patient's family, identification of the dual diagnosis has important implications for future family planning. In addition, future siblings with the familial STAT1 variant can be offered curative treatment with HSCT.
Asunto(s)
Síndromes de Inmunodeficiencia , Síndrome Nefrótico , Inmunodeficiencia Combinada Grave , Humanos , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Quinasas Janus/metabolismo , Síndrome Nefrótico/genética , Transducción de Señal , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Recién NacidoRESUMEN
Sphingosine-1-phosphate lyase (SPL) is a pyridoxal 5'-phosphate-dependent enzyme involved in the irreversible degradation of sphingosine-1-phosphate (S1P)-a bioactive sphingolipid that modulates a broad range of biological processes (cell proliferation, migration, differentiation and survival; mitochondrial functioning; and gene expression). Although SPL activity leads to a decrease in the available pool of S1P in the cell, at the same time, hexadecenal and phosphoethanolamine, compounds with potential biological activity, are generated. The increased expression and/or activity of SPL, and hence the imbalance between S1P and the end products of its cleavage, were demonstrated in several pathological states. On the other hand, loss-of-function mutations in the SPL encoding gene are a cause of severe developmental impairments. Recently, special attention has been paid to neurodegenerative diseases as the most common pathologies of the nervous system. This review summarizes the current findings concerning the role of SPL in the nervous system with an emphasis on neurodegeneration. Moreover, it briefly discusses pharmacological compounds directed to inhibit its activity.
Asunto(s)
Esfingolípidos , Esfingosina , Esfingosina/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Lisofosfolípidos/metabolismoRESUMEN
Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.