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1.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372584

RESUMO

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.


Assuntos
Bacteriólise/fisiologia , Micobacteriófagos/metabolismo , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases , Hidrólise , Mycobacterium/metabolismo , Mycobacterium/virologia , Peptidoglicano/metabolismo , Ligação Proteica
2.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752

RESUMO

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.


Assuntos
Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
3.
Biomolecules ; 11(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808760

RESUMO

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


Assuntos
Fenilalanina Hidroxilase/metabolismo , Quinolonas/química , Quinolonas/farmacologia , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Fluorometria , Células HEK293 , Humanos , Doenças Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Ressonância de Plasmônio de Superfície , Tripsina
4.
Pharmaceutics ; 13(3)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806405

RESUMO

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.

5.
Biochimie ; 183: 78-88, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33588022

RESUMO

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.


Assuntos
Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Piruvato Desidrogenase (Lipoamida)/química , Doença da Deficiência do Complexo de Piruvato Desidrogenase , Tiamina Pirofosfato/química , Substituição de Aminoácidos , Estabilidade Enzimática , Humanos , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Tiamina Pirofosfato/genética , Tiamina Pirofosfato/metabolismo
7.
Sci Rep ; 9(1): 13615, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541188

RESUMO

Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/ultraestrutura , Domínio Catalítico , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/metabolismo , Fenilcetonúrias/genética , Fenilcetonúrias/fisiopatologia , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Raios X
8.
Zookeys ; (752): 137-148, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29719477

RESUMO

The collection of the Museu Oceanográfico D. Carlos I is a historical specimen, instrument, and document collection that has been housed at the Aquário Vasco da Gama since 1935. The collection is largely the result of several scientific campaigns conducted by Dom Carlos de Bragança between 1896 and 1907. Specifically, the ichthyological collection consists of 675 surviving catalogue records of specimens caught, acquired or offered to D. Carlos I between 1892 to 1907, and includes the type specimen for Odontaspis nasutus Bragança, 1904 (junior synonym of Mitsukurina owstoni Jordan, 1898), along with several specimens of deep sea species. All specimens were captured in coastal Portuguese waters, and were preserved in alcohol, formalin, or mounted.

9.
J Biotechnol ; 253: 23-33, 2017 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-28549690

RESUMO

Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short half-life molecules. Herein, we have investigated the Zag albumin-binding domain (ABD) derived from Streptococcus zooepidemicus as a novel strategy to improve the pharmacokinetic properties of therapeutic molecules. To validate our approach, the Zag ABD was fused with an anti-TNFα single-domain antibody (sdAb). Our results demonstrated that the sdAb-Zag fusion protein was highly expressed and specifically recognizes human, rat and mouse serum albumins with affinities in the nanomolar range. Moreover, data also demonstrated that the sdAb activity against the therapeutic target (TNFα) was not affected when fused with Zag ABD. Importantly, the Zag ABD increased the sdAb half-life ∼39-fold (47min for sdAb versus 31h for sdAb-Zag). These findings demonstrate that the Zag ABD fusion is a promising approach to increase the half-life of small recombinant antibodies molecules without affecting their therapeutic efficacy. Moreover, the present study strongly suggests that the Zag ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of many others therapeutics proteins and peptides in order to improve their dosing schedule and clinical effects.


Assuntos
Proteínas de Bactérias/genética , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Domínio Único/genética , Animais , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/farmacologia , Feminino , Meia-Vida , Camundongos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/química , Albumina Sérica/metabolismo , Anticorpos de Domínio Único/farmacologia , Streptococcus equi , Fator de Necrose Tumoral alfa/imunologia
10.
Oxid Med Cell Longev ; 2017: 8940321, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28421128

RESUMO

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine ß-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.


Assuntos
Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Cristalografia por Raios X , Cistationina beta-Sintase/química , Cistationina beta-Sintase/genética , Heme/química , Heme/metabolismo , Humanos , Cinética , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , S-Adenosilmetionina/metabolismo
11.
FEBS Open Bio ; 7(2): 195-203, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28174686

RESUMO

Mammalian phenylalanine hydroxylase (PAH) has a potential allosteric regulatory binding site for l-phenylalanine (l-Phe), in addition to its catalytic site. This arrangement is supported by a crystal structure of a homodimeric truncated form of the regulatory domain of human PAH (hPAH-RD 1-118/19-118) [Patel D et al. (2016) Sci Rep doi: 10.1038/srep23748]. In this study, a fusion protein of the domain (MBP-(pepXa)-hPAH-RD 1-120) was overexpressed and recovered in a metastable and soluble state, which allowed the isolation of a dimeric and a monomeric fusion protein. When cleaved from MBP, hPAH-RD forms aggregates which are stereospecifically inhibited by l-Phe (> 95%) at low physiological concentrations. Aggregation of the cleaved dimer of the mutant form hPAH-G46S-RD was not inhibited by l-Phe, which is compatible with structurally/conformationally changed ßαßßαß ACT domain folds in the mutant.

12.
Biochemistry ; 55(51): 7086-7098, 2016 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-27976856

RESUMO

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid ß-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme's activity. The most frequent mutation in MCADD results from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein). Here, we combined in vitro and in silico approaches to assess the impact of the p.K329E mutation on the protein's structure and function. Our in silico results demonstrated for the first time that the p.K329E mutation, despite lying at the dimer-dimer interface and being deeply buried inside the tetrameric core, seems to affect the tetramer surface, especially the ß-domain that forms part of the catalytic pocket wall. Additionally, the molecular dynamics data indicate a stronger impact of the mutation on the protein's motions in dimer A/B, while dimer C/D remains similar to the wild type. For dimer A/B, severe disruptions in the architecture of the pockets and in the FAD and octanoyl-CoA binding affinities were also observed. The presence of unaffected pockets (C/D) in the in silico studies may explain the decreased enzymatic activity determined for the variant protein (46% residual activity). Moreover, the in silico structural changes observed for the p.K329E variant protein provide an explanation for the structural instability observed experimentally, namely, the disturbed oligomeric profile, thermal stability, and conformational flexibility, with respect to the wild-type.


Assuntos
Acil-CoA Desidrogenase/genética , Simulação por Computador , Erros Inatos do Metabolismo Lipídico/genética , Mutação de Sentido Incorreto , Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/deficiência , Biocatálise , Estabilidade Enzimática , Ácido Glutâmico/genética , Humanos , Cinética , Erros Inatos do Metabolismo Lipídico/enzimologia , Lisina/genética , Modelos Moleculares , Movimento (Física) , Análise de Componente Principal , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Temperatura
13.
Chem Biol Drug Des ; 88(2): 281-92, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26992026

RESUMO

The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that catalyzes the first step of mitochondrial fatty acid ß-oxidation (mFAO) pathway. Its deficiency is the most common genetic disorder of mFAO. Many of the MCAD disease-causing variants, including the most common p.K304E variant, show loss of function due to protein misfolding. Herein, we used molecular dynamics simulations to provide insights into the structural stability and dynamic behavior of MCAD wild-type (MCADwt) and validate a structure that would allow reliable new studies on its variants. Our results revealed that in both proteins the flavin adenine dinucleotide (FAD) has an important structural role on the tetramer stability and also in maintaining the volume of the enzyme catalytic pockets. We confirmed that the presence of substrate changes the dynamics of the catalytic pockets and increases FAD affinity. A comparison between the porcine MCADwt (pMCADwt) and human MCADwt (hMCADwt) structures revealed that both proteins are essentially similar and that the reversion of the double mutant E376G/T255E of hMCAD enzyme does not affect the structure of the protein neither its behavior in simulation. Our validated hMCADwt structure is crucial for complementing and accelerating the experimental studies aiming for the discovery and development of potential stabilizers of MCAD variants as candidates for the treatment of MCAD deficiency (MCADD).


Assuntos
Acil-CoA Desidrogenase/química , Animais , Domínio Catalítico , Simulação de Dinâmica Molecular , Conformação Proteica , Suínos
14.
J Biol Chem ; 291(2): 572-81, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26582199

RESUMO

Cystathionine ß-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5'-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO(•) binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (∼5-fold) and faster association (∼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO(•) binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (∼2-fold higher affinity) as compared with CO. Importantly, CO and NO(•) binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO(•). This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.


Assuntos
Monóxido de Carbono/metabolismo , Cistationina beta-Sintase/metabolismo , Óxido Nítrico/metabolismo , S-Adenosilmetionina/farmacologia , Heme/metabolismo , Humanos , Cinética , Metionina/metabolismo , Oxirredução
15.
Hum Mol Genet ; 24(25): 7339-48, 2015 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-26464485

RESUMO

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.


Assuntos
Cistationina beta-Sintase/química , Cistationina beta-Sintase/metabolismo , Arginina/genética , Arginina/metabolismo , Western Blotting , Cistationina beta-Sintase/genética , Cisteína/genética , Cisteína/metabolismo , Fluorometria , Humanos , Estrutura Secundária de Proteína
16.
JIMD Rep ; 23: 1-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25814382

RESUMO

Classic galactosemia is a rare genetic disease of the galactose metabolism, resulting from deficient activity of galactose-1-phosphate uridylyltransferase (GALT). The current standard of care is lifelong dietary restriction of galactose, which however fails to prevent the development of long-term complications. Structural-functional studies demonstrated that the most prevalent GALT mutations give rise to proteins with increased propensity to aggregate in solution. Arginine is a known stabilizer of aggregation-prone proteins, having already shown a beneficial effect in other inherited metabolic disorders.Herein we developed a prokaryotic model of galactose sensitivity that allows evaluating in a cellular context the mutations' impact on GALT function, as well as the potential effect of arginine in functionally rescuing clinically relevant variants.This study revealed that some hGALT variants, previously described to exhibit no detectable activity in vitro, actually present residual activity when determined in vivo. Furthermore, it revealed that arginine presents a mutation-specific beneficial effect, particularly on the prevalent p.Q188R and p.K285N variants, which led us to hypothesize that it might constitute a promising therapeutic agent in classic galactosemia.

17.
Hum Mutat ; 35(10): 1195-202, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25044645

RESUMO

Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.


Assuntos
Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Homocistinúria/enzimologia , Mutação , Sítio Alostérico , Sequência de Aminoácidos , Domínio Catalítico , Cistationina beta-Sintase/química , Homocistinúria/genética , Humanos , Cinética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo
18.
J Biol Chem ; 289(12): 8579-87, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24515102

RESUMO

The hexa-coordinate heme in the H2S-generating human enzyme cystathionine ß-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(•) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(•) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 µm). This discrepancy was herein reconciled by investigating the NO(•) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(•) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 µm. In line with this result, at 25 °C, NO(•) binds quickly to CBS (k on ∼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off ∼ 0.003 s(-1)). The observed rate constants for NO(•) binding were found to be linearly dependent on [NO(•)] up to ∼ 800 µm NO(•), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(•) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(•). The novel findings reported here shed new light on CBS regulation by NO(•) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(•), CO, and H2S in cell signaling.


Assuntos
Cistationina beta-Sintase/metabolismo , Óxido Nítrico/metabolismo , Monóxido de Carbono/metabolismo , Cistationina beta-Sintase/química , Heme/química , Heme/metabolismo , Humanos , Cinética , Oxirredução , Ligação Proteica
19.
J Inherit Metab Dis ; 37(2): 245-54, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23974653

RESUMO

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.


Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/enzimologia , Homocistinúria/genética , Mutação , S-Adenosilmetionina/genética , Alelos , Células Cultivadas , Cistationina beta-Sintase/metabolismo , Escherichia coli/genética , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Genótipo , Homocistinúria/metabolismo , Homocistinúria/patologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estudos Retrospectivos , S-Adenosilmetionina/metabolismo
20.
Mol Genet Genomic Med ; 2(6): 484-96, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25614870

RESUMO

Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional-structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural-functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggregation, particularly striking for the p.Q188R variant, resulting from the most frequent (∼60%) allele at a worldwide scale. The absence of major effects at the secondary and tertiary structure levels suggests that the disturbed aggregation results from subtle perturbations causing a higher and/or longer exposure of hydrophobic residues in the variants as compared to WT GALT. The results herein described indicate a possible benefit from introducing proteostasis regulators and/or chemical/pharmacological chaperones to prevent the accumulation of protein aggregates, in new avenues of therapeutic research for classic galactosemia.

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