RESUMO
ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estrutura Terciária de Proteína , Fibrose Cística/genética , Mutação , Membranas/metabolismoRESUMO
Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein-lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.
Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/tratamento farmacológico , Mutação , Dobramento de Proteína/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Pirrolidinas/farmacologia , Sítios de Ligação , Agonistas dos Canais de Cloreto/farmacologia , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Quimioterapia Combinada , Células HEK293 , Humanos , Domínios Proteicos , Estrutura Terciária de ProteínaRESUMO
C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4-NBD1 interface.
Assuntos
Brônquios/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Homozigoto , Cavidade Nasal/efeitos dos fármacos , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacologia , Brônquios/metabolismo , Brônquios/patologia , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/patologia , Humanos , Simulação de Acoplamento Molecular , Mutação , Cavidade Nasal/metabolismo , Cavidade Nasal/patologiaRESUMO
Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells. Structural analyses were also performed to study the molecular environment of the W361 residue. Western blot showed that W361R-CFTR was not efficiently processed to a mature band C, similar to F508del CFTR, but unlike F508del CFTR, it did exhibit significant transport activity at the cell surface in response to cAMP agonists. Importantly, W361R-CFTR also responded well to CFTR modulators: its maturation defect was efficiently corrected by VX-809 treatment and its channel activity further potentiated by VX-770. Based on these results, we postulate that W361R is a novel class-2 CF mutation that causes abnormal protein maturation which can be corrected by VX-809, and additionally potentiated by VX-770, two FDA-approved small molecules. At the structural level, W361 is located within a class-2 CF mutation hotspot that includes other mutations that induce variable disease severity. Analysis of the 3D structure of CFTR within a lipid environment indicated that W361, together with other mutations located in this hotspot, is at the edge of a groove which stably accommodates lipid acyl chains. We suggest this lipid environment impacts CFTR folding, maturation and response to CFTR modulators.
RESUMO
Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 µM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.
Assuntos
Benzamidas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Purinas/farmacologia , Aminofenóis/farmacologia , Benzamidas/síntese química , Benzamidas/metabolismo , Sítios de Ligação , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Sinergismo Farmacológico , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Purinas/síntese química , Purinas/metabolismo , Quinolonas/farmacologiaRESUMO
Cryo-electron microscopy (cryo-EM) has recently provided invaluable experimental data about the full-length cystic fibrosis transmembrane conductance regulator (CFTR) 3D structure. However, this experimental information deals with inactive states of the channel, either in an apo, quiescent conformation, in which nucleotide-binding domains (NBDs) are widely separated or in an ATP-bound, yet closed conformation. Here, we show that 3D structure models of the open and closed forms of the channel, now further supported by metadynamics simulations and by comparison with the cryo-EM data, could be used to gain some insights into critical features of the conformational transition toward active CFTR forms. These critical elements lie within membrane-spanning domains but also within NBD1 and the N-terminal extension, in which conformational plasticity is predicted to occur to help the interaction with filamin, one of the CFTR cellular partners.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Molecules correcting the trafficking (correctors) and gating defects (potentiators) of the cystic fibrosis causing mutation c.1521_1523delCTT (p.Phe508del) begin to be a useful treatment for CF patients bearing p.Phe508del. This mutation has been identified in different genetic contexts, alone or in combination with variants in cis. Until now, 21 exonic variants in cis of p.Phe508del have been identified, albeit at a low frequency. The aim of this study was to evaluate their impact on the efficacy of CFTR-directed corrector/potentiator therapy (Orkambi). The analysis by minigene showed that two out of 15 cis variants tested increased exon skipping (c.609C > T and c.2770G > A). Four cis variants were studied functionally in the absence of p.Phe508del, one of which was found to be deleterious for protein maturation c.1399C > T (p.Leu467Phe). In the presence of p.Phe508del, this variant was the only to prevent the response to Orkambi treatment. This study showed that some patients carrying p.Phe508del complex alleles are predicted to poorly respond to corrector/potentiator treatments. Our results underline the importance to validate treatment efficacy in the context of complex alleles.
Assuntos
Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Quinolonas/uso terapêutico , Alelos , Combinação de Medicamentos , Humanos , Mutação , Fenilalanina/genéticaRESUMO
Development of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators, targeting the root cause of cystic fibrosis (CF), represents a challenge in the era of personalized medicine, as CFTR mutations lead to a variety of phenotypes, which likely require different, specific treatments. CF drug development is also complicated by the need to preserve the right balance between stability and flexibility, required for optimal function of the CFTR protein. In this review, we highlight how structural data can be exploited in this context to understand the molecular mechanisms of disease-associated mutations, to characterize the mechanisms of action of known modulators and to rationalize the search for novel, specific compounds.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/tratamento farmacológico , Animais , Sítios de Ligação , Simulação por Computador , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenho de Fármacos , HumanosRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations. A special emphasis is placed on the CFTR-specific evolution of an ABC transporter framework towards a channel function, as well as on the understanding of the effects of disease-causing mutations and their specific modulation. This in silico work should help structure-based drug discovery and design, with a view to develop CFTR-specific pharmacotherapeutic approaches for the treatment of CF in the context of precision medicine.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Conformação ProteicaRESUMO
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animais , Sítios de Ligação/genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Citoplasma/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Homologia de Sequência de AminoácidosRESUMO
Ferroportin (SLC40A1) is the only known iron exporter in mammals and is considered a key coordinator of the iron balance between intracellular and systemic iron homeostasis. However, the structural organization of ferroportin in the lipid bilayer remains controversial and very little is known about the mechanism underlying iron egress. In the present study, we have developed an approach based on comparative modeling, which has led to the construction of a model of the three-dimensional (3D) structure of ferroportin by homology to the crystal structure of a Major Facilitator Superfamily member (EmrD). This model predicts atomic details for the organization of ferroportin transmembrane helices and is in agreement with our current understanding of the ferroportin function and its interaction with hepcidin. Using in vitro experiments, we demonstrate that this model can be used to identify novel critical amino acids. In particular, we show that the tryptophan residue 42 (p.Trp42), which is localized within the extracellular end of the ferroportin pore, is likely involved in both the iron export function and in the mechanism of inhibition by hepcidin. Thus, our 3D model provides a new perspective for understanding the molecular basis of ferroportin functions and dysfunctions.
Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Hemocromatose/genética , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Códon , Hepcidinas/química , Hepcidinas/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
BACKGROUND: CFTR is the only ABC transporter functioning as a chloride (Cl(-)) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. METHODS: Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. RESULTS: We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl(-) current of CFTR (couple E267-K1060) or ii) increases it with a change of the electrophysiological signature (couple S263-V1056). CONCLUSIONS: These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Conformação Proteica , Transportadores de Cassetes de Ligação de ATP/metabolismo , Motivos de Aminoácidos , Western Blotting , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Modelos Moleculares , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Western Blotting , Linhagem Celular , Colforsina/farmacologia , Humanos , Iodetos/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Quinolizinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (DeltaF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Moleculares , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Simulação por Computador , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.
Assuntos
Lâmina de Crescimento/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteínas/fisiologia , Hormônios Tireóideos/metabolismo , Transativadores/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Elonguina , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Lâmina de Crescimento/embriologia , Proteínas Hedgehog , Humanos , Imunoprecipitação , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Homologia de Sequência de Aminoácidos , Hormônios Tireóideos/farmacologia , Tíbia/citologia , Tíbia/efeitos dos fármacos , Tíbia/metabolismo , Transativadores/genética , Transativadores/farmacologia , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Iodotironina Desiodinase Tipo IIRESUMO
The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of kappa-caseins. In all these proteins, this linkage is observed within an exposed beta-strand-like structure, as also predicted for kappa-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the kappa-casein/chymosin complex.
Assuntos
Caseínas/química , Quimosina/química , Animais , Butiratos/química , Caseínas/antagonistas & inibidores , Bovinos , Cisteína/química , Glutamato-Amônia Ligase/química , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Inibidores de Proteases/química , Conformação Proteica , Especificidade por SubstratoRESUMO
SUMMARY: In this study, we describe a novel domain, OCRE, which is shared by the recently identified angiogenic factor VG5Q and a specific family of RNA-binding motif proteins. The OCRE domain is characterized by a 5-fold, imperfectly repeated octameric sequence, which includes a triplet of often-conserved aromatic amino acids predicted to form a beta-strand and in which the slightly modified fifth repeat might act as a repeat terminator. Although the function of this domain remains to be elucidated, the domain architecture of OCRE containing proteins and experimental data suggest a role in RNA metabolism and/or in signalling pathways activated by the tumor necrosis factor superfamily of cytokines.
Assuntos
Proteínas Angiogênicas/química , Proteínas Angiogênicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , RNA/química , RNA/metabolismo , Análise de Sequência de Proteína/métodos , Hidrocarbonetos Aromáticos/química , Hidrocarbonetos Aromáticos/metabolismo , Reconhecimento Automatizado de Padrão/métodos , Estrutura Terciária de Proteína , Alinhamento de Sequência/métodos , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
We present a new automatic algorithm, named VoTAP (Voronoï Tessellation Assignment Procedure), which assigns secondary structures of a polypeptide chain using the list of alpha-carbon coordinates. This program uses three-dimensional Voronoï tessellation. This geometrical tool associates with each amino acid a Voronoï polyhedron, the faces of which unambiguously define contacts between residues. Thanks to the face area, for the contacts close together along the primary structure (low-order contacts) a distinction is made between strong and normal ones. This new definition yields new contact matrices, which are analyzed and used to assign secondary structures. This assignment is performed in two stages. The first one uses contacts between residues close together along the primary structure and is based on data collected on a bank of 282 well-refined nonredundant structures. In this bank, associations were made between the prints defined by these low-order contacts and the assignments performed by different automatic methods. The second step focuses on the strand assignment and uses contacts between distant residues. Comparison with several other automatic assignment methods are presented, and the influence of resolution on the assignment is investigated.
Assuntos
Algoritmos , Estrutura Secundária de Proteína , Aminoácidos/química , Carbono/química , Modelos Moleculares , Estrutura Molecular , Proteínas/químicaRESUMO
Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec/Elitek), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme contains a cysteine adduct on Cys(103). In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys(35) and Cys(290)) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.
Assuntos
Cisteína/química , Urato Oxidase/química , Urato Oxidase/ultraestrutura , Aspergillus flavus/enzimologia , Ativação Enzimática , Controle de Qualidade , Proteínas Recombinantes/análise , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Viral envelope glycoproteins promote infection by mediating fusion between viral and cellular membranes. Fusion occurs after dramatic conformational changes within fusion proteins, leading to the exposure of a short stretch of mostly apolar residues, termed the fusion peptide, which is presumed to insert into the membrane and initiate the fusion process. The typical global composition of fusion peptides, rich in hydrophobic but also in small amino acids such as alanine and glycine, was used here as bait to detect other peptidic segments that can insert into membranes. We so evidenced a similar composition in several cytotoxic peptides, which promote pore formation such as peptides involved in amyloidoses and hydrophobic alpha-hairpins of pore-forming toxins. It is suggested that the structural plasticity observed for several membrane active peptides can be conferred by this particular global amino acid composition, which could be thus used to predict such functional behavior from genome data.