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1.
J Biomol Struct Dyn ; 34(3): 497-507, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26479306

RESUMEN

The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.


Asunto(s)
Sitio Alostérico , Sitios de Unión , Modelos Moleculares , Fenilalanina Hidroxilasa/química , Fenilalanina/química , Conformación Proteica , Regulación Alostérica , Dominio Catalítico , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Unión Proteica , Multimerización de Proteína , Relación Estructura-Actividad
2.
Chem Biol ; 22(2): 217-28, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25641165

RESUMEN

Human ß-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human ß-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.


Asunto(s)
Proteína-1 Reguladora de Fusión/metabolismo , beta-Defensinas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/farmacología , Biotina/química , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/metabolismo , Escherichia coli/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Proteína-1 Reguladora de Fusión/antagonistas & inhibidores , Proteína-1 Reguladora de Fusión/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Microscopía Confocal , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Resonancia por Plasmón de Superficie , beta-Defensinas/química , beta-Defensinas/farmacología
3.
CNS Neurol Disord Drug Targets ; 13(5): 896-908, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25012618

RESUMEN

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences. It is the main cause of a group of neurological diseases collectively called transmissible spongiform encephalopathies that severely affect both humans and a variety of mammals. Much effort has been directed to understanding the molecular basis of PrP activity, both in physiological and pathological terms. In this context, identification of neuronally-relevant interactors of PrP may play a crucial role. We recently discovered a specific, high-affinity (nanomolar KD) interaction with tyrosine hydroxylase (TH), a enzyme catalyzing the rate-limiting step in the synthesis of the neurotransmitter dopamine. Using molecular biological, biochemical and biophysical techniques we identified the C-terminal structured domain of PrP and the Nterminal regulatory domain of TH as interacting domains between these two proteins. This interaction does not affect TH activity in vitro, although co-expression experiments in HeLa and Chinese hamster ovary cells revealed that PrP is able to internalize TH. Moreover, TH modulated the level of expression of PrP and its localization at the plasma membrane. This novel interaction between two proteins of central importance in nervous system function may shed new light on our understanding of PrP in neurological diseases.


Asunto(s)
Enfermedades del Sistema Nervioso/metabolismo , Priones/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Humanos
4.
MAbs ; 6(3): 708-17, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24525694

RESUMEN

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85-102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C's functions in the brain.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Fructosa-Bifosfato Aldolasa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Línea Celular , Biología Computacional , Mapeo Epitopo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/genética , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neurofilamentos/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína , ARN/genética , ARN/metabolismo
5.
PLoS One ; 8(11): e79482, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244510

RESUMEN

Phenylalanine hydroxylase (PAH) catalyzes the conversion of L-Phe to L-Tyr. Defects in PAH activity, caused by mutations in the human gene, result in the autosomal recessively inherited disease hyperphenylalaninemia. PAH activity is regulated by multiple factors, including phosphorylation and ligand binding. In particular, PAH displays positive cooperativity for L-Phe, which is proposed to bind the enzyme on an allosteric site in the N-terminal regulatory domain (RD), also classified as an ACT domain. This domain is found in several proteins and is able to bind amino acids. We used molecular dynamics simulations to obtain dynamical and structural insights into the isolated RD of PAH. Here we show that the principal motions involve conformational changes leading from an initial open to a final closed domain structure. The global intrinsic motions of the RD are correlated with exposure to solvent of a hydrophobic surface, which corresponds to the ligand binding-site of the ACT domain. Our results strongly suggest a relationship between the Phe-binding function and the overall dynamic behaviour of the enzyme. This relationship may be affected by structure-disturbing mutations. To elucidate the functional implications of the mutations, we investigated the structural effects on the dynamics of the human RD PAH induced by six missense hyperphenylalaninemia-causing mutations, namely p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V. These studies showed that the alterations in RD hydrophobic interactions induced by missense mutations could affect the functionality of the whole enzyme.


Asunto(s)
Modelos Moleculares , Fenilalanina Hidroxilasa/química , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Mutación , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Conformación Proteica
6.
Nucleic Acids Res ; 41(1): 327-39, 2013 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-23104426

RESUMEN

Prion protein (PrP) is involved in lethal neurodegenerative diseases, and many issues remain unclear about its physio-pathological role. Quadruplex-forming nucleic acids (NAs) have been found to specifically bind to both PrP cellular and pathological isoforms. To clarify the relevance of these interactions, thermodynamic, kinetic and structural studies have been performed, using isothermal titration calorimetry, surface plasmon resonance and circular dichroism methodologies. Three quadruplex-forming sequences, d(TGGGGT), r(GGAGGAGGAGGA), d(GGAGGAGGAGGA), and various forms of PrP were selected for this study. Our results showed that these quadruplexes exhibit a high affinity and specificity toward PrP, with K(D) values within the range 62÷630 nM, and a weaker affinity toward a PrP-ß oligomer, which mimics the pathological isoform. We demonstrated that the NA quadruplex architecture is the structural determinant for the recognition by both PrP isoforms. Furthermore, we spotted both PrP N-terminal and C-terminal domains as the binding regions involved in the interaction with DNA/RNAs, using several PrP truncated forms. Interestingly, a reciprocally induced structure loss was observed upon PrP-NA interaction. Our results allowed to surmise a quadruplex unwinding-activity of PrP, that may have a feedback in vivo.


Asunto(s)
G-Cuádruplex , Priones/química , Sitios de Unión , Calorimetría , Dicroismo Circular , ADN/química , Cinética , Priones/metabolismo , Unión Proteica , ARN/química , Resonancia por Plasmón de Superficie , Termodinámica
7.
Biochim Biophys Acta ; 1832(1): 20-8, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23022479

RESUMEN

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.


Asunto(s)
Azul de Metileno/química , Priones/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Priones/genética , Priones/metabolismo , Conformación Proteica , Ovinos
8.
PLoS One ; 7(6): e38906, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22761713

RESUMEN

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Glucoquinasa/genética , Mutación/genética , Polimorfismo Genético/genética , Adenosina Trifosfato/metabolismo , Niño , Cromatografía Líquida de Alta Presión , Biología Computacional , Femenino , Glucoquinasa/metabolismo , Humanos , Italia , Cinética , Masculino , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica
9.
J Struct Biol ; 177(2): 506-12, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22085748

RESUMEN

Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50-105°C. The structure of the oxidized form of SsTrxA1 determined at 1.9Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl-carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with ß-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds.


Asunto(s)
Proteínas Arqueales/química , Sulfolobus solfataricus , Tiorredoxinas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dicroismo Circular , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Desnaturalización Proteica , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína
10.
Hum Mutat ; 32(12): 1460-9, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21905166

RESUMEN

Choroideremia (CHM), an X-linked degeneration of the retinal pigmented epithelium (RPE), photoreceptors, and choroid, ultimately leads to blindness. It is caused by loss-of-function of the CHM gene product, the Rab escort protein 1 (REP1) that is involved, together with its homologue REP2, in prenylation of Rab GTPases, key regulators of intracellular vesicular traffic. Here, we report the molecular characterization of 20 unrelated Italian families affected by CHM. We identified 19 different mutations, nine of which are new. In most cases, we analyzed the effect of the mutations at the mRNA level. Furthermore, we demonstrated, by in vitro trancription/translation assays, that the mutated mRNAs produced truncated proteins in all cases but one. In fact, we also identified a novel REP1 missense variant (c.1520A>G; p.H507R) associated to CHM. Thus far, only two other CHM-associated missense mutations have been identified, one of which was a splicing alteration. We investigated the impact of the p.H507R amino acid change on REP1 structure and function, thus providing the first experimental demonstration that correlates a missense mutation in CHM with a functional impairment of REP1. Overall, our results indicate that the REP1-Rab geranyl-geranyl transferase interaction and consequently REP1-mediated Rab prenylation is essential for RPE and photoreceptor function.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transferasas Alquil y Aril/metabolismo , Coroideremia/genética , Análisis Mutacional de ADN/métodos , Mutación Missense/genética , Adulto , Preescolar , Coroideremia/fisiopatología , Humanos , Italia , Masculino , Persona de Mediana Edad , Adulto Joven
11.
Biochim Biophys Acta ; 1812(11): 1435-45, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21820508

RESUMEN

Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase (PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect of BH4 addition directly in living human cells expressing wild-type PAH or p.A403V, a mild mutant associated with BH4 responsiveness in vivo. Our results demonstrate that BH4 addition promotes re-establishment of the oligomerization equilibrium, thus indicating that the dimer-to-tetramer shift in pA403V plays a key role in BH4 responsiveness. In conclusion, we show that the oligomerization process and conformational stability are altered by mutations that could affect the physiological behavior of the enzyme. This endorses the hypothesis that oligomerization and folding defects of PAH variants are the most common causes of HPAs, particularly as regards mild human phenotypes.


Asunto(s)
Mutación/genética , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/patología , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Biopterinas/análogos & derivados , Biopterinas/farmacología , Dicroismo Circular , Estabilidad de Enzimas , Células HeLa , Humanos , Immunoblotting , Mutagénesis Sitio-Dirigida , Fenotipo , Fenilalanina Hidroxilasa/genética
12.
Prog Orthod ; 11(2): 174-9, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20974455

RESUMEN

We report a case of temporomandibular disorder patient with disc displacement without reduction, myofascial pain, limited opening and a novel, never described, nonsynonimous mutation of catechol-O-methyl-transferase (COMT) gene. COMT is one of the enzymes that metabolizes catecholamines, thereby acting as a key modulator of dopaminergic and adrenergic/noradrenergic neurotransmissions, which play a key role in pain modulation. This novel mutation, p.R58S, changed a codon (58 from arginine to serine) in the COMT protein. The introduction of a serine residue in a highly organised secondary structure, in critical regions of the protein, results in a structural alteration. Therefore, we speculate an influence of the mutation on the high pain sensitivity of the patient.


Asunto(s)
Catecol O-Metiltransferasa/genética , Mutación/genética , Trastornos de la Articulación Temporomandibular/genética , Adenina , Adulto , Arginina/genética , Codón/genética , Citosina , Femenino , Humanos , Luxaciones Articulares/genética , Neurotransmisores/genética , Dimensión del Dolor , Rango del Movimiento Articular/fisiología , Serina/genética , Disco de la Articulación Temporomandibular/patología , Síndrome de la Disfunción de Articulación Temporomandibular/genética
13.
Hum Mutat ; 31(12): 1294-303, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20848650

RESUMEN

Hereditary fructose intolerance (HFI) is an autosomal recessive metabolic disease caused by impaired functioning of human liver aldolase (ALDOB). At least 54 subtle/point mutations and only two large intragenic deletions have been found in the ALDOB gene. Here we report two novel ALDOB variants (p.R46W and p.Y343H) and an intragenic deletion that we found in patients with suspected HFI. The residual catalytic activity of the recombinant p.R46W and p.Y343H variants toward F1P was particularly altered. We also characterized a large intragenic deletion that we found in six unrelated patients. This is the first report of six unrelated patients sharing the same ALDOB deletion, thus indicating a founder effect for this allele in our geographic area. Because this deletion involves ALDOB exon 5, it can mimic worldwide common pathogenic genotypes, that is, homozygous p.A150P and p.A175D. Finally, the identification of only one ALDOB mutation in symptomatic patients suggests that HFI symptoms can, albeit rarely, appear also in heterozygotes. Therefore, an excessive and continuous fructose dietary intake may have deleterious effects even in apparently asymptomatic HFI carriers.


Asunto(s)
Intolerancia a la Fructosa/enzimología , Intolerancia a la Fructosa/genética , Fructosa-Bifosfato Aldolasa/genética , Eliminación de Gen , Alelos , Secuencia de Bases , Niño , Dicroismo Circular , Resultado Fatal , Femenino , Intolerancia a la Fructosa/diagnóstico , Fructosa-Bifosfato Aldolasa/química , Frecuencia de los Genes/genética , Humanos , Lactante , Italia , Cinética , Masculino , Datos de Secuencia Molecular , Linaje , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo
14.
Gene ; 457(1-2): 35-41, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20214954

RESUMEN

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis; mutations in this gene have been associated with 1-5% of early-onset human obesity. The aim of this study was to functionally characterize MC4R mutations identified in morbidly obese subjects living in Southern Italy. We studied their ligand binding, signaling pathway and subcellular localization. As expected, mutants Q43X and S19fsX51, which produce truncated forms of receptor, were devoid of activity. The activity of mutants W174C and A175T were very different even though the mutations are adjacent and are in the same transmembrane helix (TMH). In fact, the production and expression of mutant A175T on the plasma-membrane (PM) was similar to that of the wild-type (wt) receptor and the mutant retained 70% of wt receptor activity; on the contrary, the production of W174C mutant in the cytoplasm was similar to that of the wt receptor and mutant A175T but was only barely detectable on the PM and was devoid of activity. Confocal microscopy showed that W174C remained entrapped in the endoplasmic reticulum (ER) of the cells. Structural analysis showed that substitution of Trp174, located in the middle of TMH4 and 100% conserved in all known MC4Rs, with Cys could impair the relative orientation of TMH2 and TMH4 thereby affecting the overall protein architecture. Furthermore, co-expression studies showed that mutant A175T but not W174C had a dominant negative effect on the wt receptor activity.


Asunto(s)
Obesidad Mórbida/genética , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/fisiología , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Células Cultivadas , Humanos , Italia , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor de Melanocortina Tipo 4/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Relación Estructura-Actividad , Distribución Tisular/efectos de los fármacos , Transfección , alfa-MSH/metabolismo , alfa-MSH/farmacología
16.
Biopolymers ; 91(12): 1048-55, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19603492

RESUMEN

Disulfide bonds play fundamental roles in proteins. This work is devoted to highly rare motifs containing disulfide bonds. A search for four cysteines, forming a 16-atom membered ring (4CR) embodying two disulfide bonds, was carried out against all entries in the Protein Data Bank. Searching the crystallographic subset, only few protein molecules, all dimeric, were found to embody this peculiar structural feature, which establishes a covalent link between two different polypeptide chains. In contrast, in a peptide studied in solution by NMR, the four cysteines moiety includes only residues from one chain. A comparative analysis provided evidence for similarity and difference. It emerged that 4CR motif is highly rare and may serve to gain a specialized function.


Asunto(s)
Secuencias de Aminoácidos , Cisteína/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Bovinos , Pollos , Cristalografía por Rayos X , Bases de Datos de Proteínas , Disulfuros/química , Endorribonucleasas/química , Fibrinógeno/química , Proteínas de Choque Térmico/química , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
17.
Extremophiles ; 13(5): 751-61, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19588068

RESUMEN

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.


Asunto(s)
Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Sulfolobus solfataricus/enzimología , Alcohol Deshidrogenasa/genética , Sustitución de Aminoácidos , Proteínas Arqueales/genética , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Genes Arqueales , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NAD/metabolismo , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Especificidad por Sustrato , Sulfolobus solfataricus/genética , Temperatura , Triptófano/química
18.
Artículo en Inglés | MEDLINE | ID: mdl-19478442

RESUMEN

The thioredoxin system of the archaeon Sulfolobus solfataricus involves a number of different proteins: two thioredoxin reductases (SsTrxRB2 and SsTrxRB3), two distinct thioredoxins (SsTrxA1 and SsTrxA2) and a disulfide oxidoreductase (SsPDO). Here, the crystallization and preliminary crystallographic analyses of SsTrxA1 and SsTrxA2, two dimeric proteins endowed with extraordinary thermal stability, are reported. In addition to the functional thioredoxin domain, both SsTrxA1 and SsTrxA2 present an extra N-terminal fragment of approximately 30 residues. Although crystallization trials have been conducted on both forms of the proteins, crystals that were suitable for X-ray crystallographic analyses have only been obtained for their truncated variants. The crystals of SsTrxA2 belonged to space group P2, with unit-cell parameters a = 28.27, b = 27.88, c = 62.06 A, beta = 92.34 degrees , and diffracted to 1.83 A resolution, whereas the crystals of SsTrxA1 belonged to space group P2(1), with unit-cell parameters a = 51.76, b = 75.09, c = 55.35 A, beta = 112.64 degrees , and diffracted to 1.90 A resolution. The structures of the two proteins have been solved by molecular replacement.


Asunto(s)
Proteínas Arqueales/química , Cristalografía por Rayos X , Sulfolobus solfataricus/química , Tiorredoxinas/química , Tiorredoxinas/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Cristalización , Recolección de Datos , Dimerización , Datos de Secuencia Molecular , Peso Molecular , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Estadística como Asunto , Temperatura , Tiorredoxinas/genética , Difracción de Rayos X
19.
FEBS J ; 276(7): 2048-59, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19292873

RESUMEN

Hyperphenylalaninemia (Online Mendelian Inheritance in Man database: 261600) is an autosomal recessive disorder mainly due to mutations in the gene for phenylalanine hydroxylase; the most severe form of hyperphenylalaninemia is classic phenylketonuria. We sequenced the entire gene for phenylalanine hydroxylase in 51 unrelated hyperphenylalaninemia patients from Southern Italy. The entire locus was genotyped in 46 out of 51 hyperphenylalaninemia patients, and 32 different disease-causing mutations were identified. The pathologic nature of two novel gene variants, namely, c.707-2delA and p.Q301P, was demonstrated by in vitro studies. c.707-2delA is a splicing mutation that involves the accepting site of exon 7; it causes the complete skipping of exon 7 and results in the truncated p.T236MfsX60 protein. The second gene variant, p.Q301P, has very low residual enzymatic activity (approximately 4.4%), which may be ascribed, in part, to a low expression level (8-10%). Both the decreased enzyme activity and the low expression level are supported by analysis of the 3D structure of the molecule. The putative structural alterations induced by p.Q301P are compatible with protein instability and perturbance of monomer interactions within dimers and tetramers, although they do not affect the catalytic site. In vivo studies showed tetrahydrobiopterin responsiveness in the p.Q301P carrier but not in the c.707-2delA carrier. We next investigated genotype-phenotype correlations and found that genotype was a good predictor of phenotype in 76% of patients. However, genotype-phenotype discordance occurred in approximately 25% of our patients, mainly those bearing mutations p.L48S, p.R158Q, p.R261Q and p.P281L.


Asunto(s)
Genotipo , Mutación , Fenotipo , Fenilalanina Hidroxilasa/deficiencia , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Preescolar , Dihidropteridina Reductasa/genética , Femenino , Humanos , Italia/epidemiología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina Hidroxilasa/química , Fenilcetonurias/metabolismo , Conformación Proteica
20.
Biochim Biophys Acta ; 1794(3): 554-62, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19110078

RESUMEN

Recent investigations have demonstrated that disulfide bridges may play a crucial role in the stabilization of proteins in hyperthermophilic organisms. A major role in the process of disulfide formation is played by ubiquitous proteins belonging to the thioredoxin superfamily, which includes thioredoxins (Trx), thioredoxin reductases (TrxR), and disulfide oxidases/isomerases (PDO/PDI). Here we report a characterization of the structure and stability of the TrxR (SsTrxRB3) isolated from the archaeon Sulfolobus solfataricus. This protein is particularly interesting since it is able to process different substrates (Trxs and PDO) and it is endowed with an additional NADH oxidase activity. The crystal structure of the wild-type enzyme, of its complex with NADP and of the C147A mutant provides interesting clues on the enzyme function. In contrast to what is observed for class II TrxRs, in the structure of the oxidized enzyme, the FAD binding site is occupied by a partially disordered NAD molecule. In the active site of the C147A mutant, which exhibits a marginal NADH oxidase activity, the FAD is canonically bound to the enzyme. Molecular modeling indicates that a FAD molecule can be accommodated in the site of the reduced SsTrxRB3. Depending on the oxidation state, SsTrxRB3 can bind a different cofactor in its active site. This peculiar feature has been related to its dual activity. Denaturation experiments followed by circular dichroism indicate that electrostatic interactions play an important role in the stabilization of this thermostable protein. The analysis of the enzyme 3D-structure has also provided insights into the bases of SsTrxRB3 stability.


Asunto(s)
Disulfuros/química , Sulfolobus solfataricus/enzimología , Reductasa de Tiorredoxina-Disulfuro/química , Secuencia de Aminoácidos , Sitios de Unión , Dicroismo Circular , Estabilidad de Enzimas , Flavina-Adenina Dinucleótido/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Alineación de Secuencia , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo
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