RESUMEN
Neurological heterotopic ossification (NHO) is characterized by abnormal bone growth in soft tissue and joints in response to injury to the central nervous system. The ectopic bone frequently causes pain, restricts mobility, and decreases the quality of life for those affected. NHO commonly develops in severe traumatic brain injury (TBI) patients, particularly in the presence of concomitant musculoskeletal injuries (i.e. polytrauma). There are currently no animal models that accurately mimic these combinations of injuries, which has limited our understanding of NHO pathobiology, as well as the development of biomarkers and treatments, in TBI patients. In order to address this shortcoming, here we present a novel rat model that combines TBI, femoral fracture, and muscle crush injury. Young adult male Sprague Dawley rats were randomly assigned into three different injury groups: triple sham-injury, peripheral injury only (i.e., sham-TBI + fracture + muscle injury) or triple injury (i.e., TBI + fracture + muscle injury). Evidence of ectopic bone in the injured hind-limb, as confirmed by micro-computed tomography (µCT), was found at 6-weeks post-injury in 70% of triple injury rats, 20% of peripheral injury rats, and 0% of the sham-injured controls. Furthermore, the triple injury rats had higher ectopic bone severity scores than the sham-injured group. This novel model will provide a platform for future studies to identify underlying mechanisms, biomarkers, and develop evidence based pharmacological treatments to combat this debilitating long-term complication of TBI and polytrauma.
Asunto(s)
Lesiones Traumáticas del Encéfalo , Traumatismo Múltiple , Osificación Heterotópica , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Modelos Animales de Enfermedad , Humanos , Masculino , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/etiología , Calidad de Vida , Ratas , Ratas Sprague-Dawley , Microtomografía por Rayos XRESUMEN
The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 µM and K(m[NADH]) = 399.1 ± 52.1 µM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Oxidorreductasas/química , Proteínas Bacterianas/química , Clonación Molecular , Cupriavidus/enzimología , Cianuros/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Herbicidas/metabolismo , Imidazoles/farmacología , Proteínas Hierro-Azufre/química , Rayos Láser , NADP/metabolismo , Óxido Nítrico/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Rhodococcus/enzimología , Dispersión de Radiación , Espectrofotometría Ultravioleta , Termodinámica , Tiocarbamatos/metabolismoRESUMEN
A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.
Asunto(s)
Aminas/metabolismo , Concentración de Iones de Hidrógeno , Monoaminooxidasa/metabolismo , Secuencia de Bases , Catálisis , Cartilla de ADN , Humanos , Isótopos , Cinética , Hígado/enzimología , Especificidad por SustratoRESUMEN
Human AIF-M2 is an unusual flavoprotein oxidoreductase that binds DNA, nicotinamide coenzyme, and the modified flavin 6-hydroxy-FAD. Using multiple solution methods to investigate the redox chemistry and binding interactions of AIF-M2, we demonstrate that binding of DNA and coenzyme to AIF-M2 is mutually exclusive. We also show that DNA binding does not perturb the redox chemistry of AIF-M2, but it has significant effects on the reduction kinetics of the 6-hydroxy-FAD cofactor by NAD(P)H. Based on quantitative analysis of ligand binding and redox chemistry, we propose a model for the function of AIF-M2. In this model, DNA binding suppresses the redox activity of AIF-M2 by preventing the binding of the reducing coenzyme NAD(P)H. This DNA-mediated suppression of AIF-M2 activity is expected to lower cellular levels of superoxide and peroxide, thereby lessening survival signaling by Ras, NF-kappaB, or AP-1, as suggested from knock-out studies of the related AIF in human colon cancer cell lines. We show marked differences between AIF-M2 and AIF. DNA and coenzyme binding activity is retained in the C-terminal deletion mutant AIF-M2-(Delta319-613), whereas DNA binds to the C-terminal D3 domain of AIF. Our work provides the first analysis of AIF-M2 ligand interactions and redox chemistry and identifies an important mechanistic connection between coenzyme and DNA binding, redox activity, and the apoptotic function of AIF-M2. Through its DNA binding activity, we suggest that AIF-M2 lessens survival cell signaling in the presence of foreign (e.g. bacterial and (retro)viral) cytosolic DNA, thus contributing to the onset of apoptosis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Apoptosis , ADN/química , Proteínas Mitocondriales/química , Citosol/metabolismo , Humanos , Cinética , Mutación , NAD/química , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Confinement of the heel due to the counter of the shoe is believed to influence heel pad biomechanics. Using a two-dimensional finite element model of the heel pad and shoe during a simulation of static standing, the aim of this study was to quantify the potential effect of confinement on internal heel pad stress. Non-weightbearing MRI and weightbearing MRI with plantar pressure and ground reaction force data were recorded for a single subject. The non-weightbearing MRI was used to create two FE models of the heel pad, using either homogeneous or composite material properties. The composite model included a distinction in material properties between fat pad and skin. Vertical and medial-lateral forces, as measured on the subject's heel, were applied to the models and vertical compressive strains for both models were comparable with those observed by weightbearing MRI. However, only for the composite model was the predicted plantar pressure distribution comparable with measured data. The composite model was therefore used in further analyses. In this composite model, the internal stresses were located mainly in the skin and were predominantly tensile in nature, whereas the stress state in the fat pad approached hydrostatic conditions. A representation of a running shoe, including an insole, midsole and heel counter was then added to the composite heel pad to form the shod model. In order to investigate the counter effect, the load was applied to the shod model with and without the heel counter. The effect of the counter on peak stress was to elevate compression (0-50%), reduce tension (22-34%) and reduce shear (22-28%) in the skin. In addition, the counter reduced both compressive (20-40%) and shear (58-80%) stress in the fat pad and tension in the fat pad remained negligible. Taken together the results indicate that a well-fitted counter works in sympathy with the internal structure of the heel pad and could be an effective reducer of heel pad stress. However, further research needs to be undertaken to assess the long-term effects on the soft-tissues, practicalities of achieving good fit and behavior under dynamic events.
Asunto(s)
Talón/fisiología , Modelos Biológicos , Postura/fisiología , Soporte de Peso/fisiología , Adulto , Fuerza Compresiva , Talón/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética , Masculino , Radiografía , Estrés MecánicoRESUMEN
Human methionine synthase (hMS) is a multidomain cobalamin-dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 A crystal structure of the C-terminal activation domain of hMS. The structure is C-shaped with the core comprising mixed alpha and beta regions, dominated by a twisted antiparallel beta sheet with a beta-meander region. These features, including the positions of the active-site residues, are similar to the activation domain of Escherichia coli cobalamin-dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S-adenosyl-methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild-type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 3(10)-containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Cristalografía por Rayos X/métodos , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cristalización , Dimerización , Ferredoxina-NADP Reductasa/química , Ferredoxina-NADP Reductasa/genética , Ferredoxina-NADP Reductasa/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Terciaria de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMEN
Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.
Asunto(s)
Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Ferredoxinas/química , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Monóxido de Carbono/química , Sistema Enzimático del Citocromo P-450/genética , Hemo/química , Concentración de Iones de Hidrógeno , Hierro/química , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Potenciometría , Análisis Espectral , Esterol 14-DesmetilasaRESUMEN
AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds approximately 10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor.protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of K(d) values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein.DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis.
Asunto(s)
Apoptosis/fisiología , Coenzimas/metabolismo , Proteínas de Unión al ADN/metabolismo , Flavinas/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas Reguladoras de la Apoptosis , Coenzimas/química , Proteínas de Unión al ADN/genética , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Flavinas/química , Humanos , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Estructura Molecular , NAD/metabolismo , NADP/metabolismo , Oxidorreductasas/genética , Unión ProteicaRESUMEN
In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin-NADP(+) reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild-type. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S- and F1395A-NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue.
Asunto(s)
Flavina-Adenina Dinucleótido/metabolismo , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/genética , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte de Electrón , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Ratas , Alineación de Secuencia , TermodinámicaRESUMEN
The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.
Asunto(s)
Bacillus megaterium/enzimología , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Flavoproteínas/química , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Homología de Secuencia de Aminoácido , Bacillus subtilis/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Monóxido de Carbono/metabolismo , Clonación Molecular , Coenzimas/metabolismo , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos/metabolismo , Flavoproteínas/genética , Flavoproteínas/aislamiento & purificación , Flavoproteínas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hemo/metabolismo , Hidroxilación , Cinética , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Ácido Mirístico/metabolismo , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , Oxidación-Reducción , Unión Proteica , Alineación de Secuencia , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).
Asunto(s)
Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/genética , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/metabolismo , Humanos , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutación , NADPH-Ferrihemoproteína Reductasa , Oxidación-Reducción , Alineación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14alpha-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0.2 and 0.3 micro M, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1.2 and 36.5 micro M, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K(d) values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor DeltaCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.
Asunto(s)
Antifúngicos/farmacología , Antituberculosos/farmacología , Azoles/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Mycobacterium smegmatis/efectos de los fármacos , Streptomyces/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Streptomyces/crecimiento & desarrolloRESUMEN
The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated beta-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing beta-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML-/-) as in control animals, and in PML-/- fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.
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Citomegalovirus/metabolismo , Regulación Viral de la Expresión Génica , Proteínas Nucleares , Proteínas Virales/metabolismo , Animales , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citomegalovirus/química , Femenino , Genes Inmediatos-Precoces , Vectores Genéticos , Herpesvirus Humano 1/genética , Humanos , Operón Lac , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Neuronas/metabolismo , Plásmidos , Proteína de la Leucemia Promielocítica , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor , Células Vero/metabolismo , Proteínas Virales/análisis , beta-Galactosidasa/metabolismoRESUMEN
Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH-P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Transporte de Electrón/fisiología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Sitios de Unión , Modelos Moleculares , Familia de Multigenes , NADPH-Ferrihemoproteína Reductasa , Oxidación-Reducción , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The time taken to rupture in cyclic fatigue tests, to a stress of 45 MPa, was used to compare the fatigue quality of tendons from sheep of varying ages. Muscle and tendon cross-sectional areas were used to calculate the stress-in-life of each tendon. For any given age, high-stressed plantaris tendons were of a higher fatigue quality than low-stressed extensor tendons. Both fatigue quality and stress-in-life increased with age for each tendon type. High-stressed tendons are subjected to large increases in stress-in-life during growth, and fatigue quality increased significantly with this stress. This relationship was not seen, however, in low-stressed tendons, which are not subjected to a comparable range of stresses over time. It is possible that cells modify tendon fatigue quality in response to tendon loading history. Whilst Young's modulus was seen to increase with age, no difference was detected between high- and low-stressed tendons.
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Fatiga Muscular/fisiología , Ovinos/fisiología , Tendones/fisiología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Miembro Posterior/fisiología , Músculo Esquelético/fisiología , Estrés MecánicoRESUMEN
When excised tendons are subjected to a prolonged load, whether constant or oscillatory, fatigue damage accumulates, leading eventually to rupture. 'Fatigue quality', assessed by the time-to-rupture under a given stress, was found to vary hugely among the tendons of a wallaby hind limb. This material property correlates with the varied stresses to which tendons from different anatomical sites are exposed in life. The correlation was demonstrated by subjecting each excised tendon to a load equal to the maximum isometric force that its muscle could have developed. The time-to-rupture was then approximately the same for each tendon, on average 4.2 h. A model is introduced in which damage is proposed as the trigger for adaptation of fatigue quality. The model aims, in particular, to explain why low-stressed tendons are not made of a 'better' material, although this clearly exists since it is used in high-stressed tendons. The principle of design to a minimum quality is viable in biology because of the availability of self-repair to balance routine damage. Clinical symptoms, to be included under the general heading of 'overuse injuries', will only arise when this balance fails.
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Macropodidae , Tendones/fisiología , Animales , Fenómenos Biomecánicos , Femenino , Masculino , Rotura Espontánea , Estrés Mecánico , Factores de TiempoRESUMEN
Tendon, articular cartilage and the human heel pad are all soft load-bearing collagenous tissues but are designed according to utterly different micromechanical principles. Tendon is (probably) a fibre-reinforced composite material. The mechanical properties of cartilage depend on osmotic pressure developed within an aqueous proteoglycan gel and resisted by tension in a collagenous network. The micromechanics of the heel pad have not previously been described quantitatively. Order-of-magnitude calculations are introduced to assess a model based on a fluid-filled cushion. The processes of biological design are illustrated by considering tendon. Structural design determines the tendon's cross-sectional area relative to that of its muscle and, hence, the maximum stress to which the tendon may be subjected in life. Stress-in-life varies widely between tendons. Material design includes the development of compressive stiffness in the regions where transverse loads arise. More generally, the fatigue quality of each tendon is adjusted to suit its stress-in-life. The correlation between fatigue quality and stress-in-life means that every tendon is subject, on average, to a comparable rate of fatigue damage. Homeostasis requires that routine repair can keep up with this rate of damage.
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Cartílago Articular/fisiología , Colágeno/fisiología , Talón/fisiología , Tendones/fisiología , Animales , Fenómenos Biomecánicos , Cartílago Articular/química , Colágeno/química , Humanos , Tendones/química , Resistencia a la Tracción , Soporte de PesoRESUMEN
This paper supplies quantitative data on the hind- and forelimb musculature of common chimpanzees (Pan troglodytes) and calculates maximum joint moments of force as a contribution to a better understanding of the differences between chimpanzee and human locomotion. We dissected three chimpanzees, and recorded muscle mass, fascicle length, and physiological cross-sectional area (PCSA). We also obtained flexion/extension moment arms of the major muscles about the limb joints. We find that in the hindlimb, chimpanzees possess longer fascicles in most muscles but smaller PCSAs than are predicted for humans of equal body mass, suggesting that the adaptive emphasis in chimpanzees is on joint mobility at the expense of tension production. In common chimpanzee bipedalism, both hips and knees are significantly more flexed than in humans, necessitating muscles capable of exerting larger moments at the joints for the same ground force. However, we find that when subject to the same stresses, chimpanzee hindlimb muscles provide far smaller moments at the joints than humans, particularly the quadriceps and plantar flexors. In contrast, all forelimb muscle masses, fascicle lengths, and PCSAs are smaller in humans than in chimpanzees, reflecting the use of the forelimbs in chimpanzee, but not human, locomotion. When subject to the same stresses, chimpanzee forelimb muscles provide larger moments at the joints than humans, presumably because of the demands on the forelimbs during locomotion. These differences in muscle architecture and function help to explain why chimpanzees are restricted in their ability to walk, and particularly to run bipedally.
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Brazo/anatomía & histología , Pierna/anatomía & histología , Músculo Esquelético/anatomía & histología , Pan troglodytes/anatomía & histología , Animales , Humanos , MovimientoRESUMEN
Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.
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Desoxirribonucleótidos/metabolismo , Ribonucleótido Reductasas/genética , Células Clonales , ADN Complementario , Resistencia a Antineoplásicos/genética , Humanos , Hidroxiurea/farmacología , ARN Mensajero/genética , Transfección , Células Tumorales CultivadasRESUMEN
The ribonucleotide reductase (RR) gene has been associated with malignant transformation and metastatic potential. In this report, the significance of the expression of RR mRNA and enzymatic activity to the invasive potential was examined by Boyden chamber invasion assay. Our results suggest that overexpression of RR M2 mRNA and RR enzymatic activity correlates to an increase in cell invasive potential. The drug-induced HURs clone expressed a higher level RR M2 mRNA and enzyme activity which contributes significantly to the 3-fold increase in invasive potential of the cells observed relative to the KB wild-type control. On the contrary, the HUr revertant clone decreased the RR M2 mRNA level and enzymatic activity, concomitantly decreasing their invasive potential. This phenomenon is most likely due to the return of RR to levels comparable to that of the KB wild-type cells. To confirm that this observation was not of a drug-resistance phenotype associated with multiple gene alterations, the panel of RR transfectants (M1-D transfected M1 subunit cDNA, M2-D transfected M2 subunit cDNA, X-D transfected M1/M2 cDNA) characterized in a previous study were also tested in the invasion assay. The M2-D clone expressed 6-fold higher RR M2 mRNA and RR activity and also demonstrated 6-fold higher invasive potential in vitro than either the parental or vector only transfected cell line (KB-V). The X-D clone demonstrated 3-fold higher M2 mRNA expression and revealed 4-fold higher invasive potential than control cells. The M1-D clone, in contrast, expressed a baseline level of RR M2 mRNA and higher M1 mRNA. In contrast to the X-D and M2-D cells, the invasive potential of M1-D reached an even lower level in the invasive assay than the control. These results, therefore, suggest that RR M2 overexpression plays an important role in a tumor's invasiveness.