RESUMEN
Parkinson's disease is the second most common neurodegenerative disease caused by degeneration of dopamine neurons in the substantia nigra. The origin and causes of dopamine neurodegeneration in Parkinson's disease are not well understood but oxidative stress may play an important role in its onset. Much effort has been dedicated to find biomarkers indicative of oxidative stress and neurodegenerative processes in parkinsonian brains. By using proton nuclear magnetic resonance (1H NMR) to identify and quantify key metabolites, it is now possible to elucidate the metabolic pathways affected by pathological conditions like neurodegeneration. The metabolic disturbances in the 6-hydroxydopamine (6-OHDA) hemiparkinsonian rat model were monitored and the nature and size of these metabolic alterations were analyzed. The results indicate that a unilateral injection of 6-OHDA into the striatum causes metabolic changes that not only affect the injected hemisphere but also the contralateral, non-lesioned side. We could clearly identify specific metabolic pathways that were affected, which were mostly related with oxidative stress and neurotransmission. In addition, a partial metabolic recovery by carrying out an antioxidant treatment with N-acetylcysteine (NAC) was observable.
Asunto(s)
Acetilcisteína/farmacología , Antiparkinsonianos/farmacología , Encéfalo/metabolismo , Metaboloma , Enfermedad de Parkinson/metabolismo , Animales , Encéfalo/efectos de los fármacos , Femenino , Estrés Oxidativo , Oxidopamina/toxicidad , Enfermedad de Parkinson/etiología , Ratas , Ratas Sprague-DawleyRESUMEN
Neuroinflammation is a chronic event in neurodegenerative disorders. In the rat model of Parkinson's disease, including a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA), antioxidant treatment affects the inflammatory process. Despite a heavy accumulation of microglia early after the injury, dopamine nerve fibre regeneration occurs. It remains unclear why this heavy accumulation of microglia is found early after the lesion in antioxidant-treated animals, or even more, what is the origin of these microglia. In this study magnetic resonance imaging (MRI) was used to elucidate whether the inflammatory response was generated from the blood or from activated brain microglia. Superparamagnetic iron oxide (SPIO) nanoparticles were injected intravenously prior to a striatal 6-OHDA injection to tag phagocytes in the blood. Rats were fed either with bilberry-enriched or control diet. T2*-weighted MRI scans were performed 1 week after the lesion, and hypointense areas were calculated from T2*-weighted images, to monitor the presence of SPIO particles. The results revealed that feeding the animals with bilberries significantly promoted accumulation of blood-derived immune cells. Gadolinium-enhanced MRI demonstrated no difference in leakage of the blood-brain barrier independent of diets. To conclude, bilberry-enriched diet promotes an influx of periphery-derived immune cells to the brain early after injury.
Asunto(s)
Encefalitis/fisiopatología , Imagen por Resonancia Magnética/métodos , Microglía/fisiología , Monocitos/fisiología , Neostriado/fisiopatología , Trastornos Parkinsonianos/fisiopatología , Extractos Vegetales/administración & dosificación , Vaccinium myrtillus , Animales , Barrera Hematoencefálica/metabolismo , Medios de Contraste , Modelos Animales de Enfermedad , Encefalitis/patología , Femenino , Nanopartículas de Magnetita/administración & dosificación , Microglía/metabolismo , Monocitos/metabolismo , Neostriado/metabolismo , Neostriado/patología , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Current treatments for Parkinson's disease using pharmacological approaches alleviate motor symptoms but do not prevent neuronal loss or dysregulation of dopamine neurotransmission. In this article, we have explored the molecular mechanisms underlying the neuroprotective effect of the antioxidant N-acetylcysteine (NAC) on the damaged dopamine system. METHODS: SH-SY5Y cells were differentiated towards a dopaminergic phenotype and exposed to 6-hydroxydopamine (6-OHDA) to establish an in vitro model of Parkinson's disease. We examined the potential of NAC to restore the pathological effects of 6-OHDA on cell survival, dopamine synthesis as well as on key proteins regulating dopamine metabolism. Specifically, we evaluated gene- and protein expression of tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and α-synuclein, by using qPCR and Western blot techniques. Moreover, we quantified the effect of NAC on total dopamine levels using a dopamine ELISA assay. RESULTS: Our results indicate that NAC has a neuroprotective role in SH-SY5Y cells exposed to 6-OHDA by maintaining cell proliferation and decreasing apoptosis. Additionally, we demonstrated that NAC treatment increases dopamine release and protects SH-SY5Y cells against 6-OHDA dysregulations on the proteins TH, VMAT2, and α-synuclein. CONCLUSIONS: Our findings contribute to the validation of compounds capable to restore dopamine homeostasis and shed light on the metabolic pathways that could be targeted to normalize dopamine turnover. Furthermore, our results highlight the effectiveness of the antioxidant NAC in the prevention of dopaminergic neurodegeneration in the present model. ABBREVIATIONS: DAT, dopamine transporter; 6-OHDA, 6-hydroxydopamine; NAC, N-acetylcysteine; PARP, poly (ADP-ribose) polymerase; RA; retinoic acid; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; VMAT2, vesicle monoamine transporter 2.
Asunto(s)
Acetilcisteína , Dopamina , Oxidopamina , Tirosina 3-Monooxigenasa , Proteínas de Transporte Vesicular de Monoaminas , alfa-Sinucleína , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Humanos , Oxidopamina/toxicidad , alfa-Sinucleína/metabolismo , Dopamina/metabolismo , Acetilcisteína/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Línea Celular Tumoral , Fármacos Neuroprotectores/farmacología , Supervivencia Celular/efectos de los fármacosRESUMEN
Whole-brain mapping of drug effects are needed to understand the neural underpinnings of drug-related behaviors. Amphetamine administration is associated with robust increases in striatal dopamine (DA) release. Dopaminergic terminals are, however, present across several associative brain regions, which may contribute to behavioral effects of amphetamine. Yet the assessment of DA release has been restricted to a few brain regions of interest. The present work employed positron emission tomography (PET) with [11C]raclopride to investigate regional and temporal characteristics of amphetamine-induced DA release across twenty sessions in adult female Sprague Dawley rats. Amphetamine was injected intravenously (2 mg/kg) to cause displacement of [11C]raclopride binding from DA D2-like receptors, assessed using temporally sensitive pharmacokinetic PET model (lp-ntPET). We show amphetamine-induced [11C]raclopride displacement in the basal ganglia, and no changes following saline injections. Peak occupancy was highest in nucleus accumbens, followed by caudate-putamen and globus pallidus. Importantly, significant amphetamine-induced displacement was also observed in several extrastriatal regions, and specifically in thalamus, insula, orbitofrontal cortex, and secondary somatosensory area. For these, peak occupancy occurred later and was lower as compared to the striatum. Collectively, these findings demonstrate distinct amphetamine-induced DA responses across the brain, and that [11C]raclopride-PET can be employed to detect such spatiotemporal differences.
Asunto(s)
Anfetamina , Dopamina , Femenino , Ratas , Animales , Anfetamina/farmacología , Anfetamina/metabolismo , Racloprida/farmacocinética , Dopamina/metabolismo , Ratas Sprague-Dawley , Tomografía de Emisión de Positrones/métodos , Encéfalo/metabolismo , Cuerpo Estriado/metabolismoRESUMEN
By site directed mutagenesis, we have produced recombinant mutants of human and mouse prethrombin-2 which are able to convert themselves autocatalytically into α-thrombin. We also have created a new method to amplify the signal of bioanalytical assays based on the autocatalytic activation of these mutated proenzymes. The activation of the mutants by active α-thrombin triggers an autocatalytic reaction which leads to more active thrombin resulting in the amplification of the readout signal. Addition of mutated mouse prethrombin-2 into the conventional assay for prothrombin level in human plasma, employing ecarin and the fluorogenic substrate, resulted in improvement of the detection limit by 2 orders of magnitude.
Asunto(s)
Protrombina/análisis , Espectrometría de Fluorescencia , Animales , Biocatálisis , Humanos , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Protrombina/genética , Protrombina/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodaminas/química , Especificidad por SustratoRESUMEN
This study aimed to explore the beneficial effects of the antioxidant N-acetylcysteine (NAC) on the degenerated dopamine system. The short- and long-term regulatory mechanisms of NAC on the 6-OHDA hemiparkinsonian rat model were longitudinally investigated by performing positron emission tomography (PET) imaging using the specific dopamine transporter (DAT) radioligand [18F]FE-PE2I. The results demonstrate that after a unilateral dopamine insult NAC has a strong influence on the non-lesioned hemisphere by decreasing the levels of DAT in the striatum early after the lesion. We interpret this early and short-term decrease of DAT in the healthy striatum of NAC-treated animals as a beneficial compensatory effect induced by NAC.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Femenino , Nortropanos/farmacocinética , Oxidopamina/toxicidad , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/etiología , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
We have developed an analytical assay to detect the enzymatic activity of acetylcholine esterase and alkaline phosphatase based on the generation of quantum dots by enzymatic products. Acetylcholine esterase converts acetylthiocholine into thiocholine. The latter enhances the rate of decomposition of sodium thiosulfate into H(2)S, which in the presence of cadmium sulfate yields CdS quantum dots showing a time dependent exponential growth, typical of autocatalytic processes. This assay was also applied to detect acetylcholine esterase inhibitors. Alkaline phosphatase hydrolyzes thiophosphate and yields H(2)S, which instantly reacts with Cd(2+) to give CdS quantum dots. The formation of CdS quantum dots in both reactions was followed by fluorescence spectroscopy and showed dependence on the concentration of enzyme and substrate.
Asunto(s)
Acetilcolinesterasa/metabolismo , Fosfatasa Alcalina/metabolismo , Puntos Cuánticos , Biocatálisis , Compuestos de Cadmio/química , Sulfuro de Hidrógeno/metabolismo , Sulfatos/químicaRESUMEN
Hydrogen peroxide is a very reactive byproduct of many metabolic pathways. We describe an ultra-sensitive colorimetric method to detect hydrogen peroxide based on the reconstitution of apo-horseradish peroxidase with the hemin derivative, hemin di(N,N'-acetyl-hydrazide). Oxidation of the latter by hydrogen peroxide yields hemin, which is able to reconstitute apo-horseradish peroxidase, forming an active peroxidase. We have also applied this analyte-triggered reconstitution of horseradish peroxidase to detect the activity of the enzyme glucose oxidase.
Asunto(s)
Colorimetría/métodos , Peróxido de Hidrógeno/análisis , Glucosa Oxidasa/metabolismo , Hemina/química , Peroxidasa de Rábano Silvestre/metabolismo , Oxidación-ReducciónRESUMEN
Hydrolysis of acetylthiocholine mediated by acetylcholine esterase yields the thiol-bearing compound thiocholine. At trace concentrations, thiocholine modulates the growth of Au-Ag nanoparticles on seeding gold nanoparticles in the presence of ascorbic acid. Inhibition of the enzyme by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) or by diethyl p-nitrophenyl phosphate (paraoxon) produces lower yields of thiocholine, promoting the catalytic growth of Au-Ag nanoparticles. Here, we describe the development of a simple and sensitive colorimetric assay for the detection of AChE inhibitors.
Asunto(s)
Sustancias para la Guerra Química/análisis , Inhibidores de la Colinesterasa/análisis , Colorimetría/métodos , Oro/química , Nanopartículas del Metal/química , Plata/química , Acetilcolinesterasa/química , Acetiltiocolina/química , Ácido Ascórbico/química , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/análisis , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/química , Sustancias para la Guerra Química/química , Inhibidores de la Colinesterasa/química , Hidrólisis , Paraoxon/análisis , Paraoxon/química , Tiocolina/análisis , Tiocolina/químicaRESUMEN
The water-soluble bis(bipyridine)chloro(4-picolinic acid) osmium complex, [Os(III)(bpy)2Cl(PyCOOH)]2+ (bpy=2,2'-bipyridine, Py=pyridine), is fluorescent in aqueous solution, whereas the reduced form of the complex, [Os(II)(bpy)2Cl(PyCOOH)]+, shows no significant fluorescence under the same conditions. Such reversible redox control of the fluorescence of the complex can be easily adapted to follow any enzymatic reaction to yield oxidising or reducing products that are capable of interacting with [Os(III)(bpy)2Cl(PyCOOH)]2+ or [Os(II)(bpy)2Cl(PyCOOH)]+. Based on the redox reaction between products of biocatalytic reactions and the osmium complex, we have designed a simple bioanalytical assay for the detection of nerve gases, alpha-ketoglutarate, hydrogen peroxide and glucose.
Asunto(s)
Sustancias Luminiscentes/química , Compuestos Organometálicos/química , Osmio/química , Oxidorreductasas/química , Ácidos Picolínicos/química , Biocatálisis , Glucosa/química , Peróxido de Hidrógeno/química , Ácidos Cetoglutáricos/química , Oxidación-Reducción , AguaRESUMEN
In Parkinson's disease, degeneration of substantia nigra dopaminergic neurons is accompanied by damage on other neuronal systems. A severe denervation is for example seen in the locus coerulean noradrenergic system. Little is known about the relation between noradrenergic and dopaminergic degeneration, and the effects of noradrenergic denervation on the function of the dopaminergic neurons of substantia nigra are not fully understood. In this study, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) was injected in rats, whereafter behavior, striatal KCl-evoked dopamine and glutamate releases, and immunohistochemistry were monitored at 3 days, 3 months, and 6 months. Quantification of dopamine-beta-hydroxylase-immunoreactive nerve fiber density in the cortex revealed a tendency towards nerve fiber regeneration at 6 months. To sustain a stable noradrenergic denervation throughout the experimental timeline, the animals in the 6-month time point received an additional DSP4 injection (2 months after the first injection). Behavioral examinations utilizing rotarod revealed that DSP4 reduced the time spent on the rotarod at 3 but not at 6 months. KCl-evoked dopamine release was significantly increased at 3 days and 3 months, while the concentrations were normalized at 6 months. DSP4 treatment prolonged both time for onset and reuptake of dopamine release over time. The dopamine degeneration was confirmed by unbiased stereology, demonstrating significant loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. Furthermore, striatal glutamate release was decreased after DSP4. In regards of neuroinflammation, reactive microglia were found over the substantia nigra after DSP4 treatment. In conclusion, long-term noradrenergic denervation reduces the number of dopaminergic neurons in the substantia nigra and affects the functionality of the nigrostriatal system. Thus, locus coeruleus is important for maintenance of nigral dopaminergic neurons.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Norepinefrina/fisiología , Sustancia Negra/citología , Sustancia Negra/fisiología , Animales , Bencilaminas , Proteínas de Unión al Calcio/metabolismo , Desnervación , Dopamina/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Ácido Glutámico/metabolismo , Locus Coeruleus/efectos de los fármacos , Locus Coeruleus/metabolismo , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Fibras Nerviosas/metabolismo , Neuronas/fisiología , Inhibidores de la Captación de Neurotransmisores , Ratas , Ratas Sprague-DawleyRESUMEN
We have cloned and characterized a second alpha-actinin isoform in Entamoeba histolytica. This protein, alpha-actinin2, has a N-terminal actin-binding domain, a C-terminal calcium-binding domain and an intervening rod domain containing two spectrin repeats. The protein binds and cross-links actin filaments in a calcium-dependent manner. Therefore alpha-actinin2 is a genuine alpha-actinin except for the shorter rod domain compared to the rod domain of isoforms of higher organisms. A alpha-actinin-like protein has previous been implicated in the adherence to the host cell and infection. It is therefore possible that alpha-actinin2 is involved in mechanism of infection, and in particular in reorganisation of the parasite's cytoskeleton that follows on adherence. E. histolytica alpha-actinin2 represents one of the first members of the spectrin superfamily where well defined spectrin repeats are found. The isolation and characterization of this second alpha-actinin isoform is valuable not only into the study of E. histolytica infection mechanisms, but also for understanding the evolution processes of the spectrin superfamily.
Asunto(s)
Actinina/genética , Actinina/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Actinina/química , Actinas/metabolismo , Actinas/ultraestructura , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Calcio/metabolismo , Clonación Molecular , Inmunoprecipitación , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Brain iron accumulation is a common feature shared by several neurodegenerative disorders including Parkinson's disease. However, what produces this accumulation of iron is still unknown. In this study, the 6-hydroxydopamine (6-OHDA) hemi-parkinsonian rat model was used to investigate abnormal iron accumulation in substantia nigra. We investigated three possible causes of iron accumulation; a compromised blood-brain barrier (BBB), abnormal expression of ferritin, and neuroinflammation. We identified alterations in the BBB subsequent to the injection of 6-OHDA using gadolinium-enhanced magnetic resonance imaging (MRI). Moreover, detection of extravasated IgG suggested that peripheral components are able to enter the brain through a leaky BBB. Presence of iron following dopamine cell degeneration was studied by MRI, which revealed hypointense signals in the substantia nigra. The presence of iron deposits was further validated in histological evaluations. Furthermore, iron inclusions were closely associated with active microglia and with increased levels of L-ferritin indicating a putative role for microglia and L-ferritin in brain iron accumulation and dopamine neurodegeneration.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Hierro/metabolismo , Trastornos Parkinsonianos/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Apoferritinas/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/patología , Proteínas de Unión al Calcio/metabolismo , Permeabilidad Capilar/fisiología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Inmunoglobulina G/metabolismo , Proteínas de Microfilamentos/metabolismo , Microglía/metabolismo , Microglía/patología , Neuroinmunomodulación/fisiología , Oxidopamina , Trastornos Parkinsonianos/diagnóstico por imagen , Trastornos Parkinsonianos/patología , Ratas Sprague-DawleyRESUMEN
We have cloned, expressed and characterized a alpha-actinin-like protein of Entamoeba histolytica. Analysis of the primary structure reveals that the essential domains of the alpha-actinin protein family are conserved: an N-terminus actin-binding domain, a C-terminus calcium-binding domain and a central helical rod domain. However, the rod domain of this Entamoeba protein is considerably shorter than the rod domain in alpha-actinins of higher organisms. The cloned Entamoeba 63 kDa protein is recognized by conventional alpha-actinin antibodies as well as binds and cross-links filamentous actin and calcium ions in the same manner as alpha-actinins. Despite the shorter rod domain this protein has conserved the most important functions of alpha-actinins. Therefore, it is suggested that this 63 kDa protein is an atypical and ancestral alpha-actinin.
Asunto(s)
Actinina , Entamoeba histolytica/metabolismo , Actinina/química , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Animales , Calcio/metabolismo , Dicroismo Circular , Clonación Molecular , Entamoeba histolytica/ultraestructura , Microscopía Electrónica de Transmisión , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson's disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.
Asunto(s)
Hierro/metabolismo , Imagen por Resonancia Magnética , Adrenérgicos/farmacología , Adrenérgicos/uso terapéutico , Animales , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Oxidopamina/farmacología , Oxidopamina/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Radiografía , Ratas , Ratas Sprague-DawleyRESUMEN
Alpha-actinin is a ubiquitous actin-binding protein, composed of 3 domains; an actin-binding domain and a calcium-binding domain at the termini, connected by a rod domain composed by 1, 2, or 4 spectrin repeats (SRs). To understand how the rod domain has evolved during evolution, we have analyzed and compared the amino acid residue heterogeneity and phylogeny of the SRs of alpha-actinins of vertebrates, invertebrates, fungi, and several protozoa. The repeats of vertebrate alpha-actinins show a high degree of similarity, whereas repeats of invertebrates, fungi, and, in particular, of protozoa are more divergent. In the phylogeny, SR1 of all species were clustered together, independent of the number of repeats in the protein. It was also obvious that the second and last repeat in fungi (SR2) grouped with the fourth and last repeat of vertebrates and invertebrates (SR4). Therefore, the phylogeny implied that the rod domain of the cenancestral alpha-actinin only contained one SR. It was also obvious that SR2 of fungi are related to SR4 of vertebrates and invertebrates, implying that in the second intragenic duplication 2 repeats (i.e., what become SR2 and SR3) were inserted between the initial 2 repeats that become SR1 and SR4.
Asunto(s)
Actinina/química , Actinina/clasificación , Estructura Secundaria de Proteína , Actinina/genética , Animales , Secuencia de Bases , Proteínas Fúngicas/química , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Alineación de SecuenciaRESUMEN
The N-terminal actin-binding domain of alpha-actinin is connected to the C-terminal EF-hands by a rod domain. Because of its ability to form dimers, alpha-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells. In the prototypic alpha-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats. We have found some atypical alpha-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one and two spectrin repeats, respectively. This implies that the four repeats present in modern alpha-actinins arose after two consecutive intragenic duplications from an alpha-actinin with a single repeat. Further, the evolutionary gene tree of alpha-actinins shows that the appearance of four distinct alpha-actinin isoforms may have occurred after the vertebrate-invertebrate split. The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early in the vertebrate radiation. The phylogeny also considers these atypical isoforms as the most basal to alpha-actinins of vertebrates and other eukaryotes. The analysis also positioned alpha-actinin of the fungi Encephalitozoo cuniculi close to the protozoa, supporting the suggestion that microsporidia are early eukaryotes. Because alpha-actinin is considered the basal member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily.