RESUMEN
MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G(1) and the growing bud tip during S and G(2) phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Microfilamentos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Activadoras de ras GTPasa , Alelos , División Celular , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Microfilamentos/genética , Mitosis/fisiología , Mutagénesis , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/genética , Fenotipo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiaeRESUMEN
BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. RESULTS: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs. We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction. CONCLUSIONS: Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.
Asunto(s)
Ácido Anhídrido Hidrolasas , Aminohidrolasas/química , Caenorhabditis elegans/química , Proteínas de Neoplasias , Proteínas/química , Secuencia de Aminoácidos , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Animales , Fusión Artificial Génica , Sitios de Unión , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismoRESUMEN
Histidine triad (HIT) proteins were until recently a superfamily of proteins that shared only sequence motifs. Crystal structures of nucleotide-bound forms of histidine triad nucleotide-binding protein (Hint) demonstrated that the conserved residues in HIT proteins are responsible for their distinctive, dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites. Hint-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Hint homologs are intracellular receptors for purine mononucleotides whose cellular function remains elusive. Fhit homologs bind and cleave diadenosine polyphosphates (Ap(n)A) such as ApppA and AppppA. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues. In invertebrates, Fhit homologs are encoded as fusion proteins with proteins related to plant and bacterial nitrilases that are candidate signaling partners in tumor suppression.
Asunto(s)
Hidrolasas , Nucleótidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Mammalian DNA polymerase beta is a crucial enzyme in cell genomic maintenance. Its structure is highly conserved. Some splice variants of beta-pol mRNA were observed. One alternative splice DNA polymerase beta mRNA, generated by 87 nt deletion (exon 11) in the catalytic domain of this enzyme, was suggested to be responsible for genomic instability in tumorigenesis and in genetic disorder (Werner syndrome). Here, we show that exon-11-deleted beta-pol mRNA is present in all examined normal and tumor tissues as well as in resting or PHA-stimulated peripheral-blood mononuclear cells. This finding proves that the presence of the exon-11 alternative splicing variant of beta-pol mRNA is not tumor-specific.
Asunto(s)
Empalme Alternativo , ADN Polimerasa I/genética , Exones/genética , Isoenzimas/genética , ARN Mensajero/genética , Artefactos , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Neoplasias Testiculares/genética , Transcripción GenéticaRESUMEN
We have used a PCR based method to analyse TCR gamma chain repertoire and clonality of gamma delta T cells in the CSF and blood of II MS patients. Samples collected from nine patients with other neurological diseases were used as a control. Five controls had central nervous system inflammation and four had non-inflammatory processes. We have observed a decreased percentage of gamma delta T cells expressing TCR gamma with V gamma 9 and J gamma P fragments in the CSF samples in comparison with the blood. We did not final clonal expansion of the gamma delta T cells in any control case. Clonal expansion of gamma delta T cells occurred in five of II MS cases in the CSF but not in the blood. Two of these clones expressed TCR gamma rearranged with V gamma 9 and J gamma 1 fragments, two others used V gamma 10 and J gamma P1, and one used V gamma 9 and J gamma P fragments. We found no correlation between clonality and clinical state of patients, duration of the disease or number of cells in CSF. Our study provides additional evidence for the possible role of the gamma delta T cells in the MS pathogenesis.
Asunto(s)
Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos T/inmunología , Adolescente , Adulto , Clonación Molecular , Cartilla de ADN , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T gamma-delta/sangre , Linfocitos T/químicaRESUMEN
We present an unusual case of the myelodysplastic syndrome (subtype refractory anemia with the excess of blasts in transformation--RAEB-t) associated with significant increase of IgG (4,700 mg/dl), lambda (160 U/dl) in blood serum and circulating clone of B lymphocytes SIgG, lambda, manifesting clonal rearrangement of JH domain. Peripheral blood cells of the patient showed two different chromosomal abnormalities: 47,XY, + del/8/p? and 47,XY, +22, +14, -19. We suppose that two independent neoplastic clones are developed in the described case, i.e. a population displaying markers of myeloblasts and monoblasts, and a clone of B lymphocytes.