RESUMEN
A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.
Asunto(s)
Edema/inducido químicamente , Fabaceae/química , Lectina de Unión a Manosa , Simulación del Acoplamiento Molecular , Lectinas de Plantas , Semillas/química , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Edema/patología , Femenino , Glicosilación , Inflamación/inducido químicamente , Inflamación/patología , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/toxicidad , Espectrometría de Masas , Lectinas de Plantas/química , Lectinas de Plantas/toxicidad , Huella de Proteína , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Endometriosis is a chronic gynecological condition that affects 10-32% of women of reproductive age and may lead to infertility. The study of protein profiles in follicular fluid may assist in elucidating possible biomarkers related to this disease. For this, follicular fluid samples were obtained from women with tubal factor or minimal male factor infertility who had pregnancy outcomes after in vitro fertilization (IVF) treatment (control group, n = 10), women with endometriosis (endometriosis group, n = 10), along with the endometrioma from these same patients were included (endometrioma group, n = 10). For proteomic analysis, samples were pooled according to their respective groups and normalized to protein content. Proteins were analyzed by in tandem mass spectrometry (MS(E)) Spectra processing and the ProteinLynx Global Server v.2.5. was used for database searching. Data was submitted to the biological network analysis using Cytoscape 2.8.2 with ClueGO plugin. As a result, 535 proteins were identified among all groups. The control group differentially or uniquely expressed 33 (6%) proteins and equal expression of 98 (18%) proteins was observed in the control and endometriosis groups of which 41 (7%) proteins were further identified and/or quantified. Six (1%) proteins were observed in both the endometriosis and endometrioma groups, but 212 (39%) proteins were exclusively identified and/or quantified in the endometrioma group. There were 9 (1%) proteins observed in both the control and endometrioma groups and there were 139 (25%) proteins common among all three groups. Distinct differences among the protein profiles in the follicular fluid of patients included in this study were found, identifying proteins related to the disease progression and IVF success. Thus, some pathways related to endometriosis are associated with the presence of specific proteins, as well as the absence of others. This study provides a first step to the development of more sensitive diagnostic tests and treatment.
Asunto(s)
Endometriosis/metabolismo , Líquido Folicular/metabolismo , Adulto , Biomarcadores/metabolismo , Femenino , Fertilización In Vitro , Humanos , Embarazo , Huella de Proteína , ProteómicaRESUMEN
The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.