RESUMEN
Burdening guilt refers to the belief that one's emotions, needs, and ways of being are a burden to others, and is one type of interpersonal guilt proposed by the control-mastery theory (CMT). The aim of this article is to validate two new measures of burdening guilt. In the two studies conducted, we examined the psychometric properties of these scales and the relationship between burdening guilt and self-perceived burden (burdensomeness), self-esteem, shame, anxiety, depression, mental health, attachment insecurity, adverse childhood experiences, social desirability, empathy, and suicidal ideation. In Study 1, we presented a newly developed Burdening Guilt Rating Scale (BGRS) and its correlation with measures of the abovementioned dimensions. In Study 2 we verified, through confirmatory factor analysis and correlation techniques, the possibility of expanding the Interpersonal Guilt Rating Scale-15 with a shorter, 5-item burdening guilt scale derived from the BGRS, and showed that this shorter scale correlates similarly to the longer one. Findings allowed us to validate these new scales providing empirical measures of burdening guilt-a theoretical concept with important clinical implications.
Asunto(s)
Culpa , Vergüenza , Humanos , Emociones , Ideación Suicida , Ansiedad/psicologíaRESUMEN
Introduction According to Control-Mastery Theory (CMT)-a cognitive-dynamic relational theory of mental functioning, psychopathology, and psychotherapy-patients come to therapy with an unconscious plan to disprove their pathogenic beliefs and achieve adaptive goals. One of the primary ways patients work to disconfirm their pathogenic beliefs is by testing them within the therapeutic relationship. Objectives: The present study aimed to replicate and expand the results of previous studies suggesting that therapists' responses that disconfirmed patient's pathogenic beliefs were predictive of patients' within-session progress. Moreover, we wanted to investigate whether these interventions correlated with the therapeutic alliance. Methods: Transcriptions of 81 sessions from five brief psychodynamic psychotherapies were assessed by 11 independent raters. For each case, the patient's plan was formulated and tests identified, the accuracy of the therapist's responses to these tests was rated, and the impact of the therapist's interventions on the patient's subsequent communications and their relationship with the therapeutic alliance was measured. Results: The results supported the central hypothesis of the CMT that when the therapist's interventions passed the patient's tests, the patient showed signs of improvement. Moreover, the ability of the therapist to pass the patient's tests correlated with the therapeutic alliance. Conclusions: The clinical implications and the limitations of these findings are discussed, together with the relevance of a good case formulation for clinicians' optimal responsiveness.
Asunto(s)
Psicoterapia Breve , Psicoterapia Psicodinámica , Alianza Terapéutica , Humanos , Relaciones Profesional-Paciente , Psicoterapia/métodos , Actitud del Personal de SaludRESUMEN
The aim of this article is to empirically investigate the relationships among interpersonal guilt, as conceived within control-mastery theory (CMT), and attachment, altruism, and personality pathology in an English-speaking sample. An online sample of 393 participants was recruited to complete the Interpersonal Guilt Rating Scale self-report version-15 (IGRS-15s), together with other empirically validated measures for the assessment of attachment, altruism, and personality pathology. On the basis of previous studies conducted in Italian-speaking samples, we hypothesized that survivor guilt, separation/disloyalty guilt, and omnipotent responsibility guilt would be associated with attachment anxiety and avoidance, altruism, and personality pathology; self-hate was hypothesized to be associated only with attachment anxiety and avoidance and personality pathology. Analyses examined bivariate associations as well as the network of partial correlations among variables. The results largely confirmed hypothesized associations, with self-hate evincing the strongest unique association with personality dysfunction. Findings provide a basis for further research regarding interpersonal guilt and personality and relational functioning, with potential implications for clinical conceptualizations of the role of guilt in psychopathology.
Asunto(s)
Altruismo , Relaciones Interpersonales , Humanos , Culpa , Personalidad , Trastornos de la PersonalidadRESUMEN
Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ß3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.
Asunto(s)
Proteínas de Drosophila , N-Acetilglucosaminiltransferasas , Mapeo Peptídico , Péptidos , Receptores Notch , Animales , Línea Celular , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Fucosa/química , Fucosa/genética , Fucosa/metabolismo , Glicosilación , N-Acetilglucosaminiltransferasas/biosíntesis , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a â¼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Glucosiltransferasas/genética , Células Fotorreceptoras/metabolismo , Retinitis Pigmentosa/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas del Ojo/metabolismo , Técnicas de Sustitución del Gen , Glucosa/metabolismo , Glucosiltransferasas/metabolismo , Glicosilación , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras/patología , Receptores Notch/genética , Retinitis Pigmentosa/patología , Transducción de Señal/genéticaRESUMEN
One of the evolutionarily conserved posttranslational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a specific consensus sequence by the protein O-glucosyltransferase Rumi (POGLUT1 in human). Loss of rumi in flies results in a temperature-sensitive loss of Notch signaling. To demonstrate that the Notch receptor itself is the biologically relevant target of Rumi in flies, and to determine the role of the 18 Rumi target sites on Notch in regulating Notch signaling, we have performed an in vivo structure-function analysis of Drosophila Notch. In this chapter, we provide a detailed protocol for this analysis. To avoid the potential artifacts associated with overexpression of Notch and random insertion of transgenes, we have used recombineering and site-specific integration technologies, which have been adapted for usage in Drosophila in recent years. Using gene synthesis and site-directed mutagenesis, we generated a series of Notch genomic transgenes which harbor mutations in all or specific subsets of Notch O-glucose sites. Gene dosage and rescue experiments in animals raised at various temperatures allowed us to dissect the contribution of O-glucosylation sites to the regulation of the Notch signaling strength. The reagents and methods presented here can be used to address similar questions about other posttranslational modifications of Notch or other Drosophila proteins.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transgenes , Animales , Secuencia de Bases , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/química , Glucosiltransferasas/metabolismo , Glicosilación , Mutagénesis Sitio-Dirigida , Mutación , Receptores Notch/química , Recombinación Genética , Transducción de SeñalRESUMEN
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14-20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster , Glucosiltransferasas/genética , Receptores Notch/genética , Xilosa/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Glucosa/metabolismo , Glucosiltransferasas/metabolismo , Humanos , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Receptores Notch/metabolismo , Secuencias Repetitivas de Aminoácido , Transducción de Señal , Xilosa/genética , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Glucosiltransferasas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Glucosa/metabolismo , Glucosiltransferasas/genética , Mutación , Receptores Notch/genética , Temperatura , TransgenesRESUMEN
Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C(1)-X-S-X-P-C(2) consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi(-/-) mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1(+/-) (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi(-/-) embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.
Asunto(s)
Desarrollo Embrionario/fisiología , Glucosiltransferasas/metabolismo , Receptores Notch/metabolismo , Anomalías Múltiples/embriología , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Animales , Conductos Biliares Intrahepáticos/anomalías , Conductos Biliares Intrahepáticos/metabolismo , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Anomalías Cardiovasculares/embriología , Anomalías Cardiovasculares/genética , Anomalías Cardiovasculares/metabolismo , Línea Celular , Proteínas de Drosophila , Desarrollo Embrionario/genética , Factor de Crecimiento Epidérmico/genética , Femenino , Dosificación de Gen , Glucosiltransferasas/deficiencia , Glucosiltransferasas/genética , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Hígado/anomalías , Hígado/metabolismo , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Fenotipo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Serrate-Jagged , Transducción de SeñalRESUMEN
The evolutionarily conserved Notch signaling pathway plays broad and important roles during embryonic development and in adult tissue homeostasis. Unlike most other pathways used during animal development, Notch signaling does not rely on second messengers and intracellular signaling cascades. Instead, pathway activation results in the cleavage of the Notch intracellular domain and its translocation into the nucleus, where it functions as a transcriptional co-activator of the Notch target genes. To ensure tight spatial and temporal regulation of a pathway with such an unusually direct signaling transduction, animal cells have devised a variety of specialized modulatory mechanisms. One such mechanism takes advantage of decorating the Notch extracellular domain with rare types of O-linked glycans. In this review, we will discuss the genetic and biochemical data supporting the notion that carbohydrate modification is essential for Notch signaling and attempt to provide a brief historical overview of how we have learned what we know about the glycobiology of Notch. We will also summarize what is known about the contribution of specific nucleotide-sugar transporters to Notch biology and the roles-enzymatic and non-enzymatic-played by specific glycosyltransferases in the regulation of this pathway. Mutations in the Notch pathway components cause a variety of human diseases, and manipulation of Notch signaling is emerging as a powerful tool in regenerative medicine. Therefore, studying how sugar modification modulates Notch signaling provides a framework for better understanding the role of glycosylation in animal development and might offer new tools to manipulate Notch signaling for therapeutic purposes.
Asunto(s)
Glicosiltransferasas/metabolismo , Polisacáridos/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , HumanosRESUMEN
Telomerase is the ribonucleoprotein complex that adds telomeric repeats to the ends of chromosomes. Its protein subunit TERT is highly conserved among eukaryotes, whereas the RNA subunit varies greatly in size and sequence, hindering the identification of telomerase RNAs in some important model organisms. Here we report the identification and functional characterization of TER1, the telomerase RNA component from fission yeast Schizosaccharomyces pombe. Deletion of ter1+ caused progressive shortening of telomeres and cellular senescence followed by chromosome circularization. Interactions between Est1 and Trt1, the two known protein components of fission yeast telomerase, were dependent on TER1, supporting its role as a scaffold for the assembly of protein subunits. Using a series of template mutations, we show that translocation or dissociation site variability and template-primer slippage account for the sequence heterogeneity of fission yeast telomeres.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN de Hongos/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Telomerasa/genética , Telomerasa/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/química , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fenotipo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/química , Eliminación de Secuencia , Telomerasa/química , Telómero/química , Telómero/genéticaRESUMEN
The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.