RESUMEN
In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.
Asunto(s)
Señales de Clasificación de Proteína , Recombinación V(D)J , Señales de Clasificación de Proteína/genética , Proteínas de Homeodominio/metabolismo , Receptores de Antígenos/genética , Pirimidinas , PurinasRESUMEN
V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs. Each RAG2 construct was labeled with GFP at the N-terminus. Compared to the T490A mutant, cells expressing FL RAG2 exhibited elevated apoptosis by 24 h following irradiation, and this coincided with a greater amount of Caspase 3 cleavage measured in cell lysates. Pre-B cells expressing either RAG2 protein exhibited similar increases in phospho-p53 levels following irradiation. Interestingly, FL RAG2-expressing cells exhibited elevated division relative to the T490A clone beginning ~24 h following irradiation, as well as an increased percentage of cells proceeding through mitosis, suggesting an improved rate of recovery following the initial burst in apoptosis. Altogether, these data show that FL RAG2, but not its stable nuclear export-defective T490A mutant, participates in pre-B cell decisions between apoptosis versus DNA repair and cell cycle progression following DNA damage.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/genética , Expresión Génica , Proteínas Nucleares/genética , Células Precursoras de Linfocitos B/metabolismo , Ciclo Celular , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Mutación , Proteínas Nucleares/metabolismo , Recombinación V(D)JRESUMEN
RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood. Here, we used cell imaging to measure the effect of mutating the RAG2 noncore region on properties of the full length protein. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant in the noncore region, which has unique regulatory properties. This showed that FL, T490A, and Core localized to nuclear domains that were adjacent to DAPI-rich heterochromatin, and that contained the active chromatin marker H3K4me3. Within the RAG2-enriched regions, T490A exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, colocalization of H3K4me3 with FL and T490A, but not Core, increased in conditions that increased H3K4me3 levels. Superresolution imaging showed H3K4me3 was distributed as puncta that RAG2 abutted, and mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating Trp453 of the T490A mutant (W453A,T490A), which blocks PHD-dependent interactions with H3K4me3, abolished the T490A-mediated increased colocalization with H3K4me3 and slower mobility compared to FL. Altogether, these data show that Thr490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Animales , Línea Celular , Núcleo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Mutación/genéticaRESUMEN
All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth.
Asunto(s)
Apolipoproteína A-I/biosíntesis , Proteínas Aviares/biosíntesis , Coroides/metabolismo , Proteínas del Ojo/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Ácido Retinoico/biosíntesis , Tretinoina/farmacología , Animales , Apolipoproteína A-I/química , Proteínas Aviares/química , Pollos , Coroides/química , Proteínas del Ojo/química , Receptores de Ácido Retinoico/química , Tretinoina/químicaRESUMEN
V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , Complejos Multiproteicos/química , Animales , Sitios de Unión , Dominio Catalítico , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Humanos , Ratones , Complejos Multiproteicos/genética , Unión Proteica , Estructura Terciaria de Proteína , VDJ Recombinasas/química , VDJ Recombinasas/genéticaRESUMEN
Since the inception of the fluid mosaic model, cell membranes have come to be recognized as heterogeneous structures composed of discrete protein and lipid domains of various dimensions and biological functions. The structural and biological properties of membrane domains are represented by CDM (cholesterol-dependent membrane) domains, frequently referred to as membrane 'rafts'. Biological functions attributed to CDMs include signal transduction. In T-cells, CDMs function in the regulation of the Src family kinase Lck (p56lck) by sequestering Lck from its activator CD45. Despite evidence of discrete CDM domains with specific functions, the mechanism by which they form and are maintained within a fluid and dynamic lipid bilayer is not completely understood. In the present chapter, we discuss recent advances showing that the actomyosin cytoskeleton has an integral role in the formation of CDM domains. Using Lck as a model, we also discuss recent findings regarding cytoskeleton-dependent CDM domain functions in protein regulation.
Asunto(s)
Colesterol/metabolismo , Antígenos Comunes de Leucocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Microdominios de Membrana/metabolismo , Linfocitos T/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Colesterol/química , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Humanos , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Microdominios de Membrana/ultraestructura , Microtúbulos/química , Microtúbulos/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/ultraestructuraRESUMEN
V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr(490). In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.
Asunto(s)
Núcleo Celular/metabolismo , Centrosoma/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Células Cultivadas , ADN/efectos de los fármacos , ADN/efectos de la radiación , Reparación del ADN , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Fluorescente , Mutación , Proteínas Nucleares/genética , Radiación Ionizante , Fracciones Subcelulares/metabolismo , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismoRESUMEN
T cells become polarized during initial interactions with an APC to form an Ag-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with costimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAPK ERK that potentiated Ca²âº signaling in response to CD3 cross-linking. CD45 also enriched in AISs but to modulate Src kinase activity, because localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca²âº signaling by an Erk-dependent mechanism and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to Ag by CD28.