RESUMEN

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

Asunto(s)

Cobre , Metaloproteínas , Cobre/química , Metaloproteínas/metabolismo , Unión Proteica , Ubiquitinas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Zinc/metabolismo , Dominios RING Finger/fisiología

RESUMEN

PHRF1 is involved in transforming growth factor ß (TGF-ß) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1Δ/Δ, in the forebrain region. PHRF1Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-ß1 treatment showed a reduced synaptic plasticity in PHRF1Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-ß signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.

Asunto(s)

Dendritas/química , Hipocampo/metabolismo , Proteínas de la Membrana/fisiología , Células Piramidales/metabolismo , Dominios RING Finger/fisiología , Memoria Espacial/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Dendritas/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Plasticidad Neuronal , Células Piramidales/citología , Transducción de Señal , Factor de Crecimiento Transformador beta/genética

RESUMEN

Potato cold-induced sweetening (CIS) is a major drawback restricting potato process industry. Starch degradation and sucrose decomposition are considered to be the key pathways in potato CIS. Our previous study showed that the RING finger gene SbRFP1 could slow down starch degradation and the accumulation of reducing sugars (RS) through inhibiting amylase and invertase activity in cold-stored tubers. However, the regulation mechanism of SbRFP1 is not clear. In this paper, we first proved that SbRFP1 could promote starch synthesis and modify the shape of starch granules. By further yeast two hybrid, GST-pull down and inhibition of enzyme activity assays, we confirmed that SbRFP1 could slow down the transformation of starch to RS in tubers mainly through the inhibition of ß-amylase StBAM1 activity. SbRFP1 was also proved to possess E3 ubiquitin ligase activity by ubiquitination assay. Thus, SbRFP1 may regulate the accumulation of RS in cold-stored tubers by ubiquitination and degradation of StBAM1. Therefore, our study reveals the regulatory mechanism of SbRFP1 in the process of CIS and provides more powerful evidence for the effect of starch degradation on potato CIS.

Asunto(s)

Proteínas de Plantas/fisiología , Tubérculos de la Planta/crecimiento & desarrollo , Dominios RING Finger/fisiología , Solanum tuberosum/crecimiento & desarrollo , Amilasas/metabolismo , Frío , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología , Proteínas de Plantas/genética , Dominios RING Finger/genética , Almidón/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5-TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains.

Asunto(s)

Multimerización de Proteína/fisiología , Dominios RING Finger/fisiología , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Factor 5 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo

RESUMEN

The MDM2 RING domain harbors E3 ubiquitin ligase activity critical for regulating the degradation of tumor suppressor p53, which controls many cellular pathways. The MDM2 RING domain also is required for an interaction with MDMX. Mice containing a substitution in the MDM2 RING domain, MDM2C462A, disrupting MDM2 E3 function and the MDMX interaction, die during early embryogenesis that can be rescued by p53 deletion. To investigate whether MDM2C462A, which retains p53 binding, has p53-suppressing activity, we generated Mdm2C462A/C462A ;p53ER/- mice, in which we replaced the endogenous p53 alleles with an inducible p53ER/- allele, and compared survival with that of similarly generated Mdm2-/-;p53ER/- mice. Adult Mdm2-null mice died ∼7 days after tamoxifen-induced p53 activation, indicating that in the absence of MDM2, MDMX cannot suppress p53. Surprisingly, Mdm2C462A/C462A ;p53ER/- mice died ∼5 days after tamoxifen injection, suggesting that p53 activity is higher in the presence of MDM2C462A than in the absence of MDM2. Indeed, in MDM2C462A-expressing mouse tissues and embryonic fibroblasts, p53 exhibited higher transcriptional activity than in those expressing no MDM2 or no MDM2 and MDMX. This observation indicated that MDM2C462A not only is unable to suppress p53 but may have gained the ability to enhance p53 activity. We also found that p53 acetylation, a measure of p53 transcriptional activity, was higher in the presence of MDM2C462A than in the absence of MDM2. These results reveal an unexpected role of MDM2C462A in enhancing p53 activity and suggest the possibility that compounds targeting MDM2 RING domain function could produce even more robust p53 activation.

Asunto(s)

Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios Proteicos/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Dominios RING Finger/fisiología , Activación Transcripcional/fisiología , Proteína p53 Supresora de Tumor/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación

RESUMEN

The Arabidopsis constitutive photomorphogenic/de-etiolated 1/FUSCA (COP/DET1/FUS) proteins repress photomorphogenesis by degrading positive regulators of photomorphogenesis, such as the transcription factor long hypocotyl5 (HY5). The gain-of-function mutant ted3, which partially suppresses the det1 mutant, contains a missense mutation of a Val-to-Met substitution before the C-terminal RING finger domain of the peroxisomal membrane protein peroxin2 (PEX2). We hypothesized that a truncated PEX2 protein, which only contains the C-terminal RING domain, is initiated by the ted3 mutation and by-passes the function of DET1 in the nucleus. Although we have not been able to detect this hypothetic peptide in vivo, we show in this study that, when fused with a fluorescent protein and overexpressed, the PEX2 RING domain can localize to the nucleus, where it is able to interact with HY5, and PEX2 RING domain overexpression in det1 also partially suppresses the det1 phenotype. Compared with det1, ted3 det1 plants have significantly decreased levels of the HY5 protein and the expression of most of the analyzed HY5 target genes is altered to levels comparable to those in hy5. We conclude that compromised activity of HY5 may have been mainly responsible for the partial reversal of the det1 phenotype in ted3 det1. Our data support the notion that, when appropriately localized, some RING finger domains may be able to achieve neomorphic effects in the cell.

Asunto(s)

Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Proteínas de la Membrana/fisiología , Proteínas Nucleares/genética , Dominios RING Finger/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Núcleo Celular/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes Reporteros , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Morfogénesis/efectos de la radiación , Mutación , Mutación Missense , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Peroxinas , Fenotipo , Plantas Modificadas Genéticamente , Mapeo de Interacción de Proteínas , Dominios RING Finger/genética , Proteínas Recombinantes de Fusión/metabolismo , Plantones , Técnicas del Sistema de Dos Híbridos

RESUMEN

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein-Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.

Asunto(s)

FN-kappa B/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Regulación hacia Abajo/fisiología , Células HEK293 , Humanos , Interleucina-8/metabolismo , Dominios RING Finger/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Proteínas de la Matriz Viral

RESUMEN

Almost all eukaryotic mRNAs must be polyadenylated at their 3' ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3'-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3'-end processing.

Asunto(s)

Dominios RING Finger/fisiología , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Proteínas Asociadas a Pancreatitis , Poliadenilación , Polinucleotido Adenililtransferasa/metabolismo , Estabilidad del ARN , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinación , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

Polycomb group proteins play essential roles in the epigenetic control of gene expression in plants and animals. Although some components of Polycomb repressive complex 1 (PRC1)-like complexes have recently been reported in the model plant Arabidopsis, how they contribute to gene repression remains largely unknown. Here we show that a putative PRC1 RING-finger protein, AtRING1A, plays a hitherto unknown role in mediating the transition from vegetative to reproductive development in Arabidopsis. Loss of function of AtRING1A results in the late-flowering phenotype, which is attributed to derepression of two floral repressors, MADS AFFECTING FLOWERING 4/5 (MAF4/5), which in turn downregulate two floral pathway integrators, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. Levels of the H3K27me3 repressive mark at MAF4 and MAF5 loci, which is deposited by CURLY LEAF (CLF)-containing PRC2-like complexes and bound by LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), are affected by AtRING1A, which interacts with both CLF and LHP1. Levels of the H3K4me3 activation mark correlate inversely with H3K27me3 levels at MAF4 and MAF5 loci. Our results suggest that AtRING1A suppresses the expression of MAF4 and MAF5 through affecting H3K27me3 levels at these loci to regulate the floral transition in Arabidopsis.

Asunto(s)

Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Dominio MADS/genética , Proteínas Represoras/genética , Arabidopsis/crecimiento & desarrollo , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Mutación , Plantas Modificadas Genéticamente , Dominios RING Finger/genética , Dominios RING Finger/fisiología , Proteínas Represoras/metabolismo , Transducción de Señal

RESUMEN

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.

Asunto(s)

Proteínas de Arabidopsis/química , Arabidopsis/fisiología , Gravitropismo , Fosfoglucomutasa/química , Fosfolipasas/química , Brotes de la Planta/química , Plastidios/química , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Centrifugación , Gravitropismo/genética , Hipergravedad , Microscopía de Polarización , Mutación , Fosfoglucomutasa/genética , Fosfoglucomutasa/fisiología , Fosfolipasas/genética , Fosfolipasas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Plastidios/genética , Plastidios/fisiología , Dominios RING Finger/genética , Dominios RING Finger/fisiología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología

RESUMEN

BACKGROUND: By using genome-wide methylation screening, the authors identified ring finger protein 180 (RNF180) as preferentially methylated in cancer. This study was undertaken to clarify its structure and functional role in gastric cancer. METHODS: The transcription start site and core functional promoter region of RNF180 were revealed by 5' rapid amplification of cDNA ends and luciferase activity assays. Promoter methylation was detected by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell growth was detected by colony formation assay, apoptosis by annexin V assay, and RNF180 target genes by cDNA microarray. RESULTS: The authors revealed the transcription start site of RNF180 gene and identified the functional core promoter region (-202/+372) in the CpG island, which could be silenced by in vitro methylation assay. RNF180 was silenced in 6 of 7 gastric cancer cell lines and significantly down-regulated in primary gastric cancers compared with adjacent normal tissues (P = .001). Loss of gene expression was associated with promoter methylation. Re-expression of RNF180 suppressed cell growth (P < .001) and induced apoptosis (P < .05), which were mediated by up-regulating the antiproliferation regulators MTSS1 and CDKN2A and the proapoptotic mediator TIMP3. Promoter methylation of RNF180 was detected in 76% (150 of 198) of primary gastric cancers and 55% (11 of 20) of intestinal metaplasia, but in none of 23 normal gastric tissues. Methylated RNF180 DNA was detected in the plasma of 56% of gastric cancer patients, but not in healthy controls (P = .003). Patients with low or loss of RNF180 expression had significantly poorer overall survival. CONCLUSIONS: RNF180 is a novel potential tumor suppressor in gastric carcinogenesis and has potential clinical utility as a biomarker for gastric cancer patients.

Asunto(s)

Apoptosis/genética , Apoptosis/fisiología , Proliferación Celular , Dominios RING Finger/genética , Dominios RING Finger/fisiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/fisiopatología , Anciano , Secuencia de Bases , Estudios de Casos y Controles , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Regulación hacia Abajo , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Pronóstico , Neoplasias Gástricas/mortalidad , Tasa de Supervivencia , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología

RESUMEN

KLF5 (Krüppel-like factor 5) is a multifunctional transcription factor involved in cell proliferation, differentiation and carcinogenesis. In addition to frequent inactivation in different types of human cancers, including breast cancer, KLF5 has been identified as an essential co-factor for the TGF-ß (transforming growth factor ß) tumour suppressor. In our previous study demonstrating a negative regulation of ER (oestrogen receptor α) function by KLF5 in breast cancer cells [Guo, Dong, Zhao, Sun, Li and Dong (2010) Int. J. Cancer 126, 81-89], we noticed that oestrogen reduced the protein level of KLF5. In the present study, we have tested whether and how oestrogen/ER signalling regulates KLF5 protein. We found that oestrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors, but not other inhibitors. The oestrogen-inducible E3 ligase EFP (oestrogen-responsive finger protein) was identified as a key player in oestrogen-mediated degradation of KLF5, as knockdown and overexpression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. Although only unubiquitinated EFP interacted with KLF5, overexpression of EFP appeared to prevent the ubiquitination of KLF5, while resulting in heavy ubiquitination of the E3 itself. Furthermore, ubiquitination of EFP interrupted its interaction with KLF5. Although the mechanism for how EFP degrades KLF5 remains to be determined, the results of the present study suggest that oestrogen causes the degradation of KLF5 protein by inducing the expression of EFP in ER-positive breast cancer cells.

Asunto(s)

Neoplasias de la Mama/fisiopatología , Receptor alfa de Estrógeno/fisiología , Estrógenos/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción/biosíntesis , Ubiquitina-Proteína Ligasas/biosíntesis , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Humanos , Dominios RING Finger/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress the genes that are not required in a specific differentiation status [1]. In animals, the two best-characterized PcG complexes are PRC2 and PRC1, which respectively possess histone 3 lysine 27 (H3K27) trimethyltransferase [2-4] and histone 2A lysine 119 (H2AK119) E3 ubiquitin ligase activities [5-7]. In Arabidopsis, PRC2 activity is also required for the gene silencing mechanism [8]; however, the existence of PRC1 has been questioned, because plant genomes do not encode clear PRC1 components and H2A monoubiquitination has not been detected [6, 9]. Conversely, recent reports have unveiled the presence of homologs to PRC1 components that together with plant-specific proteins could be part of the long-sought PRC1-like complexes [10, 11]. Here we show that the PRC1 RING-finger homologs AtBMI1A and AtBMI1B are implicated in the repression of embryonic and stem cell regulators. Plants impaired in AtBMI1A and AtBMI1B show derepression of embryonic traits in somatic cells, displaying a phenotype similar to plants mutant in PRC2 components [12-14]. Our data demonstrate that the AtBMI1A/B proteins mediate H2A monoubiquitination in Arabidopsis and that this mark, together with PRC2-mediated H3K27 trimethylation, plays a key role in maintaining cell identity.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Desdiferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Glucosiltransferasas/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/metabolismo , Dominios RING Finger/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación

RESUMEN

The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.

Asunto(s)

Cromatina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/fisiología , Dominios RING Finger/fisiología , Sitios de Unión , Línea Celular , Proteínas de Homeodominio/química , Humanos , Mutagénesis Sitio-Dirigida , Recombinación Genética , Ubiquitinación

RESUMEN

While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi. This gene was specific to T. cruzi and was designated spring (secretory protein with RING finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro. These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas Protozoarias/metabolismo , Dominios RING Finger/fisiología , Trypanosoma cruzi/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Western Blotting , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Dominios RING Finger/genética , Trypanosoma cruzi/fisiología , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación

RESUMEN

LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/fisiología , Dedos de Zinc/fisiología , Animales , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , Humanos , Conformación Proteica , Pliegue de Proteína , Dominios RING Finger/fisiología , Homología de Secuencia de Aminoácido

RESUMEN

The ubiquitin proteasome system (UPS) plays a fundamental role in maintaining the correct balance of protein levels inside all living cells. Degradation of proteins by this pathway is essential for most cellular processes including cell signalling, DNA repair, apoptosis and gene transcription. Any disruption to the system is likely to have severe consequences which may lead to disorders including neurodegeneration and cancer. Ubiquitin protein ligases are a group of UPS proteins of particular importance because these proteins determine targeting specificity via recognition of a 'target' protein and its' subsequent 'tagging' with ubiquitin. The 26S proteasome recognises these mutli-ubiquitylated proteins, allowing the correct protein to be degraded at the correct time and place within each cell. Several types of ubiquitin protein ligase have now been identified, however, the largest group by far are those proteins containing a 'RING' motif. In this review, examples will be given whereby abnormal protein ubiquitylation due to absence or inefficiency of a RING protein ligase is proposed to be a key regulator of the disease process. Ways in which we may be able to reverse these effects or manipulate these proteins to restore function will be discussed.

Asunto(s)

Diseño de Fármacos , Dominios RING Finger/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Humanos , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación/fisiología

RESUMEN

The RING (Really Interesting New Gene) family is the largest type of E3 ubiquitin ligases. RING finger domains bind two zinc ions in a unique "cross-brace" arrangement through a defined motif of cysteine and histidine residues. This arrangement endows the RING domain with a globular conformation, characterized by a central alpha-helix and variable-length loops separated by several small beta-strands. RING E3 ubiquitin ligases, play an essential role in the regulation of many biologic processes and defects in some of them are involved in cancer development. Furthermore, some RING E3 ligases are frequently overexpressed in human cancers. Today, RING ligases represent potentially molecular targets for disease intervention and could act as prognostic biomarkers. Targeting specific RING E3 ligases could lead to the development of a novel class of anticancer drugs. However RING fingers exhibit remarkable variations in their sequence and their topology characteristics. Structure determination of new RING finger domain is in the core of the design of new pharmaceuticals and what is presented in this article is a thorough review of achievements on the NMR or Xray structure determinations. Protein preparation protocols along with analysis of the structural features of known RING finger are also presented.

Asunto(s)

Sistemas de Liberación de Medicamentos , Dominios RING Finger/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/farmacología , Diseño de Fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Conformación Proteica

RESUMEN

E3 ubiquitin ligases regulate many dynamic cellular processes important for cancer cell survival. Together with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s), E3s catalyze the ubiquitination of numerous protein substrates that are subsequently targeted to the 26S proteasome for degradation. The clinical success of the proteasome inhibitor bortezomib has encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention. Targeting specific E3s is particularly attractive because there is the potential to selectively block the degradation of certain cellular proteins and possibly avoid unwanted effects on other proteins. The cullin-RING ubiquitin E3 ligases (CRLs) represent the largest subfamily of E3s. The requirement that CRLs be activated by NEDD8 modification on the cullin protein offers an "achilles heel" for modulating this entire subfamily. NEDD8-activating enzyme (NAE) catalyzes the first step in the NEDD8 pathway and as such controls the activity of CRLs. In this article, we describe the role of the NEDD8 pathway in activating CRLs and discuss the preclinical findings with a first-in-class NAE inhibitor that is currently in phase I clinical trials for both solid tumor and hematological malignancies. In addition, we speculate where NAE inhibitors may find clinical utility.

Asunto(s)

Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirazinas/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Ácidos Borónicos/metabolismo , Bortezomib , Proteínas Cullin/metabolismo , Ciclopentanos/metabolismo , Humanos , Proteína NEDD8 , FN-kappa B/metabolismo , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Pirazinas/metabolismo , Pirimidinas/metabolismo , Dominios RING Finger/fisiología , Transducción de Señal , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo

RESUMEN

The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Ubiquitinación/fisiología , Ligasas de Carbono-Nitrógeno/metabolismo , Células Cultivadas , Histona Demetilasas , Humanos , Histona Demetilasas con Dominio de Jumonji , Metilación , Oxidorreductasas N-Desmetilantes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios RING Finger/fisiología , Proteínas Represoras , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo