RESUMEN
Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.
Asunto(s)
Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/fisiología , Proteasas Ubiquitina-Específicas/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Complejo del Señalosoma COP9/fisiología , Humanos , Inmunidad Innata , Masculino , Ratones , FN-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Dominios Proteicos , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Precursores del ARN/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteasas Ubiquitina-Específicas/química , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismoRESUMEN
Ubiquitination and deubiquitination play a critical role in all aspects of cellular processes, and the enzymes involved are tightly regulated by multiple factors including posttranslational modifications like most other proteins. Dysfunction or misregulation of these enzymes could have dramatic physiological consequences, sometimes leading to diseases. Therefore, it is important to have a clear understanding of these regulatory processes. Here, we have reviewed the posttranslational modifications of deubiquitinating enzymes and their consequences on the catalytic activity, stability, abundance, localization, and interaction with the partner proteins.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Procesamiento Proteico-Postraduccional , Catálisis , Humanos , Fosforilación , Transporte de Proteínas , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome.
Asunto(s)
Antígenos de Neoplasias/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión al ARN/genética , Proteasas Ubiquitina-Específicas/genética , Enzimas Reparadoras del ADN/genética , Células HeLa , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Factores de Empalme de ARN/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Empalmosomas/genética , Ubiquitinación/genéticaRESUMEN
Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) is a U4/U6 recycling factor as well as a targeting factor of USP4 and USP15. However, the details of how SART3 recognizes these deubiquitinases and how they get subsequently translocated into the nucleus are not known. Here, we present the crystal structures of the SART3 half-a-tetratricopeptide (HAT) repeat domain alone and in complex with the domain present in ubiquitin-specific protease (DUSP)-ubiquitin-like (UBL) domains of ubiquitin specific protease 4 (USP4). The 12 HAT repeats of SART3 are in two sub-domains (HAT-N and HAT-C) forming a dimer through HAT-C. USP4 binds SART3 at the opposite surface of the HAT-C dimer interface utilizing the ß-structured linker between the DUSP and the UBL domains. The binding affinities of USP4 and USP15 to SART3 are 0.9 µM and 0.2 µM, respectively. The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of SART3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity of USP4.
Asunto(s)
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Empalmosomas/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Cristalografía por Rayos X , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Unión al ARN/genética , Relación Estructura-Actividad , Secuencias Repetidas en Tándem , Ubiquitina Tiolesterasa/genética , Proteasas Ubiquitina-Específicas/metabolismo , alfa Carioferinas/química , alfa Carioferinas/metabolismoRESUMEN
TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt which contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU-repeat motifs impair TDP-43 function.
RESUMEN
TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt that contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU-repeat motifs impair TDP-43 function.
RESUMEN
A series of 36 pyrazol-4-yl pyridine derivatives (8a-i, 9a-i, 10a-i, and 11a-i) was designed, synthesized, and evaluated for its antiproliferative activity over NCI-60 cancer cell line panel and inhibitory effect against JNK isoforms (JNK1, JNK2, and JNK3). All the synthesized compounds were tested against the NCI-60 cancer cell line panel. Compounds 11b, 11c, 11g, and 11i were selected to determine their GI50s and exerted a superior potency over the reference standard SP600125 against the tested cell lines. 11c showed a GI50 of 1.28 µM against K562 leukemic cells. Vero cells were used to assess 11c cytotoxicity compared to the tested cancer cells. The target compounds were tested against hJNK isoforms in which compound 11e exhibited the highest potency against JNK isoforms with IC50 values of 1.81, 12.7, and 10.5 nM against JNK1, JNK2, and JNK3, respectively. Kinase profiling of 11e showed higher JNK selectivity in 50 kinase panels. Compounds 11c and 11e showed cell population arrest at the G2/M phase, induced early apoptosis, and slightly inhibited beclin-1 production at higher concentrations in K562 leukemia cells relative to SP600125. NanoBRET assay of 11e showed intracellular JNK1 inhibition with an IC50 of 2.81 µM. Also, it inhibited CYP2D6 and 3A4 with different extent and its hERG activity showed little cardiac toxicity with an IC50 of 4.82 µM. hJNK3 was used as a template to generate the hJNK1 crystal structure to explore the binding mode of 11e (PDB ID: 8ENJ) with a resolution of 2.8 °A and showed a typical type I kinase inhibition against hJNK1. Binding energy scores showed that selectivity of 11e towards JNK1 could be attributed to additional hydrophobic interactions relative to JNK3.
Asunto(s)
Azoles , Proteínas Quinasas JNK Activadas por Mitógenos , Animales , Chlorocebus aethiops , Células Vero , Azoles/farmacología , Isoformas de Proteínas , Piridinas/farmacología , Proliferación CelularRESUMEN
Cardiac muscle contraction is initiated by the entry of Ca²âº ions from the extracellular spaces and the Ca²âº stores in the sarcoplasmic reticulum into the myoplasm. Calcium signaling is the most important factor in cardiac cell homeostasis. In this study, we investigated the effect of caffeine, an inducer of intracellular Ca²âº accumulation, on HL-1 cardiomyocytes by using a proteomic approach. Following the separation of the cell lysates and visualization of the protein spots using two-dimensional gel electrophoresis and silver staining, respectively, we identified 24 differentially expressed protein spots in the caffeine-treated group as compared with the controls by using MALDI-TOF/TOF MS. Of these 24 spots, 8 proteins were up-regulated and 16 proteins were down-regulated. These differentially expressed proteins are predominantly involved in cellular metabolism, cellular organization, and ion/protein transport. Furthermore, we found that Hsp25, one of the differentially expressed proteins, is modified by caffeine treatment. Depletion of Hsp25 transcripts by siRNA increased caffeine-mediated signaling, including ERK activation, and decreased the Ca²âº transient peak and expression of calsequestrin 2 in HL-1 cardiomyocytes. These results suggest that proteins having various functions are involved in the regulation of Ca²âº homeostasis, and that Hsp25 plays an important role in regulating cardiac function during caffeine response.
Asunto(s)
Cafeína/farmacología , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Calcio/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Línea Celular , Proteínas de Choque Térmico/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Chaperonas Moleculares , Contracción Muscular , Miocitos Cardíacos/citología , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The deubiquitinating enzyme USP15 is implicated in several human cancers by regulating different cellular processes, including splicing regulation. However, the underlying molecular mechanisms of its functional relevance and the successive roles in enhanced tumorigenesis remain ambiguous. Here, we found that USP15 and its close paralog USP4 are overexpressed and facilitate lung cancer cell proliferation by regulating the alternative splicing of SRSF1. Depletion of USP15 and USP4 impair SRSF1 splicing characterized by the replacement of exon 4 with non-coding intron sequences retained at its C-terminus, resulting in an alternative isoform SRSF1-3. We observed an increased endogenous expression of SRSF1 in lung cancer cells as well, and its overexpression significantly enhanced cancer cell phenotype and rescued the depletion effect of USP15 and USP4. However, the alternatively spliced isoform SRSF1-3 was deficient in such aspects for its premature degradation through nonsense-mediated mRNA decay. The increased USP15 expression contributes to the lung adenocarcinoma (LUAD) development and shows significantly lower disease-specific survival of patients with USP15 alteration. In short, we identified USP15 and USP4 as key regulators of SRSF1 alternative splicing with altered functions, which may represent the novel prognostic biomarker as well as a potential target for LUAD.
RESUMEN
In the current article, we introduce design of a new series of 4-(imidazol-5-yl)pyridines with improved anticancer activity and selective B-RAFV600E/p38α kinase inhibitory activity. Based on a previous work, a group of structural modifications were applied affording the new potential antiproliferative agents. Towards extensive biological assessment of the target compounds, an in vitro anticancer assay was conducted over NCI 60-cancer cell lines panel representing blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers. Compounds 7c, 7d, 8b, 9b, 9c, 10c, 10d, and 11b exhibited the highest potency among the tested compounds and demonstrated sub-micromolar or one-digit micromolar GI50 values against the majority of the employed cell lines. Compound 10c emerged as the most potent agent with nano-molar activity over most of the cells and incredible activity against melanoma (MDA-MB-435) cell line (GI50 70 nM). It is much more potent than sorafenib, the clinically used anticancer drug, against almost all the NCI-60 cell lines. Further cell-based mechanistic assays showed that compound 10c induced cell cycle arrest and promoted apoptosis in K562, MCF-7 and HT29 cancer cell lines. In addition, compound 10c induced autophagy in the three cancer cell lines. Kinase profiling of 10c showed its inhibitory effects and selectivity towards B-RAFV600E and p38α kinases with IC50 values of 1.84 and 0.726 µM, respectively. Docking of compound 10c disclosed its high affinity in the kinases pockets. Compound 10c represent a promising anticancer agent, that could be optimized in order to improve its kinase activity aiming at developing potential anticancer agents. The conformational stability of compound 10c in the active site of B-RAFV600E and p38α kinases was studied by applying molecular dynamic simulation of the compound in the two kinases for 600 ns in comparison to the native ligands.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
The formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer's disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de Drosophila/genética , MicroARNs/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas tau/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ojo/metabolismo , Ojo/patología , Humanos , Lisosomas/metabolismo , Ratones , MicroARNs/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas tau/metabolismoRESUMEN
Deubiquitinating enzymes have key roles in diverse cellular processes whose enzymatic activities are regulated by different mechanisms including post-translational modification. Here, we show that USP15 is phosphorylated, and its localization and activity are dependent on the phosphorylation status. Nuclear-cytoplasmic fractionation and mass spectrometric analysis revealed that Thr149 and Thr219 of human USP15, which is conserved among different species, are phosphorylated in the cytoplasm. The phosphorylation status of USP15 at these two positions alters the interaction with its partner protein SART3, consequently leading to its nuclear localization and deubiquitinating activity toward the substrate PRP31. Treatment of cells with purvalanol A, a cyclin-dependent kinase inhibitor, results in nuclear translocation of USP15. USP4, another deubiquitinating enzyme with a high sequence homology and domain structure as USP15, also showed purvalanol A-dependent changes in activity and localization. Collectively, our data suggest that modifications of USP15 and USP4 by phosphorylation are important for the regulation of their localization required for cellular function in the spliceosome.