RESUMEN
Protein degradation by the ubiquitin-proteasome system is central to cell homeostasis and survival. Defects in this process are associated with diseases such as cancer and neurodegenerative disorders. The 26S proteasome is a large protease complex that degrades ubiquitinated proteins. Here, we show that ADP-ribosylation promotes 26S proteasome activity in both Drosophila and human cells. We identify the ADP-ribosyltransferase tankyrase (TNKS) and the 19S assembly chaperones dp27 and dS5b as direct binding partners of the proteasome regulator PI31. TNKS-mediated ADP-ribosylation of PI31 drastically reduces its affinity for 20S proteasome α subunits to relieve 20S repression by PI31. Additionally, PI31 modification increases binding to and sequestration of dp27 and dS5b from 19S regulatory particles, promoting 26S assembly. Inhibition of TNKS by either RNAi or a small-molecule inhibitor, XAV939, blocks this process to reduce 26S assembly. These results unravel a mechanism of proteasome regulation that can be targeted with existing small-molecule inhibitors.
Asunto(s)
Drosophila melanogaster/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Tanquirasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT-ß-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3-5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT-ß-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.
Asunto(s)
Biocatálisis , Microscopía por Crioelectrón , Polimerizacion , Tanquirasas , beta Catenina , Tanquirasas/química , Tanquirasas/metabolismo , Tanquirasas/ultraestructura , Activación Enzimática , Dominio Catalítico , Vía de Señalización Wnt , Secuencias de AminoácidosRESUMEN
Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Anafase/fisiología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Tanquirasas/metabolismo , Aneuploidia , Segregación Cromosómica , Daño del ADN/genética , ADN Ribosómico/genética , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.
Asunto(s)
Luxación de la Cadera , Osteosclerosis , Tanquirasas , Humanos , Tanquirasas/genética , Tanquirasas/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Vía de Señalización Wnt/genética , Osteosclerosis/genética , beta Catenina/metabolismoRESUMEN
The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.
Asunto(s)
Querubismo/metabolismo , Tanquirasas/química , Tanquirasas/metabolismo , Secuencia de Aminoácidos , Animales , Repetición de Anquirina , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Querubismo/metabolismo , Transducción de Señal , Tanquirasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Querubismo/genética , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Estabilidad Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Eliminación de Secuencia , Quinasa Syk , Tanquirasas/genética , Factor de Necrosis Tumoral alfa/metabolismo , UbiquitinaciónRESUMEN
Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.
Asunto(s)
Desarrollo de Músculos , Fibras Musculares Esqueléticas , Miogenina , ARN Mensajero , Tanquirasas , Tanquirasas/metabolismo , Tanquirasas/genética , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Desarrollo de Músculos/genética , Animales , Fibras Musculares Esqueléticas/metabolismo , Ratones , Miogenina/genética , Miogenina/metabolismo , Nucleofosmina , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Estabilidad del ARN/genética , Poli ADP Ribosilación/genética , Línea Celular , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Diferenciación Celular/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células HEK293RESUMEN
Axin (also known as AXIN1) is a central negative regulator of the proto-oncogenic Wnt/ß-catenin signaling pathway, as axin condensates provide a scaffold for the assembly of a multiprotein complex degrading ß-catenin. Axin, in turn, is degraded through tankyrase. Consequently, tankyrase small-molecule inhibitors block Wnt signaling by stabilizing axin, revealing potential for cancer therapy. Here, we discovered that axin is phosphorylated by casein kinase 1 alpha 1 (CSNK1A1, also known as CK1α) at an N-terminal casein kinase 1 consensus motif, and that this phosphorylation is antagonized by the catalytic subunit alpha of protein phosphatase 1 (PPP1CA, hereafter referred to as PP1). Axin condensates promoted phosphorylation by enriching CK1α over PP1. Importantly, the phosphorylation took place within the tankyrase-binding site, electrostatically and/or sterically hindering axin-tankyrase interaction, and counteracting tankyrase-mediated degradation of axin. Thus, the presented data propose a novel mechanism regulating axin stability, with implications for Wnt signaling, cancer therapy and self-organization of biomolecular condensates.
Asunto(s)
Neoplasias , Tanquirasas , Humanos , Proteína Axina/metabolismo , Fosforilación , Tanquirasas/metabolismo , Condensados Biomoleculares , beta Catenina/metabolismo , Vía de Señalización WntRESUMEN
Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/ß-catenin signalling. However, we show that nPSCs depleted of ß-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.
Asunto(s)
Angiomotinas , Células Madre Pluripotentes , Tanquirasas , Proteínas Señalizadoras YAP , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , beta Catenina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Tanquirasas/metabolismo , Vía de Señalización Wnt , Células Madre Pluripotentes/citologíaRESUMEN
During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked ß-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation (PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Inmunidad Innata , Infecciones por Virus ARN , Virus ARN , Tanquirasas , Ubiquitina-Proteína Ligasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células HEK293 , Humanos , Inmunidad Innata/genética , Ratones , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , Tanquirasas/genética , Tanquirasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of ß-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of ß-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/ß-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/ß-catenin pathway.
Asunto(s)
Estabilidad de Enzimas/genética , Tanquirasas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Vía de Señalización Wnt/fisiología , Repetición de Anquirina , Proteína Axina/metabolismo , Línea Celular , Cristalografía por Rayos X , Células HCT116 , Células HEK293 , Humanos , Mutación , Unión Proteica , Tanquirasas/química , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética , Vía de Señalización Wnt/genéticaRESUMEN
BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.
Asunto(s)
Neoplasias Colorrectales , Tanquirasas , Animales , Ratones , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Tanquirasas/genética , Tanquirasas/metabolismo , Línea Celular Tumoral , beta Catenina/genética , beta Catenina/metabolismo , Ratones Endogámicos NOD , Vía de Señalización Wnt/genética , Biomarcadores , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismoRESUMEN
The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-ß-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active ß-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive ß-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.
Asunto(s)
Motivo alfa Estéril , Tanquirasas/metabolismo , Vía de Señalización Wnt , Proteína Axina/metabolismo , Sitios de Unión , Dominio de Reclutamiento y Activación de Caspasas , Catálisis , Cristalografía , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Tanquirasas/química , Tanquirasas/genética , TransfecciónRESUMEN
p21WAF1/Cip1 acts as a key negative regulator of cell cycle progression, which can form complexes with cyclin-dependent kinases together with specific cyclins to induce cell cycle arrest at specific stages. p21 protein levels have been shown to be regulated primarily through phosphorylation and ubiquitination during various stages of the cell cycle. Although phosphorylation and ubiquitin-dependent proteasomal degradation of p21 have been well established, other post-translational modifications that contribute to regulation of p21 stability and function remain to be further elucidated. Here, we show that p21 degradation and its function are controlled by tankyrases, which are members of the poly(ADP-ribose) polymerase (PARP) protein family. p21 interacts with tankyrases via newly defined tankyrase-binding motifs and is PARylated by tankyrases in vitro and in vivo, suggesting that PARylation is a new post-translational modification of p21. Up-regulation of tankyrases induces ubiquitin-dependent proteasomal degradation of p21 through an E3 ligase RNF146, thus promoting cell cycle progression in the G1/S phase transition. On the contrary, inhibition of tankyrases by knockdown or inhibitor treatment stabilizes p21 protein and leads to cell cycle arrest in the G1 phase. Together, our data demonstrate that tankyrase may function as a new molecular regulator that controls the protein levels of p21 through PARylation-dependent proteasomal degradation. Hence, a novel function of the tankyrase-p21 axis may represent a new avenue for regulating cell cycle progression.
Asunto(s)
Tanquirasas , Tanquirasas/química , Tanquirasas/metabolismo , Poli ADP Ribosilación , Ubiquitinación , Ciclo Celular , Ubiquitinas/metabolismoRESUMEN
PTEN [phosphatidylinositol (3,4,5)-trisphosphate phosphatase and tensin homolog deleted from chromosome 10], a phosphatase and critical tumor suppressor, is regulated by numerous post-translational modifications, including phosphorylation, ubiquitination, acetylation, and SUMOylation, which affect PTEN localization and protein stability. Here we report ADP-ribosylation as a new post-translational modification of PTEN. We identified PTEN as a novel substrate of tankyrases, which are members of the poly(ADP-ribose) polymerases (PARPs). We showed that tankyrases interact with and ribosylate PTEN, which promotes the recognition of PTEN by a PAR-binding E3 ubiquitin ligase, RNF146, leading to PTEN ubiquitination and degradation. Double knockdown of tankyrase1/2 stabilized PTEN, resulting in the subsequent down-regulation of AKT phosphorylation and thus suppressed cell proliferation and glycolysis in vitro and tumor growth in vivo. Furthermore, tankyrases were up-regulated and negatively correlated with PTEN expression in human colon carcinomas. Together, our study revealed a new regulation of PTEN and highlighted a role for tankyrases in the PTEN-AKT pathway that can be explored further for cancer treatment.
Asunto(s)
Neoplasias del Colon/fisiopatología , Neoplasias Colorrectales/fisiopatología , Fosfohidrolasa PTEN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Tanquirasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Glucólisis , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Proteína Oncogénica v-akt/metabolismo , Fosfohidrolasa PTEN/genética , Fosforilación , Procesamiento Proteico-Postraduccional , UbiquitinaciónRESUMEN
Tankyrases, a versatile protein group within the poly(ADP-ribose) polymerase family, are essential for post-translational poly(ADP-ribosyl)ation, influencing various cellular functions and contributing to diseases, particularly cancer. Consequently, tankyrases have become important targets for anti-cancer drug development. Emerging approaches in drug discovery aim to disrupt interactions between tankyrases and their binding partners, which hinge on tankyrase-binding motifs (TBMs) within partner proteins and ankyrin repeat cluster domains within tankyrases. Our study addresses the challenge of identifying and ranking TBMs. We have conducted a comprehensive review of the existing literature, classifying TBMs into three distinct groups, each with its own scoring system. To facilitate this process, we introduce TBM Hunter-an accessible, web-based tool. This user-friendly platform provides a cost-free and efficient means to screen and assess potential TBMs within any given protein. TBM Hunter can handle individual proteins or lists of proteins simultaneously. Notably, our results demonstrate that TBM Hunter not only identifies known TBMs but also uncovers novel ones. In summary, our study offers an all-encompassing perspective on TBMs and presents an easy-to-use, precise, and free tool for identifying and evaluating potential TBMs in any protein, thereby enhancing research and drug development efforts focused on tankyrases.
Asunto(s)
Tanquirasas , Tanquirasas/metabolismo , Repetición de Anquirina , Poli ADP RibosilaciónRESUMEN
Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-ß catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/ß-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.
Asunto(s)
Proteína Axina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Intestinos/fisiología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Células Madre/citología , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt1/metabolismo , Animales , Sistemas CRISPR-Cas , Proliferación Celular , Cruzamientos Genéticos , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Masculino , Modelos Genéticos , Mutación , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tanquirasas/metabolismoRESUMEN
Poly(ADP-ribose) polymerases (PARP) act as DNA damage sensors that produce poly(ADP-ribose) (PAR) chains at double-strand breaks, facilitating the recruitment of repair factors. Cancers with homologous recombination defects are sensitive to small molecule PARP inhibitors. Despite PARP5B gene copy number changes in many cancers, the effects of this genetic alteration on tumor phenotype are largely unknown. To better understand this clinical finding, we characterized a PARP5B null mutation in a carcinogen-induced in vivo head and neck squamous cell carcinoma (SCC) model. Reduced PARP5B expression inhibited tumor growth, induced primary tumor differentiation and apoptosis, and inhibited cell proliferation and metastasis. Loss of PARP5B expression-induced ataxia telangiectasia and Rad3 related (ATR) activation and depleted the cancer stem cell fraction. PARP5B null tumor cells lacked 53BP1+ double-strand break foci, ATM activation, and p53 induction compared to PARP5B+/+ cancers. PARP5B null SCC expresses a multiprotein complex containing PML, pRPA, Rad50, Rad51, XRCC1, proliferating cell nuclear antigen (PCNA), and Mcm2, suggesting an HR-mediated repair mechanism at DNA replication foci. Low doses of etoposide combined with the PARP5B inhibitor XAV939 induced senescence and apoptosis in human SCC lines. NBS1 overexpression in these cells inhibited the effects of low-dose etoposide/XAV939 treatment. Our results indicate that PARP5B inhibition is new targeted cancer therapy.
Asunto(s)
Carcinógenos/toxicidad , Regulación hacia Abajo , Neoplasias de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Tanquirasas/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Etopósido/administración & dosificación , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/inducido químicamente , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Mutación con Pérdida de Función , Ratones , Invasividad Neoplásica , Carcinoma de Células Escamosas de Cabeza y Cuello/inducido químicamente , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Tanquirasas/metabolismoRESUMEN
Due to the frequent mutations, influenza A virus (IAV) becomes resistant to anti-viral drugs targeting influenza viral proteins. There are increasing interests in anti-viral agents that target host cellular proteins required for virus replication. Tankyrase (TNKS) has poly (ADP-ribose) polymerase activity and is a negative regulator of many host proteins. The objectives of this study are to study the role of TNKS2 in IAV infection, identify the microRNAs targeting TNKS2, and to understand the mechanisms involved. We found that TNKS2 expression was elevated in human lung epithelial cells and mouse lungs during IAV infection. Knock-down of TNKS2 by RNA interference reduced viral replication. Using a computation approach and 3'-untranslation regions (3'-UTR) reporter assay, we identified miR-206 as the microRNA that targeted TNKS2. Overexpression of miR-206 reduced viral protein levels and virus production in cell culture. The effect of miR-206 on IAV replication was strain-independent. miR-206 activated JNK/c-Jun signalling, induced type I interferon expression and enhanced Stat signalling. Finally, the delivery of an adenovirus expressing miR-206 into the lung of mice challenged with IAV increased type I interferon response, suppressed viral load in the lungs and increased survival. Our results indicate that miR-206 has anti-influenza activity by targeting TNKS2 and subsequently activating the anti-viral state.
Asunto(s)
Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , MicroARNs/metabolismo , MicroARNs/farmacología , Tanquirasas/genética , Tanquirasas/metabolismo , Replicación Viral/efectos de los fármacos , Regiones no Traducidas 3' , Células A549 , Animales , Línea Celular , Perros , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Interacciones Microbiota-Huesped , Humanos , Gripe Humana/virología , Pulmón/metabolismo , Pulmón/virología , Sistema de Señalización de MAP Quinasas , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/virología , Interferencia de ARN , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Carga ViralRESUMEN
The directing group assisted decarboxylative ortho-benzoylation of N-aryl-7-azaindoles with α-keto acids has been achieved by synergistic visible light promoted photoredox and palladium catalysis. The approach tenders rapid entry to aryl ketone architectures from simple α-keto acid precursors via the in situ generation of a benzoyl radical intermediate. The transformation provides a range of ortho-benzoylated N-aryl-7-azaindoles, with excellent site-selectivity and good functional group compatibility under mild reaction conditions. Biological target predictions indicate that these molecules may serve as potential anti-cancer and anti-viral agents.