Добірка наукової літератури з теми "RIOK2"

The RIO kinases (RIOKs) are a universal family of atypical kinases that are essential for assembly of the pre-40S ribosome complex. Here, we present the crystal structure of human RIO kinase 2 (RIOK2) bound to a specific inhibitor. This first crystal structure of an inhibitor-bound RIO kinase reveals the binding mode of the inhibitor and explains the structure–activity relationship of the inhibitor series. The inhibitor binds in the ATP-binding site and forms extensive hydrophobic interactions with residues at the entrance to the ATP-binding site. Analysis of the conservation of active site residues reveals the reasons for the specificity of the inhibitor for RIOK2 over RIOK1 and RIOK3, and it provides a template for inhibitor design against the human RIOK family.

Abstract Here we report the discovery of a new master regulator of cell fate during hematopoietic differentiation, one whose function has major implications for the treatment of blood disorders such as anemia. Anemia is a major comorbidity in aging, chronic diseases such as renal failure and inflammation, bone marrow failure disorders and in hematologic neoplasms such as myelodysplastic syndromes (MDS), affecting roughly one third of the world population. Anemia is also often diagnosed in patients treated with chemotherapy or other cytotoxic agents. The comorbidities of peripheral blood cytopenias especially in elderly patients with MDS often outweigh the treatment benefits from allogeneic stem cell transplants leaving only a handful of FDA-approved drugs/therapies for treatment of such disorders. There is thus a dire need to revisit the origins of hematopoietic differentiation defects underlying these hematologic disorders to identify additional targets for novel therapies in treating anemia. We present evidence establishing that right open reading frame kinase 2 (RIOK2), an understudied atypical kinase associated with pre-40S ribosome biogenesis (Ferreira-Cerca et al., Nat. Str. Biol. 2012), is also a master transcriptional regulator of hematopoietic lineage commitment that simultaneously drives erythroid differentiation and represses myeloid and megakaryocytic lineages. We show that ablation of RIOK2 expression leads to hematopoietic differentiation defects in primary human hematopoietic stem and progenitor cells, the cells of origin for hematologic neoplasms. We identity RIOK2 as an integral player in governing major blood cell differentiation processes: erythropoiesis, megakaryopoiesis and myelopoiesis. Analyses in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) revealed that CRISPR/Cas9-mediated depletion of RIOK2 led to impaired erythropoiesis and a concomitant elevation in megakaryopoiesis and myelopoiesis. A more comprehensive analysis revealed that RIOK2 regulates the transcriptomic profiles of several key transcription factors that determine hematopoietic cell fate, including GATA1, GATA2, SPI1, RUNX3 and KLF1. Most importantly, we also observed a significant correlation between mRNA levels of RIOK2 and GATA1, GATA2, RUNX3 and KLF1 in MDS patient-derived bone marrow cells. We also demonstrate that loss of RIOK2 causes massive alterations in chromatin accessibility, both globally and specifically at the promoters of its putative target genes. This places RIOK2 at the apex of a transcriptional regulatory network controlling hematopoietic differentiation. We identify a previously unappreciated DNA-binding winged helix-turn-helix (wHTH) domain in RIOK2 conferring the protein with the properties and activities of a transcription factor. Transcriptomic profiling, structural modeling, chromatin immunoprecipitation-sequencing and a range of domain-deleted mutants reveal that RIOK2 functions as a bona-fide master transcription factor in hematopoiesis. We also identify two transactivation domains within the wHTH motif of RIOK2 that play integral roles in associating with the core transcriptional complex at promoter regions of genes. To the best of our knowledge, we present the first evidence of a protein that not only controls 40S ribosome biogenesis governing translation but also functions in the nucleus as a master transcription factor by regulating the expression of key transcription factors that determine hematopoietic cell fate. Our discovery of a novel master transcriptional regulator governing a multitude of hematopoietic lineages significantly advances our current understanding of the transcriptomic landscape underlying hematopoietic differentiation. We hope that our findings may lead to new approaches to target these newly identified regulatory networks in hematopoiesis that may be relevant not just for malignancies, but for other hematologic disorders as well, such as the anemia of aging, chronic and inflammatory diseases and aplastic anemias. We are hopeful that this study will also lay a foundation to discovering how proteins, like RIOK2, may integrate transcriptional processes with translational outcomes to drive cellular functions. Disclosures Raundhal: Jnana Therapeutics: Current Employment. Petsko: Amicus Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics, and Proclara Bioscience: Membership on an entity's Board of Directors or advisory committees; Denali Therapeutics, MeiraGTx, Annovis Bio, Retromer Therapeutics and Proclara Biosciences: Current equity holder in publicly-traded company. Glimcher: Kaleido Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Former Director; Repare Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Abpro Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK stimulates cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which open a hitherto poorly explored area of ribosome biogenesis.

Up to now, the chemotherapy approaches for glioblastoma were limited. 1-[2-Thiazolylazo]-2-naphthol (named as NSC139021) was shown to significantly inhibit the proliferation of prostate cancer cells by targeting the atypical protein kinase RIOK2. It is documented that RIOK2 overexpressed in glioblastoma. However, whether NSC139021 can inhibit the growth of glioblastoma cells and be a potential drug for glioblastoma treatment need to be clarified. In this study, we investigated the effects of NSC139021 on human U118MG, LN-18, and mouse GL261 glioblastoma cells and the mouse models of glioblastoma. We verified that NSC139021 effectively inhibited glioblastoma cells proliferation, but it is independent of RIOK2. Our data showed that NSC139021 induced cell cycle arrest at G0/G1 phase via the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway in G1/S checkpoint regulation. In addition, NSC139021 also increased the apoptosis of glioblastoma cells by activating the p53 signaling pathway and increasing the levels of Bax and cleaved caspase 3. Furthermore, intraperitoneal administration of 150 mg/kg NSC139021 significantly suppressed the growth of human and mouse glioblastoma in vivo. Our study suggests that NSC139021 may be a potential chemotherapy drug for the treatment of glioblastoma by targeting the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway.

La biogenèse des ribosomes assure l'approvisionnement en ribosomes dans la cellule pour subvenir aux besoins en synthèse protéique. La production des ribosomes est un processus très couteux en énergie. Elle mobilise les trois ARN polymérases (Pol), la machinerie de traduction, le transport nucléo-cytoplasmique, ainsi qu'une succession complexe d'étapes de maturations qui implique plus de 200 facteurs d'assemblage et de maturation (AMF). Dans les cellules de mammifères en prolifération, le taux de synthèse des ribosomes a été estimé à 7500 sous-unités ribosomiques par minute, ce qui nécessiterait l'apport de ~300 000 protéines ribosomiques (RP), 150 petits ARN nucléolaires (snoARN) par ARN précurseur, et un nombre élevé d'AMF. Ce processus très énergivore est finement régulé par des mécanismes qui coordonnent de manière dynamique les niveaux de production de ribosomes aux besoins de la cellule. Ces régulations permettent notamment d'éviter des pertes importantes en énergie cellulaire qui résulteraient d'une production inutile de ribosomes. Au cours de mon doctorat, j'ai étudié des évènements précis de régulation de la biogenèse des ribosomes orchestrés par la voie de signalisation Ras-MAPK/ERK. Cette voie de signalisation est l'un des principaux acteurs de la croissance et de la prolifération cellulaire. ERK et sa kinase effectrice RSK stimulent trois événements clés de la biogénèse des ribosomes : la transcription Pol I/Pol III, le transport nucléo-cytoplasmique et la traduction. Cependant, aucun substrat de cette voie n'a été clairement identifié dans les étapes post-transcriptionnelles de la biogenèse des ribosomes, à savoir les étapes d'assemblage et de maturation des ribosomes. Mes travaux ont permis d'identifier la kinase atypique RIOK2 comme une nouvelle cible de la kinase effectrice RSK. Nous avons découvert que la phosphorylation de RIOK2 par RSK favorise sa dissociation des particules pré-40S, facilitant ainsi la production de la particule mature 40S. Outre ces résultats, nous avons également identifié et caractérisé des partenaires proches de RIOK2 dans la cellule. Cette analyse ouvre la voie vers de nouvelles connexions entre RIOK2, ou d'autres AMF, et des processus intracellulaires clés autres que la synthèse des ribosomes. Dans l'ensemble, ma thèse aura contribué à la découverte de nouvelles perspectives de recherche dans la régulation des étapes de maturation des ribosomes. L'identification de nouveaux mécanismes de régulation pourrait aider à mieux intégrer les phénotypes associés à des dérégulations de la biogenèse des ribosomes, en conditions physiologiques ou pathologiques
Ribosome biogenesis feeds the cellular needs in protein synthesis by synthetizing translation-competent ribosomes. This highly energy-consuming process mobilizes the three RNA polymerases (Pol) and the translational machinery, active import and export through the nucleo-cytoplasmic network, as well as an intricate maturation pathway that involves more than 200 assembly and maturation factors (AMFs). In proliferating mammalian cells, the synthesis rate of ribosomes has been estimated at 7500 ribosomal subunits per minutes, requiring ~300 000 ribosomal proteins (RPs), 150 small nucleolar RNAs (snoRNAs) per pre-rRNA and an even higher number of AMFs. This fuel-consuming cellular process is tightly regulated by mechanisms that dynamically coordinate ribosome levels with cell requirements, thereby preventing energy waste due to production of unnecessary ribosomes. During my thesis, I studied discrete ribosome biogenesis regulatory events orchestrated by the Ras-MAPK/ERK signaling pathway. This signaling pathway is one of the main actor of cell growth and proliferation. ERK and its downstream effector kinase RSK stimulate three key events of ribosome biogenesis: Pol I/Pol III transcription, nucleo-cytoplasmic transport, and translation. However, no substrate of this pathway has been clearly identified in the post-transcriptional steps of ribosome biogenesis, namely ribosome assembly and maturation. My study identified the kinase RIOK2 as a new target of RSK kinase. We found that phosphorylation of RIOK2 by RSK promotes its dissociation from pre-40S particles, thereby facilitating the completion of small ribosomal subunit synthesis. Beside these findings, we have characterized the RIOK2 proximal interactome. Analysis of the proteins spatially close to RIOK2 paves the way to new connections between RIOK2, as well as other AMFs, and key intracellular processes other than ribosome biogenesis. Altogether, my thesis contributed to the discovery of novel insights into the regulation of ribosome maturation steps. Identification of novel regulatory events may help better integrating phenotypes associated with deregulation of ribosome biogenesis, during both physiological changes and diseases

Nos últimos anos, inúmeros avanços têm sido alcançados no que diz respeito aos mecanismos moleculares que participam na indução e manutenção da dor crônica, incluindo ativação glial. Já foi demonstrado que alguns receptores de reconhecimento padrão (PRRs), como os receptores do tipo Toll (TLRs) participam desse processo e, que em modelos de inflamação/infecção do Sistema Nervoso Central, os TLRs e os receptores do tipo NOD (NLRs) cooperam na ativação das células da glia, o que nos levou a hipotetizar que os receptores NOD1 e NOD2 também possam desempenhar um papel importante no processo de dor crônica. O NOD2 é responsável pela detecção intracelular do muramil dipeptídeo (MDP) enquanto que NOD1 reconhece o ácido diaminopimélico (iE-DAP), ambos padrões moleculares associados a patógenos (PAMPs) encontrados no peptideoglicano de bactérias. Após o reconhecimento, NLRs recrutam diretamente RIPK2 (proteína 2 de interação com o receptor RICK), uma serina-treonina quinase importante na ativação do fator nuclear kB (NF-kB). Assim, o objetivo do presente estudo foi avaliar a participação de NOD1 e NOD2, via RIPK2, no desenvolvimento da hipersensibilidade mecânica neuropática focando principalmente nos mecanismos espinais envolvidos. Dessa maneira, foi observado que os animais NOD1-/-, NOD2-/- e RIPK2-/- apresentaram redução significativa da hipersensibilidade nociceptiva mecânica quando comparado aos animais selvagens após indução de neuropatia periférica pelo modelo experimental de lesão limitada do nervo isquiático (SNI, Spared Nerve Injury). Ao contrário, a hipersensibilidade inflamatória induzida pelo adjuvante completo de Freud (CFA) não foi reduzida nesses animais. A redução da dor neuropática em NOD1-/-, NOD2-/- e RIPK2-/- foi associada a uma diminuição da expressão de IBA-1, GFAP, IL-1, TNF- e P2X4 na medula espinal quando comparado ao grupo WT. In vitro, foi observado que culturas primárias de micróglia não induziram liberação de IL-1, TNF-, IL-6 em resposta ao MDP (10g/mL) e iE-DAP (100ng/mL). No entanto, quando o MDP foi administrado juntamente com uma baixa concentração de lipopolissacarídeo (LPS) (0,1ng/mL), apresentou uma forte produção destas citocinas. Além disso, também foi demonstrado que células periféricas que infiltram na medula espinal podem expressar NOD1 e NOD2 e portanto serem capazes de induzir hipersensibilidade mecânica e ativação microglial após a indução de neuropatia. Dessa maneira, os resultados sugerem que NOD1 e NOD2, via RIPK2, contribuem para a gênese da dor neuropática, possivelmente mediando a liberação de citocinas pró-nociceptivas e a ativação de células gliais. Além disso, os resultados apontam ação potencial de NOD2 com TLR4 no intuito de estimular a ativação glial. Estes mecanismos representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica e um possível alvo para o desenvolvimento de drogas para o tratamento da dor neuropática.
In the last years, many advances have been made related to the molecular mechanisms involved in the induction and maintenance of chronic pain, including glial activation. It has been shown that some pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) are involved in this process, and that in inflammation/infection models of the CNS, the TLRs and Nod-like receptors (NLRs) cooperate in activation of glial cells, which led us to hypothesize that NOD1 and NOD2 receptors may also play an important role in chronic pain process. NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP) and NOD1 detects meso-diaminopimelic acid (iE-DAP), pathogen-associated molecular patterns (PAMPs) found in the peptidoglycan from bacteria. Upon recognition, NLRs recruit directly RIPK2, an adaptor protein, important in NLRs-mediated NFB activation. In the present study, we aimed to evaluate the participation of NOD1 and NOD2, via RIPK2, in the development of neuropathic mechanical hypersensitivity focusing mainly on spinal mechanisms involved. The results demonstrate that NOD1-/-, NOD2-/-, RIPK2-/- showed a significant reduction in mechanical hypersensitivity when compared to WT mice, after submitted to an experimental model of neuropathic pain Spared Nerve Injury (SNI). Interestingly, CFA-induced chronic inflammatory hypersensitivity was not decreased in these mice. The reduction in neuropathic pain in NOD1-/-, NOD2-/- and RIPK2-/- mice was associated with a decrease in the expression of IBA-1, GFAP, IL-1, TNF- and P2X4 in spinal cord when compared with WT. In vitro, it was observed that primary cultures of microglia did not produce IL-1, TNF-, IL-6 in response to MDP (3g/mL) or iE-DAP (100ng/mL). However, MDP, together with an ineffective concentration of LPS (0.1ng/mL), produced a robust production of these cytokines. Moreover, it was also demonstrated that peripheral cells infiltrating the spinal cord could express NOD1 and NOD2 and thus, be able to induce mechanical hypersensitivity and microglial activation after induction of peripheral neuropathy. The results suggest that NOD1 and NOD2, via RIPK2, contribute to the genesis of neuropathic pain, possibly by mediating the release of pronociceptive cytokines and increased glial cells activation. Moreover, the results indicate potential action of NOD2 with TLR4 in attempt to stimulate glial cells activation. These mechanisms represent a novel approach for elucidating the pathophysiology of chronic pain, and a target for the development of drugs for the treatment of neuropathic pain.

博士
國立成功大學
基礎醫學研究所
107
The homeostasis of innate immunity is critical for the primary defense mechanism against infection and regulating immune response in metazoans. It has been proven that aberrant up-regulation of innate immune signaling pathways can be detrimental to the host. The p38 MAPK/PMK-1 innate immune signaling pathway has been demonstrated to play essential roles in cellular defenses against numerous infections in metazoans, including Caenorhabditis elegans. However, the negative regulators that maintain the homeostasis of this important innate immune pathway remain largely understudied. By screening a focused kinome RNAi library of C. elegans, we identified a human RIO kinase homolog RIOK-1 as a novel suppressor of the p38 MAPK/PMK-1 signal pathway. We showed that the suppression of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. Using riok-1 reporter worms and qRT-PCR, we found the expression levels of riok-1 were significantly up-regulated in A. dhakensis infected worms. With genetic epistasis analysis of many immune pathways in C. elegans, we also discovered that riok-1 acts on the upstream of the p38 MAPK/pmk-1genetic pathway. Moreover, the suppression of riok-1 enhanced the activity of p38 MAPK, suggesting that riok-1 is a negative regulator of this innate immune pathway in C. elegans. The epistatic results also put riok-1 upstream of skn-1, which encodes a p38 MAPK downstream transcription factor, participates as an upstream activator to riok-1 and serves as a feedback loop to the p38 MAPK pathway during A. dhakensis infection. In conclusion, we proposed riok-1 is a novel innate immune suppressor and a negative feedback loop model involving p38 MAPK cascade, SKN-1, and RIOK-1 in C. elegans.

Orientação: Manuel da Mota dos Santos Coelho ; co-orientação: Cândida Leonor Pinto Rocha
Ultimamente, a ocorrência de graves catástrofes naturais tem demonstrado o problema do ambiente ecológico global. O modo com que atualmente produzimos bens e serviços é insustentável, contribuindo claramente para muitos problemas ambientais atuais. Para tornar o desenvolvimento e o ambiente sustentáveis, os programas experimentais e as campanhas para fortalecer a consciência ambiental devem ser destacados como programas fundamentais preventivos. Apesar de o conceito ser comummente aceite e relativamente fácil de entender, a dificuldade surge na aplicação efetiva dos princípios do desenvolvimento sustentável. Uma vez que a Conferência Rio+20 mobilizou diversos quadrantes da sociedade, levou a debate muitos países de todo o mundo e procura alcançar o desenvolvimento sustentável, e tendo em consideração que a comunicação social representa um pertinente meio de transmissão de informação e as entidades oficiais constituem os principais agentes participativos, procurou-se efetuar uma análise multidimensional das notícias difundidas por alguns destes veículos informativos, de janeiro a julho de 2012, tendo como objeto de estudo a respetiva Conferência, de forma a avaliar o impacte que a mesma provocou nos diversos atores sociais e se existem diferenças expressivas entre estes. A execução deste trabalho obedeceu a uma determinada metodologia, nomeadamente, pesquisa bibliográfica, seleção da informação, tratamento de dados com o QSR Nvivo 10, onde foram categorizadas individualmente todas as notícias, tendo em consideração várias vertentes, e elaboração dos gráficos com o Microsoft Excel 2010. Os resultados deste estudo indicam, claramente, que a Conferência Rio+20 tem inerente a si uma conotação negativa proeminente, pelo que a maioria dos atores sociais revelaram-se pessimistas e/ou insatisfeitos com a mesma. Foi ainda possível constatar a existência de algumas divergências significativas relativamente à origem das notícias, tendo em consideração os setores das fontes mais presentes nas notícias, a incidência dos temas mais abordados, a cobertura efetuada em relação às fontes e o discurso predominante.
Lately, the occurrence of several natural disasters have demonstrated the problem of global ecological environment. The way we currently produce goods and services is unsustainable, clearly contributing to many current environmental problems. To have sustainable development and environment, experimental programs and campaigns to strengthen environmental awareness should be highlighted as fundamental preventive programs. Although the concept is widely accepted and relatively easy to understand, the difficulty arises in the effective application of the principles of sustainable development. Once the Rio+20 Conference mobilized several sectors of society, led to debate many countries around the world and search to achieve sustainable development and taking into account that the media is a relevant means of transmitting information and official bodies are the main participating agents, we tried to make a multidimensional analysis of the news broadcast by some of these informational vehicles from January to July 2012, with the object of study the respective Conference, to assess the impact that it has caused in many social actors and if there are significant differences between them. The execution of this work followed a certain methodology, including literature, selection of information, processing of data with QSR NVivo 10, which were categorized individually all the news, taking into consideration several aspects and preparation of the graphs with Microsoft Excel 2010. The results of this study clearly indicate that the Rio+20 Conference has inherent in itself a prominent negative connotation, so most social actors have proved pessimistics and/or dissatisfied with it. It was also possible to confirm the existence of some significant differences regarding the source of the news, taking into account the sectors of the sources most present in the news, the incidence of the most discussed topics, the coverage provided in relation to sources and mostly dominant discourse.