RESUMO
Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.
Assuntos
Membrana Celular , Movimento Celular , Imunoprecipitação , Células MDA-MB-231 , HumanosRESUMO
Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.
Assuntos
Proteína Semelhante a ELAV 1 , Lipopolissacarídeos , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Macrófagos/metabolismo , RNA/metabolismo , RNA Mensageiro/genéticaRESUMO
YTHDF proteins bind the N 6-methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.
RESUMO
Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.
Assuntos
Acinetobacter baumannii , Motivo de Reconhecimento de RNA , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Animais , Proteínas de Transporte/metabolismo , Humanos , Ligação Proteica/genética , Proteoma/metabolismo , RNA/metabolismo , Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The Human antigen R (HuR) protein is an RNA-binding protein, ubiquitously expressed in human tissues, that orchestrates target RNA maturation and processing both in the nucleus and in the cytoplasm. A survey of known modulators of the RNA-HuR interactions is followed by a description of its structure and molecular mechanism of action - RRM domains, interactions with RNA, dimerization, binding modes with naturally occurring and synthetic HuR inhibitors. Then, the review focuses on HuR as a validated molecular target in oncology and briefly describes its role in inflammation. Namely, we show ample evidence for the involvement of HuR in the hallmarks and enabling characteristics of cancer, reporting findings from in vitro and in vivo studies; and we provide abundant experimental proofs of a beneficial role for the inhibition of HuR-mRNA interactions through silencing (CRISPR, siRNA) or pharmacological inhibition (small molecule HuR inhibitors).
Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/metabolismo , Neoplasias/fisiopatologia , RNA/metabolismo , RNA/farmacologia , Animais , Sistemas de Liberação de Medicamentos/métodos , Inativação Gênica , Humanos , Mediadores da Inflamação/metabolismo , Peso Molecular , Neoplasias/tratamento farmacológico , RNA Mensageiro/farmacologia , RNA Interferente Pequeno/farmacologiaRESUMO
Matrin3 (MATR3) is a nuclear RNA/DNA-binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells, and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced pluripotent stem cells (hiPSCs). MATR3 downregulation affects hiPSCs' differentiation potential by altering key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts.
RESUMO
RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Ribonucleoproteínas/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Regiões não TraduzidasRESUMO
The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ligação Proteica/efeitos dos fármacos , Quinonas/farmacologia , RNA Mensageiro/metabolismo , Abietanos , Linhagem Celular , Desenho de Fármacos , Humanos , Simulação de Dinâmica Molecular , Mimetismo Molecular , Quinonas/síntese química , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Cancer therapies are associated with increased infertility risk due to accelerated reproductive aging. Oxidative stress (OS) is a potential mechanism behind ovarian toxicity by cyclophosphamide (CPM), the most ovotoxic anticancer drug. An important sensor of OS is SIRT1, a NAD+-dependent deacetylase which regulates cellular defence and cell fate. This study investigated whether the natural carotenoid crocetin and the synthetic compound AS101 protect the ovary against CPM by modulating SIRT1 and mitochondrial markers. We found that the number of primordial follicles of female CD1 mice receiving crocetin plus CPM increased when compared with CPM alone and similar to AS101, whose protective effects are known. SIRT1 increased in CPM mouse ovaries revealing the occurrence of OS. Similarly, mitochondrial SIRT3 rose, whilst SOD2 and the mitochondrial biogenesis activator PGC1-α decreased, suggesting the occurrence of mitochondrial damage. Crocetin and AS101 administration prevented SIRT1 burst suggesting that preservation of redox balance can help the ovary to counteract ovarian damage by CPM. Decreased SIRT3 and increased SOD2 and PGC1-α in mice receiving crocetin or AS101 prior to CPM provide evidence for mitochondrial protection. Present results improve the knowledge of ovarian damage by CPM and may help to develop interventions for preserving fertility in cancer patients.
Assuntos
Carotenoides/uso terapêutico , Ciclofosfamida/efeitos adversos , Mitocôndrias/metabolismo , Ovário/efeitos dos fármacos , Sirtuína 1/metabolismo , Telúrio/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Ovário/metabolismo , Vitamina A/análogos & derivadosRESUMO
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.