RESUMO
mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of Nrn1, Hmgb1, Actb, and Gap43 mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to Nrn1 and Hmgb1, revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration.
Assuntos
Axônios/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Motivos de Nucleotídeos/genética , RNA Mensageiro/genética , Regulon/genética , Regiões 5' não Traduzidas/genética , Animais , Transporte Axonal/genética , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Masculino , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Transporte de RNA/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.
Assuntos
Anti-Inflamatórios/farmacologia , Budesonida/farmacologia , Feto/efeitos dos fármacos , Pulmão/embriologia , Anti-Inflamatórios/metabolismo , Budesonida/metabolismo , Quimiocinas/metabolismo , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pneumonia/genética , Pneumonia/patologia , Tensão Superficial/efeitos dos fármacos , Fatores de TempoRESUMO
Critical functions of intra-axonally synthesized proteins are thought to depend on regulated recruitment of mRNA from storage depots in axons. Here we show that axotomy of mammalian neurons induces translation of stored axonal mRNAs via regulation of the stress granule protein G3BP1, to support regeneration of peripheral nerves. G3BP1 aggregates within peripheral nerve axons in stress granule-like structures that decrease during regeneration, with a commensurate increase in phosphorylated G3BP1. Colocalization of G3BP1 with axonal mRNAs is also correlated with the growth state of the neuron. Disrupting G3BP functions by overexpressing a dominant-negative protein activates intra-axonal mRNA translation, increases axon growth in cultured neurons, disassembles axonal stress granule-like structures, and accelerates rat nerve regeneration in vivo.
Assuntos
Axônios/metabolismo , Grânulos Citoplasmáticos/metabolismo , Regeneração Nervosa/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Feminino , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Masculino , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Regeneração Nervosa/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Background: Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. Methods: To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. Results: An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. Conclusions: These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stress responses are highly nuanced and variable, but how this diversity is achieved by modulating receptor function is largely unknown. Corticotropin-releasing factor receptors (CRFRs), class B G protein-coupled receptors, are pivotal in mediating stress responses. Here we show that the two known CRFRs interact to form heteromeric complexes in HEK293 cells coexpressing both CRFRs and in vivo in mouse pancreas. Coimmunoprecipitation and mass spectrometry confirmed the presence of both CRF1R and CRF2ßR, along with actin in these heteromeric complexes. Inhibition of actin filament polymerization prevented the transport of CRF2ßR to the cell surface but had no effect on CRF1R. Transport of CRF1R when coexpressed with CRF2ßR became actin dependent. Simultaneous stimulation of cells coexpressing CRF1R+CRF2ßR with their respective high-affinity agonists, CRF+urocortin2, resulted in approximately twofold increases in peak Ca2+ responses, whereas stimulation with urocortin1 that binds both receptors with 10-fold higher affinity did not. The ability of CRFRs to form heteromeric complexes in association with regulatory proteins is one mechanism to achieve diverse and nuanced function.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Hormônio Liberador da Corticotropina/genética , Células HEK293 , Humanos , Camundongos , Pancrelipase , Transdução de Sinais , Estresse Fisiológico/fisiologia , Técnicas de Cultura de Tecidos/métodosRESUMO
In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.
Assuntos
GTP Fosfo-Hidrolases/genética , Melanócitos/metabolismo , Melanoma/patologia , Proteínas de Membrana/genética , Proteômica/métodos , Neoplasias Cutâneas/patologia , Caseína Quinase II/metabolismo , Humanos , Espectrometria de Massas/métodos , Melanoma/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinase/metabolismo , Fosfopeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genéticaRESUMO
Cell surface receptors are central to the cell's ability to generate coordinated responses to the multitude of biochemical and physical cues in the microenvironment. However, the mechanisms by which receptors enable this concerted cellular response remain unclear. To investigate the effect of cellular tension on cell surface receptors, we combined novel high-resolution imaging and single particle tracking with established biochemical assays to examine TGFß signaling. We find that TGFß receptors are discretely organized to segregated spatial domains at the cell surface. Integrin-rich focal adhesions organize TßRII around TßRI, limiting the integration of TßRII while sequestering TßRI at these sites. Disruption of cellular tension leads to a collapse of this spatial organization and drives formation of heteromeric TßRI/TßRII complexes and Smad activation. This work details a novel mechanism by which cellular tension regulates TGFß receptor organization, multimerization, and function, providing new insight into the mechanisms that integrate biochemical and physical cues.