RESUMEN
mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.
Asunto(s)
Regulación de la Expresión Génica , Lipogénesis , Procesamiento Postranscripcional del ARN , Transducción de Señal , Animales , Núcleo Celular/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Femenino , Xenoinjertos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.
Asunto(s)
Agonismo Inverso de Drogas , Neuralgia , Ratas , Animales , Calidad de Vida , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/uso terapéutico , Neuralgia/tratamiento farmacológico , Fenómenos ElectrofisiológicosRESUMEN
Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair.
Asunto(s)
Daño del ADN , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efectos de la radiación , Cromatina/metabolismo , Cromatina/efectos de la radiación , Reparación del ADN , Células HeLa , Humanos , Técnicas In Vitro , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas del Grupo Polycomb , Proteómica , Rayos Ultravioleta/efectos adversosRESUMEN
Proteomic analyses via tandem mass spectrometry have been greatly enhanced by the recent development of fast, highly accurate instrumentation. However, successful application of these developments to high-throughput experiments requires careful optimization of many variables which adversely affect each other, such as mass accuracy and data collection speed. We examined the performance of three shotgun-style acquisition methods ranging in their data collection speed and use of mass accuracy in identifying proteins from yeast-derived complex peptide and phosphopeptide-enriched mixtures. We find that the combination of highly accurate precursor masses generated from one survey scan in the FT-ICR cell, coupled with ten data-dependent tandem MS scans in a lower-resolution linear ion trap, provides more identifications in both mixtures than the other examined methods. For phosphopeptide identifications in particular, this method identified over twice as many unique phosphopeptides as the second-ranked, lower-resolution method from triplicate 90-min analyses (744 +/- 50 vs. 308 +/- 50, respectively). We also examined the performance of four popular peptide assignment algorithms (Mascot, Sequest, OMSSA, and Tandem) in analyzing the results from both high-and low-resolution data. When compared in the context of a false positive rate of approximately 1%, the performance differences between algorithms were much larger for phosphopeptide analyses than for an unenriched, complex mixture. Based upon these findings, acquisition speed, mass accuracy, and the choice of assignment algorithm all largely affect the number of peptides and proteins identified in high-throughput studies.