RESUMO
Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.
Assuntos
Modelos Animais de Doenças , Melanoma/metabolismo , RNA Longo não Codificante/metabolismo , Peixe-Zebra , Animais , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Proteínas de Ligação a RNA/metabolismo , Testículo/metabolismoRESUMO
A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone-protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix. Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N-terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)). Strong synergistic interactions were observed between the CLP protease system (clpr1-2, clpr2-1, clpc1-1, clpt1, clpt2) and both PREP homologs (prep1, prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N-terminal proteomes showed that many nuclear encoded chloroplast proteins have alternatively processed N-termini in prep1prep2, clpt1clpt2 and prep1prep2clpt1clpt2. Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Aminas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Peptídeo Hidrolases/metabolismo , Proteoma/metabolismoRESUMO
Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,ß) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.
Assuntos
Citocinas/isolamento & purificação , Receptores Proteína Tirosina Quinases/isolamento & purificação , Motivos de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Citocinas/química , Citocinas/fisiologia , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Células PC12 , Multimerização Proteica , Ratos , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/metabolismoRESUMO
Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Vias Biossintéticas/genética , Vias Biossintéticas/efeitos da radiação , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Immunoblotting , Luz , Espectrometria de Massas/métodos , Microscopia Eletrônica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Estresse Oxidativo/efeitos da radiação , Pigmentação/genética , Pigmentação/efeitos da radiação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tilacoides/genética , Tilacoides/metabolismo , Tilacoides/ultraestruturaRESUMO
Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.
Assuntos
Aminoácidos Essenciais/metabolismo , Afídeos/microbiologia , Afídeos/fisiologia , Buchnera/metabolismo , Proteoma , Aminoácidos Essenciais/biossíntese , Animais , Afídeos/genética , Afídeos/crescimento & desenvolvimento , Dieta , Metionina/biossíntese , Metionina/metabolismo , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Ninfa/fisiologiaRESUMO
The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Cloroplastos/fisiologia , Endopeptidase Clp/genética , Regulação da Expressão Gênica de Plantas , Proteômica , Alelos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação para Baixo , Endopeptidase Clp/metabolismo , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Homeostase , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Fenótipo , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia , Deleção de Sequência , Regulação para CimaRESUMO
Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions.
Assuntos
Arabidopsis/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas das Membranas dos Tilacoides/metabolismo , Aclimatação , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Senescência Celular , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrilinas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Interações Hidrofóbicas e Hidrofílicas , Immunoblotting , Ponto Isoelétrico , Metaloproteases/genética , Metaloproteases/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Fotossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteoma/genética , Terpenos/metabolismo , Proteínas das Membranas dos Tilacoides/genéticaRESUMO
Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.
Assuntos
Afídeos/metabolismo , Proteínas de Bactérias/metabolismo , Buchnera/metabolismo , Proteínas de Insetos/metabolismo , Proteoma/metabolismo , Algoritmos , Aminoácidos/metabolismo , Animais , Afídeos/enzimologia , Afídeos/microbiologia , Buchnera/enzimologia , Fracionamento Celular , Chaperonina 60/metabolismo , Análise por Conglomerados , Gluconeogênese , Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas , Pisum sativum , Purinas/metabolismo , Análise de Sequência de Proteína , Simbiose , Espectrometria de Massas em TandemRESUMO
Myeloid-derived suppressor cells (MDSCs) promote tumor progression. The mechanisms of MDSC development during tumor growth remain unknown. Tumor exosomes (T-exosomes) have been implicated to play a role in immune regulation, however the role of exosomes in the induction of MDSCs is unclear. Our previous work demonstrated that exosomes isolated from tumor cells are taken up by bone marrow myeloid cells. Here, we extend those findings showing that exosomes isolated from T-exosomes switch the differentiation pathway of these myeloid cells to the MDSC pathway (CD11b(+)Gr-1(+)). The resulting cells exhibit MDSC phenotypic and functional characteristics including promotion of tumor growth. Furthermore, we demonstrated that in vivo MDSC mediated promotion of tumor progression is dependent on T-exosome prostaglandin E2 (PGE2) and TGF-beta molecules. T-exosomes can induce the accumulation of MDSCs expressing Cox2, IL-6, VEGF, and arginase-1. Antibodies against exosomal PGE2 and TGF-beta block the activity of these exosomes on MDSC induction and therefore attenuate MDSC-mediated tumor-promoting ability. Exosomal PGE2 and TGF-beta are enriched in T-exosomes when compared with exosomes isolated from the supernatants of cultured tumor cells (C-exosomes). The tumor microenvironment has an effect on the potency of T-exosome mediated induction of MDSCs by regulating the sorting and the amount of exosomal PGE2 and TGF-beta available. Together, these findings lend themselves to developing specific targetable therapeutic strategies to reduce or eliminate MDSC-induced immunosuppression and hence enhance host antitumor immunotherapy efficacy.
Assuntos
Exossomos/fisiologia , Células Mieloides/fisiologia , Neoplasias/patologia , Animais , Antígeno CD11b/análise , Linhagem Celular Tumoral , Dinoprostona/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Quimiocinas/análise , Fator de Crescimento Transformador beta/fisiologiaRESUMO
BACKGROUND: Human seminal fluid contains small exosome-like vesicles called prostasomes. Prostasomes have been reported previously to play an important role in the process of fertilization by boosting survivability and motility of spermatozoa, in addition to modulating acrosomal reactivity. Prostasomes have also been reported to present with sizes varying from 50 to 500 nm and to have multilayered lipid membranes; however, the fine morphology of prostasomes has never been studied in detail. METHODS: Sucrose gradient-purified prostasomes were visualized by cryo-electron microscopy (EM). Protein composition was studied by trypsin in-gel digestion and liquid chromatography/mass spectrometry. RESULTS: Here we report for the first time the detailed structure of seminal prostasomes by cryo-EM. There are at least three distinct dominant structural types of vesicles present. In parallel with the structural analysis, we have carried out a detailed proteomic analysis of prostasomes, which led to the identification of 440 proteins. This is nearly triple the number of proteins identified to date for these unique particles and a number of the proteins identified previously were cross-validated in our study. CONCLUSION: From the data reported herein, we hypothesize that the structural heterogeneity of the exosome-like particles in human semen reflects their functional diversity. Our detailed proteomic analysis provided a list of candidate proteins for future structural and functional studies.
Assuntos
Exossomos/química , Exossomos/ultraestrutura , Sêmen/química , Microscopia Crioeletrônica , Humanos , Masculino , Microscopia Eletrônica , Proteínas/química , Proteínas/isolamento & purificação , Valores de ReferênciaRESUMO
Recurrent point mutations in SPOP define a distinct molecular subclass of prostate cancer. Here, we describe a mouse model showing that mutant SPOP drives prostate tumorigenesis in vivo. Conditional expression of mutant SPOP in the prostate dramatically altered phenotypes in the setting of Pten loss, with early neoplastic lesions (high-grade prostatic intraepithelial neoplasia) with striking nuclear atypia and invasive, poorly differentiated carcinoma. In mouse prostate organoids, mutant SPOP drove increased proliferation and a transcriptional signature consistent with human prostate cancer. Using these models and human prostate cancer samples, we show that SPOP mutation activates both PI3K/mTOR and androgen receptor signaling, effectively uncoupling the normal negative feedback between these two pathways.
Assuntos
Mutação , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias da Próstata/etiologia , Receptores Androgênicos/fisiologia , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Proliferação de Células , Humanos , Masculino , Camundongos , Coativador 3 de Receptor Nuclear/fisiologia , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-ets/fisiologiaRESUMO
Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Peptidomiméticos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Embrião de Galinha , DNA/metabolismo , Humanos , Masculino , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Biblioteca de Peptídeos , Peptidomiméticos/química , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Domínios Proteicos , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , RNA Longo não Codificante/metabolismo , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , RNA Longo não Codificante/genética , Receptores de Estrogênio , Tamoxifeno/farmacologiaRESUMO
The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.
Assuntos
Hepacivirus/enzimologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Long noncoding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes including cancer. To delineate genome-wide lncRNA expression, we curated 7,256 RNA sequencing (RNA-seq) libraries from tumors, normal tissues and cell lines comprising over 43 Tb of sequence from 25 independent studies. We applied ab initio assembly methodology to this data set, yielding a consensus human transcriptome of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% were previously unannotated. About 1% (597) of the lncRNAs harbored ultraconserved elements, and 7% (3,900) overlapped disease-associated SNPs. To prioritize lineage-specific, disease-associated lncRNA expression, we employed non-parametric differential expression testing and nominated 7,942 lineage- or cancer-associated lncRNA genes. The lncRNA landscape characterized here may shed light on normal biology and cancer pathogenesis and may be valuable for future biomarker development.
Assuntos
RNA Longo não Codificante/genética , Transcriptoma , Linhagem Celular , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Neoplasias/genética , Análise de Sequência de RNA/métodosRESUMO
Polycomb repressive complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency and neoplastic progression. Here we show that the polycomb group protein EED, a core component of PRC2, physically interacts with and functions as part of PRC1. Components of PRC1 and PRC2 compete for EED binding. EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1-mediated H2A ubiquitin E3 ligase activity. Taken together, we suggest an integral role for EED as an epigenetic exchange factor coordinating the activities of PRC1 and 2.
Assuntos
Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Anticorpos/farmacologia , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste , Técnicas de Silenciamento de Genes , Loci Gênicos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos SCID , Modelos Biológicos , Complexo Repressor Polycomb 1/metabolismo , Ligação Proteica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Long noncoding RNAs (IncRNAs) are increasingly implicated in cancer biology, contributing to essential cancer cell functions such as proliferation, invasion, and metastasis. In prostate cancer, several lncRNAs have been nominated as critical actors in disease pathogenesis. Among these, expression of PCGEM1 and PRNCR1 has been identified as a possible component in disease progression through the coordination of androgen receptor (AR) signaling (Yang et al., Nature 2013, see ref. [1]). However, concerns regarding the robustness of these findings have been suggested. Here, we sought to evaluate whether PCGEM1 and PRNCR1 are associated with prostate cancer. Through a comprehensive analysis of RNA-sequencing data (RNA-seq), we find evidence that PCGEM1 but not PRNCR1 is associated with prostate cancer. We employ a large cohort of >230 high-risk prostate cancer patients with long-term outcomes data to show that, in contrast to prior reports, neither gene is associated with poor patient outcomes. We further observe no evidence that PCGEM1 nor PRNCR1 interact with AR, and neither gene is a component of AR signaling. Thus, we conclusively demonstrate that PCGEM1 and PRNCR1 are not prognostic lncRNAs in prostate cancer and we refute suggestions that these lncRNAs interact in AR signaling.