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Background: Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. Methods: We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells. Results: Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells. Conclusion: The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.
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The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K-mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E-mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti-PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. SIGNIFICANCE: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.
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Neoplasias Encefálicas , Melanoma , Encéfalo/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-rafRESUMEN
Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.
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Síndrome de Angelman/metabolismo , Síndrome de Angelman/fisiopatología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen gag/química , Proteínas de Unión al ARN/metabolismo , Retroviridae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Movimiento Celular , Preescolar , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Retroelementos/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/ultraestructura , Transcriptoma/genéticaRESUMEN
Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1ß, il-33 and tgf-ß were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.
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Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Susceptibilidad a Enfermedades , Yodatos/efectos adversos , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas del Sistema Complemento/genética , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata , Inmunohistoquímica , Ratones , Degeneración Retiniana/patologíaRESUMEN
The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.
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Portadores de Fármacos/química , Vesículas Extracelulares/química , Leche/citología , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos/administración & dosificación , Administración Oral , Animales , Composición de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Neuronas , Oligonucleótidos/farmacocinética , Oligonucleótidos Antisentido/farmacocinética , Células Madre Pluripotentes , Cultivo Primario de Células , Distribución TisularRESUMEN
Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action. A loss-of-function screen for genes required for BRD4 degradation revealed strong dependence on the E2 and E3 ubiquitin ligases as well as for members of the COP9 signalosome complex for both cereblon- and Von Hippel-Lindau-engaging BRD4 degraders. Cancer cell lines raised to resist BRD4 degraders manifested a degrader-specific mechanism of resistance, resulting from the loss of components of the ubiquitin proteasome system. In addition, degrader profiling in a cancer cell line panel revealed a differential pattern of activity of Von Hippel-Lindau- and cereblon-based degraders, highlighting the need for the identification of degradation-predictive biomarkers enabling effective patient stratification.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Azepinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Dipéptidos/farmacología , Células HEK293 , Humanos , Ftalimidas/farmacología , Prueba de Estudio Conceptual , Proteolisis , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients. Mouse models are commonly used to evaluate new therapeutic agents or regimens. However, to make clinical translation more successful, better characterized preclinical models are required. We therefore extensively investigated two immune-competent orthotopic HCC mouse models, namely transplanted Hep-55.1c and transgenic iAST, with respect to morphological, immunological and genetic traits and evaluated both models' responsiveness to immunotherapies. Hep-55.1c tumors were characterized by rich fibrous stroma, high mutational load and pronounced immune cell infiltrates, all of which are features of immune-responsive tumors. These characteristics were less distinct in iAST tumors, though these were highly vascularized. Cell depletion revealed that CD8+ T cells from iAST mice do not affect tumor growth and are tumor tolerant. This corresponds to the failure of single and combined ICB targeting PD-1 and CTLA-4. In contrast, combining anti-PD-1 and anti-CTLA-4 showed significant antitumor efficacy in the Hep-55.1c mouse model. Collectively, our data comprehensively characterize two immune-competent HCC mouse models representing ICB responsive and refractory characteristics. Our characterization confirms these models to be suitable for preclinical investigation of novel cancer immunotherapy approaches that aim to either deepen preexisting immune responses or generate de novo immunity against the tumor.
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Antineoplásicos Inmunológicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Modelos Animales de Enfermedad , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Antígenos Transformadores de Poliomavirus/genética , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Línea Celular Tumoral/trasplante , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.
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Terapia Genética/métodos , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos Antisentido/metabolismo , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa H/metabolismo , Animales , Apolipoproteínas B/genética , Sitios de Unión/genética , Células Cultivadas , Femenino , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9/genéticaRESUMEN
The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.
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Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.
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Técnicas Biosensibles/métodos , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Isoproterenol/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Ratas Sprague-Dawley , Sarcómeros/metabolismo , Sarcómeros/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.
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Modelos Animales , Oligonucleótidos/toxicidad , Porcinos Enanos , Animales , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Femenino , Oligonucleótidos/administración & dosificación , Oligonucleótidos/farmacocinética , Porcinos , Distribución Tisular , ToxicocinéticaRESUMEN
BACKGROUND: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development. RESULTS: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies. CONCLUSIONS: Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.
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Genoma , Porcinos Enanos/genética , Envejecimiento/genética , Animales , Cromosomas , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Hígado/metabolismo , Preparaciones Farmacéuticas/metabolismo , Seudogenes , Especificidad de la Especie , Porcinos , Transcripción GenéticaRESUMEN
Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active-site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)-2-methylpiperidine in 98% ee (71% yield) and (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in >98% ee (82% yield).
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Proteínas Bacterianas/genética , Iminas/química , Modelos Moleculares , Oxidorreductasas/genética , Proteínas Bacterianas/química , Dominio Catalítico , Estructura Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PURPOSE: Cell surface proteins are the primary means for a cell to sense and interact with its environment and their dysregulation has been linked to numerous diseases. In particular, the identification of proteins specific to a single tissue type or to a given disease phenotype may enable the characterization of novel therapeutic targets. We tested here the feasibility of a cell surface proteomics approach to identify pertinent markers directly in a clinically relevant tissue. EXPERIMENTAL DESIGN: We analyzed the cell surface proteome of freshly isolated primary heptatocytes using a glycocapture-specific approach combined with a robust bioinformatics filtering. RESULTS: Using primary lung epithelial cell cultures as negative controls, we identified 32 hepatocyte-specific cell surface proteins candidates. We used mRNA expression to select six markers that may provide adequate specificity for targeting therapeutics to the liver. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate the feasibility and the importance of conducting such studies directly in a clinically relevant tissue. In particular, the cell surface proteome of freshly isolated hepatocytes differed substantially from cultured cell lines.
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Sistemas de Liberación de Medicamentos , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Glicómica , Glicoproteínas/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Especificidad de Órganos , Péptidos/metabolismo , Proteoma/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0-7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0-3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA-Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene-coding for a transcription factor that activates signaling pathways in response to environmental stimuli-satisfied the differential expression criteria (fold change ≥ 1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.
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Cobre/toxicidad , Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Animales , Células CHO , Línea Celular , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Cricetinae , Cricetulus , Factores de Transcripción de la Respuesta de Crecimiento Precoz/análisis , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Prostaglandina-Endoperóxido Sintasas/análisis , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The propagation of phosphorylation downstream of receptor tyrosine kinases is a key dynamic cellular event involved in signal transduction, which is often deregulated in disease states such as cancer. Probing phosphorylation dynamics is therefore crucial for understanding receptor tyrosine kinases' function and finding ways to inhibit their effects. MS methods combined with metabolic labeling such as stable isotope labeling with amino acids in cell culture (SILAC) have already proven successful in deciphering temporal phosphotyrosine perturbations. However, they are limited in terms of multiplexing, and they also are time consuming, because several experiments need to be performed separately. Here, we introduce an innovative approach based on 5-plex SILAC that allows monitoring of phosphotyrosine signaling perturbations induced by a drug treatment in one single experiment. Using this new labeling strategy specifically tailored for phosphotyrosines, it was possible to generate the time profiles for 318 unique phosphopeptides belonging to 215 proteins from an erlotinib-treated breast cancer cell line model. Hierarchical clustering of the time profiles followed by pathway enrichment analysis highlighted epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 signaling as the major pathways affected by erlotinib, thereby validating the method. Moreover, based on the similarity of its time profile to those of other proteins in the ErbB pathways, the phosphorylation at Tyr453 of protein FAM59A, a recently described adaptor of EGFR, was confirmed as tightly involved in the signaling cascade. The present investigation also demonstrates the remote effect of EGFR inhibition on ErbB3 phosphorylation sites such as Tyr1289 and Tyr1328, as well as a potential feedback effect on Tyr877 of ErbB2. Overall, the 5-plex SILAC is a straightforward approach that extends sample multiplexing and builds up the arsenal of methods for tyrosine phosphorylation dynamics.
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Marcaje Isotópico/métodos , Proteómica/métodos , Tirosina/química , Tirosina/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatografía Liquida/métodos , Receptores ErbB/química , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Femenino , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Whole transcriptome analyses are an essential tool for understanding disease mechanisms. Approaches based on next-generation sequencing provide fast and affordable data but rely on the availability of annotated genomes. However, there are many areas in biomedical research that require non-standard animal models for which genome information is not available. This includes the Syrian hamster Mesocricetus auratus as an important model for dyslipidaemia because it mirrors many aspects of human disease and pharmacological responses. We show that complementary use of two independent next generation sequencing technologies combined with mapping to multiple genome databases allows unambiguous transcript annotation and quantitative transcript imaging. We refer to this approach as "triple match sequencing" (TMS). RESULTS: Contigs assembled from a normalized Roche 454 hamster liver library comprising 1.2 million long reads were used to identify 10'800 unique transcripts based on homology to RefSeq database entries from human, mouse, and rat. For mRNA quantification we mapped 82 million SAGE tags (SOLiD) from the same RNA source to the annotated hamster liver transcriptome contigs. We compared the liver transcriptome of hamster with equivalent data from human, rat, minipig, and cynomolgus monkeys to highlight differential gene expression with focus on lipid metabolism. We identify a cluster of five genes functionally related to HDL metabolism that is expressed in human, cynomolgus, minipig, and hamster but lacking in rat as a non-responder species for lipid lowering drugs. CONCLUSIONS: The TMS approach is suited for fast and inexpensive transcript profiling in cells or tissues of species where a fully annotated genome is not available. The continuously growing number of well annotated reference genomes will further empower reliable transcript identification and thereby raise the utility of the method for any species of interest.
Asunto(s)
Metabolismo de los Lípidos/genética , Hígado/metabolismo , Mesocricetus/genética , Animales , Cricetinae , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Humanos , Macaca fascicularis/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/genética , Ratas , Sus scrofa/genéticaRESUMEN
The stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation and bimolecular fluorescence complementation approaches identify an α-helical stretch of rapsyn to be crucial for binding to the dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn α-helical sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking the α-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse maintenance.
Asunto(s)
Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , Proteínas Musculares/metabolismo , Receptores Colinérgicos/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/química , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Unión Neuromuscular/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3'-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.
Asunto(s)
Disparidad de Par Base , Factor VII/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Regiones no Traducidas 3' , Animales , Sitios de Unión , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Clonación Molecular , Genes Reporteros , Células HEK293 , Células Hep G2 , Humanos , Nucleótidos/genética , Nucleótidos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Receptores de Glucocorticoides/metabolismo , Factores de Tiempo , Activación Transcripcional , TransfecciónRESUMEN
The long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads. The sixfold sequence coverage of the draft genome sequence enabled discovery of about 2.1 million potential single-nucleotide polymorphisms based on occurrence of a dimorphic nucleotide at a given position in the genome sequence. Homology-based annotation allowed us to identify 17,387 orthologs of human protein-coding genes in the M. fascicularis draft genome, and the predicted transcripts enabled the design of a M. fascicularis-specific gene expression microarray. Using liver samples from 36 individuals of different geographic origin we identified 718 genes with highly variable expression in liver, whereas the majority of the transcriptome shows relatively stable and comparable expression. Knowledge of the M. fascicularis draft genome is an important contribution to both the use of this animal in disease models and the safety assessment of drugs and their metabolites. In particular, this information allows high-resolution genotyping and microarray-based gene-expression profiling for animal stratification, thereby allowing the use of well-characterized animals for safety testing. Finally, the genome sequence presented here is a significant contribution to the global "3R" animal welfare initiative, which has the goal to reduce, refine, and replace animal experiments.