RESUMO
Demand for healthcare services is unprecedented. Society is struggling to afford the cost. Pricing of biopharmaceutical products is under scrutiny, especially by payers and Health Technology Assessment agencies. As we discuss here, rapidly advancing technologies, such as Real-World Data (RWD), are being utilized to increase understanding of disease. RWD, when captured and analyzed, produces the Real-World Evidence (RWE) that underpins the economic case for innovative medicines. Furthermore, RWD can inform the understanding of disease, help identify new therapeutic intervention points, and improve the efficiency of research and development (R&D), especially clinical trials. Pursuing precompetitive collaborations to define shared requirements for the use of RWD would equip service-providers with the specifications needed to implement cloud-based solutions for RWD acquisition, management and analysis. Only this approach would deliver cost-effective solutions to an industry-wide problem.
Assuntos
Biofarmácia/economia , Animais , Ensaios Clínicos como Assunto/economia , Análise Custo-Benefício/economia , Humanos , Pesquisa/economiaRESUMO
Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.
Assuntos
Complexo Mediador/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Peptídeos/química , Proteínas de Ligação a RNA/química , Animais , Ataxina-1 , Ataxinas , Células COS , Chlorocebus aethiops , Escherichia coli/genética , Humanos , Complexo Mediador/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Plasmídeos , Polimerização , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
IRF-1 is a tumor suppressor protein that activates gene expression from a range of promoters in response to stimuli spanning viral infection to DNA damage. Studies on the post-translational regulation of IRF-1 have been hampered by a lack of suitable biochemical tools capable of targeting the endogenous protein. In this study, phage display technology was used to develop a monoclonal nanobody targeting the C-terminal Mf1 domain (residues 301-325) of IRF-1. Intracellular expression of the nanobody demonstrated that the transcriptional activity of IRF-1 is constrained by the Mf1 domain as nanobody binding gave an increase in expression from IRF-1-responsive promoters of up to 8-fold. Furthermore, Mf1-directed nanobodies have revealed an unexpected function for this domain in limiting the rate at which the IRF-1 protein is degraded. Thus, the increase in IRF-1 transcriptional activity observed on nanobody binding is accompanied by a significant reduction in the half-life of the protein. In support of the data obtained using nanobodies, a single point mutation (P325A) involving the C-terminal residue of IRF-1 has been identified, which results in greater transcriptional activity and a significant increase in the rate of degradation. The results presented here support a role for the Mf1 domain in limiting both IRF-1-dependent transcription and the rate of IRF-1 turnover. In addition, the data highlight a route for activation of downstream genes in the IRF-1 tumor suppressor pathway using biologics.
