RESUMEN
Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrP(C)). They propagate by recruiting host-encoded PrP(C) although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrP(C) expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrP(C) deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state.
Asunto(s)
Diferenciación Celular/genética , Matriz Extracelular/metabolismo , Redes Reguladoras de Genes/genética , Modelos Moleculares , Proteínas PrPC/metabolismo , Priones/genética , Transcriptoma/genética , Animales , Clonación Molecular , Citometría de Flujo , Humanos , Ratones , Análisis por Micromatrices , Microscopía Fluorescente , Proteínas PrPC/genética , Priones/química , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrofotometría , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report the quantification of the resource load imposed by different mammalian genetic components and identify construct designs with increased performance and reduced resource footprint. We use these to generate improved synthetic circuits and optimise the co-expression of transfected cassettes, shedding light on how this can be useful for bioproduction and biotherapeutic applications. This work provides the scientific community with a framework to consider resource demand when designing mammalian constructs to achieve robust and optimised gene expression.