文章基本信息

标题：Transplantation of prokaryotic two-component signaling pathways into mammalian cells
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作者：Jonathan Hansen ; Erik Mailand ; Krishna Kumar Swaminathan 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：44
页码：15705-15710
DOI：10.1073/pnas.1406482111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceSynthetic biology and genetic engineering would greatly benefit from engineered genetic elements that are orthogonal to the host in which they operate. Two-component signaling pathways are the prevalent signal processing modality in prokaryotes that is also found in low eukaryotes and plants but absent from vertebrate cells. Here we investigate whether the elements of prokaryotic two-component pathways are operational in mammalian cells. We find that the core biochemical processes are maintained, whereas the capacity to sense chemical ligands is diminished or obscured. We use the pathways for multiinput gene regulation and show that they can serve as a rich source of orthogonal building blocks for gene expression control in mammalian cells. Our findings open new avenues in synthetic circuit design. Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.
关键词：synthetic biology ; two-component signaling ; biological computing