Proteostasis Engineering

Protein homeostasis networks and their biotechnological application  

In all cell types, protein homeostasis or “proteostasis” is maintained by sophisticated quality control (QC) networks that regulate protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. For example, E. coli cells employ a proteostasis system that determines whether substrates of the twin-arginine translocation (Tat) pathway are correctly folded and thus suitable for transport across the tightly sealed cytoplasmic membrane. Our early publications provided evidence that the Tat translocase itself discriminates folded proteins from those that are misfolded and/or aggregated (PNAS 2003). More recently, our laboratory isolated genetic suppressors that inactivate this folding QC mechanism and provide direct evidence for the participation of the Tat translocase in structural proofreading of its protein substrates (PNAS 2012). Our group has leveraged this folding QC mechanism for the creation of clever genetic screens and selections that permit engineering of stability-enhanced antibodies and enzymes (J Mol Biol 2015; J Mol Biol 2012; J Mol Biol 2009), mining of the human genome for well expressed proteins (Protein Sci 2009), and isolating factors such as chemical probes that stabilize aggregation-prone proteins (Protein Sci 2009). Our group has also shown that the Tat folding sensor together with its inbuilt “hitchhiker” mechanism can be exploited for genetic selection of protein-protein interactions in the cytoplasm of living cells (PNAS 2009). This technology platform is now spawning a collection of engineered binding proteins for use as therapeutic leads or as proteomic tools for dissection of molecular networks. 

Notable contributions include: 

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We have repurposed cellular proteostasis mechanisms in a number of other creative ways:

 

(i) the first reported biosynthesis of full-length antibodies in the cytoplasm of bacteria (Nat Commun 2015)

 

(ii) one of the first examples of ordered arrangement (“scaffolding”) of metabolic enzymes for improved pathway performance (Nucleic Acid Res 2012; Metab Eng 2008)

 

(iii) a novel approach for engineering water-soluble variants of integral membrane proteins (Nat Commun 2015) that were subsequently used to build an unnatural pathway for disulfide bond formation in the cytoplasm of bacteria (Nat Chem Biol 2017)

 

(iv) creation of a generalizable protein knockout method by engineering protein chimeras called “ubiquibodies” that combine the activity of E3 ubiquitin ligases with designer binding proteins to steer virtually any protein to the proteasome for degradation (J Biol Chem 2014; Curr Protoc Chem Biol 2018)

Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University

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