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Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies. A synthetic yeast-based therapeutic that secretes an ATP-degrading enzyme in response to pro-inflammatory extracellular ATP in the gut reduces intestinal inflammation, fibrosis and dysbiosis in mouse models of colitis and enteritis., Author(s): Benjamin M. Scott [sup.1] [sup.2] [sup.3] [sup.9] , Cristina Gutiérrez-Vázquez [sup.4] , Liliana M. Sanmarco [sup.4] , Jessica A. da Silva Pereira [sup.4] , Zhaorong Li [sup.4] , Agustín [...]

α1‐Antitrypsin (α1‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α1‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α1‐AT, specific mutants of interest, and providing an appended catalog of the >200 α1‐AT mutants published to date. [ABSTRACT FROM AUTHOR]

The synthesis of two 2-formylquinolines is reported via the Skraup method followed by SeO2 oxidation. Each aldehyde is condensed with (1R,2R)-diaminocyclohexane and (R)-BINAM, yielding four enantiomerically-pure bis(imine-quinoline) ligands. The neutral ligands are reacted with ZnCl2 to give complexes with bis(bidentate) coordination of ZnCl2 units. X-Ray structural characterization of three complexes shows them to have a single-stranded helical motif, with M helicity, except in one case where a 1 : 1 mixture of M and P helices is seen. The ligands and complexes are further characterized spectroscopically by solution ¹H and 13CNMR, UV-vis and ECD. [ABSTRACT FROM AUTHOR]

α 1 -Antitrypsin (α 1 -AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α 1 -AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure-function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α 1 -AT, specific mutants of interest, and providing an appended catalog of the >200 α 1 -AT mutants published to date., (© 2019 The Protein Society.)

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease., (Copyright © 2019 by the Genetics Society of America.)

Phage display is a protein engineering approach that involves construction of libraries of variant proteins displayed on the surface of bacteriophage as capsid fusion proteins and their screening for binding and inhibitory function through the use of bait proteins. Recently, we adapted a commercially available T7 phage display system to create phage-displayed serpin libraries hypervariable in up to five positions in their reactive center loop (RCL). The RCL is a key determinant in serpin specificity, the relationship between the structure of a given serpin and which target proteinase(s) it inhibits. In this chapter, we describe protocols to assess the feasibility of this method for different serpin/proteinase combinations and share experience with this technology gathered in the course of studying two serpins and multiple proteinases with this powerful iterative screening approach.

The ability to sense and process cues about changing environments is fundamental to life. Cells have evolved elaborate signaling pathways in order to respond to both internal and external stimuli appropriately. These pathways combine protein receptors, signal transducers, and effector genes in highly connected networks. The numerous interactions found between signaling proteins are essential to maintain strict regulation and produce a suitable cellular response. As a result, a signaling protein's activity in isolation can differ greatly from its activity in a native context. This is an important consideration when studying or engineering signaling pathways. Fortunately, the difficulty of studying network interactions is fading thanks to advances in library construction and cell sorting. In this chapter, we describe two methods for generating libraries of mutant proteins that exhibit altered network interactions: whole-gene point mutagenesis and domain shuffling. We then provide a protocol for using fluorescence-activated cell sorting to isolate interesting variants in live cells by focusing on the unicellular eukaryotic model organism Saccharomyces cerevisiae, using as an example recent work that we have done on its G protein-coupled receptor Ste2.