Structural and functional profile of phytases across the domains of life.

Phytase enzymes are a crucial component of the natural phosphorus cycle, as they help make phosphate bioavailable by releasing it from phytate, the primary reservoir of organic phosphorus in grain and soil. Phytases also comprise a significant segment of the agricultural enzyme market, used primarily as an animal feed additive. At least four structurally and mechanistically distinct classes of phytases have evolved in bacteria and eukaryotes, and the natural diversity of each class is explored here using advances in protein structure prediction and functional annotation. This graphical review aims to provide a succinct description of the major classes of phytase enzymes across phyla, including their structures, conserved motifs, and mechanisms of action.

Scaling up Functional Analyses of the G Protein-Coupled Receptor Rhodopsin.

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.

Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae.

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.

Predicted coronavirus Nsp5 protease cleavage sites in the human proteome.

BACKGROUND: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. RESULTS: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. CONCLUSIONS: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of coronavirus infection.

Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

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.

Correction: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin.

[This corrects the article DOI: 10.1371/journal.pone.0084491.].

Engineering the serpin α1 -antitrypsin: A diversity of goals and techniques.

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.

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease.

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.

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.

Serpin Phage Display: The Use of a T7 System to Probe Reactive Center Loop Libraries with Different Serine Proteinases.

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.

Directed Evolution Methods to Rewire Signaling Networks.

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.

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer serpins" with novel reactivity and/or specificity.

Facile synthesis of Raman active phospholipid gold nanoparticles.

Gold nanoparticle-based surface-enhanced Raman scattering (SERS) probes have shown promise for disease detection and diagnosis. To improve their structural and functional stability for in vivo applications, we synthesized a colloidal SERS gold nanoparticle that encapsulates Raman molecules adsorbed on 60 nm gold with a nonthiol phospholipid coating. Transmission electron microscopy and Raman and UV spectroscopy validated its reproducibility and stability. This novel lipid-based SERS probe provides a viable alternative to the PEGylation and silica coating strategies.

Establishing amide...amide reliability and synthon transferability in the supramolecular assembly of metal-containing one-dimensional architectures.

On the basis of a combination of new structural data (eleven single-crystal structure determinations are presented) and information from the Cambridge Structural Database, it has been shown that self-complementary hydrogen-bond based amide...amide dimers can be relied upon as effective supramolecular synthons for the assembly and organization of acac- and paddle-wheel complexes of a variety of metal(II) ions. The targeted molecular recognition event and intended extended one-dimensional motif appear with a supramolecular yield of 78% (a total of 28 structures were examined). Despite the fact that the hydrogen bonds that give rise to the R2(2)(8) motif can be disrupted by both carboxylate- and acac-ligands, as well as by solvent molecules, they remain remarkably resilient and therefore represent useful synthetic tools in inorganic crystal engineering.

Dinuclear zinc(II) complexes of symmetric Schiff-base ligands with extended quinoline sidearms.

The synthesis of two 2-formylquinolines is reported via the Skraup method followed by SeO(2) 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 ZnCl(2) to give complexes with bis(bidentate) coordination of ZnCl(2) 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 (1)H and (13)C NMR, UV-vis and ECD.

Balancing supramolecular reagents for reliable formation of co-crystals.

The rational design and synthesis of a supramolecular reagent (SR) composed of two distinct hydrogen bonding sites (pyrazole-benzamide), and four co-crystals resulting from reactions between this SR and a variety of carboxylic acids are described; the observed primary intermolecular interaction is consistent and predictable in each case.