Monitoring mis‐acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site‐specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run‐off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser‐scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl‐tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell type

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chem. aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amts. of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately det. suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control expts. showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential exptl. parameters for expanding the scope of the suppressor tRNA technol. to different cell types. [on SciFinder (R)

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

In this thesis, two different fluorescent labeling techniques for in vivo investigations on the 5-HT3 receptor (5-HT3R) functions are presented. This plasma membrane protein contains five subunits surrounding an ion channel that opens after binding of a 5-HT3-specific neurotransmitter. The first technique, described in the first part of this report, focuses on receptor labeling via genetic fusion to spectral variants of the green fluorescent protein. I found that the resulting chimeras containing one fluorescent protein per subunit exhibit preserved ligand binding and channel activity, opening a wide range of biological research applications. Among these, I present the possibility to follow the 5-HT3R trafficking and localization during its entire life cycle by multicolor imaging in live cells, starting with its cytoplasmic biogenesis and ending with its ligand-induced internalization. The intracellular subunit assembly is shown to occur in the endoplasmic reticulum, and the importance of the cytoskeleton microtubules for proper membrane targeting is unraveled. The utility of bioluminescent 5-HT3R contructs was also demonstrated in another approach using the chemical disruption of cellular actin filaments to produce vesicular fractions of cells, in the order of 0.1 to a few micrometers in diameter. These so-called native vesicles, containing the labeled receptors in their membrane, were shown to be suitable for measurements using fluorescence confocal microscopy of ligand binding and of ion influx, opening new possibilities for miniaturized bioanalytics. In a third approach, I demonstrated that after detergent-solubilization of the receptor, the green fluorescent protein (GFP) inserted into the receptor sequence permitted to monitor ligand binding via fluorescence resonance energy transfer (FRET). Furthermore, I could observe a spatial reorientation of the receptor GFPs upon binding an agonist to the receptor. In the second part of this thesis, I adapted the mis-acylated suppressor tRNA technology to mammalian cells, permitting the introduction of unnatural amino acids at specific positions in the protein of interest. I achieved an efficiency of amino acid incorporation using in vitro aminoacylated suppressor tRNA in the order of 15% in CHO cells. A novel methodology for the quantification of background natural nonsense codon readthrough in different cell lines was also developed, permitting to select the most suitable codon-anticodon pair for this suppressor tRNA technique in various cell lines. Finally, I present a general strategy to increase the aforementioned artificial incorporation efficiency by down-regulating the competing eukaryotic release factor 1 (eRF1) using small interfering RNAs

Mechanistic Investigations of Receptor Signaling via Canonical and Non-Canonical Amino Acid Mutagenesis

This dissertation primarily describes investigations of the mechanisms by which pentameric ligand-gated ion channels (pLGICs) activate ("gating") using canonical and non-canonical amino acid mutagenesis. Chapter 1 provides an introduction to the systems being studied, their physiological roles, and the techniques that we have used to study them. Chapter 2 describes a series of experiments comparing the roles of amino acid residues proximal to the neurotransmitter binding site in the type 3 serotonin receptor (5-HT3R) to the aligning residues of the muscle-type nicotinic acetylcholine receptor (nAChR). The findings presented in Chapter 3 assess the functional roles of proline residues in the prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC). Chapter 4 describes an extensive investigation of salient details of 5-HT3R gating using canonical and non-canonical amino acid mutagenesis of amino acid residues at the interface of the extracellular domain and transmembrane domain of this receptor. Chapter 5 applies a photocrosslinking strategy employing the non-canonical amino acid p-azidophenylalanine to study dimerization and cofactor interactions of the estrogen receptor α.</p

The Development of Bicyclic Peptide Library Scaffolds and the Discovery of Biostable Ligands using mRNA Display

Peptides are a promising class of therapeutic candidates due to their high specificity and affinity for cellular protein targets. However, peptides are susceptible to protease degradation and are typically not cell-permeable. In efforts to design more effective peptide drug discovery systems, investigators have discovered that incorporation of non-canonical amino acids (ncAAs) and macrocyclization overcome these limitations, making peptides more drug-like. In this work, we exploit the promiscuity of wild-type aminoacyl-tRNA synthetases (aaRSs) to ‘mischarge’ ncAAs onto tRNA and ribosomally incorporate them into peptides using a cell-free translation system. We have demonstrated the ability to incorporate five ncAAs into a single peptide with near-wild type yield and fidelity. We also demonstrated the in situ incorporation of ncAAs containing azide and alkyne functionalities, enabling the use of CuAAC (click chemistry) to generate triazole-bridged cyclic peptides. When combined with bisalkylation of peptides containing two cysteines via an α,α’-dibromo-m-xylene linker, we created bicyclic peptides which are structurally similar to the highly bioactive knotted peptide natural products. Biological display methods, such as mRNA display, are powerful peptide discovery tools based on their ability to generate libraries of \u3e1014 unique peptides. We combined our ability to incorporate ncAAs with our bicyclization technique adapted for use with mRNA display to create knotted peptide library scaffolds. We performed side-by-side monocyclic and bicyclic in vitro selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance. We used a new library that enables the generation of a diverse collection of linear, monocyclic and bicyclic scaffolds in one pot, increasing the likelihood of target-ligand conformational alignment. We performed a second selection against streptavidin and revealed a nearly unanimous preference for linear peptides containing an HPQ motif, a known streptavidin-binding sequence. However, when we used these libraries for in vitro selection against a biological target, DNA repair protein XRCC4, we did not observe convergence. In summary, we have developed a novel technique for production of bicyclic peptide libraries. These highly-constrained protease-stable scaffolds can be used as platforms to identify high affinity, drug-like ligands using mRNA display

Selection of a Non-Phosphorylated Peptide Inhibitor of BRCA1’s (BRCT)2 Domain

A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (220 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10^13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in selection of non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1

Adaptation of Chemical Biology Approaches for Dissecting Disordered Protein Dynamics

Proteins containing intrinsic disorder often undergo dynamic conformational change in response to ligand binding or post-translational modification. Chemical dissection of these dynamic protein perturbations presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein (IDP) that both folds into a short alpha-helix upon binding to its protein ligand, eIF4E, and a four-stranded beta-sheet upon hyperphosphorylation. Post-translational modifications (PTMs) are an important mode of regulation for IDPs and intrinsically disordered regions (IDRs); however, the enzymatic generation of uniformly modified proteins in vitro remains challenging. Studying PTMs in IDPs is further complicated by their instability and markedly dynamic nature. Chemical methods of site-specific PTM incorporation have been developed in attempt to circumvent this problem. Here, we evaluate a chemical mutagenesis-based approach for generating pCys as a phosphomimetic in the 4E-BPs, a family of proteins that acts to repress cap-dependent translation, and whose function is regulated by a hierarchy of phosphorylation events. Using NMR and CD spectroscopy, we have characterized pCys in two unique contexts within 4E-BPs: induction and destabilization of secondary structure. Understanding the applicability of pCys in these unique contexts is important for expanding its use to answer biological questions in complex protein systems. Additionally, biophysical methods for analysis of binding-induced structural changes are low throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the alpha-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147481/1/otjohn_1.pd