Non-Canonical Amino Acids As Biochemical Probes of Ligand-Gated Ion Channel Structure and Function

This dissertation describes several different chemical-scale studies of proteins involved in cellular signaling. The primary focus of this work is on ligand-gated ion channels, an important family of membrane receptors. In each study, the incorporation of structurally diverse non-canonical amino acids have been used to attain a high level of precision in probing the mechanisms of molecular processes. The first chapter provides an introduction to the nonsense suppression methodology used to genetically encode these probes, and surveys the classes of proteins studied herein. The second and third chapters are concerned with the mechanism of activation of a prokaryotic receptor, Gloeobacter violaceus ligand-gated ion channel. In these experiments, novel histidine derivatives were designed, synthesized, and incorporated to test the functional importance of acid-base titration at several positions in the receptor. Then, a battery of proline analogs were used to identify necessary structural features of several critical proline residues, providing clues to conformational changes that occur during receptor activation. The fourth chapter discusses studies of a different class of receptors, the Acid-Sensing Ion Channels. Several fluorinated aspartic acid and glutamic acid derivatives were targeted to modulate the acidity of putative proton binding sites. Attempts at preparation of these compounds for incorporation into proteins are detailed. Additional sections describe, efforts to elucidate factors in the binding selectivity of the tarantula venom psalmotoxin and selectivity of cation permeability. In the final chapter, early efforts at developing crosslinking assays for protein-protein interactions in mammalian cells are outlined. These assays involve the introduction of orthogonal tRNA/synthetase pairs for genetically encoding photoreactive phenylalanine analogs. The most successful studies involve the ligand-dependent dimerization of the soluble nuclear receptor, estrogen receptor α. However, the ultimate goal of this work is to to probe protein-protein interactions among membrane receptors and other proteins. Progress toward extension of the photocrosslinking assay into membrane receptors is described.</p

Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism

Functional constraints on the constitutive androstane receptor inferred from human sequence variation and cross-species comparisons

<p>Abstract</p> <p>Members of the NR1I subfamily of nuclear receptors play a role in the transcriptional activation of genes involved in drug metabolism and transport. NR1I3, the constitutive androstane receptor (CAR), mediates the induction of several genes involved in drug response, including members of the <it>CYP3A</it>, <it>CYP2B </it>and <it>UGT1A </it>subfamilies. Large inter-individual variation in drug clearance has been reported for many drug metabolising enzyme genes. Sequence variation at the <it>CAR </it>locus could potentially contribute to variation in downstream targets, as well as to the substantial variation in expression level reported. We used a comparative genomics-based approach to select resequencing segments in 70 subjects from three populations. We identified 21 polymorphic sites, one of which results in an amino acid substitution. Our study reveals a common haplotype shared by all three populations which is remarkably similar to the ancestral sequence, confirming that CAR is under strong functional constraints. The level and pattern of sequence variation is approximately similar across populations, suggesting that interethnic differences in drug metabolism are not likely to be due to genetic variation at the <it>CAR </it>locus. We also identify several common non-coding variants that occur at highly conserved sites across four major branches of the mammalian phylogeny, suggesting that they may affect <it>CAR </it>expression and, ultimately, the activity of its downstream targets.</p

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors.

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.This work was supported by grants from the NIH (NS 34407 to DAD) and the Wellcome Trust (WT 81925 to SCRL), and an NIH/NRSA training grant (GM07616 to MR).This is the final version of the article. It first appeared from American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.69437

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN