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Thesis
Peer Reviewed

Structural and functional study of Adenosine deaminase acting on RNA

UC Davis Electronic Theses and Dissertations (2022)

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the conversion of adenosine to inosine in double stranded RNA. Inosine selectively base pairs with cytidine and is recognized like guanosine by cellular machinery. A-to-I editing has a myriad of downstream effects and dysregulated editing by ADARs is observed in multiple neurological and oncogenic diseases. In humans there are three ADAR enzymes, named ADAR1, ADAR2 and ADAR3, each with their own unique polypeptide sequences and RNA substrate preferences. There is great interest in uncovering the structural basis of RNA editing by ADAR with the potential to accelerate the development of targeted therapeutics to restore homeostatic levels of RNA editing in diseased patients. This thesis describes the effort to understand the structural basis behind ADAR and RNA interactions. Chapter 1 will provide an overview of what is known about RNA editing and describe the ways in which it is implicated in human disease. Chapter 2 describes the structural elucidation of an asymmetric protein-protein dimer of ADAR and how its formation aids in forming an RNA-bound complex. The biophysical characteristics of this protein-protein dimer will also be discussed, in addition to the identification of key conserved amino acid residues that are important for the formation of the protein-protein dimer. Chapter 3 describes the work towards the structural elucidation of the deaminase domain of ADAR1 bound to double stranded RNA. Current strategies to solve the crystal structure of ADAR1 bound to RNA will be discussed. A low resolution data set was collected. Information gained from the low-resolution model, how it was generated and strategies to optimize crystallization and diffraction to obtain a high-quality diffraction dataset will also be discussed. Chapter 4 will describe structural studies of ADAR2’s deaminase domain and an RNA duplex with a non-ideal nearest neighbor. Additionally, this chapter will discuss collaborative efforts towards the study of full-length ADAR2 bound to a double stranded RNA will be discussed.

Article
Peer Reviewed

Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aβ.

UC Irvine Previously Published Works (2018)

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimers disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ17-36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

Cover page: Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aβ.

Article
Peer Reviewed

Controlling the Oligomerization State of Aβ-Derived Peptides with Light.

UC Irvine Previously Published Works (2018)

A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic β-hairpin peptides that are derived from amyloidogenic peptides and contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aβ16-36 or Aβ17-36 constrained into β-hairpins. The N-2-nitrobenzyl group is appended to the amide backbone of Gly33 to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that N-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the N-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic β-hairpin peptides. SDS-PAGE studies show that removing the N-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating N-2-nitrobenzyl groups into macrocyclic β-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers.

Cover page: Controlling the Oligomerization State of Aβ-Derived Peptides with Light.

Article
Peer Reviewed

Correction to “Repurposing Triphenylmethane Dyes To Bind to Trimers Derived from Aβ”

UC Irvine Previously Published Works (2018)

There was an error in listing the PDB ID numbers in the captionof Figure 7. The corrected caption should read as follows: (Figure Presented).

Cover page: Correction to “Repurposing Triphenylmethane Dyes To Bind to Trimers Derived from Aβ”

Creative Commons 'BY' version 4.0 license

Article
Peer Reviewed

Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition.

Cancer Research Coordinating Committee (CRCC) Funded Publications (2020)

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Article
Peer Reviewed

Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition

UC Davis Previously Published Works (2020)

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.