MicroRNAs (miRNAs) control translation of certain proteins through degradation or suppression of its messenger RNA. These non-coding RNAs are often dysregulated in many forms of cancer and can inhibit the expression of tumor suppressor proteins. The PUF domain of the human Pumilio1 protein is the only known RNA-binding protein with a defined nucleotide recognition code. The goal of this project is to engineer this domain so that it can bind to and inhibit the biogenesis of miRNA-21, an oncogene which is overexpressed in many cancers. The wild type PUF domain has previously been shown to exhibit strong binding to a specific eight nucleotide sequence. We are engineering this domain to target a semi-conserved eight nucleotide sequence on the loop region of the precursor form of miRNA-21. Amino acid site changes were introduced into the PUF domain to generate a protein which will inhibit production of the mature miRNA-21 in cancer cells. This project involves the application of various methods in biochemistry, including PCR site-directed mutagenesis, cell transfection, DNA cloning, protein expression and purification, RNA transcription and purification as well as gel electrophoresis assays and 3-Dimensional structural analyses of protein and RNA through nuclear magnetic resonance studies. Currently, both wild type and mutant PUF domain proteins have been expressed and will be purified to test stability. The precursor species of miRNA-21 will be prepared to characterize its interaction with the engineered protein biochemically, biophysically and structurally.

Ubiquitin is a 76-amino-acid protein that has important signaling roles in the cell. Attachment of ubiquitin to protein substrates signals a variety of cellular processes, including protein degradation, DNA repair, cell cycle control, and endocytosis. Due to these diverse cellular roles, ubiquitin has important implications for human disease. Indeed, the ubiquitin-proteasome system (UPS) has been implicated in neurodegenerative disorders, cardiac disease, and cancer. The UPS includes three enzyme classes that facilitate ubiquitination in a step-wise manner: ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). E2 and E3 enzymes can be very diverse and exhibit specificity for different protein substrates. However, their unique substrates remain poorly understood. For example, there are as many as 13 E2 enzymes and 42 E3 enzymes in yeast, each with unknown protein substrates and corresponding regulatory pathways. By starting with E2 substrate characterization, we can increase our understanding of E2 specificity and develop an approach for studying the more numerous (and more specific) E3s. In order to characterize E2 substrates, we have over-expressed nine different E2 enzymes in Saccharomyces cerevisiae and observed changes in ubiquitination of the proteome. Specifically, E2 over-expression vectors were transformed into yeast cells and protein content was extracted, digested into peptides, and enriched for diglycine remnants that indicate ubiquitination. Each E2 over-expression proteome was compared to a normal control via stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry (MS). We analyzed MS-identified peptides that exhibited elevated ubiquitination when a certain E2 was over-expressed, which allowed us to characterize E2-specific protein substrates and ubiquitination sites. Identifying the functional classes of proteins ubiquitinated by certain E2s increases our understanding of the cellular processes these E2s regulate and their implications for human disease.

The attachment of the small protein ubiquitin to lysine residues in other proteins is important for their proteasome-mediated degradation. Ubiquitin is attached to the lysines of proteins by the action of ubiquitin ligases. However, if ubiquitin ligases spuriously add ubiquitin to themselves, they could also be destroyed by the proteasome. Therefore, it is important that ubiquitin ligases have the means of preventing autoubiquitination. One particular ubiquitin ligase (San1) is involved in the degradation of misfolded proteins in the nucleus of yeast cells. Misfolded proteins exposed hydrophobic residues that are normally buried in their core. Exposure of hydrophobicity can lead to aggregation, and eventually cell death. Protein aggregation is thought to cause diseases such as Alzheimer’s and Lou Gehrig's. We previously found that San1 prevents autoubiquitination from occurring by not having lysine residues in the N-terminal and C-terminal region of the protein. However when lysine residues are introduced to the N- and C-terminal regions of San1, it undergoes autoubiquitination, both within itself (in cis) and by other San1 ligases (in trans). The introduction of lysine residues also causes degradation of San1 by an additional protein. The premise of my experiments is to identify the second form of degradation as well as the enzyme involved. In order to know if the mechanism of degradation is proteasome dependent, proteasome activity is blocked and levels of San1 are measured with western blots. Retained stability of San1 within cells with blocked proteasomes indicates that the mechanism is proteasome dependent. Subsequently ubiquitin is knocked down and San1 levels are measured to determine whether it is ubiquitin dependent.

The elucidation of specific intracellular signaling pathways is one of the greatest challenges of modern day cellular biology; understanding these systems, and the individual events that comprise a signaling network, is integral to advancing knowledge of human diseases such as cancer and diabetes. Much of the difficulty in studying these systems lies in the importance of spatial and temporal regulation of the individual components that comprise such signaling cascades. Small molecule pharmacological agents are desirable tools for studying these systems due to their capability for fast, dose-dependent control of proteins within the cell. Unfortunately, designing and synthesizing small molecule agents with the necessary specificity is a daunting task, and even though small molecules have a high temporal resolution the existing agents lack spatial control within the cell. While powerful, RNAi and gene knockout studies have a complete lack of temporal control and compensatory up-regulation of other proteins may mask phenotypes. Our lab is looking to develop a novel system of readily expandable chemical-genetic tools with the temporal resolution and dose dependent control intrinsic to small molecule systems. Our toolkit will be generated by adapting existing small molecule mediated protein-protein interactions, such as chemical inducers of dimerization, to allosterically regulate protein inhibitor scaffolds, namely Designed Ankyrin Repeat Proteins (DARPins) and ubiquitin variant (Ubv) inhibitors. In this system, the scaffold is generated as two distinct protein fragments, with each being genetically fused to a component of a dimerization platform. When the halves are expressed in the cell they do not retain any inhibitory activity, but when the bio-orthogonal inducer of dimerization is added, the fused domains will dimerize and the DARPin or Ubv halves will refold and regain their inhibitory function. This system will allow for unprecedented spatial and temporal control over any protein of interest in the cell.

Pseudomonas aeruginosa is the major pathogen found in many polymicrobial diseases such as cystic fibrosis and chronic obstructive pulmonary disease. P. aeruginosa antagonistically targets other species of bacteria by a novel pathway termed type VI secretion system (T6SS). The T6SS directly injects antibacterial toxins into competing bacterial cells, which confers a fitness advantage on P. aeruginosa over other cells in a polymicrobial environment. While the T6SS is known to exhibit antibacterial activity, the mechanisms by which the toxins secreted by the T6SS exert their detrimental effects on target bacteria are poorly understood. One of the T6-exported toxic effector proteins identified in P. aeruginosa is named Tse6, which upon injection into the cytoplasm of recipient cells results in growth arrest. In order to determine the mechanism of toxicity of Tse6, I determined its 1.4 Å X-ray crystal structure. Analysis of the Tse6 structure suggests that this protein utilizes nicotinamide adenine dinucleotide (NAD⁺) as a substrate to exert its toxicity in cells. To test this hypothesis, I performed further biochemical experiments and discovered that Tse6 exhibits potent NAD⁺ glycohydrolase activity both in vitro and in vivo. Therefore, my results suggest that Tse6 causes growth arrest in target cells by depleting cellular NAD⁺ levels, which would adversely affect many aspects of central metabolism including respiration. Discovering the function of toxins involved in interbacterial competition is crucial for understanding bacterial interactions in polymicrobial environments such as the human microbiome and sites of infection.

The distribution of organelles in a cell is important, especially in asymmetric cells such as neurons, where the axon can be up to a meter away from the nucleus in humans. Mitochondria function in many critical cellular activities including the maintenance of calcium ion homeostasis, ATP production, lipid biosynthesis, cell cycle progression, and cell death. These activities are critical for axonal function and mitochondrial transport is required to distribute mitochondria down the axon. Mitochondrial movement occurs via microtuble directed transport, but the mechanism and regulation of this transport is still not well understood. Mitochondrial movement on microtubules requires membrane bound and adaptor proteins to attach to motor proteins, kinesin and dynein, which bind to the microtubule and "walk" along the cytoskeleton using ATP. Miro1 (mitochondrial Rho1) and Miro2 are anchored in the outer mitochondrial membrane and have Ca²⁺-binding motifs and two GTPase domains, which suggests Miro may be involved in the regulation process. Trak1 (trafficking kinesin protein) and Trak2 simultaneously bind to Miro1/2 and kinesin and dynein, acting as an adaptor protein. Characterization of these proteins will provide insight into the mechanism and regulation of mitochondrial movement. My goal is to clone the cDNA of these proteins into bacterial expression vectors to facilitate purification of these proteins. With pure protein in hand, I aim to rebuild the transport machinery in vitro with microtubules and liposomes to investigate the function of each protein individually and their effects on each other in a simple system.