Chemistry & Biochemistry Theses and Dissertations
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Item USING DNA LOOPING PROTEINS TO ENHANCE HOMOLOGY DIRECTED REPAIR IN VIVO FOLLOWING A CAS9 INDUCED DOUBLE STRAND BREAK(2024) Ferencz, Ian Theodore; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genome engineering methods that start with a CRISPR/Cas9 targeted genomic DNA double strand break proceed through cellular DNA repair mechanisms after the induction of the break. Imprecise nonhomologous end-joining (NHEJ) is useful for knockouts, but precise homology-directed repair (HDR) is necessary for gain of function changes. NHEJ tends to be more efficient, so directing the cell to knock in a precise sequence via HDR is an active area of research. The system we have designed uses a bivalent protein to recruit HDR donor DNA to the site of a specific DNA double strand break induced by a Cas9/sgRNA nuclease. Previously described leucine zipper dual-binding (LZD) proteins areused because they are small and stable. The system was designed to reduce the effort needed for screening, shorten the time required for the repair process, and/or decrease the amount of donor DNA needed, reducing potential off-target effects. We developed a model system in Saccharomyces cerevisiae to measure gene disruption and HDR frequencies in yeast that contain combinations of non-replicating donor DNA plasmid or linear DNA, expression plasmids of four LZD variants, and a plasmid expressing Cas9 and an sgRNA targeting either the AGC1 or ADE2 genes. The donor DNA includes a gene coding for G418 resistance in yeast. It also includes an INV-2 site recognized by the C-terminal DNA binding domain of LZDs adjacent to suboptimal regions of homology to the target gene. The N-terminal DNA binding domain of the LZDs recognizes an endogenous CREB site near the target gene. The desired recombinants are scored by their inability to grow on acetate as a sole carbon source (for AGC1) or their red color (ADE2), accompanied by resistance to G418. We believe that LZD enhancement can become a simple and valuable adjunct to any other method of improving the efficiency of HDR, in any system. We were able to show that the inclusion of LZD73 in recombination experiments increased the number of colonies presenting with the desired phenotype and genotype nearly eight-fold in the absence of a designed DNA break. We also provide evidence suggesting that the presence of LZD73 has a slight positive effect on the efficiency of Cas9 targeting. Desired recombinants were recovered after Cas9/sgRNA cleavage in an experiment where there was no apparent recombination in the absence of LZD73. Future work on this project includes optimization of the homologous sequences to improve background recombination so a more quantitative measure of the improvement observed in the presence of LZD proteins. This work can be transferred laterally to enhance other recombination-based methods in other organisms: the LZD proteins could be analogous to an adjuvant that increases overall efficiency.Item DISCOVERYING THE ROLES OF SOLUBLE DHH-DHHA1 TYPE PHOSPHODIESTERASES IN RNA DEGRADATION AND CYCLIC DINUCLEOTIDE SIGNALING(2023) Myers, Tanner Matthew; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis and degradation of RNA is a fundamental and essential process for all life forms. It is imperative that cells utilize multiple mechanisms to modulate the lifetimes of RNA molecules to ultimately control protein synthesis, to maintain homeostasis or adapt to environmental challenges. One mechanism to directly alter the stability or half-life of an RNA is through the direct enzymatic activity of ribonucleases (RNases). Bacterial organisms encode for many different RNases that possess distinct functions in RNA metabolism. It is through the actions of multiple cellular RNases that a long RNA polymer corresponding to an mRNA, rRNA, tRNA, or sRNA can be fully degraded back into nucleotide monophosphate precursors. The nucleoside monophosphate precursors are recognized by kinases that ultimately recycle these molecules for use in the synthesis of other long RNA polymers. The processing of RNA polymers by endo- and exoribonucleases generates nucleoside monophosphates as well as short RNA oligonucleotides ranging from 2-6 nucleotides in length. Previously, a subset of enzymes broadly referred to as “nanoRNases” were found to process these short RNA fragments. Oligoribonuclease (Orn), NanoRNase A (NrnA), NanoRNase B (NrnB), and NanoRNase C (NrnC) were previously ascribed the function of indiscriminately processing nanoRNA substrates. However, recent analyses of the evolutionarily related DnaQ-fold containing proteins Orn and NrnC have provided compelling evidence that some nanoRNase protein families possess distinct dinucleotide substrate length preferences (Kim et al., 2019; Lormand et al., 2021). To determine whether all nanoRNases are diribonucleotide-specific enzymes, we utilized a combination of in vitro and in vivo assays to conclusively elucidate the substrate specificity and intracellular roles of the DHH-DHHA1 family proteins NrnA and NrnB. Through an in vitro biochemical survey of many NrnA and NrnB protein homologs, including from organisms of varying degrees of relatedness, we have determined that there are many functional dissimilarities contained within the DHH-DHHA1 protein family. Furthermore, we have conducted a rigorous investigation into the biological and biochemical functions of NrnA and NrnB in the Gram-positive model organism Bacillus subtilis. These analyses have shown that B. subtilis NrnA and NrnB are not redundant in biochemical activities or intracellular functions, as previously believed. In fact, we have found that B. subtilis NrnA is a 5’-3’ exoribonuclease that degrades short RNAs 2-4 nucleotides in length during vegetative growth, while B. subtilis NrnB is specifically expressed within the developing forespore and functions as a 3’-5’ exoribonuclease that processes short RNA in addition to longer RNA substrates (>40-mers). Our collective data provide a strong basis for the subdivision of the DHH-DHHA1 protein family on the basis on their diverse substrate preferences and intracellular functions.Item The Ribosome and Beyond: Understanding the role of translational fidelity in rare genetic disorders(2022) Olson, Alexandra; Dinman, Jonathan D.; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)With the increasing accessibility of patient genome sequencing, causative mutations for rare genetic diseases are being uncovered at an unprecedented rate. Among these are disorders resulting from mutations in protein synthesis machinery, including the ribosome and translation factors. Originally described in 1999, the accumulation of new information brings new questions regarding their tissue-specific and otherwise paradoxical nature. Explored here are investigations into two classes of genetic disorders, describing several novel diseases that illustrate the commonalities and differences between their classes. Specifically, two variants of RPL9 are shown to cause disparate clinical presentations despite both causing pre-rRNA processing defects, including Diamond Blackfan anemia (DBA) from a 5’UTR variant and multiple cancer incidences from a missense mutation. The 5’UTR variant is shown to result in haploinsufficiency and p53 activation, while the missense variant impairs translational fidelity because of defective stop codon recognition. Additionally, evidence is presented that correlates several de novo missense mutations in EEF2 to neurodevelopmental disorders, building on research connecting eEF2 dysfunction to neurological disease. These mutations are shown to also cause translational fidelity loss and implicate eEF2-ribosome interactions in reading frame maintenance. All of the disease-causing mutants of eEF2 were found to map to sites of interaction with critical features of ribosomal RNA. These eEF2 sites of ribosome contact were further investigated using a panel of rationally designed mutations intended to probe the relationships between biophysical interactions of eEF2 and the ribosome, and biological function. These mutants exhibited translational fidelity defects and were demonstrated to have lower catalytic activity in vitro. Overall, this work highlights salient points about ribosomopathies and translationopathies, their molecular mechanisms, and the relevance of translational fidelity to human health.Item Investigations of Substrate Recognition of the Biofilm Glycosidase Enzyme Dispersin B(2022) Peterson, Alexandra Breslawec; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms, which are comprised of bacterial cells embedded in a thick extracellular polymeric substance (EPS), is a type of survival mechanism used by a variety of medically relevant bacteria to endure harsh conditions, the immune system of the host organism, and medical intervention such as antibiotics. Biofilms confer additional protection to these bacteria, protecting them from a number of stressors, and contribute to the growing problem of antibiotic-resistant infections. Biofilm EPS is comprised of extracellular polysaccharides, proteins, DNA, and other small molecules such as enzymes and nutrients, and the strength and structure of biofilms are often attributed to extracellular polysaccharides such as poly-β-D-(1→6)-N-acetyl-glucosamine (PNAG). Glycoside hydrolase enzymes that are produced as part of the biofilm’s life cycle are being explored as possible anti-biofilm compounds, due to their ability to destabilize biofilms through degradation of the polysaccharide components. The enzyme Dispersin B (DspB), a family 20 glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans, hydrolyzes partially de-N-acetylated PNAG (dPNAG), and shows promise as a potential anti-biofilm agent. Here, we use a variety of techniques to investigate the interactions between DspB and PNAG, leading to a greater understanding of the binding interactions and mechanisms used by DspB to hydrolyze PNAG (Chapter 2). First, the activity of DspB on a monosaccharide probe, 4-methylumbelliferone-GlcNAc (4muGlcNAc) was observed over a pH range to determine the ideal conditions for DspB activity (2.2). Specifically acetylated PNAG trisaccharide analogs were then used to determine the substrate specificity of DspB, which supported the existing hypothesis that DspB uses a substrate-assisted mechanism to hydrolyze PNAG (2.4-2.6). These studies also indicated the possibility of electrostatic interactions between anionic amino acids on the binding surface of DspB and cationic deacetylated residues on PNAG that stabilize the substrate-binding interactions and allow for additional cleavage activities of DspB, namely improved cleavage of partially deacetylated PNAG and the ability to perform endo- or exoglycosidic cleavage activity, dependent on the substrate acetylation patterns present (2.5). Mutagenesis of amino acid residues on the binding surface of DspB was performed to investigate these interactions (Chapters 3-4), resulting in the discovery of an improved DspB mutant. This E248Q mutant of DspB also has an improved ability to clear Staphylococcus epidermidis biofilms, indicating that it may have improved anti-biofilm activity (3.3). Finally, a high-throughput assay for anti-PNAG activity has been developed for use with a degenerate DspB mutant library in order to identify additional DspB mutants with improved anti-biofilm activity.Item BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF NUSG PARALOG LOAP(2021) Elghondakly, Amr; Winkler, Wade; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The NusG family of transcription factors is the only universally conserved family of transcription elongation regulators in all three domains of life. NusG proteins exert ubiquitous genetic regulatory effects by reversibly binding RNA-polymerase (RNAP) during transcription elongation and modulate its function. A phylogenetic analysis of the NusG family of proteins identified several distinct subfamilies of NusG paralogs that are widespread amongst bacterial species. These different NusG paralogs are likely to exert regulatory control over distinct subsets of genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Additionally, the regulatory mechanisms that these transcription elongation factors employ are likely to differ between one another to allow for specific recruitment to target operons and prevent competition with the housekeeping NusG factor. The LoaP subfamily of NusG proteins is primarily encoded by Actinobacteria, Firmicutes and Spirochaetes. While regulons for the LoaP subfamily have only been identified in a few organisms, the loaP gene is oftentimes found adjacent to long operons encoding for biosynthesis of secondary metabolites suggesting a regulatory relationship with these pathways. In Bacillus velezensis, LoaP promotes transcription antitermination of two long biosynthetic operons which encode for two different polyketide antibiotics: difficidin and macrolactin. Intriguingly, the cis-determinants for LoaP antitermination include a small RNA hairpin (~26 nts) located within the 5’ leader region of target operons. LoaP associates with the RNA hairpin in vitro with nanomolar affinity and high specificity via basic residues that are highly conserved within the C-terminal KOW domain, in contrast to other well-characterized bacterial NusG proteins which do not exhibit RNA-binding activity. These data indicate that LoaP employs a distinct regulatory mechanism to achieve targeted regulation of large biosynthetic operons in bacteria. Furthermore, this discovery expands the repertoire of macromolecular interactions exhibited by bacterial NusG proteins during transcription elongation to include an RNA ligand. Crystallographic studies of LoaP-RNA complex are in progress, and recent results will be discussed.Item PLASTICITY IN PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS(2021) He, Chenlu; Beckett, Dorothy; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is defined as the functional regulation at one site in a protein by activity at a distant site. Because of the ubiquitous occurrence and diverse cellular roles of allosteric proteins, designing novel allosteric proteins is of great interest for applications in synthetic biological and disease treatment. However, the engineering of allostery is often hindered by our limited understanding of the protein sequence- function relationship, especially at residue positions that are distant from functional sites or evolutionarily nonconserved. In this dissertation, the sequence-function relationship was investigated in the Escherichia coli biotin protein ligase (BirA) system, which serves as both an essential metabolic enzyme and a transcription regulator. In its repressor function, binding to the vitamin biotin allosterically activates BirA dimerization and the resulting repression complex assembly on the biotin operator sequence. Although the allosteric regulation is conserved among bifunctional biotin protein ligases such as BirA, their sequences, even those of functional importance, are highly divergent. The in vitro characterization of BirA super repressor variants reveals that the sensitivity of transcription repression response to input biotin concentration can be altered solely through substitution-perturbed dimerization. These single amino acid substitutions are located at sites scattered throughout the protein structure including some that are distal from the BirA dimerization surface. Computational simulations indicate that the long-range effect of substitutions on dimerization results from rearrangement of a residue network that contributes to the allosteric activation in BirA. Several loops on the BirA dimerization surface were characterized for their roles in the corepressor-induced dimerization. The study of nonconserved amino acid positions spanning these surface loops reveals that a broad range of functional response in dimerization and transcription repression can be achieved by sequence variations at the nonconserved residues. Surprisingly, the substitution outcomes poorly correlate with amino acid chemistry or evolutionary frequencies, which deviates from canonical expectations based on conserved residues. Combined, these results illustrate the plastic nature of protein sequence-function relationship and provide insight into how this plasticity functions in the mechanism and evolution of allostery in BirA. Our deepened understanding of allostery in BirA and in general may facilitate the development of synthetic allosteric proteins in the future.Item Yeast Pseudo-Haploinsufficiency as a Model System for Human Ribosomopathies(2015) Kobylarz, Ryan; Dinman, Jonathan D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribosomopathies belong to a class of human diseases caused by mutations in genes that encode ribosomal proteins, ribosomal biogenesis factors, ribosomal RNA (rRNA) or rRNA post-transcriptional modifying factors. Ribosomal Protein S19 (RPS19) is the gene linked to Diamond Blackfan Anemia, the first identified ribosomopathy. Paradoxically, patients suffering from this disorder initially present with insufficient blood cells but later exhibit a proclivity toward developing hyper-proliferative blood cell formation. The other two most common ribosomopathies include Isolated Congenital Asplenia (linked to mutations in the gene encoding for RPS0) and 5q- syndrome (a somatically acquired haploinsufficiency of RPS14). Despite originating in the ribosome, the unique phenotypes that are symptomatic of under-developed cells and the tissue specificity of ribosomopathies are not compatible with ribosomal biogenesis defect etiologies. The unique clinical presentations of each of these diseases are consistent with the presence of “specialized” ribosomes, where each tissue type may require a certain subset of ribosomes. Recent studies into another ribosomopathy, X-linked dyskeratosis congenita (X-DC), revealed that defects in rRNA pseudouridylation patterns result in defects in translational fidelity. In order to study the translational effects of ribosomal protein haploinsufficient ribosomes, we used the Saccharomyces cerevisiae yeast as model for human ribosomopathies. Haploid yeast cells harbor two functional paralogs of RPS0, RPS14 and RPS19, in addition to other ribosomal proteins, due to an ancient whole genome duplication event. The yeast model enables the generation of single knockout of either the A or B paralogous ribosomal protein gene. Yeas also provides the ability to monitor gene specific differences in translational fidelity relative to isogenic wild-type cells. Ribosomal protein gene haploinsufficiency confers gene-specific translational fidelity defects. In assays that monitor recoding event frequencies, the most notable result was an increase in stop codon readthrough for all haploinsufficient strains. -1 or +1 Programmed Ribosomal Frameshift (PRF) recoding events were shown to exhibit isoform and sequence specific events, e.g. one isoform of RPS0 exhibits an increased -1 PRF recoding efficiency while the other demonstrates a decreased -1 PRF efficiency. Steady state mRNA abundance measurements reveals that RPS19 gene pseudo-haploinsufficiency confers a global decrease in mRNA abundance. In steady state mRNA abundance measurements of genes involved in telomere length maintenance, RPS0 and RPS14 were shown to exhibit sequence specific effects, only presenting an increase in mRNA abundance for CDC13 while exhibiting a decrease for others. These idiosyncratic results challenge the prevailing notion of the “monolithic ribosome”. Here, we present a novel model whereby the transcriptome is translated and regulated by a heterogeneous population of ribosomes.Item Elevated Temperature Effects on Carotenoid Biosynthesis in the Diploid Strawberry, Fragaria vesca(2015) Jackson, Melantha E.; Sintim, Herman; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Carotenoids, a subfamily of the isoprenoids, are one of the most diverse classes of secondary metabolites distributed throughout nature. They are lipophilic in nature, and include over 600 tetraterpenoid compounds synthesized by plants, bacteria, and fungi. Carotenoids, as the major pigment responsible for the red, yellow, and orange colors of fruits and vegetable, help promote human health and wellness by serving as antioxidants and precursors to vitamin A. Climate changes that threaten plant reproduction, negatively impact crop production worldwide. Little is understood about the chemistry of carotenoids in plant reproductive structures. Insight into the metabolic roles and functions of carotenoids in plant reproduction and, the effects of abiotic stresses on carotenoid biosynthesis in these structures would globally impact agriculture production by reducing yield loss. The potential for these metabolites to protect the reproductive structures under elevated temperature stress was assessed using biochemical analysis, genomics, and genetic studies. Fourteen candidate genes involved in carotenoid biosynthesis were identified, revealing three small gene families. Quantitative real-time polymerase chain reaction (qPCR) expression analysis of these genes and targeted metabolic profiling using liquid chromatography-high resolution mass spectrometry (LC-HRMS) throughout plant development under control and moderately elevated temperature stress showed that gene expression and metabolite accumulation are tissue specific and differentially responsive to elevated temperature stress. Three phytoene synthase genes were identified and characterized. Genomic analyses revealed that the PSY gene family exhibits functional diversity in plant tissues, both with respect to location and stage of development, as well as in response to abiotic stress.Item Epigenetics of Neurodegeneration: Quantification of Histone Deacetylase Isoforms and Post-translational Modifications of Histones in Alzheimer’s Disease(2015) Anderson, Kyle; Fenselau, Catherine; Turko, Illarion V; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Histone post-translational modifications have been implicated in many biological functions and diseases and serve an important role in epigenetic regulation of gene expression. Aberrant modulations in histone post-translational have been suggested to occur in the brain as part of Alzheimer’s disease (AD) pathology, consistent with the epigenetic blockade of neurodegeneration. This dissertation details the development and optimization of unique protein standards for quantification, called quantification concatamers, for the absolute quantification of histone deacetylase isoforms in human frontal cortex with AD, human neural retina with AD and age-related macular degeneration, and whole brain hemisphere of a 5XFAD mouse model of AD. Histone deacetylases are enzymes responsible for the deacetylation of histones, which can directly regulate transcription, and have been implicated in AD pathology. In addition to measuring isoforms of histone-modifying enzymes, measurements of post-translational modifications on histones were also obtained for whole hemispheres of brain from 5XFAD mice and frontal cortex from human donors affected with AD. For the changes in post-translational modifications observed, structural mechanisms were proposed to explain alterations in the DNA-histone affinity in the nucleosome, which can modulate gene expression. Measurements and structural mechanisms were consistent with the global decrease in gene expression observed in AD, which supports the data. This body of work aims to better elucidate the epigenetic pathology of AD and to aid in identification of histone-modifying enzymes involved in AD pathology for drug targets and treatment options. Currently, there are no treatments that prevent, delay, or ameliorate AD, stressing the crucial importance of AD pathology research and the promise of epigenetics as the solution.Item The Core of Eukaryotic Ribosomal Protein uS19 Functions as a Pivot Point Enhancing Eukaryotic Ribosome Flexibility(2015) Bowen, Alicia Marie; Dinman, Jonathan D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While most ribosomal elements are highly conserved in the three domains of life, over the course of evolution, significant differences have emerged as ribosomes have been subjected to different types of selective pressure. In prokaryotes and archaea, a single small subunit protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges, respectively. In eukaryotes, it appears that the small subunit component was split into two separate proteins during the course of evolution. One of these, also known as uS13 (previously known as S18), only participates in bridge B1b/c with uL5 in eukaryotes (previously known as L11). The other, called uS19 (previously known as S15) is the small subunit partner in the B1a bridge with H38. Here, poly-alanine mutants of the uS19/Us13 interface of uS19 were used to elucidate the evolutionary advantage of this split. A previously described chemical protection profile of the B7a bridge was utilized in order to determine the ribosomal rotational status of the selected mutants. rRNA structure probing analyses reveal that uS19/uS13 interface mutations shift the ribosomal rotational equilibrium toward the unrotated state. This perturbation of ribosomal rotational equilibrium also affected the ribosomes affinity for two intrinsic ligands: unrotated ribosomes exhibit increased affinity for ternary complex and disfavor binding of the translocase, eEF2. We posit that this mutation causes the normally flexible head region of the small subunit to "stiffen", thereby decreasing the ribosomes range of motion. A model is presented in which residues L112GH114 at the uS19/uS13 interface act as the ball in a "ball-and-socket joint", providing the increased flexibility required in the head region of the eukaryotic SSU as a consequence of the evolutionary process.