Alterations in protein stability and propensity for aggregation are hallmarks of many prevalent and costly age-related diseases, and are actively investigated in cases like Alzheimer’s and cataracts. However, as expensive clinical trials of drugs based on the native targets fail, we must reconsider what the toxic species are within these protein aggregation diseases. One hypothesis focuses on the alteration of aggregative properties by spontaneous protein modifications, which may generate more toxic polymorphs. One such age-related post-translational modification, the isomerization of aspartate residues (L-isoaspartate, L-isoAsp), is a pervasive modification where preliminary evidence for such aggregation enhancement has been obtained. The primary goal of this thesis has been to characterize how this form of protein damage is linked to aggregation of proteins associated with specific aging diseases, as well as how this modification is maintained in the functioning cell. In the context of the water-soluble extracts of aging mammalian lens, this work has shown that L-isoAsp accumulates to high levels, primarily within aggregated low molecular weight species. In vitro experiments based on our localization of L-isoAsp in the lens revealed that introduction of the L-isoAsp modification in a water-soluble derived peptide was accompanied by decreased aggregative properties, while the opposite result was observed for a water-insoluble derived peptide. In further experiments with segments of the Alzheimer’s disease-associated amyloid-β (Aβ) peptide it was demonstrated that the rate of Aβ fibril formation is greatly accelerated with an isoaspartyl residue in agreement with earlier literature. Structural studies revealed the L-isoAsp residue facilitates the formation of a novel protofilament interface, which may represent a distinct polymorph of the full-length Aβ peptide. Here we also begin the characterization of a novel L-isoAsp maintenance pathway distinct from the canonical L-isoAsp repair enzyme – the protein-L-isoaspartyl (D-aspartyl) O-methyltransferase or protein carboxyl methyltransferase (PCMT1) - through the protein carboxyl domain-containing proteins (PCMTDs). We have shown that these proteins assemble with cullin-RING ligase proteins and lower detectable levels of L-isoAsp in vitro. From this work, it is clear that L-isoAsp residues have significant consequences in protein aggregation diseases, and are attenuated in cells through multiple pathways.

Methylation is an essential post translational modification that can be found on a variety of proteins in higher and lower species. The effects of these modifications are diverse and include targeting genes for activation or silencing and protein stability. The most extensively characterized methylation reaction is that of histone tails. In the past decade researchers became interested in identifying non-histone protein substrates and the enzymes responsible for methylating them (methyltransferases). In the yeast Saccharomyces cerevisiae, a substantial number of methyltransferases have been identified for modifying mitochondrial proteins, translational release factors, ribosomal proteins and translational elongation factors.

When I started my doctoral journey the newest methyltransferase of the yeast translational apparatus was found to modify elongation factor 1 alpha (EF1A), the protein responsible for bringing the aminoacyl-tRNA to the ribosome. This enzyme, Efm7, was the fifth elongation factor methyltransferase (Efm) responsible for methylating EF1A! EF1A methylation by five methyltransferases is unique as other elongation factors, EF2 and EF3, only have two methyltransferases that modify them both. Additionally, each of these five EF1A methyltransferases appears to target specific residues on EF1A. This has not been seen in any other protein of the translational apparatus that is methylated. Thus I was captivated by this uniqueness of EF1A methylation.

Although it is known that EF1A and other translational proteins are methylated and the enzymes responsible for its methylation, we still don’t know why it is methylated. GTP-bound EF1A functions as the courier of aminoacylated tRNA to the ribosome. When a correct codon-anticodon match is made, a conformational change in the ribosome results in the hydrolysis of GTP to GDP and the release of EF1A. Additionally, GDP bound-EF1A functions outside of the ribosome including being able to bind and bundle filamentous actin. This dissertation focused on identifying whether or not there was a direct link between methylation and functionality of EF1A. In a separate study, I examined the functional role of ribosomal protein methyltransferases.

My studies lead to the development and characterization of the first quintuplet knockout yeast strain for the enzymes responsible for methylating EF1A. These strains have been confirmed to be methylation deficient at the respective EF1A lysine sites, using mass spectrometry. I found that EF1A methylation appears to be necessary for helping yeast to survive and adapt to differences in its cellular environment but may not be necessary for the integrity of the actin cytoskeleton as well as EF1A induced actin binding.

Over the last decade, protein arginine methyltransferases (PRMTs) have emerged as key regulators in epigenetic processes and have been shown to be crucial targets for drug therapies in various types of cancers. As drug-development is progressing, however, it is vital to understand the biochemical nature of these enzymes and what makes them different from other posttranslational modifiers in the epigenetic landscape. To that end, the studies conducted in this work provide novel insights into how PRMTs specify the products they make, how one of the mammalian PRMTs—PRMT7—exhibits a peculiar preference for sub-physiological temperatures, and the first evidence of positive cooperativity observed in PRMTs and potential crosstalk among them. Specifically, a model has been developed which describes the unique aspects of PRMT active sites in way that highlights the role of sterics and overall volume differences between the different types of PRMTs and allows them to produce different methylarginine derivates. Secondly, biophysical techniques in this work show that human PRMT7 preference for colder, sub-physiological temperatures may be caused due to incredibly minute structural changes that need to be more finely measured in vivo and replicated in vitro. Finally, as the field of epigenetic research expanded and the general knowledge of enzymes in this area has grown, more and more evidence for the regulation of these regulators in the way of enzyme crosstalk has been documented. In the case of PRMTs, however, this work shows for the first time that both PRMT1 and PRMT5 display positive cooperativity when methylating histone H4 N-terminal peptides; furthermore, PRMT5’s ability to methylate histone H4 R3 seems to be greatly affected by a prior methylation of H4 R17—a modification carried out by PRMT7.

Protein arginine methylation is an important posttranslational modification in eukaryotes, shown to be involved in the regulation of transcription, the splicing machinery, signaling, and DNA repair. Mammalian protein arginine methyltransferases (PRMT) include a family of nine enzymes that transfer methyl groups onto the omega nitrogen atoms of the guanidino groups of arginine residues, producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA) and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While the other PRMTs have been extensively studied, the roles and activities of two members of this family, PRMT7 and PRMT9, had been less well investigated. Both PRMT7 and PRMT9 are distinguished from other family members with having two methyltransferase-like domains and having acidic residues in an otherwise well-conserved “double-E” substrate-binding motif. My work confirms PRMT7 as the only type III enzyme in the group, with an unusual low temperature optimum for activity and preference for basic residues in an RXR sequence for methylation. I found that mutations of the acidic residues in the double-E motif result in a loss of the specific RXR substrate recognition motif and the appearance of a RG specificity motif typical of many of the other PRMTs. The physiological substrate(s) of PRMT7 remain to be determined, although I found that histone H2B is an effective in vitro substrate. PRMT9, on the other hand, had no reported activity, until I was able to show in a pulldown experiment using HeLa cells that it was associated with two RNA splicing factors. I was able to determine by amino acid analysis that PRMT9 is very specific for methylating the RNA splicing factor SF3B2. This PRMT9-dependent modification reaction produces both MMA and SDMA and thus makes PRMT9 the second example of a type II enzyme in mammals. I found that the position of the methylated arginine residue in SF3B2 is important for PRMT9 recognition, and that the acidic residues in the substrate-binding motif also play an important role in substrate recognition. In addition, mutagenesis studies in the active site cavity of the PRMT7 and PRMT9 enzymes uncovered conserved residues in the substrate binding double-E loop that are important for substrate recognition and residues in the conserved THW motif that are responsible for conferring the methylation activity type. Lastly, I examined the orthologous PRMT7 and PRMT9 enzymes in the nematode Caenorhabditis elegans. I found that the C. elegans PRMT-7 has a distinct substrate preference from the mammalian ortholog, while C. elegans PRMT-9 appears to be biochemically indistinguishable from its human ortholog.

Post translational modifications of proteins alter the biological landscape creating functional diversity. One modification, arginine methylation, was first identified in 1968 from calf thymus hydrolysates producing guanidino-methylated arginine derivatives. However, the enzymes that produce these modifications were poorly characterized until 1996 when the genes of the first protein arginine methyltransferases were cloned from yeast and mammalian cells. At this time, a family of nine mammalian genes has been identified that encode protein arginine methyltransferases (PRMTs). In vitro experiments identified three distinct types. Type I PRMTs catalyze asymmetric dimethylarginine (ADMA) (PRMTs 1-4, 6 and 8), Type II PRMTs catalyze symmetric dimethylarginine (SDMA) (PRMT5 and 9), and the only type III PRMT that catalyzes monomethylarginine (MMA) (PRMT7). The active sites of each of the major enzymes that form ADMA, SDMA and MMA have distinct structural architectures allowing for their specificity.

In this dissertation I have focused my work on the major type I enzyme, PRMT1, the major type II enzyme, PRMT5, and the type III enzyme, PRMT7. I showed that each of these human enzymes behave differently under physiological stress conditions associated with temperature, pH, and ionic strength thus potentially leading to alterations in the proteomic arginine methylation landscape. In particular, PRMT7 is maximally active at sub-physiological temperatures and at nonphysiological pH and ionic strength, suggesting regulatory roles. I then characterized the unusual substrate specificity of the PRMT7 enzyme with peptide substrates to demonstrate the exquisite dependence upon variations of the Arg-X-Arg motif.With the identification of a PRMT7 motif in the human Fhod1 and Fhod3 actin binding proteins, I characterized methylation reactions that were dependent upon the phosphorylation state of an adjacent serine residue. These results pointed to the cross-talk that can occur between phosphorylation and methylation reactions. Interestingly, I found little or no effect of methylation on ROCK1 protein kinase activity.

PRMT enzymes have been identified to be oncogenic and closely associated with cancer progression. Surprisingly, it was found that methionine-dependent malignant cancer cells had no detectable alteration of protein arginine methylation than methionine-independent less malignant cells, suggesting that the methionine effect maybe be regulated through alternative pathways.

Protein methylation has recently emerged as an abundant and important posttranslational modification, affecting many cellular functions including transcription and translation. The budding yeast Saccharomyces cerevisiae has become an important model organism for studying methylation reactions and the enzymes responsible for catalyzing the reaction (methyltransferases). Generally, either unfractionated lysates from cells grown under fermentation or specific protein substrates of well-characterized methyltransferases have been investigated for protein methylation. As a result of this, the methylation state of proteins inside the mitochondria remains largely unknown. The goal of this dissertation was two-fold: 1.) determine the experimental requirements for thoroughly identifying the methylation state of proteins in yeast mitochondria and 2.) identify methylated proteins in mitochondria that may have been previously overlooked.

Only one example in the scientific literature has investigated the global level of protein methylation in the mitochondria of any organism and only two proteins in the yeast mitochondria are known to be methylated. I set out to determine mitochondrial protein methylation by detecting differences between wild type and methyltransferase gene deletion yeast strains using a combination of commonly used biochemical and molecular biology assays. When I found that these methods were ill-suited for identification of mitochondrial protein methylation, I turned to mass spectrometry approaches.

From my initial mass spectrometric analyses of proteolytic digests of yeast proteins, I learned that it was not sufficient to simply rely on computer algorithms to identify novel methylated peptides. It is important to use isotopic labeling to validate the mass shifts that occur as a result of methylation on identified peptides. This led me to use to adapt heavy methyl stable isotope labeling with amino acids in cell culture (SILAC) for use in yeast with my identification of a strain better suited for these experiments. From this approach, I have been able to curate a list of proteins comprising the mitochondrial methyl proteome. Using this method, I have also compared the cytosolic methyl proteome between yeast cells grown under fermentative or respiratory conditions.

I conclude with future directions for the work included in this dissertation. The functional implications of the mitochondrial methylation reactions have yet to be discovered and the methyltransferases responsible remain to be identified.

This dissertation contains three supplemental Excel spreadsheets. These materials are available on ProQuest.com. The first supplemental Excel table is an expanded version of Table 2-1 with a list of the known and putative methyltransferases in yeast, their confirmed and predicted substrate type, the substrate and residue methylated if known, and descriptions of the methyltransferase. The second Excel table contains data from nine separate mass spectrometry experiments, attempting to identify the methylation state of mitochondrial ribosomal proteins from wild type and methyltransferase gene deletion strains, including peptides identified and descriptions. The third supplemental Excel table contains the peptides identified as methylated in cytosol isolated from fermenting and respiring yeast cells.