Protein arginine methylation is an abundant post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Arginine methylation plays important roles in a variety of cellular pathways and human diseases. PRMT1, the predominant PRMT, catalyzes approximately 85% of all protein arginine methylation in vivo. While many details of how PRMT1 functions have been uncovered through the past two decades, there are many details which remain unclear, including how arginine methylation is regulated, how PRMT1 binds substrates, and what role PRMTs play in RNA surveillance. Our recent data presented in this thesis showed that reduction of the PRMT1 enzyme, following recombinant expression and purification, changes both enzymatic activity and oligomeric state. A cysteine residue(s) was found to be responsible for the observed redox chemistry in PRMT1 and at least one parameter in the kinetic mechanism, S-adenosylmethionine (AdoMet) binding, was faster with a reduced enzyme. This work suggests exciting potential for the regulation of PRMTs in vivo by oxidative stress. In addition to studying the effects of reduction/oxidation on PRMT1, a foundation for future experiments was laid. These experiments investigate substrate recognition by PRMTs and what the role arginine methylation may play in RNA processing and surveillance. To better understand how PRMTs selectively bind a wide variety of substrates, I have designed and preliminarily characterized several Hmt1 (the S. cerevisiae homologue of PRMT1) variants. These variants will be used for crystallization trials of a homogeneous complex, containing Hmt1, AdoMet, and a peptide substrate, capable of revealing specific chemical interactions between Hmt1 and the peptide substrate. To further our understanding of Hmt1's role in RNA processing and surveillance, particularly in RNA degradation pathways, I extracted yeast RNA from both wild type and Hmt1-null cells. The RNA was probed using a S. cerevisiae whole-genome microarray. This analysis revealed that Hmt1 exhibits statistically significant effects in several broad areas including molecular function, biological processes, cellular components, and some KEGG pathways. The presented studies have revealed the exciting potential for an in vivo regulatory mechanism of PRMT1 and each study is primed for further investigation both in vivo and in vitro.

Rapid technological advances are providing unprecedented insights in the biologicalsciences, with massive amounts of data generated on genomic and protein sequences.These data continue to grow exponentially, and they are extremely valuable for com-putational tools where the effect of genomic variants on human health is predicted.State of the art tools in this field give varying results and only tend to agree in thecase of single variants that are strongly correlated to disease. The aim of this workis to increase the reliability of these methods, as well as our understanding of theunderlying biological mechanisms that lead to disease. We first developed machinelearning (ML) based structural bioinformatics predictors that are able to predictmolecular features of proteins from the sequence alone. We then used these tools forin silico analysis of the molecular effects of known variants on the affected proteins,and integrated these data with other sources heterogenous sources of information,such as the essentiality of a gene, that put the variants into their broader biologicalcontext. With this information we created DEOGEN, a novel predictor in this field,which is able to deal with the two most common forms of genomic variation, namelySingle Nucleotide Variants (SNVs) and short Insertions and DELetions (INDELs).DEOGEN performs at least on par with other state of the art methods in this fieldon different datasets. The method was then extended with additional contextualdata and is now available as DEOGEN2 via a web server, which visualizes the pre-dicted results for all variants in most human proteins through an interactive interfacetargeted to both bioinformaticians and clinicians. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The reversible oxidation of cysteine by reactive oxygen species (ROS) is both a mechanism for cellular protein signaling as well as a cause of cellular injury and death through the generation of oxidative stress. The study of cysteine oxidation is complicated by the methodology currently available to isolate and enrich oxidized-cysteine containing proteins. We sought to simplify this process by reducing the time needed to process samples and reducing sample loss and contamination risk. We accomplished this by eliminating precipitation steps needed for the protocol by (a) introducing an in-solution NEM-quenching step prior to reduction and (b) replacing soluble dithiothreitol reductant with a series of newly-developed high-capacity polyacrylamide-based solid-phase reductants that could be easily separated from the lysate through centrifugation. These modifications, collectively called resin-assisted reduction and capture (RARC), reduced the time needed to perform the RAC method from 2-3 days to 4-5 hours, while the overall quality and quantity of previously-oxidized cysteines captured was increased. In order to demonstrate the RARC method’s utility in studying complex cellular oxidants, the optimized methodology was used to study cysteine oxidation caused by the redox-active metals arsenic, cadmium, and chromium. As(III), Cr(VI), and Cd(II) were all found to increase cysteine oxidation significantly, with As(III) and Cd(II) inducing more oxidation than Cr(VI) following a 24-hour exposure to cytotoxic concentrations. Label-free proteomic analysis and western blotting of RARC-isolated oxidized proteins found a high degree of commonality between the proteins oxidized by these metals, with cytoskeletal, translational, stress response, and metabolic proteins all being oxidized. Several previously-unreported redox-active cysteines were also identified. These results indicate that cysteine oxidation by As(III), Cr(VI), and Cd(II) may play a significant role in these metals’ cytotoxicity and demonstrates the utility of the RARC method as a strategy for studying reversible cysteine oxidation by oxidants in oxidative signaling and disease. The RARC method is a simplification and improvement upon the current state of the art which decreases the barrier of entry to studying cysteine oxidation, allowing more researchers to study this modification. We predict that the RARC methodology will be critical in expanding our understanding of reactive cysteines in cellular function and disease.