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The metabolism of tyrosine is essential for many critical biochemical events including catecholamine synthesis, melanogenesis and insect cuticle sclerotization. These pathways are highly regulated in both insects and mammals by many well-characterized enzymes including dopa decarboxylase and tyrosine hydroxylase. On the other hand, there are still many enzymes involved in these processes that we know very little about. Dopachrome tautomerase (DCT), dopachrome conversion enzyme (DCE) and α-methyldopa resistant protein (AMD) fall into the category of the less characterized enzymes. Dopachrome is a pivotal intermediate in melanogenesis. Mammalian DCT and insect DCE both use dopachrome as a substrate. DCE catalyzes a decarboxylative structural rearrangement of dopachrome to 5,6-dihydroxyindole (DHI), whereas DCT mediates the isomerization/tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHI is oxidized easily, leading to the production of melanin, as well as reactive oxygen species (ROS). DHICA is less reactive, relative to DHI, and consequently produces less toxic byproducts during melanogenesis; therefore DCT plays an important role in detoxification of DHI and ROS. Purification and MS analysis of DCE and DCT determined that N-glycosylation is a primary post-translational modification. Q-TOF mass spectrometry was used to determine N-glycosylation patterns from Aedes aegypti DCE and MALDI-TOF/TOF was used to determine multiple glycosylation sites in DCT. N-glycosylation is critical for the folding and trafficking of secreted proteins in the endomembrane system. The analysis of glycosylation sites in DCE and DCT therefore is essential toward achieving a comprehensive understanding of their structure and function. Like DCT, AMD also plays a protective role. The AMD protein was originally identified in Drosophila mutants hypersensitive to α-methyldopa, an inhibitor of dopa decarboxylase (DDC). Production of dopamine by DDC is critical for developing insects because dopamine conjugates are used as crosslinking agents for cuticle sclerotization. Although there has been much discussion into the function of AMD, what exactly this protein does has been unknown. AMD shares 48% sequence identity with DDC, however we have found that AMD is an enzyme, which possesses a different catalytic activity. GC-MS analysis of AMD enzymatic reaction components revealed that AMD catalyzes the oxidative decarboxylation of L-DOPA to DOPAL, and also the oxidative decarboxlation of α-methyldopa to 3,4-dihydroxyphenylacetone. In summary, multiple N-glycosylation sites were characterized in DCT and DCE, furthermore a new protein function has been demonstrated for AMD. These experiments were performed using classical biochemistry techniques in combination with mass spectrometry. / Ph. D.

Yes / 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used Tiparp–/– mice to determine Tiparp’s role in the development of the prefrontal cortex. Loss of Tiparp resulted in an aberrant organization of the mouse cortex, where the upper layers presented increased cell density in the knock-out mice compared with wild type. Tiparp loss predominantly affected the correct distribution and number of GABAergic neurons. Furthermore, neural progenitor cell proliferation was significantly reduced. Neural stem cells (NSCs) derived from Tiparp–/– mice showed a slower rate of migration. Cytoskeletal components, such as α-tubulin are key regulators of neuronal differentiation and cortical development. α-tubulin mono-ADP ribosylation (MAR) levels were reduced in Tiparp–/– cells, suggesting that Tiparp plays a role in the MAR of α-tubulin. Despite the mild phenotype presented by Tiparp–/– mice, our findings reveal an important function for Tiparp and MAR in the correct development of the cortex. Unravelling Tiparp’s role in the cortex, could pave the way to a better understanding of a wide spectrum of neurological diseases which are known to have increased expression of TIPARP. / European Union Seventh Framework Program (FP7-PEOPLE-2013-COFUND) Grant n609020-Scientia Fellows (to G.G.) and by the Johan Throne Holst Foundation and the University of Oslo (J.M.).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

E2F-1 has a central role in cell cycle orchestration, and its activity is tightly regulated. One of the ways E2F-1 activity is controlled is by direct modification by post translational modifications such as acetylation, ubiquitination and phosphorylation. Here it was demonstrated that E2F-1 is targeted by two novel modifications, namely methylation by Set7/9 and NEDDylation, both within the DNA binding and heterodimerisation domain of the protein. NEDDylation and methylation of E2F-1 both decrease the stability and diminish the transcriptional activity of E2F-1. Lysine residues in E2F-1 involved in NEDDylation are also targeted by methylation, allowing the potential for interplay between these modifications. Methylation of E2F-1 was demonstrated to be a prerequisite for its NEDDylation and the multi-domain protein UHRF1 implicated in mediating this effect. The results define a new level of control on E2F-1 and suggest a protein code with pleiotropic effects involved in E2F-1 regulation.

Histone post-translational modifications (PTMs), as the histone code assumes, are related with regulation of gene transcription, an important mechanism of cells in the differentiation process. Many PTMs are simultaneously present in histone proteins, and changes in the PTM stoichiometric ratios can have several effects, like changes in the chromatin structure leading to a transcriptionally active or repressive state. Significant progresses were made to map variations of histone PTMs by mass spectrometry (MS), and although many protocols were developed there are still some drawbacks. Incomplete and side reactions were identified, which can directly affect the quantification of histone PTMs, because both (incomplete and side reactions) can be misinterpreted as endogenous histone post translational modifications. Therefore, a protocol for derivatization of histones with no noticeable undesired reactions (<10%) was required. In this thesis a new chemical modification methodology is presented, which allows the improvement of sequence coverage by acylation with propionic anhydride of lysine residues and N-terminal (free ε- and α- amino groups) and trypsin digestion. more than 95% of complete reaction was achieved with the new derivatization methodology. This strategy (chemical derivatization of histones), in combination with bottom-up MS approach, allows the quantification of lysine methylation (Kme) and acetylation (Kac) in histones from Saccharomyces cerevisiae (S.cerevisiae), mouse embryonic stem cells (mESCs) and human cell lines. The results showed histone H3 PTM pattern as the most variable profile regarding histone Kme and Kac across the three different organisms and experimental conditions. Therefore, it was concluded that quantification of H3 PTM pattern can be used to examine changes in chromatin states when cells are subjected to any kind of perturbation.