The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses.

Cells respond to their environment through dynamic posttranslational modification of their existing proteins. There are more than 20 posttranslational modifications that occur on eukaryotic proteins. Several of these modifications, with phosphorylation being the hallmark, participate in signal transduction. Generally, glycosylation is not thought to participate directly in signaling. Complex N-and 0-linked glycosylation occurs on membrane-bound or secreted proteins that are synthesized in the endoplasmic reticulum and Golgi apparatus. The lumenal or extracellular localization ofthese glycans restricts their potential for dynamic responsiveness to signals. In contrast, 0-GlcNAc is a simple monosaccharide modification that is abundant on serine or threonine residues ofnucleocytoplasmic proteins. An 0-GlcNAc site consensus motif has not yet been identified. However, many attachment sites are identical to those used by serine/threonine) kinases, and a neural network program has been developed to predict 0GlcNAc sites. The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by 0-linked beta-N-acetylglucosamine (0-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, 0-GlcNAc and 0-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. Many proteins have been identified that carry this modification, including transcription factors, cytoskeletal proteins, nuclear pore proteins, oncogene products, and tumor suppressors. 0-GlcNAc appears to modify a large number of nucleocytoplasmic proteins· One of important regulatory proteins on which this project concentrates is β-catenin. Here, we examined where does this type ofposttranslational modification takes place on the protein. Our results indicated that P-catenin is 0-glycosylated on both the N-terminus and Cterminus, but not at the ARMADILLO segment. Further, we show that the known phosphorylation sites located at theN-terminal "destruction box" of this protein are not involved in 0-glycosylation. Furthermore, we demonstrated that the threonines adjacent to phosphorylation-site Threonin41 are not essential in 0-glycosylation process. In addition, treatment ofprostate cancer lines with PUGNAc, a non-cytotoxic reversible inhibitor ofOGlcNAcase, caused a decrease in the expression oftransfected P-catenin in the nucleus with increasing cellular 0-glycosylation ofthe protein suggesting that 0-glycosylation was hindering P-catenin's nuclear translocation. Additional studies showed that 0-glycosylation of P-catenin decreased transcriptional activity of a TopFlash reporter plasmid. In summary, our results show that P-catenin is 0-glycosylated on theN-and C-terminus, but not on ARMADILLO segment, and that phosphorylation sites are not the critical for 0-glycosylation. Furthermore, our data show that 0-glycosylation of P-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of P-catenin. / Thesis / Master of Science (MSc)

No description available.

The MDM2 homolog MDMX is an important regulator of p53 during mouse embryonic development. DNA damage promotes MDMX phosphorylation, nuclear translocation, and degradation by MDM2. Here we show that MDMX copurifies with 14-3-3, and DNA damage stimulates MDMX binding to 14-3-3. Chk2-mediated phosphorylation of MDMX on S367 is important for stimulating 14-3-3 binding, MDMX nuclear import by a cryptic NLS, and degradation by MDM2. Mutation of MDMX S367 inhibits ubiquitination and degradation by MDM2, and prevents MDMX nuclear import. Expression of 14-3-3 stimulates the degradation of phosphorylated MDMX. Chk2 and 14-3-3 cooperatively stimulate MDMX ubiquitination and overcome the inhibition of p53 by MDMX. These results suggest that MDMX-14-3-3 interaction plays a role in p53 response to DNA damage by regulating MDMX localization and stability. We also show the identification of a cryptic nuclear localization sequence within the C-terminus RING finger domain MDMX. Mutation of MDMX on one lysine residue at position 468 to glutamic acid completely abrogates the nuclear import after DNA damage. This mutation had no effect on MDM2-mediated nuclear import of MDMX in cotransfection assays, suggesting that it is specifically required for the MDM2-independent nuclear import. Interestingly, the MDMX- K468E mutant induces the expression of p21 more efficiently than the wild-type MDMX after ionizing radiation (IR). Furthermore, the K468E mutant induction of p21 is associated with enhanced G1 arrest after DNA damage. These results indicate an important function of MDMX nuclear import in regulating p53 activity after DNA damage.

Ilf3 et NF90, deux protéines de liaison aux ARN localement structurés en double-brin, sont générées par épissage mutuellement exclusif à partir du gène ILF3. Pour chacune d’entre-elles, un épissage alternatif permet la synthèse de deux isoformes, une longue et une courte, qui diffèrent par la présence ou non d’une séquence de 13 acides aminés localisée à leur extrémité N-terminale et qui correspond à un signal de localisation nucléolaire. La particularité de ces deux protéines est de présenter une forte hétérogénéité avec au moins 20 isoformes produites à partir du même gène, 12 pour Ilf3 et 8 pour NF90. Elle est générée par deux mécanismes complémentaires, l’épissage alternatif et les modifications post-traductionnelles dont deux ont été mises en évidence au laboratoire, la diméthylation asymétrique de l’arginine 609/622 présente dans une séquence consensus de type RGG et catalysée par PRMT1 (« protein arginine N-methyltransferase 1 ») ainsi qu’une phosphorylation sur la sérine 190/203. Ce polymorphisme pourrait être à l’origine des différences de localisation subcellulaire observées selon l’isoforme considérée et/ou aurait comme fonction de réguler soit leurs interactions avec leurs partenaires protéiques ou nucléiques, soit leur activité biologique. Par des expériences d’immunofluorescence et de « GST pull-down », il a été montré que ces deux modifications post-traductionnelles d’Ilf3 et de NF90 ne semblent impliquées ni dans leur localisation subcellulaire, ni dans la régulation de leurs interactions avec leurs partenaires protéiques. Du fait des nombreuses fonctions associées aux protéines Ilf3 et NF90 dans la littérature, les modifications identifiées pourraient être impliquées dans la régulation de leurs interactions avec leurs partenaires nucléiques, ADN ou ARN. / Ilf3 and NF90, two double stranded RNA-binding proteins, are generated by exclusive splicing from the ILF3 gene. For each one, a 5? alternative splicing leads to the synthesis of a long and a short isoforms that differ by the presence or not of 13 amino acid sequence at their N-terminus corresponding to a nucleolar localization signal. The characteristic of these two proteins is to exhibit a high degree of heterogeneity with at least 20 isoforms produced from the same gene, 12 for Ilf3 and 8 for NF90. It is generated by two complementary mechanisms, alternative splicing and posttranslational modifications which two have been identified in the laboratory, the arginine 609/622 asymmetric dimethylation present in a RGG consensus sequence and catalyzed by PRMT1 ("protein arginine N-methyltransferase 1") and the serine 190/203 phosphorylation. This polymorphism could explain the various cellular functions described for both proteins and could regulate their subcellular localization and the interaction with protein or nucleic partners. By immunofluorescence and GST pull-down experiments, it was shown that these two posttranslational modifications of Ilf3 and NF90 neither seem involved in their subcellular localization, nor in the regulation of interactions with their protein partners. Because of the many functions associated with Ilf3 and NF90 proteins in the literature, the identified modifications may be implicated in regulating the interactions with their nucleic partners, DNA or RNA.

Type 1 diabetes (T1D) is the result of the autoimmune-mediated destruction of the pancreatic beta-cells leading to the inability to produce insulin sufficiently and, in turn, regulate blood glucose levels. Abnormal levels of blood glucose, specifically hyperglycemia, have been linked to many diabetic complications, with Brownlee proposing decreased GAPDH activity and the resultant increase in four main pathways as the mechanism(s) leading to these complications. Though skeletal muscles play a major role in glucose uptake, they are believed to be relatively protected against these complications as they are able to regulate their glucose uptake. However, evidence is accumulating that skeletal muscles are adversely affected in T1D, particularly with respect to their mitochondrial function. This led us to consider that the skeletal muscles of those with T1D would experience substrate overload (high intracellular lipids and recurrent, high levels of intracellular glucose), which would initiate a negative spiral whereby substrate excess would damage mitochondria - leading to an impaired ability to utilize these substrates - further worsening the substrate overload. Therefore, the objective of this study was to investigate glycogen and intramyocellular lipid (IMCL) content in the muscles of mice and humans with T1D, as well as the potential downstream effects in the form of post-translational modifications (PTMs), mitochondrial content, and lipofuscin accumulation. The Akita T1D mouse model was used to assess substrate overload in uncontrolled diabetes, whereas human participants were used to investigate substrate overload in the presence of insulin therapy. Assessment of glycogen and IMCL content revealed no difference between controls and diabetic cohorts in both the rodent and human study, indicating the lack of substrate overload. Post-translational modifications did not significantly change between Akita and wild-type mice; however, there was a main effect of diabetes on acetylation levels within Akita mice. Lastly, most mitochondrial properties, except for subsarcolemmal pixel density, did not differ either between diabetic and non-diabetic subjects in the human study. Thus, despite mitochondrial complex impairments in diabetic subjects, its extent was not significant enough to cause alterations to the mitochondria as a whole and result in mitochondrial degradation and lipofuscin formation. This study has provided novel insight into the metabolic properties of skeletal muscle during diabetes. Although there was no indication of substrate overload, diabetes still resulted in some changes to PTM levels and mitochondrial pixel density. However, the effects of these changes did not significantly alter the muscle and resulted in pathway impairments of those that were studied. This could be due to an adaptive mechanism in mice, although future studies are needed to confirm this hypothesis. In the human study, healthy, well-controlled individuals could explain why there was hardly any difference seen, suggesting that controlling glycemic levels was imperative in preventing diabetic complications in muscle. / Thesis / Master of Health Sciences (MSc)

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications. In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA. In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications. Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface. N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation. The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

The influences of posttranslational modifications on the functions of the versatile serpin protein C inhibitor (PCI) were studied. PCI is a serine protease inhibitor that is expressed in many tissues and secreted to various fluids in human, including blood plasma, seminal plasma, and urine. PCI in blood can act both as an anticoagulant and a procoagulant and is believed to play a role in pathogen defence. PCI in reproductive tissues is believed to regulate human reproduction at several steps, including the fertilization process. Due to the broad protease specificity and the contradictory activities, the physiological role of PCI is elusive. In this work the inhibitor was purified from blood and seminal plasma by immunoaffinity chromatography. Blood-derived PCI was found to be highly heterogeneous, due to variations in posttranslational modifications. The occupancy and structures of N- and O-glycans attached to blood plasma PCI and N-glycans of seminal plasma PCI were determined by mass spectrometry. An O-glycosylation site at Thr 20 was identified in PCI derived from blood. N-glycan structures of PCI isolated from blood and seminal plasma differed markedly, demonstrating that they are expressed in a tissue-specific manner. Proteolytic processing also appeared to be tissue-specific, since N-terminally cleaved PCI was found in PCI isolated both from blood and seminal plasma, but the length of the lacking segment differed. The effects of the N-linked glycans and the N-terminus of PCI on protease inhibition were determined using enzymatic measurements with chromogenic substrates. The N-glycans and the N-terminus had different effects on the inhibition of thrombin, factor Xa and prostate specific antigen, demonstrating that posttranslational modifications of PCI affect its functional specificity. These findings enhance the understanding of the regulation of the various functions of PCI and may potentially be used for the production of specialized PCI variants for medical purposes.

No description available.

We did functional characterisation of Drosophila melanogaster linker histone, dH1. In the mutant state for this protein, we observed structural changes in polytene chromosomes chromocenter and nucleoli of mutant larvae. In addition, we performed a microarray analysis in H1 mutant background in order to determine contribution of dH1 to gene expression regulation. We determined effects of dH1 loss in different types of chromatin and we identified groups of differentially expressed (DE) genes, groups in sense of physical clusters of genes and genomic elements rather than groups of functionally related genes. We found that dH1 affects in greater extent expression of heterochromatin genes compared to its effect on euchromatin genes; that dH1 regulates transcription in a regional manner, since the genes physically nearest to the most DE genes tend to be upregulated as well; and that dH1 is negatively regulating expression of transposable elements and members of certain gene families. In addition, we found that dH1 is necessary for preserving genome stability. Among DE transposable elements we detected R1 and R2 retrotransposons, elements that are integrating specifically in rRNA locus. We showed that activation of their transcription is also upregulating expression of aberrant, transposon-inserted, rDNA units of the locus. In this regard we observed an accumulation of extra-chromosomal rDNA circles, increased γ-H2Av content, stop in cell proliferation and activation of apoptosis. Altogether, these results are revealing so far unknown role of histone H1 in preserving genome stability and its effects on cell proliferation.