4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin

The emergence of tandem mass spectrometry (MS/MS) technology has significantly accelerated protein identification and quantification in proteomics. It enables high-throughput analysis of proteins and their quantities in a complex protein mixture. A mass spectrometer can easily and rapidly generate large volumes of mass spectral data for a biological sample. This bulk of data makes manual interpretation impossible and has also brought numerous challenges in automated data analysis. Algorithmic solutions have been proposed and provide indispensable analytical support in current proteomic experiments. However, new algorithms are still needed to either improve result accuracy or provide additional data analysis capabilities for both protein identification and quantification. Accurate identification of proteins in a sample is the preliminary requirement of a proteomic study. In many cases, a mass spectrum cannot provide complete information to identify the peptide without ambiguity because of the inefficiency of the peptide fragmentation technique and the prevalent existence of noise. We propose ADEPTS to this problem using the complementary information provided in different types of mass spectra. Meanwhile, the occurrence of posttranslational modifications (PTMs) on proteins is another major issue that prevents the interpretation of a large portion of spectra. Using current software tools, users have to specify possible PTMs in advance. However, the number of possible PTMs has to be limited since specifying more PTMs to the software leads to a longer running time and lower result accuracy. Thus, we develop DeNovoPTM and PeaksPTM to provide efficient and accurate solutions. Glycosylation is one of the most frequently observed PTMs in proteomics. It plays important roles in many disease processes and thus has attracted growing research interest. However, lack of algorithms that can identify intact glycopeptides has become the major obstacle that hinders glycoprotein studies. We propose a novel algorithm, GlycoMaster DB, to fulfil this urgent requirement. Additional research is presented on protein quantification, which studies the changes of protein quantity by comparing two or more mass spectral datasets. A crucial problem in the quantification is to correct the retention time distortions between different datasets. Heuristic solutions from previous research have been used in practice but none of them has yet claimed a clear optimization goal. To address this issue, we propose a combinatorial model and practical algorithms for this problem.

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.

The emergence of tandem mass spectrometry (MS/MS) technology has significantly accelerated protein identification and quantification in proteomics. It enables high-throughput analysis of proteins and their quantities in a complex protein mixture. A mass spectrometer can easily and rapidly generate large volumes of mass spectral data for a biological sample. This bulk of data makes manual interpretation impossible and has also brought numerous challenges in automated data analysis. Algorithmic solutions have been proposed and provide indispensable analytical support in current proteomic experiments. However, new algorithms are still needed to either improve result accuracy or provide additional data analysis capabilities for both protein identification and quantification. Accurate identification of proteins in a sample is the preliminary requirement of a proteomic study. In many cases, a mass spectrum cannot provide complete information to identify the peptide without ambiguity because of the inefficiency of the peptide fragmentation technique and the prevalent existence of noise. We propose ADEPTS to this problem using the complementary information provided in different types of mass spectra. Meanwhile, the occurrence of posttranslational modifications (PTMs) on proteins is another major issue that prevents the interpretation of a large portion of spectra. Using current software tools, users have to specify possible PTMs in advance. However, the number of possible PTMs has to be limited since specifying more PTMs to the software leads to a longer running time and lower result accuracy. Thus, we develop DeNovoPTM and PeaksPTM to provide efficient and accurate solutions. Glycosylation is one of the most frequently observed PTMs in proteomics. It plays important roles in many disease processes and thus has attracted growing research interest. However, lack of algorithms that can identify intact glycopeptides has become the major obstacle that hinders glycoprotein studies. We propose a novel algorithm, GlycoMaster DB, to fulfil this urgent requirement. Additional research is presented on protein quantification, which studies the changes of protein quantity by comparing two or more mass spectral datasets. A crucial problem in the quantification is to correct the retention time distortions between different datasets. Heuristic solutions from previous research have been used in practice but none of them has yet claimed a clear optimization goal. To address this issue, we propose a combinatorial model and practical algorithms for this problem.

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)

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.

No description available.

In der Hefe Saccharomyces cerevisiae können Proteine entweder co- oder posttranslational durch die Membran des Endoplasmatischen Retikulum transportiert werden. Sie besitzen eine Signalsequenz, die sie zu einem hydrophilen Kanal in der Membran bringt, durch den der Transport erfolgt. Die zentrale Komponente des Translokationsapparates in der Membran ist der aus den Untereinheiten Sec61p, Sbh1p und Sss1p bestehende Sec61p-Komplex. Beim Proteintransport wirkt der Sec61p-Komplex zusammen mit anderen Faktoren: Im cotranslationalen Transport geht er eine feste Bindung mit Ribosomen ein; der posttranslationale Transport erfordert die Assoziation mit dem tetrameren Sec62/63p-Komplex unter Bildung des sogenannten Sec-Komplexes. In der vorliegenden Arbeit wurde die Struktur des Sec61p-Komplexes durch Elektronenmikroskopie analysiert. Er liegt in Detergenzlösung in ringförmigen Strukturen mit einem Durchmesser von ~82Å und einer zentralen Pore von ~21Å vor. Jeder Ring besteht aus drei oder vier heterotrimeren Sec61p-Komplexen. Die oligomeren Ringstrukturen des Sec61p-Komplexes entsprechen vermutlich proteinleitenden Kanälen der Membran des Endoplasmatischen Retikulum. In Membranen wird ihre Bildung durch die Bindung von Ribosomen oder die Interaktion mit dem Sec62/63p-Komplex induziert. Eine dreidimensionale Struktur, die durch Kryo-Elektronenmikroskopie erhalten wurde, zeigt, daß das Ribosom so an den Sec61p-Komplex bindet, daß der Tunnel im Ribosom, durch den die naszierende Polypeptidkette das Ribosom verläßt, genau in die zentrale Pore des Sec61p-Oligomers mündet. Es existiert also ein kontinuierlicher Kanal, der sich vom Peptidyltransferase-Zentrum im Ribosom durch die zentrale Pore des Sec61p-Oligomers erstreckt, durch den naszierende Polypeptidketten cotranslational direkt in das Lumen des Endoplasmatischen Retikulum transportiert werden könnten. In dieser Arbeit wurde ein dem Sec61p-Komplex verwandter heterotrimerer Komplex in der Membran des Endoplasmatischen Retikulum identifiziert, der aus den Untereinheiten Ssh1p, Sbh2p und Sss1p besteht. Sss1p ist beiden trimeren Komplexen gemein; Ssh1p und Sbh2p sind homolog zu Sec61p bzw. Sbh1p. Durch Deletion von Ssh1p und Sbh2p wurde gezeigt, daß der Ssh1p-Komplex wie der Sec61p-Komplex am Transport von Proteinen in das Endoplasmatische Retikulum beteiligt ist. Der Ssh1p-Komplex ist mit membrangebundenen Ribosomen assoziiert und bildet in Detergenzlösung oligomere Ringstrukturen, aber interagiert nicht mit dem Sec62/63p-Komplex. Wir postulieren daher, daß der Ssh1p-Komplex ausschließlich den cotranslationalen Transport von Proteinen vermittelt. Beim posttranslationalen Transport interagiert das vollständig synthetisierte Modellsubstrat Prepro-Alphafaktor mit vielen cytosolischen Proteinen. Die cytosolischen Chaperone Hsp70 und TRiC konnten als Interaktionspartner identifiziert werden. Bei der Bindung des Prepro-Alphafaktors an die Membran werden die cytosolischen Proteine freigesetzt. Wir verwendeten einen Photoquervernetzungsansatz, um zu untersuchen, wie die Signalsequenz des Prepro-Alphafaktors im Bindungsschritt durch den Sec-Komplex erkannt wird. Die Signalsequenz-bindungsstelle wird hauptsächlich von Sec61p gebildet und befindet sich an der Grenzfläche zur Lipiddoppelschicht. Die gebundene Signalsequenz ist in einer helikalen Struktur fixiert und wird auf gegenüberliegenden Seiten von den Transmembrandomänen 2 und 7 des Sec61p umgeben. Sec62p und Sec71p, zwei Untereinheiten des Sec62/63p-Komplexes, flankieren gemeinsam eine Seite der Signalsequenzhelix, befinden sich aber in größerer Entfernung zur Signalsequenz als Sec61p. Es wird ein Modell vorgeschlagen, das beschreibt, wie die Bindung der Signalsequenz den Translokationskanal für den Transport öffnen könnte. / Protein transport across the membrane of the endoplasmic reticulum occurs either co- or posttranslationally in the yeast Saccharomyces cerevisiae. In both cases, polypeptides are directed to a translocation apparatus in the membrane by virtue of their signal sequences and then transported across the lipid bilayer through a protein-conducting channel. The major component of the protein translocation apparatus in the membrane is the heterotrimeric Sec61p complex consisting of the subunits Sec61p, Sbh1p and Sss1p. During translocation the Sec61p complex associates with other factors: In the cotranslational mode it interacts with ribosomes, whereas in the posttranslational mode it associates with the tetrameric Sec63/62p complex to form the so-called Sec complex. Here, we have analyzed the structure of the Sec61p complex by electron microscopy. In detergent this complex forms ring-like structures with a diameter of about 82Å and a central pore of about 21Å. Each ring contains 3 or 4 heterotrimeric Sec61p complexes. In membranes the formation of ring structures of the Sec61p complex is induced by its association with ribosomes or the Sec62/63p complex. We propose that the ring-like Sec61p oligomers represent protein-conducting channels of the endoplasmic reticulum membrane. A 3-dimensional structure of the ribosome-Sec61p complex obtained by electron-cryo-microscopy and single particle reconstruction showed, that the central pore of the Sec61p oligomer aligns precisely with the exit of a tunnel traversing the large ribosomal subunit that forms the passageway for the nascent chain. Thus, in cotranslational translocation a continuous channel extending from the ribosome through the Sec61p oligomer could guide the nascent chain directly into the lumen of the endoplasmic reticulum. Furthermore, we have discovered a trimeric protein complex in the yeast endoplasmic reticulum membrane that is structurally related to the Sec61p complex. This so-called Ssh1p complex consists of Ssh1p, a distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability, but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential for cell viability, but cells lacking both proteins are impaired in their growth at elevated temperature and accumulate precursors of secretory proteins in the cytosol. Like the Sec61p complex, the Ssh1p complex forms ring-like structures in detergent and interacts with membrane-bound ribosomes, but it does not associate with the Sec62/63p complex. We therefore postulate that the Ssh1p complex functions exclusively in the cotranslational pathway of protein translocation. In the posttranslational transport process the newly synthesized translocation substrate prepro-a-factor associates with a large number of cytosolic proteins including the chaperones Hsp70 and TRiC. Upon binding of prepro-a-factor to the Sec complex all cytosolic proteins are released. Using a photo-crosslinking approach and a unique mapping technique we have investigated, how the signal sequence of prepro-a-factor is recognized by the Sec complex during the binding step. The signal sequence contacts primarily the multispanning membrane protein Sec61p. The bound signal sequence adopts a helical structure that interacts on opposite sides with transmembrane domains 2 and 7 of Sec61p, respectively. Sec62p and Sec71p, two subunits of the Sec62/63p complex, contact one side of the signal sequence, but are further away than Sec61p. Our data show, that the signal sequence binding site is located at the interface of the protein channel and the lipid bilayer. We suggest that binding of the signal sequence could open the channel for polypeptide transport.

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.