Studies of the N-end Rule Pathway in Bacteria and Mammals

Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a minute or so. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS), in conjunction with molecular chaperones, autophagy, and lysosomal proteolysis. The N-end rule pathway, the first specific pathway of UPS to be discovered, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Physiological functions of the N-end rule pathway are strikingly broad and continue to be discovered. In bacteria and in eukaryotic organelles mitochondria and chloroplasts all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. What is the main biological function of this metabolically costly, transient, and not strictly essential modification of N-terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the formyl groups of N-terminal Met in Nt formylated bacterial proteins may signify a proteolytic role of Nt-formylation. My colleagues and I addressed this hypothesis experimentally, as described in Chapter 3 of this thesis. Among the multitude of biological functions of the mammalian Arg/N-end rule pathway are its roles in the brain, including the regulation of synaptic transmission and the regulation of brain’s G-protein circuits. This regulation is mediated, in part, by the its Ate1-mediated arginylation branch of the Arg/N-end rule pathway. One role of the Ate1 arginyltransferase (R-transferase) is to mediate the conditional degradation of three G-protein down-regulators, Rgs4, Rgs5, and Rgs16. Ate1-/- mice, which lack the Ate1 R-transferase, exhibit a variety of abnormal phenotypes. Chapter 4 describes our studies of neurological abnormalities in Ate1-/- mice (and also in mice that express Ate1 conditionally, upon the addition of doxycycline), with an emphasis on the propensity of these mice to epileptic seizures. </p

Structural characterization of the siderophore rhodochelin from Rhodococcus jostii RHA1 and elucidation of its biosynthetic machinery

Rhodococci represent an important genus of industrial interest, both because of their role in bioremediation and biocatalysis, as well as for their potential as producers of natural products. In this context, the genome sequencing of the biphenyl-degrading soil bacterium Rhodococcus jostii RHA1 represents the first attempt to harness the biosynthetic metabolic potential of the genus Rhodococcus, by enabling the systematic exploration of its natural product-producing capabilities. The genome of R. jostii RHA1 contains 23 secondary metabolite gene clusters, all considered to be orphan with respect to their product, including two clusters putatively involved in siderophore biosynthesis. In this study, the isolation, structural characterization and genetic analysis of the biosynthetic origin of rhodochelin, a unique mixed-type catecholate-hydroxamate siderophore isolated from R. jostii RHA1, which represents the first characterized NRPS-derived natural product of the strain, is reported. Structure elucidation of rhodochelin was accomplished via MSn- and NMR-analysis and revealed the tetrapeptide to contain an unusual ester bond between an L-δ-N-formyl-δ-N-hydroxyornithine (L-fhOrn)moiety and the side chain of a threonine residue. Bioinformatic analysis of the R. jostii RHA1 genome revealed the enzymes responsible for siderophore biosynthesis to be encoded in three distant NRPS gene clusters. Single gene deletions within the three putative biosynthetic gene clusters abolished rhodochelin production, proving that the ORFs responsible for rhodochelin biosynthesis are located in different chromosomal loci. Biochemical characterization of the monooxygenase Rmo and the formyltransferase Rft established a route for the biosynthesis of the nonproteinogenic amino acid L-fhOrn, prior to its incorporation into the peptide scaffold by the NRPS-assembly line. The insights gained from the structural and functional characterization of rhodochelin, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed the proposal of a biosynthetic model for rhodochelin assembly. Finally, the efficient and, in this work, first reported cross-talk between three distantly located secondary metabolite gene clusters provides deep insights into natural product biosynthesis that may facilitate future attempts to isolate new natural products

Structural Characterization of the Heterobactin Siderophores from Rhodococcuserythropolis PR4 and Elucidation of their Biosynthetic Machinery

Summary The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior

Filter-wrapper combination and embedded feature selection for gene expression data

Biomedical and bioinformatics datasets are generally large in terms of their number of features - and include redundant and irrelevant features, which affect the effectiveness and efficiency of classification of these datasets. Several different features selection methods have been utilised in various fields, including bioinformatics, to reduce the number of features. This study utilised Filter-Wrapper combination and embedded (LASSO) feature selection methods on both high and low dimensional datasets before classification was performed. The results illustrate that the combination of filter and wrapper feature selection to create a hybrid form of feature selection provides better performance than using filter only. In addition, LASSO performed better on high dimensional data

The computational analysis of post-translational modifications

The post-translational modification (PTMs) of proteins presents a means to increase the proteome size and diversity of an organism through the inclusion of structural elements not encoded at the sequence-level alone. Their erroneous inclusion or exclusion has been linked to a variety of diseases and disorders thus their characterisation has the potential to present viable drug targets. The proliferation of newer high-throughput methods, such as mass spectrometry, to identify such modifications has led to a rapid increase in the number of databases and tools to display and analyse such vast amounts of data effectively. This study covers the development of one such tool; PTM Browser, and the construction of the underlying database that it is based upon. This new database was initially seeded with annotations from the Swiss-Prot and Phospho.ELM resources. The initial database of PTMs was then expanded to include a large repertoire of previously unannotated proteins for a selection of topical species (e.g. Danio rerio and Tetraodon nigroviridis). Orthologue assignments have also been added to the database – to allow for queries to be performed regarding the conservation of modifications between homologous proteins. The PTM Browser tool allows for a full exploration of this new database of PTMs – with a special focus on allowing users to identify modifications that are both shared between and are specific to particular species. This tool is freely available for non-commercial use at the following URL: http://www.ptmbrowser.org. An analysis is presented on the conservation of modifications between members of the tumour suppressor family, p53, using this new tool. This tool has also been used to analysis the conservation of modifications between super-kingdoms and Eukaryote species

Chemoenzymatic Synthesis of Chromodepsipeptides and Natural Product Discovery via Genome Mining

Recent advances in the development of sequencing technologies have enabled the identification of a multitude of bacterial gene clusters, putatively involved in the biosynthesis of nonribosomal peptides (NRPs). Peptides of nonribosomal origin constitute a class of structurally and functionally diverse natural products, which are assembled by multimodular nonribosomal peptide synthetases (NRPSs). These compounds exhibit a broad pharmacological spectrum, ranging from antibacterial- to immunosuppressive properties. Understanding the assembly mechanisms in combination with rational genome mining approaches will provide opportunities for the discovery of new bioactive natural products. Within this study one approach was utilized to generate thiocoraline analogs via chemoenzymatic synthesis and the second strategy focused on the de novo natural product discovery via genome mining. Thiocoraline represents a pseudosymmetrical chromophore-capped octathiodepsipeptide, in which the symmetrical halves are linked via thioester bonds. In this study, the cyclodimerization potential of the thioesterase domain of the thiocoraline biosynthetic machinery (TioS PCP-TE) was investigated to obtain further insights into the iterative assembly of chromodepsipeptides. To address this objective, the recombinant enzyme was incubated with synthetically derived tetrapeptidyl substrates, resembling thiocoraline precursors. It was shown that the enzyme catalyzes the cyclodimerization of linear precursor molecules and an unprecedented macrothiolactonization. Evaluation of the biocombinatorial potential established the thioesterase as a robust and versatile catalyst for the generation of chromodepsipeptide analogs, harbouring thioester- or ester-linkages. As thiocoraline attains its antitumor activity from DNA-bisintercalation, the chemoenzymatically generated macrocycles were isolated and investigated towards DNA-bisintercalation activity in vitro. In the second part of this study, bioinformatic analysis of the 8.2 Mb Saccharopolyspora erythraea genome revealed two cryptic NRPS gene clusters related to hydroxamate-type siderophore biosynthesis. Detailed analysis of adenylation domain substrate-specificity and module organization enabled the establishment of a highly selective and sensitive radio-LCMS-guided genome mining approach. Application of this approach resulted in the discovery of the siderophore erythrochelin. Structure elucidation of erythrochelin was accomplished via NMR- and MSn-analysis and revealed the sequence of the tetrapeptide siderophore to be: α-N-acetyl-δ-N-acetyl-δ-N-hydroxy-D-ornithine-D-serine-cyclo(δ-N-hydroxy-L-ornithine-δ-N-acetyl-δ-N-hydroxy-L-ornithine). Erythrochelin assembly requires the proliferation of δ-N-hydroxy-L-ornithine (L-hOrn) and δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn). The corresponding modifying enzymes, the FAD-dependent monooxygenases EtcB and Sace_1309 together with the bifunctional malonyl-CoA decarboxylase/N-acetyltransferase were identified and biochemically characterized. In vitro studies revealed EtcB and Sace_1309 to exclusively catalyze the δ-N-hydroxylation of free L-ornithine. The second tailoring enzyme, Mcd, was shown to catalyze malonyl-CoA decarboxylation and subsequent acetyltransfer onto the δ-hydroxamino group of L-hOrn, affording L-haOrn. Based on the elucidation of precursor biosynthesis (L-haOrn), a model for the entire erythrochelin assembly is presented

Targeting Peptides: The New Generation of Targeted Drug Delivery Systems

Peptides can act as targeting molecules, analogously to oligonucleotide aptamers and antibodies. They are particularly efficient in terms of production and stability in physiological environments; in recent years, they have been increasingly studied as targeting agents for several diseases, from tumors to central nervous system disorders, also thanks to the ability of some of them to cross the blood–brain barrier. In this review, we will describe the techniques employed for their experimental and in silico design, as well as their possible applications. We will also discuss advancements in their formulation and chemical modifications that make them even more stable and effective. Finally, we will discuss how their use could effectively help to overcome various physiological problems and improve existing treatments

Prevention and prediction of production instability of CHO-K1 cell lines by the examination of epigenetic mechanisms

The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.Die CHO-K1 Zelllinie ist das am häufigsten verwendete Expressionssystem für therapeutische Proteine innerhalb der pharmazeutischen Industrie. Aus wirtschaftlichen Gründen wird die verwendete Zelllinie sowie die eingesetzten Vektoren ständig verbessert um die Produktqualität und -quantität zu erhöhen. Während der Kultivierungsphase neigen Produktionszelllinien dazu an Produktivität zu verlieren. Dabei wird der Produktivitätsverlust häufig mit einer Reduktion der Kopienzahl oder dem Silencing von Transgenen assoziiert. Während der Zelllinienentwicklung werden vielversprechende Zelllinien ausgewählt und im großen Ansatz kultiviert. Ein Produktivitätsverlust innerhalb solcher Zellen ist somit sehr kostenintensiv. Um diese Gefahr zu minimieren entwickelte ich unterschiedliche Stategien, welche darauf abzielen den Produktivitätsverlust zu vermeiden. Produktionsinstabilität konnte von unserer Gruppe schon anhand des CpG Methylierungsgrades am CMV Promoter vorhergesagt werden. Die DNA Methylierung wird wahrscheinlich zur Aufrechterhaltung eines inaktiven Chromatinstatus benötigt und steht am Ende einer epigentischen Kaskade. Im Gegensatz dazu erscheinen Histonmodifikationen früher in der Signalkaskade und könnten deswegen eine höhere Aussagekraft über die Stabilität haben. Aus diesem Grunde wurden von mir Histonemodifkationen am hCMV-MIE Promoter und Enhancer zu Beginn der Kultivierungsphase gemessen. H3K4me3, H3ac sind Histonmodifikationen die mit Expression assoziiert werden wohingegen H3K27me3 und H3K9me3 grundsätzlich mit einem inaktiven Chromatinstatus in Verbindung gebracht werden. Der Grad der unterschiedlichen Modifikationen wurde mit dem über zwei Monate entstehenden Produktivitätsverlust verglichen. Dabei stellte sich heraus, dass der Grad der Histon H3 Acetylierung die höchste Korrelation mit der Stabilität aufwies. Des Weiteren konnte ich einen Grenzwert für die H3 Acetylierung definieren der einen Ausschluss der meisten instabilen Produktionszelllinien ermöglicht. Im zweiten Projekt wurde das Vector Design unter epigenetischen Aspekten verändert. Ich erstellte eine zielgerichtete Histonacetyltransferase, um in dem Chromatinbereich des Transgenes einen offenen und aktiven Status zu induzieren. Desweiteren mutierte ich methylierungsanfällige CpGs des hCMV-MIE Promoters und Enhancers um eine Methylierung und daraus folgend einen inaktiven Chromatinstatus zu verhindern. Die C zu G Konversion an dem 179 Basenpaar oberhalb der Transkriptionsstartstelle führte zu einer bemerkenswert stabilen Antikörperexpression in klonalen Zelllinien. Desweiteren konnte ich bei gleicher Promotervariante in eGFP exprimierenden Zellpools eine geringere Methylierung und Genamplifikation feststellen. Somit konnte zum ersten Mal die Effektsensitivität eines einzelnen CpGs verdeutlicht werden. Im letzten Projekt wurde die Expressionsstabilität abhängig von der Integrationsstelle des Transgenes untersucht. Dabei konnte ich zeigen, dass die standardmäßig durchgeführte zufällige Integration entweder bevorzugt in inaktiven Bereichen des Euchromatin stattfindet oder dass die Selektionsdruck induzierte Genamplifikation hauptsächlich im Heterochromatin stattfindet. Weiterhin vermute ich, dass beide Ereignisse hintereinander geschaltet sind, bei der die geringe Aktivität des Transgenes im inaktiven Euchromatin die Genamplifikation im Heterochromatin fördert. Bei der Untersuchung der Chromatinlandschaft und den enthaltenden Transgenen konnte ich vielversprechende aktive Regionen identifizieren, die wahrscheinlich die Stabilität der Expression fördern. Jedoch müssten diese Ergebnisse in weiteren Experimenten bestätigt werden. Bei der Betrachtung der drei Projekte zeigt sich, dass das Wechselspiel zwischen der Belastung des Stoffwechsels der Zelle und dem Selektionsdruck in der frühen Kultivierungsphase ausschlaggebend ist für deren weitere Entwicklung. Dabei können kleine Veränderungen des Selektionsdruckes die Zellen maßgebend beeinflussen. Stabil exprimierende Zellen sind dabei weniger angreifbar als schwach exprimierende Zellen. Bei einer Erhöhung des Selektionsdruckes kompensieren die schlechteren Produktionszelllinien ihren Nachteil durch Genamplifikation. Die Anpassung der Produktivität überdeckt die stabilen Zellen welches die richtige Auswahl erschwert. Aus diesem Grunde sollte der Selektiondruck, die Kopienzahl, sowie die Wachstumsrate in den Selektionskriterien mit einbezogen werden, um reprimierende Effekte zu minimieren