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Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment.
Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states – while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation.
Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in small-molecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25.
This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors.
Deubiquitinases are regulators of the ubiquitin proteasome system that counteract the ubiquitination cascade by removing ubiquitin from substrates and cleaving ubiquitin chains. Due to their involvment in various important pathways, they are associated with several diseases and may thus present promising drug targets. The two related ubiquitin specific proteases USP25 and USP28 share a highly conserved amino acid sequence but perform distinct biological functions. USP28 plays roles in cell cycle regulation and was also linked to several types of cancer. It adopts oncogenic functions by rescuing the oncoproteins MYC and JUN from proteasomal degradation, which is induced by the E3-ligase SCF (FBW7). Opposingly, USP28 also regulates the stability of the tumor suppressor FBW7 itself. USP25 contributes to a balanced innate immune system by stabilizing TRAF3 and TRAF6 and lately was found to promote Wnt-signaling by deubiquitinating TNKS.
Due to the high level of identity of both proteases, a recent attempt to inhibit USP28 led to cross reactivity against USP25. In our study, we characterized both USP25 and USP28 structurally and functionally using x-ray crystallography, biochemical as well as biophysical approaches to determine similarities and differences that can be exploited for the development of specific inhibitors.
The crystal structure of the USP28 catalytic domain revealed a cherry-couple like dimer that mediates self-association by an inserted helical subdomain, the USP25/28 catalytic domain inserted domain (UCID). In USP25, the UCID leads to formation of a tetramer composed of two interlinked USP28-like dimers. Structural and functional analysis revealed that the dimeric USP28 is active, whereas the tetrameric USP25 is auto inhibited. Disruption of the tetramer by a cancer-associated mutation or a deletion-variant activates USP25 through dimer formation in in vitro assays and leads to an increased stability of TNKS in cell studies. Furthermore, in vitro data showed that neither ubiquitin nor substrate binding led to the activation of the USP25 tetramer construct. With the structure of the C-terminal domain of USP25, we determined the last unknown region in the enzyme as a separately folded domain that mediates substrate interactions.
Combined the structures of the USP25 and USP28 catalytic domains and the functional characterization of both enzymes provide novel insights into the regulation of USPs by oligomerization. Furthermore, we identified individual features of each protease that might be explored for the development of specific small molecule inhibitors.
The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively.
BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety.
The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target.
In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann’s reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann’s reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above.
We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB.
The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme.
Our structural analysis revealed that despite its low sequence identity of 28%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD.
With 9.6 million new cases and 1.5 million deaths in 2014, tuberculosis (TB) is alongside with AIDS the most deadly infection. Foremost, the increased prevalence of resistant strains of M. tuberculosis among the TB-infected population represents a serious thread. Hence, in the last decades, novel drug targets have been investigated worldwide. So far a relatively unexplored target is the cell wall enzyme β-ketoacyl-ACP-synthase “KasA”, which plays a crucial role in maintaining the membrane impermeability and hence the cell ability to resist to the immune response and drug therapy. KasA is a key enzyme in the fatty acid synthase “FAS-II” elongation cycle, responsible for the extension of the growing acyl chain within the biosynthesis of precursors for the most hydrophobic constituents of the cell wall – mycolic acids. Design of the novel KasA inhibitors, performed in the research group of Prof. Sotriffer by C. Topf and B. Schaefer, was based on the recently published crystal structure of KasA in complex with its known inhibitor thiolactomycin (TLM). Considering the essential ligand-enzyme interactions, a pharmacophore model was built and applied in the virtual screening of a modified ZINC database. Selected hits with the best in silico affinity data have been reported by Topf and Schaefer.
In this work, two of the obtained hits were synthesized and their structure was systematically varied. First, a virtual screening hit, chromone-2-carboxamide derivative GS-71, was modified in the amide part. Since the most of the products possessed a very low solubility in the aqueous buffer medium used in biological assays, polar groups (nitro, succinamidyl and trimethyl-amino substituent in position 6 of the chromone ring or hydroxyl group on the benzene ring in the amide part have been inserted to the molecule. Further variations yielded diaryl ketones, diaryl ketone bearing a succinamidyl substituent, carboxamide bearing a methylpiperazinyl-4-oxobutanamido group and methyl-malonyl ester amides. Basically, the essential structural features necessary for the ligand-enzyme interactions have been maintained. The latter virtual screening hit, a pyrimidinone derivative VS-8 was synthesized and the structure was modified by substitution in positions 2, 4, 5 and 6 of the pyrimidine ring. Due to autofluorescence, detected in most of the products, this model structure was not further varied.
Simultaneously, experiments on solubilization of the first chromone-2-carboxamides with cyclodextrins, cyclic oligosacharides known to form water-soluble inclusion complexes, were performed. Although the assessed solubility of the chromone 3b/DIMEB (1:3) mixture exceeded 14-fold the intrinsic one, the achieved 100 µM solubility was still not sufficient to be used as a stock solution in the binding assay. The experiments with cyclodextrin in combination with DMSO were ineffective. Owing to high material costs necessary for the appropriate cyclodextrin amounts, the aim focused on structural modification of the hydrophobic products.
Precise structural data have been obtained from the solved crystal structures of three chromone derivatives: the screening hit GS-71 (3b), its trimethylammonium salt (18) and 6-nitro-substituted N-benzyl-N-methyl-chromone-2-carboxamide (9i). The first two compounds are nearly planar with an anti-/trans-rotamer configuration. In the latter structure, the carboxamide bridge is bent out of the chromone plane, showing an anti-rotamer, too. Considering the relatively low partition coefficient of compound 3b (cLogP = 2.32), the compound planarity and correlating tight molecular packing might be the factors significantly affecting its poor solubility.
Regarding the biological results of the chromone-based compounds, similar structure-activity correlations could be drawn from the binding assay and the whole cell activity testing on M. tuberculosis. In both cases, the introduction of a nitro group to position 6 of the chromone ring and the presence of a flexible substituent in the amide part showed a positive effect. In the binding study, the nitro group at position 4 on the N-benzyl residue was of advantage, too. The highest enzyme affinity was observed for N-(4-nitrobenzyl)-chromone-2-carboxamide 4c (KD = 34 µM), 6-nitro substituted N-benzyl-chromone-2-carboxamide 9g (KD = 40 µM) and 6‑nitro-substituted N-(4-nitrobenzyl)-chromone-2-carboxamide 9j (KD = 31 µM), which could not be attributed to the fluorescence quenching potential of the nitro group. The assay interference potential of chromones, due to a covalent binding on the enzyme sulfhydryl groups, was found to be negligible at the assay conditions. Moderate in vivo activity was detected for 6‑nitro-substituted N-benzyl-chromone-2-carboxamide 9g and its N-benzyl-N-methyl-, N‑furylmethyl-, N-cyclohexyl- and N-cyclohexylmethyl derivatives 9i, 9d, 9e, 9f, for which MIC values 20 – 40 µM were assessed. Cytotoxicity was increased in the N‑cyclohexylmethyl derivative only. None of the pyrimidine-based compounds showed activity in vivo. The affinity of the model structure, VS-8, surpassed with KD = 97 µM the assessed affinity of TLM (KD = 142 µM).
Since for the model chromone compound GS-71 no reliable KasA binding data could be obtained, a newly synthesized chromone derivative 9i was docked into the KasA binding site, in order to derive correlation between the in silico and in vitro assessed affinity. For the 6‑nitro-derivative 9i a moderate in vivo activity on M. tuberculosis was obtained. The in silico predicted pKi values for TLM and 9i were higher than the corresponding in vitro results, maintaining though a similar tendency, i.e., the both affinity values for compound 9i (pKi predicted = 6.64, pKD experimental = 4.02) surpassed those obtained for TLM (pKi predicted = 5.27, pKD experimental = 3.84). Nevertheless, the experimental pKD values are considered preliminary results.
The binding assay method has been improved in order to acquire more accurate data. Owing to the method development, limited enzyme batches and solubility issues, only selected compounds could be evaluated. The best hits, together with the compounds active on the whole cells of M. tuberculosis, will be submitted to the kinetic enzyme assay, in order to confirm the TLM-like binding mechanism. Regarding the in vivo testing results, no correlations could be drawn between the predicted membrane permeability values and the experimental data, as for the most active compounds 9e and 9f, a very low permeability was anticipated (0.4 and 0.7 %, respectively). Further biological tests would be required to investigate the action- or transport mode.
KasA is a key enzyme which plays an essential part in the biosynthetic pathway of mycolic acids, the building block of cell wall in Mycobacterium tuberculosis. Its importance was demonstrated by the finding that the depletion of KasA leads to the cell lysis of Mycobacterium tuberculosis. Since Mycobacterium tuberculosis is a pathogen of tuberculosis, the second leading cause of death from an infectious disease worldwide, KasA has drawn attention as one of the attractive drug targets against tuberculosis. Due to the emergence of extensively drug-resistant strains which make most of the known antibiotics for treating tuberculosis ineffective, it became an urgent issue to develop new drugs against tuberculosis. In chapter 3.1, the protonation state of the catalytic residues in the resting state was mainly addressed. The FEP computation and MD simulations were employed for this investigation, and the results showed that the zwitterionic state is most probable. To underpin this conclusion with more solid data, The PESs for the proton transfer between the neutral and zwitterionic state were computed in the context of QM/MM. However, due to the strong dependency of the QM/MM optimization on the initial structure, it was not possible to obtain consistent results from these computations. To circumvent this problem, QM/MM based umbrella sampling was carried out with a semi-empirical method (RM1), and the resulting PMF surface indicated that the zwitterionic state is more stable than the neutral state. In chapter 3.2, the protonation state of significant residues in the acyl-enzyme state was investigated. Unlike other catalytic residues, the protonation state of His311 is ambiguous in the acyl-enzyme state, and different decarboxylation mechanisms can be derived depending on the protonation state of His311 in the acyl-enzyme state. Therefore, FEP computations were carried out to find most probable protonation state of His311 in terms of free energy, and the results showed that the pKa value at Nδ is considerably lowered by the enzyme environment while that of Nε is not. Additionally, the PMF profiles for the proton transfer between Lys340 and Glu354 were computed using QM/MM based umbrellas sampling method, and the results showed that the property of the Lys340/Glu354 pair is neutral rather than ionic when His311 is protonated at Nε. Moreover, a relatively larger ionic character of the Lys340/Glu354 pair when His311 is doubly protonated provides a valuable insight into how the Lys340/Glu354 pair plays a role in shifting the protonated state from Nδ to Nε in His311 after the acyl-transfer step. Overall, the results demonstrated that His311 is neutral and protonated at Nε, and the Lys340/Glu354 pair is also neutral in the acyl-enzyme state. Those computational results lead to the conclusion that the decarboxylation reaction is facilitated by an oxyanion hole which is comprised of two catalytic histidines. In chapter 3.3, the protonation state of catalytic residues in the resting state was revisited because a recent benchmark study showed that the employed semi-empirical method (RM1) in chapter 3.1 tends to overestimate the stabilization of the zwitterionic state. Furthermore, the Lys340/Glu354 pair was considered as purely ionic in chapter 3.1, while it actually has a mixed neutral and ionic character as demonstrated in chapter 3.2. The new investigations employed a larger QM region including the Lys340/Glu354 pair with the BLYP/6-31G** approach, which was proven to be accurate enough for the present purpose by benchmark computations. The new results from the QM/MM MD and FEP computations indicated the catalytic residues to be neutral most probably in the resting state, and this in turn brought up the question how KasA can be activated to initiate the catalytic reaction. On the basis of the results from the MD simulations and FEP computations for the His311Ala mutant in chapter 3.1, we hypothesized that the open conformation of Phe404 would trigger the activation of the catalytic residues by the formation of a strong hydrogen bond. The QM/MM MD simulation proved that the activation of the catalytic residues can indeed be accomplished by the open conformation of Phe404 we suggested, and the corresponding force field based PMF profile also indicated that this conformational change is energetically feasible. The distribution of hydrophilic and hydrophobic residues in the malonyl binding pocket in conjunction with our computational results further provided a valuable insight into the detailed process how the catalytic residues is activated upon the substrate entering.
The SARS virus is the etiological agent of the severe acute respiratory syndrome, a deadly disease that caused more than 700 causalities in 2003. One of its viral proteins, the SARS coronavirus main protease, is considered as a potential drug target and represents an important model system for other coronaviruses. Despite extensive knowledge about this enzyme, it still lacks an effective anti-viral drug. Furthermore, it possesses some unusual features related to its active-site region. This work gives atomistic insights into the SARS coronavirus main protease and tries to reveal mechanistic aspects that control catalysis and inhibition. Thereby, it applies state-of-the-art computational methods to develop models for this enzyme that are capable to reproduce and interpreting the experimental observations. The theoretical investigations are elaborated over four main fields that assess the accuracy of the used methods, and employ them to understand the function of the active-site region, the inhibition mechanism, and the ligand binding. The testing of different quantum chemical methods reveals that their performance depends partly on the employed model. This can be a gas phase description, a continuum solvent model, or a hybrid QM/MM approach. The latter represents the preferred method for the atomistic modeling of biochemical reactions. A benchmarking uncovers some serious problems for semi-empirical methods when applied in proton transfer reactions. To understand substrate cleavage and inhibition of SARS coronavirus main protease, proton transfer reactions between the Cys/His catalytic dyad are calculated. Results show that the switching between neutral and zwitterionic state plays a central role for both mechanisms. It is demonstrated that this electrostatic trigger is remarkably influenced by substrate binding. Whereas the occupation of the active-site by the substrate leads to a fostered zwitterion formation, the inhibitor binding does not mimic this effect for the employed example. The underlying reason is related to the coverage of the active-site by the ligand, which gives new implications for rational improvements of inhibitors. More detailed insights into reversible and irreversible inhibition are derived from in silico screenings for the class of Michael acceptors that follow a conjugated addition reaction. From the comparison of several substitution patterns it becomes obvious that different inhibitor warheads follow different mechanisms. Nevertheless, the initial formation of a zwitterionic catalytic dyad is found as a common precondition for all inhibition reactions. Finally, non-covalent inhibitor binding is investigated for the case of SARS coranavirus main protease in complex with the inhibitor TS174. A novel workflow is developed that includes an interplay between theory and experiment in terms of molecular dynamic simulation, tabu search, and X-ray structure refinement. The results show that inhibitor binding is possible for multiple poses and stereoisomers of TS174.
Macromolecular complexes, also termed molecular machines, facilitate a large spectrum of biological reactions and tasks crucial to the survival of cells. These complexes are composed of either protein only, or proteins bound to nucleic acids (DNA or RNA). Prominent examples for each class are the proteosome, the nucleosome and the ribosome. How such units are assembled within the context of a living cell is a central question in molecular biology. Earlier studies had indicated that even very large complexes such as ribosomes could be reconstituted from purified constituents in vitro. The structural information required for the formation of macromolecular complexes, hence, lies within the subunits itself and, thus, allow for self- assembly. However, increasing evidence suggests that in vivo many macromolecular complexes do not form spontaneously but require assisting factors (“assembly chaperones”) for their maturation. In this thesis the assembly of RNA-protein (RNP) complexes has been studied by a combination of biochemical and structural approaches. A resourceful model system to study this process is the biogenesis pathway of the uridine-rich small nuclear ribonucleoproteins (U snRNPs) of the spliceosome. This molecular machine catalyzes pre-mRNA splicing, i.e. the removal of non-coding introns and the joining of coding exons to functional mRNA. The composition and architecture of U snRNPs is well defined, also, the nucleo-cytoplasmic transport events enabling the formation of these particles in vivo have been analyzed in some detail. Furthermore, recent studies suggest that the formation of U snRNPs in vivo is mediated by an elaborate assembly machinery consisting of protein arginine methyltransferase (PRMT5)- and survival motor neuron (SMN)-complexes. The elucidation of the reaction mechanism of cellular U snRNP assembly would serve as a paradigm for our understanding of how RNA-protein complexes are formed in the cellular environment. The following key findings were obtained as part of this study: 1) Efforts were made to establish a full inventory of the subunits of the SMN-complex. This was achieved by the biochemical definition and characterization of an atypical component of this complex, the unrip protein. This protein is associated with the SMN-complex exclusively in the cytoplasm and influences its subcellular localization. 2) With a full inventory of the components in hand, the architecture of the SMN-complex was defined on the basis of an interaction map of all subunits. This study elucidated that the proteins SMN, Gemin7 and Gemin8 form a backbone, onto which the remaining subunits adhere in a modular manner. 3) The two studies mentioned above formed the basis to elucidate the reaction mechanism of cellular U snRNP assembly. Initially, an early phase in the SMN-assisted formation of U snRNPs was analyzed. Two subunits of the U7 snRNP (LSm10 and 11) were found to interact with the PRMT5-complex, without being methylated. This report suggests that the stimulatory role of the PRMT5-complex is independent of its methylation activity. 4) Key reaction intermediates in U snRNP assembly were found and characterized by a combination of biochemistry and structural studies. Initially, a precursor to U snRNPs with a sedimentation coefficient of 6S is formed by the pICln subunit of the PRMT5-complex and Sm proteins. This intermediate was shown to constitute a kinetic trap in the U snRNP assembly reaction. Progression towards the assembled U snRNP depends on the activity of the SMN-complex, which acts as a catalyst. The formation of U snRNPs is shown to be structurally similar to the way clamps are deposited onto DNA to tether poorly processive polymerases. 5) The human SMN-complex is composed of several subunits. However, it is unknown whether all subunits of this entity are essential for U snRNP assembly. A combination of bioinformatics and biochemistry was applied to tackle this question. By mining databases containing whole-genome assemblies, the SMN-Gemin2 heterodimer is recognized as the most ancestral form of the SMN-complex. Biochemical purification of the Drosophila melanogaster SMN-complex reveals that this complex is composed of the same two subunits. Furthermore, evidence is provided that the SMN-Gemin2 heterodimer is necessary and sufficient to promote faithful U snRNP assembly. Future studies will adress further details in the reaction mechanism of cellular U snRNP assembly. The results obtained in this thesis suggest that the SMN and Gemin2 subunits are sufficient to promote U snRNP formation. What then is the function of the remaining subunits of the SMN-complex? The reconstitution schemes established in this thesis will be instrumental to address this question. Furthermore, additional mechanistic insights into the U snRNP assembly reaction will require the elucidation of structures of the assembly machinery trapped at various states. The prerequisite for these structural studies, the capability to generate homogenous complexes in sufficient amounts, has been accomplished in this thesis.
Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli.