Charakterisierung redundanter Kerntransportsignale des minoren Kapsidproteins L2 humaner Papillomviren

Das minore Kapsidprotein L2 humaner Papillomviren wird im Laufe des HPV-Lebenszyklus zweimal in den Zellkern importiert und akkumuliert dort an den Nukleären Domänen 10 (ND10). Der erste Kernimport erfolgt in der frühen Phase der Infektion zusammen mit der Virus-DNA. Für den Zusammenbau von Virionen wird neu synthetisiertes L2-Protein ein weiteres Mal in den Kern transportiert. Im Rahmen dieser Arbeit konnte die Domäne von L2 identifiziert werden, die für den Kernimport von HPV16 L2 absolut notwendig ist. Dabei gelang es diesen Bereich auf 25 Aminosäuren einzuengen. Sowohl während der frühen Phase der Infektion als auch während der Morphogenese scheint die zentrale, basische Aminosäureregion 291-315 (mNLS) hauptverantwortlich für die Interaktion mit Kernimportrezeptoren zu sein. Möglicherweise leisten dabei flankierende Sequenzen einen Beitrag zur Stabilisierung der notwendigen Konformation. Des Weiteren gelang die Identifizierung der Aminosäuren, die für die Funktionalität des mNLS essentiell sind. Hierbei handelt es sich um ein zentrales Arginin-Motiv, bestehend aus vier dicht beieinander liegenden Argininen, dessen Mutation den Kernimport von L2 während Infektion und Morphogenese verhindert. Untersuchungen mit HPV16 und HPV18 L2-Proteinen verdeutlichten, dass es möglicherweise ein universelles Motiv zu sein scheint und in verschiedenen HPV-Typen konserviert ist. Flankiert wird dieses Arginin-Motiv von konservierten Serinen und Threoninen. Wie die Analyse von Punktmutationen zeigte, sind diese Aminosäuren für den Kernimport von L2 ohne Bedeutung. Interessanterweise verhinderte aber die Mutation TS295/6A die Kolokalisation von L2 mit ND10 im Zellkern. L2wt rekrutiert den transkriptionellen Regulator Daxx. Auch diese Funktion ging bei der Mutante TS295/6A verloren. Diese Ergebnisse zeigen, dass nicht nur die ND10-Lokalisationsdomäne (AS 390-420) in L2 sondern auch weitere Aminosäuren oder Domänen für die Assoziation mit ND10 und die Rekrutierung von Daxx verantwortlich sein könnten. Auf der Suche nach zellulären Faktoren, die eine Rolle im mNLS-vermittelten Kernimport spielen, wurde zunächst die Bedeutung von Hsc70 untersucht. Während der Morphogenese maskiert Hsc70 den C-Terminus von L2 und verhindert damit unerwünschte Interaktionen mit Mikrotubuli im Zytoplasma. Es existieren aber weitere noch unbekannte Hsc70-Bindedomänen in L2, die möglicherweise den Kernimport ebenfalls beeinflussen können. Wie die Untersuchungen deutlich machten, ist der zentrale, basische Bereich von L2 aber nicht mit Hsc70 assoziiert und der mNLS-vermittelte Kernimport findet unabhängig von Hsc70 statt. In einem siRNA-Screen wurde anschließend die Rolle von Karyopherinen während der Infektion untersucht. Sowohl Kapß2-siRNA als auch Kapß3-siRNA waren in der Lage unabhängig voneinander die Infektion von HPV16-Pseudovirionen zu reduzieren. Für beide Karyopherine konnte in der Vergangenheit in vitro die Interaktion mit HPV16 L2 nachgewiesen werden. Das L2-Protein ist das einzige virale Protein, das während der Infektion die Virus-DNA in den Kern begleitet (Day et al., 2004). Demzufolge ist es auch das einzige virale Protein, das mit Importinen während der Infektion interagiert. Möglicherweise sind also beide Karyopherine in der Lage sein L2 während der Infektion in den Kern zu importieren. Abschließend wurden Präzipitationsversuche durchgeführt, die zur Identifizierung möglicher Bindungspartner des mNLS führen sollten. In diesen Versuchen konnte eine erhöhte Bindungsaffiniät zu den beiden Importinen Kapß1 und Kapß2 festgestellt werden. Möglicherweise ist das L2-Protein mit seiner mNLS in der Lage mehrere Importrezeptoren zu binden und für den Kernimport zu nutzen. Eines dieser Importine ist Kapß2. Dieser Importrezeptor scheint sowohl bei der Infektion als auch während der Morphogenese den Kernimport von L2 durch die Bindung an das mNLS zu vermitteln.

Detailed analyses of mutations affecting structural formation of neuromuscular synapses in Drosophila melanogaster

The establishment of appropriate synapses between neurons and their target cells is an essential requirement for the formation of functional neuronal circuits. However, there is very little insight into the mechanisms underlying de novo formation of synapses and synaptic terminals. To identify novel genes involved in signalling or structural aspects of these processes I capitalised on possibilities provided by the model organism Drosophila. Thus, I contributed to a screen of a collection of third chromosomal mutations (Salzberg et al., 1997, Genetics 147, 1723ff.) selecting those mutant strains displaying structural defects of Drosophila neuromuscular junctions (NMJ). Carrying out genetic mapping experiments, I could assign 7 genes to interesting candidate mutations. All 7 mutations selected in this process cause size alterations of the embryonic NMJ, and one shows additional disturbances in the distribution of synaptic markers. 4 of these turned out to be transcription factors, not falling into the remit of this project. Only for one of these, the neuronal transcription factor Castor, I could show that its overgrown mutant NMJ phenotype is due to an increase in the number of motorneurons. The remaining genes encode a potential nitrophenylphosphatase, the translation initiation factor eIF4AIII, and a novel protein Waharan. Unfortunately, the nitophenylphosphatase gene was identified too late to carry out functional studies in the context of this project, but potential roles are discussed. eIF4AIII promotes NMJ size tempting to speculate that local translation at the NMJ is affected. I found that the synaptic scaffolding molecule Discs large (Dlg; orthologue of PSD95) is upregulated at eIF4AIII mutant NMJs. Targeted upregulation of Dlg can not mimic the eIF4AIII mutant phenotype, but dlg mutations suppress it. Therefore, Dlg function is required but not sufficient in this context. My findings are discussed in detail, pointing out future directions. The main focus of this work is the completely novel gene waharan (wah), an orthologue of the human gene KIAA1267 encoding a big brain protein of likewise unknown structure and function. My studies show that mutations or RNAi knock-down of wah cause NMJ overgrowth and reveal additional crucial roles in the patterning of wing imginal discs. RNAi studies suggest Wah to be required pre- and postsynaptically at NMJs and, consistently, wah is transcribed in the nervous system and muscles. Anti-Wah antisera were produced but could no longer be tested here, but preliminary studies with newly generated HA-targeted constructs suggest that Wah localises at NMJs and in neuronal nuclei. In silico analyses predict Wah to be structurally related to the Rad23-family of proteins, likely to target ubiquitinated proteins to the proteasome for degradation (Chen et al., 2002, Mol Cell Biol 22, 4902ff.) . In agreement with this prediction, poly-ubiquitinated proteins were found to accumulate in the absence of wah function, and wah-like mutant phenotypes were induced in NMJs and wing discs by knocking down proteasome function. My analysis further revealed that poly-ubiquitinated proteins are reduced in nuclei of wah mutant neurons and muscles, suggesting that Wah may play additional roles in ubiquitin-mediated nuclear import. Taken together, this study has uncovered a number of interesting candidate genes required for the de novo formation of Drosophila NMJs. 3 of these genes fell into the focus of this project. As discussed in detail, discovery of these genes and insights gained into their function have high potential to be translatable into vertebrate systems.

Anaerobic pathways in the Porifera: Strombine dehydrogenase, an opine dehydrogenase, from the sponge Suberites domuncula

The study presented here encompasses identification, analysis and characterization of the strombine dehydrogenase (StDH) from the sponge S. domuncula, on the gene and protein level. StDH is an opine dehydrogenase which is involved in opine production pathways found mainly in marine invertebrates. These anaerobic pathways are regarded as analogues to the classical anaerobic glycolytic pathway (lactate production pathway), which is predominant in vertebrates. The StDH was previously annotated as a tauropine dehydrogenase (TaDH) on the basis of its 68% identity with the TaDH protein from Halichondria japonica. Subsequent enzymatic assays showed that S. domuncula opine dehydrogenase is in fact strombine dehydrogenase which possesses specific characteristics not found in other proteins of the same family. It is described here for the first time the StDH gene in Eukaryotes. Two allelic variants have been identified which are present in the different specimens either as a homozygotic or a heterozygotic. Phylogenetic analyses supported with enzymatic assays indicate that S. domuncula StDH is only distantly related to the opine dehydrogenases from marine invertebrates. StDH showed that the protein is highly specific to glycine and inhibited by the substrate pyruvate. Furthermore, S. domunucla StDH has a dimeric structure (~75 kDa) which is not observed in so far described OpDHs that are monomeric proteins. This enzyme showed similarities to the OCD/mu-cristallyin protein family. Results showed that a sponge StDH is unusual enzyme that belongs to the independent enzyme class. In addition, expression studies revealed that the StDH is down-regulated with aeration. Immunohistology analyses showed high expression of the protein in almost all sponge cells. A strong accumulation of the enzyme was seen around the bacteria indicating that under aerobic conditions the bacteria might metabolize strombine (end product of the reaction). In conclusion, the data documented here shed new light on the anaerobic pathways in marine invertebrates. Potential mutual influences between bacteria and sponge are discussed as well. Hopefully, these results could have a small but important contribution to the better understanding of the evolution in the animal kingdom.

LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.