Novel multinary nitrides of gallium are synthesized and characterized. Different synthesis approaches are shown. Novel compounds are characterized regarding their crystal structures and their properties.

No description available.

The Ski2-Ski3-Ski8 (SKI) complex is a conserved multi-protein assembly required for the cytoplasmic functions of the exosome, including messenger RNA (mRNA) turnover, surveillance and interference. The helicase Ski2, the tetratricopeptide repeat (TPR) protein Ski3 and the �-propeller Ski8 assemble in a heterotetramer with 1:1:2 stoichiometry. While the function of the Ski2-Ski3-Ski8 complex as a general cofactor of the cytoplasmic exosome has been well established, it remains largely unclear how it contributes to the regulation of the exosome. The PhD thesis at hand addresses this question by investigating the structural and biochemical properties of the Ski2-Ski3-Ski8 complex. Solving the crystal structure of the 113 kDa helicase region of S. cerevisiae Ski2 by experimental phasing revealed the presence of a canonical DExH core and an atypical accessory domain that is inserted in the helicase core. This insertion domain binds ribonucleic acid (RNA) unspeci�cally and is located at the RNA entry site of the helicase core. The overall architecture of Ski2 including the presence of an accessory domain is similar to the structure of the related helicase Mtr4, but the structural and biochemical properties of the accessory domains from both proteins are di�erent. The Ski2 insertion domain is not required for formation of the Ski2-Ski3-Ski8 complex. Its removal allowed to crystallize a Ski2�insert-Ski3-Ski8 complex from S. cerevisiae, and the crystal structure of this 370 kDa core complex was determined experimentally. It shows that Ski3 forms an array of 33 TPR motifs, creating a sca�old for the other subunits. Ski3 and the two Ski8 subunits bind the helicase core of Ski2 and position it centrally within the complex. This creates an extended internal RNA channel and modulates the enzymatic properties of the Ski2 helicase. Both Ski8 subunits are bound through a structurally conserved motif. A similar motif is present and functional in yeast Spo11, a protein that initiates deoxyribonucleic acid (DNA) double strand breaks during meiotic recombination. Association of Ski8 to either complex is mutually exclusive, rationalizing how Ski8 can perform its two distinct roles in mRNA metabolism and meiotic recombination. Biochemical studies suggest that the SKI complex can thread RNAs directly to the exosome, coupling the helicase and the exoribonuclease through a continuous channel of 43-44 nucleotides length. Finally, an internal regulatory mechanism in the Ski2-Ski3-Ski8 complex was identi�ed. Both the Ski2-insertion domain and the Ski3 N-terminus cooperate to inhibit ATPase and helicase activity of Ski2 when bound in the SKI complex. Thus, the SKI complex regulates exosome activity in two ways. First by a direct substrate channeling mechanism to the exosome that connects helicase and nuclease activities of both complexes which may activate the exosome towards certain substrates. Second, by an inhibitory mechanism that regulates substrate access to the helicase complex, which is a prerequisite for controlling the exosome's substrate speci�city. This doctoral thesis provides the �rst structural description of the entire yeast SKI complex and identi�es two mechanisms that may contribute to regulation of the activity of the cytoplasmic exosome.

No description available.

Verschiedene Neuropeptidhormone, welche einerseits sehr klein sind oder als intrinsisch unstrukturiert vorhergesagt werden, werden als größere Vorläufer-Proteine synthetisiert. Diese enthalten neben der N-terminalen Signalsequenz eine Prodomäne, welche später im sekretorischen Biosyntheseweg abgespalten wird und nicht Teil des aktiven Hormons ist. Im Rahmen dieser Doktorarbeit wurde die Rolle der Prodomänen in der Biogenese der intrinsisch unstrukturiert vorhergesagten Neuropeptidhormone Thyrotropin-Releasing Hormon (TRH), Somatostatin (Som) und Gonadotropin-Releasing Hormon (GnRH) untersucht. Die hier dargelegten Ergebnisse zeigen erstmals, dass die Prodomänen dieser Proteine für eine effiziente Translokation der intrinsisch unstrukturierten Hormondomänen in das ER-Lumen verantwortlich sind. Bisher wurde eine Funktion der Prodomäne von Peptidhormonen erst nach der Translokation bei der Proteinfaltung, Prozessierung und Sortierung der Proteine im ER-Golgi-Kompartment beschrieben. Zudem deuten die durchgeführten Domänen-Austausch-Experimente daraufhin, dass für die beschriebene translokationsfördernde Aktivität der Prodomäne ihre alpha-helikale Struktur entscheidend ist. Durch die in vitro durchgeführten Zielsteuerungsexperimente konnte gezeigt werden, dass ein Fehlen der Prodomäne bzw. von alpha-helikalen Domänen im Allgemeinen die Translokation in das ER-Lumen und nicht die kotranslationale Zielsteuerung der naszierenden Polypeptidkette an die ER-Membran negativ beeinflusst. Durch die Analyse des weiteren Schicksals der nicht-translozierten, intrinsisch unstrukturierten Proteine konnte ein neuartiger, dualer Zielsteuerungsmechanismus charakterisiert werden, wobei die ER-Signalsequenzen von Somatostatin (Som), Shadoo (Sho) und APP (Amyloid-Vorläuferprotein) eine Weiterleitung der Proteine zum Mitochondrium vermitteln. Es zeigte sich, dass die Zielsteuerungsrichtung der genannten bivalenten Signalsequenzen von der Sekundärstruktur der Polypeptidkette abhängt, wobei intrinsisch unstrukturierte Domänen eine mitochondriale Lokalisation und α-helikale Domänen präferentiell eine ER-Translokation vermittelten. Die Erkenntnisse dieser Arbeit bekräftigen eine Regulation der ER-Translokation durch die Sekundärstruktur der maturen Domäne des Proteins. Die hier gewonnenen Daten zeigen zudem, dass eine duale Zielsteuerung von Proteinen zwischen ER und Mitochondrium durch die Sekundärstruktur der maturen Domäne koordiniert werden kann. Diese Erkenntnisse können möglicherweise in Zukunft zu einem tieferen Verständnis von einigen Erkrankungen beitragen, welche auf einer Mislokalisation von sekretorischen Proteinen beruhen.

In the present study the mechanisms leading to acquired chemoresistance, as well as new treatment strategies implying the prevention evading of tumor cells were addressed. Resistance formation is one of the major hurdles in cancer therapy. Metronomic antiangiogenic treatment of xenografted prostate cancer tumors in mice with cyclophosphamide (CPA) results in the appearance of resistant tumors. To investigate the complex molecular changes occurring during resistance formation, a comprehensive gene expression analysis of the resistant tumors in vivo was performed. A multitude of differentially expressed genes, e.g. PAS domain containing protein 1 (PASD1), annexin A3 (ANXA3), neurotensin (NTS) or plasminogen activator tissue (PLAT), were observed, when comparing resistant to in vivo passaged tumor samples. Moreover, tumor cells from in vivo and in vitro conditions showed a significant difference in target gene expression. For clarification of the mechanisms leading to the survival of tumor cells during maintained anti-angiogenic CPA therapy the differentially expressed genes were assigned to functional pathways like: axon guidance, steroid biosynthesis and complement and coagulation cascades. As blood flow might play a crucial role during maintained anti-angiogenic therapy, further analysis was focused on the genes grouped in complement and coagulation cascades. pregulation of anti-coagulatory ANXA3 and PLAT and downregulation of SERPIN A1 and other SERPIN-family members was shown by qPCR analysis. In contrast coagulation factor F3 was upregulated, accompanied by the expression of an altered gene product. Taken together, a potential role of anticoagulation as a resistance mechanism for anti-angiogenic CPA therapy could be described. Furthermore, the role of archazolid, a novel myxobacterial V-ATPase inhibitor in cancer treatment and in particular its action on the secreted cellular proteome was evaluated. As extracellular protein secretion may have an impact on invasive properties of tumor cells, the changes of the secretome profile of highly migratory urinary bladder carcinoma cells upon archazolid treatment were analyzed. An induced secretion of prometastatic lysosomal proteins such as the cathepsin family was observed. Interestingly, intracellular cathepsin B activity however strongly decreases and mature cathepsin B protein diminishes. It could be shown that archazolid inhibits the mannose-6-phosphate receptor mediated trafficking of procathepsin B from the trans-Golgi network to prelysosomal compartments, leading to an impaired cathepsin B maturation process. This results in an unnatural secretion of the inactive proenzyme and a dramatic decrease in intracellular cathepsin B activity. Importantly, also in vivo an archazolid induced reduction of cathepsin B activity was proven and archazolid treatment resulted in a reduced formation of distant metastases in the lungs. In summary these results indicate that archazolid in addition to its known anti-migratory properties might exert an anti-metastatic effect by reducing the activity of pro metastatic proteases like cathepsin B.

No description available.

No description available.

No description available.

No description available.