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Protein quality control systems are essential for the viability and growth of all living organisms. They protect the cell from irreversible protein aggregation. Because the frequency of protein misfolding, which ultimately results in protein aggregation, varies with the environmental conditions, the amount and activity of protein quality systems have to be accurately adapted to the rate of protein misfolding. The main goal of this thesis was to gain detailed molecular insights into the transcriptional and post-translational regulation of these protein quality control networks in the ecologically, medically and industrially important phylum of low GC, Gram-positive bacteria. In these bacteria the core protein quality control systems are under the transcriptional control of the global repressor CtsR. In a first study it was demonstrated that the arginine kinase McsB is not responsible for the regulation of CtsR activity during heat stress, as was concluded by others on the basis of previous in vitro data. Rather, it was demonstrated that CtsR acts as an intrinsic thermosensor that adapts its activity to the surrounding temperature. CtsR displays a decreased DNA binding at higher temperatures, which leads to induction of transcription of the protein quality control systems under these conditions. This CtsR feature is conserved in all low GC, Gram-positive bacteria. However, the CtsR proteins of various low GC, Gram-positive species do not have the same temperature optima. CtsR responds to heat in a species-specific manner according to their corresponding growth temperature. Detailed analysis revealed that a highly conserved tetra-glycine loop within the winged helix-turn-helix domain of CtsR is responsible for thermosensing. Dual control of CtsR activity during different stresses was demonstrated for the first time in this work. In addition to heat-dependent de-repression, CtsR is inactivated by thiol-specific stress conditions. This latter de-repression depends on a molecular redox-switch that is independent of CtsR auto-regulation. In Bacillus subtilis and its closest relatives the McsA/McsB stress-sensing complex is responsible for CtsR de-repression during redox stress conditions. McsA is able to sense the redox state of the cell via its highly conserved cysteine residues. When these cysteines are reduced, McsA is able to bind and inhibit McsB. But when these cysteine residues are oxidized, McsB is released from McsA. Thereby, McsB is activated and removes CtsR from the DNA. However, the McsA/McsB complex is not present in all low GC, Gram-positive bacteria. In the species lacking this complex, ClpE is able to act as a redox-sensor probably via its highly conserved N-terminal zinc finger domain. When these cysteine residues are oxidized, ClpE is activated which results in CtsR de-repression. In addition to the transcriptional regulation of CtsR low GC, Gram-positive protein quality control systems are regulated post-transcriptionally. The expression of the McsA/McsB adaptor pair is regulated by CtsR. However, McsB activity is also tightly regulated by three different regulatory proteins (McsA/ClpC/YwlE). McsB is needed to target specific substrates to ClpC, either for refolding or degradation by the ClpCP protease. It was demonstrated that only the auto- phosphorylated form of McsB is able to bind to its substrates. This McsB function is inhibited in non-stressed cells by a direct interaction with ClpC. Consequently, McsB is activated by a release from ClpC during protein stress. In addition, McsB activation depends on the presence of its activator McsA. Accordingly, McsB cannot be activated as an adaptor protein during thiol-specific stress because McsA is no longer able to bind to McsB under these conditions. However, also active McsB is subject to post-translational control. Activated McsB is either de-phosphorylated by McaP or degraded by ClpCP ensuring an appropriate shut-down of the McsB adaptor. Both McaP and ClpC inhibit McsB activity with different intensities. ClpC possesses a stronger impact on McsB activity than McaP but both proteins are needed for an adequate silencing of McsB activity. In addition, it was shown for the first time that B. subtilis McsB is a global adaptor that influences the stability of multiple proteins. The B. subtilis ClpC protein is unlike most members of the Hsp100 family because it not only requires several adaptor proteins for substrate recognition but also for its general ATP- dependent activity. Biochemical analysis revealed how ClpC is activated by distinct adaptor proteins. McsB modulates ClpC activity by regulatory phosphorylation of arginine residues. Moreover, McaP (formerly YwlE) was identified as an arginine phosphatase that modulates the McsB mediated ClpC activity. MecA, another known adaptor protein for ClpC, activates ClpC independently of these arginine phosphorylations, which demonstrates the existence of multiple pathways for ClpC activation.
Einleitung Das kolorektale Karzinom ist eine der häufigsten Tumorerkrankungen der westlichen Welt und die zweithäufigste tumorassoziierte Todesursache bezogen auf die Gesamtbevölkerung. Im Stadium IV beträgt das 5-Jahresüberleben unbefriedigende 12%. Minnelide™ ist das wasserlösliche Prodrug des pflanzlichen Wirkstoffs Triptolid, der vielversprechende Ergebnisse in der Behandlung des Pankreaskarzinoms zeigt. Über die Effektivität von Minnelide™ im Kolonkarzinom und den Mechanismus von Triptolide ist nur wenig bekannt. Material und Methoden Der Einfluss von Minnelide™ auf das Tumorwachstum, das Fortschreiten der Metastasierung und das Gesamtüberleben wurde in einem subkutanen und einem Lebermetastasen-Xenograftmodell mit humanen HCT116-Zellen in nu/nu-Mäusen untersucht. In vitro Studien zum Mechanismus von Triptolid wurden in HCT116 und HT29 Zelllinien durchgeführt und mit Pankreaskarzinomzelllinien S2-VP10 und MiaPaCa-2 verglichen. Zellviabilität wurde mittels CCK-8, Caspaseaktivität fluorometrisch und der Zellzyklusarrest mittels FACS bestimmt. Autophagie und die Expression von anti-apoptotischen und proliferationsfördernden Proteinen XIAP, Survivin, BCL-Xl, c-Myc, Cyclin D1 und Cdk-4 in Zelllysaten und Tumorhomogenaten wurden im Western Blot bestimmt. C-myc mRNA wurde mittels Real-Time-PCR gemessen. Jak-2-Aktivierung wurde durch Bestimmung von P-Jak-2 im Western Blot bestimmt und die physische Interaktion zwischen Jak-2 und STAT-3 in Co-Immunopräzipitationen untersucht. Der Einfluss auf die STAT-3-Aktivität wurde mit STAT-3-Dual-Luciferase-Reporter-Assay erfasst. Als spezifischer Jak2-Inhibitor kam WP1066 zum Einsatz. Ergebnisse Minnelide™ hemmt das Wachstum von subkutanen Tumoren und von Lebermetastasen signifikant und verlängert das Überleben von Mäusen mit Lebermetastasen. Triptolid führt dosisabhängig zu Zelltod durch Apoptose und G1-Zellzyklusarrest in dem es die Expression von XIAP, Survivin, BCL-Xl und c-Myc, nicht jdeoch Cyclin D1 und Cdk-4 hemmt. Dies geht mit einem Verlust von P-Jak-2 und STAT-3-Aktivität einher und der Effekt läßt sich durch Verwendung eines Jak2-Inhibitors validieren. Die Inhibtion des Jak-2/STAT-3-Signalweg ist in Kolon- und Pankreaskarzinomzellen gleichermaßen nachvollziehbar. Diskussion In dieser Arbeit wird gezeigt, dass Minnelide™ in Analogie zu den Ergebnissen in Pankreaskarzinommodellen das Tumorwachstum signifkant hemmt, die Metastasierung bremst und das Überleben der Versuchstiere verbessert und damit eine vielversprechende Substanz für die Behandlung des metastasierten Kolonkarzinoms darstellt. Weitere Studien zum Vergleich mit Standardsubstanzen und in nicht-immundefizienten Tiermodellen sind jedoch notwendig. Eine Inhibtion von Jak-2 durch Triptolid war bisher weder für das Kolon- noch für das Pankreaskarzinom beschrieben. Jak-2, als Zielstruktur von Triptolid, reiht sich damit in eine ständig wachsende Liste von möglichen Wirkmechanismen ein. In welcher Weise Triptolid mit Jak-2 und anderen Signalwegen interagiert, bleibt weiter unverstanden und bedarf weiterer Forschung.
Activation of trace amine-associated receptor 1 (TAAR1) in endocrine pancreas is involved in weight regulation and glucose homeostasis. The purpose of this study was the identification and characterization of potential TAAR1 variants in patients with overweight/obesity and disturbed glucose homeostasis. Screening for TAAR1 variants was performed in 314 obese or overweight patients with impaired insulin secretion. The detected variants were functionally characterized concerning TAAR1 cell surface expression and signaling properties and their allele frequencies were determined in the population-based Study of Health in Pomerania (SHIP). Three heterozygous carriers of the single nucleotide missense variants p.Arg23Cys (R23C, rs8192618), p.Ser49Leu (S49L, rs140960896), and p.Ille171Leu (I171L, rs200795344) were detected in the patient cohort. While p.Ser49Leu and p.Ille171Leu were found in obese/overweight patients with slightly impaired glucose homeostasis, p.Arg23Cys was identified in a patient with a complete loss of insulin production. Functional in vitro characterization revealed a like wild-type function for I171L, partial loss of function for S49L and a complete loss of function for R23C. The frequency of the R23C variant in 2018 non-diabetic control individuals aged 60 years and older in the general population-based SHIP cohort was lower than in the analyzed patient sample. Both variants are rare in the general population indicating a recent origin in the general gene pool and/or the consequence of pronounced purifying selection, in line with the obvious detrimental effect of the mutations. In conclusion, our study provides hints for the existence of naturally occurring TAAR1 variants with potential relevance for weight regulation and glucose homeostasis.