Chemie | Biochemie | Medizin
Thierry Grimm, 2001 | Binningen, BL
When nutrient supply becomes limited, one of the primary responses in cells is bulk autophagy, a low-level cellular self-digestion mechanism. Autophagy is a highly regulated pathway and also acts as an intracellular waste-disposal machinery. The findings of this thesis indicate that Vac8, one player in autophagy, is of great importance in the vacuolar morphology of yeast and binds the Atg13 protein, which serves as a platform in the process of pre-autophagosomal structure (PAS) formation.
The primary question of this thesis was whether Vac8 interacts with Atg1 complex members, as Vac8 seems to have similar binding partners. Additionally, the project examined whether such a potential interaction influences the PAS formation and the role of Vac8 therein.
PAS can be tracked and measured using different assays. With a western blot, the phosphorylation of Atg13, the activation of Atg1 and the lipidation of Atg8 were assessed. Through sectional fluorescence microscopy and a yeast transformation, the cellular localisation of autophagosomes in relation to the vacuole, proteins of interest and organelles could be identified and evaluated. With an in-vitro fusion assay, samples from transformed yeast clones were used to assess the role of Vac8 in fusion events. With a plated and liquid yeast-2-hybrid assay, different protein constructs of Vac8 and Atg proteins were qualitatively tested for their binding affinity. And with a fractionation assay and succeeding western blot, the cellular localisation of Vac8 was determined and compared to controls. As a complete simulation of protein dynamics and interactions is not viable, computational heuristics such as structure prediction methods through both evolutionary couplings (ECs) and template-based modelling (TBM) were used to gauge binding modes and loci. And through systematic annotation, Gene Ontology (GO) was used to predict the intracellular function and compartment of Vac8 through the interaction partner’s primary protein sequences.
It was found that Vac8 interacts with the Atg1 complex subunit of Atg13. As there were significantly more Atg13 dots at the vacuole when Vac8 was fully functional, Atg13 most likely acts as a platform for Vac8 to bind to the vacuole. The findings also suggest that Vac8 is involved in vacuole-autophagosome fusion, as there were significantly fewer fusion events when Vac8 was deleted. Vac8 is unique to yeast, but orthologues, such as the mammalian plakoglobin junction protein, raise questions, as autophagy also plays a pivotal role in certain neurodegenerative diseases. The motility of autophagosomes through the cytoskeleton is one such factor. Finally, Vac8 also seems to be involved in vacuolar morphology as Vac8-deficient cells present multilobular and fragmented vacuoles.
The Atg1 complex in yeast lies at the core of starvation-induced assembly of the pre-autophagosomal structure (PAS). While it was previously unclear what the role of Vac8 in the yeast was, it appears that Vac8 might recruit Atg13 at the vacuole to promote their fusion. The multimeric vesicle assembly is mostly speculative in character, as individual interactions are scarcely indicative of the entire complex structure. Nevertheless, reasonable estimations can be made that fit these observations and deepen our understanding of the molecular mechanisms in autophagy. Deregulation and malfunctions of the individual steps of the autophagy pathway and the mutation are believed to cause neurodegenerative diseases and to promote the growth of some cancers. Thus, new developments in research have found that targeted autophagy may be a viable therapeutic gateway in fighting cancer. In the model organism of yeast, Vac8 lies at the core of many of these functional pathways also found in mammals, but this is still poorly understood. This thesis has both made a contribution but also raised more questions about the intricacies of PAS formation, autophagosome fusion and a potential connection to mammalian junction proteins.
The findings of this thesis indicate that Vac8 is of great importance in the PAS and vacuolar morphology of yeast and that it binds Atg13, which likely serves as a platform for Vac8 to bind to the vacuole. Vac8 is unique to yeast, but homologues, such as the plakoglobin junction protein, suggest either a common ancestor or a partial conservation of function. Future research has to measure the binding affinity quantitatively to assess whether the interaction of Vac8 is permanent or transitionary, how Vac8 interacts with actin-cables or the cytoskeleton and how it relates to the mammalian junction proteins.
Würdigung durch den Experten
Prof. Jörn Dengjel
Autophagie ist ein Recycling Mechanismus, der für das zelluläre Gleichgewicht von grundlegender Bedeutung ist. So kann eine Störung der Autophagie zu menschlichen Erkrankungen führen. Herr Grimm hat in seiner Arbeit grundlegende, evolutionär-konservierte, zelluläre Mechanismen in Hefe Saccharomyces cerevisiae untersucht. In seinem ambitionierten Projekt hat Herr Grimm die Funktion von einzelnen Proteinen charakterisiert. Hierfür hat er hochmoderne, interdisziplinäre experimentelle und Computer-basierte Methoden verwendet. Diese Interdisziplinarität zeichnet zukünftige Forscher aus!
Lehrer: Dr. Samuel Zschokke