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Investigating the Mechanistic role of Ykt6 in Wnt Trafficking and Cell cycle Progression

Investigating the Mechanistic role of Ykt6 in Wnt Trafficking and Cell cycle Progression

dc.contributor.advisorGross, Julia Prof. Dr.
dc.contributor.authorManivannan, Pradhipa Karuna
dc.date.accessioned2021-09-28T12:39:43Z
dc.date.available2022-09-13T00:50:33Z
dc.date.issued2021-09-28
dc.identifier.urihttp://hdl.handle.net/21.11130/00-1735-0000-0008-591F-3
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-8843
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc610
dc.titleInvestigating the Mechanistic role of Ykt6 in Wnt Trafficking and Cell cycle Progressionde
dc.title.alternativeInvestigating the Mechanistic role of Ykt6 in Wnt Trafficking and Cell cycle Progressionde
dc.typecumulativeThesisde
dc.contributor.refereeGross, Julia Prof. Dr.
dc.date.examination2021-09-15
dc.description.abstractengYkt6 is an evolutionarily conserved R-SNARE that mediates vesicle fusion events at diverse cellular compartments of secretory, endocytic and autophagy pathways. It is known to be required for the secretion of Wnts on small extracellular vesicles termed exosomes. However, the exact molecular mechanism of Ykt6 in exosomal Wnt sorting remains unclear. Employing proximity labelling, in vivo genetics and biochemical approaches, we identified that Ykt6 acts at the level of sorting endosome where it trafficks Wnts to the PM for extracellular Wnt release. Ykt6 is a unique SNARE that lacks a transmembrane domain and cycles between cytosol and membranes to initiate fusion events. Strikingly, blocking acidification led to an increased association of Ykt6 with membranes. Thus, it is plausible that Ykt6 gets recruited to de-acidified endosomal compartments to traffic Wnt proteins. The exact mechanism that governs this conformational switch of Ykt6 from closed, cytosolic form to open, membranebound form is unknown. We identified putative phosphosites in the SNARE domain, that triggers the conformational switch, thereby regulating its membrane recruitment and activity. Furthermore, we identified PDK1 to phosphorylate Ykt6 in vitro and successfully confirmed PDK1 to regulate its membrane recruitment. PDK1, a nutrient-sensing axis, could sense upstream metabolic cues, which in turn recruits Ykt6 to traffic Wnts. Ykt6 is an essential gene in yeast and lack of Ykt6 leads to growth defects in vivo. Ykt6 is highly upregulated in aggressive and metastatic tumours. Furthermore, we identified that Ykt6 KD leads to a delay in cell growth and proliferation in human colon cancer cells, which in turn leads to a delay in cell cycle progression. By single-cell transcriptomics, we identified novel Ykt6-regulated genes involved in membrane trafficking, cell cycle, and polyamine metabolism. Polyamines – putrescine, spermidine and spermine are indispensable for cell growth and proliferation. Remarkably, Ykt6 KD led to reduced expression levels of a key metabolic enzyme that acts as a global polyamine rheostat: SAT1. Concordantly, metabolomic profiling revealed lower levels of polyamines and precursor amino acids upon Ykt6 KD cells and one of the polyamines, putrescine, partially rescued the growth defects of Ykt6 KD. In conclusion, this study elucidates a hitherto unknown function of Ykt6 in modulating polyamine homeostasis during cell cycle progression, possibly via the trafficking of polyamine transporters. This fits with the previously established role of Ykt6 in the trafficking of nutrient transporters to PM and growth factors such as Wnt proteins onto exosomes. Taken together, we propose that Ykt6 acts as a valve that adapts Wnt and metabolite levels required for cell growth and therefore, a proper cell cycle progression.de
dc.contributor.coRefereeBastians, Holger Prof. Dr.
dc.subject.engYkt6, Wnt, Cell cycle, PDK1, Polyaminesde
dc.identifier.urnurn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-591F-3-0
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullGOK-MEDIZINde
dc.description.embargoed2022-09-13
dc.identifier.ppn1771998318


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