In objective 1, we aim to map how widespread O-Mannosylation is in yeast. We have now applied our methodology and made significant progress in this objective, and have identified more than 1000 yeast proteins that undergo O-Mannosylation. This progress has brought novel knowledge on which additional biochemical processes are controlled by O-Mannosylation in yeast and we are now preparing a manuscript to disseminate these results. We will continue improving our techniques to enable in-depth analyses of yeast O-Mannosylations; these methods will be applicable in other domains of mass spectrometry based-glycoproteomics and advance our knowledge in related areas of cell biology.
In addition, we have manipulated yeast to assess the dynamics of O-Mannosylation. Using state-of-the-art quantitative mass spectrometry, we have been able to identify specific proteins that undergo dynamic O-Mannosylation in response to stress, which has unveiled new clues that may improve our understanding on how cellular processes adapt to stress in yeast, and by extension, how these analogous processes work in humans.
In objective 2, we aim to identify the biosynthetic machineries (enzymes) that enable O-Mannosylation in yeast. We have employed biochemihal methods to identify enzymatic activities in yeast combined with purification schemes to isolate the specific enzymes responsible for O-Mannosylation. Using this approach, we've made partial progress and identified the sub-cellular fractions with the highest enzyme activities but we have not been able to identify the specific enzymes responsible for O-Mannosylation yet. In parallel, we have also adapted a bioinformatic approach and identified promising candidates that we believed are responsible for the O-Mannosylation in yeast. We are currently analyzing these candidates with biochemical methods to test this hypothesis.
In objective 3, we aim to transfer knowledge gained through our work on O-Mannosylation and apply it to the analogous O-GlcNAcyation processes that takes place in higher eukaryotes. We have now developed analytical methods that are transferable and may be used for analysis of O-GlcNAcylation in other model organisms. We have, as proof-of-principle, applied our techniques to map and understand O-GlcNAcylation in fruit flies and made significant progress. We are currently preparing a manuscript to disseminate these results.
Dissemination:
Our work related to protein O-Mannosylation in eukaryotes has been disseminated in four separate publications, two original research articles and two review articles. These results have been disseminated in Journal of Biological Chemistry (JBC) and Proceedings of National Academy of Sciences USA (PNAS). Of note, the work published in JBC was selected by the editorial board as the representative cell biology paper of 2017, acknowledging the importance and impact of our research.
In addition, the MSCA fellow has been invited to write two review articles, both now published in Current opinion in Structural Biology.
