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Nucleocytoplasmic O-glycosylation in Yeast

Periodic Reporting for period 2 - Yeast-Glyco (Nucleocytoplasmic O-glycosylation in Yeast)

Reporting period: 2018-10-01 to 2019-09-30

Saccharomyces cerevisiae, more commonly known as yeast, are single-cell organism predominantly responsible for breakdown (fermentation) of simple carbohydrates in nature. In science, yeast have for a long time been one of the best characterized organism from the genetic, biochemical and physiological point of view and are continuously used by scientist to explore and understand biological processes in higher eukaryotic organisms, which include e.g. plants, mammals and humans. Yeast utilize a biochemical process known as phosphorylation to control biological processes; specific biological functions, e.g. cell division or carbohydrate fermentation may be activated or deactivated in yeast by phosphorylation. Surprisingly, yeast lack the co-regulatory mechanisms known as O-GlcNAcylation, which is so commonly used by many other eukaryotic organisms (plants, mammals, humans etc.), to control many important biological functions. Dysregulation of O-GlcNAcylation is associated with human illness and it has remained a longstanding conundrum how yeast control biological functions without the involvement of O-GlcNAcylation.
This project is based on our discovery demonstrating that yeast indeed have a co-regulatory mechanisms for controlling biological processes based on a similar mechanisms known as O-Mannosylation and aims to improve our understanding of O-Mannosylations in eukaryotes. More specifically, this research project aims to explore when and where yeast utilize the O-Mannosylation mechanism and how it functions in yeast cell biology. In addition, this project aims to identify and characterize the biosynthetic machineries responsible for O-Mannosylation in yeast. This research will bring novel knowledge on how yeast orchestrates biological processes and advance our understanding on how essential cellular functions are controlled in these organisms. In addition, this will allow us to extrapolate and understand how O-Mannosylation and O-GlcNAcylation functions in higher organisms (e.g. humans) which will improve our understanding of human diseases including diabetes and cancer.
The overall objective is to use biochemical methods and state-of-art technologies (mass spectrometry) to: 1)identify which proteins undergo O-Mannosylation, 2) identify the biosynthetic machineries (enzymes) responsible for O-Mannosylation and 3) transfer knowledge for comparative studies on protein modifications (O-glycosylation) higher eukaryotes.
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.

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.
The project has progressed beyond the state-of-art, and revealed the identity of more than 1000 proteins undergoing O-Mannosylation in yeast. We project that we will be able to collect a larger dataset in the near future and obtain a more detailed map over O-Mannosylations in yeast. Furthermore, we anticipate that we will be able to improve our understanding on how yeast regulate biological processes by understanding the dynamics of the O-Mannosylation system. Identification of the enzymes enabling these modifications will enable us to modify yeast which will bring unique opportunities to improve bio-processing for wide applications in industry and biotechnology.

Furthermore, this knowledge has further expanded our understanding of O-Mannosylations in humans: by applying our technologies to human cells, we have discovered a novel O-Mannosylation pathway and identified the biosynthetic machineries responsible for these modifications. These efforts have led to major breakthroughs in our understanding of O-Mannosylation in humans; dysregulation of this unique type of O-Mannosylation is connected to (previously inexplainable) neurological diseases which cause brain malformations or loss of hearing. Deficiencies in a subset of genes with unknown functions have previously been linked to these disorders but the cause of disease remained unknown. Our discoveries now provide the molecular explanation by identifying the gene products as enzymes responsible for O-Mannosylation on major classes of plasma membrane proteins. We now believe that this unique form of O-Mannosylation is an important functional component, with critical but unexplored roles in human health and disease. The project has thus opened new avenues in biomedical research, revolving around O-Mannosylations, which may transform the understanding of biological processes in humans and open new paths to drug discovery and treatments.
Illustration of mannose modifications (in orange) on cellular components (proteins; white/purple)