Aufklärung des Glykoms von Kragengeißeltierchen zur Evaluierung des Einflusses von Glykosylierungen bei der Entstehung der Multizellularität
Zusammenfassung der Projektergebnisse
The aim of the project was to understand the role of protein glycosylation in the model organism choanoflagellates, which during their development go from a single cellular to multicellular state. To this end a glycoproteomic approach called Isotope Targeted Glycoproteomics (IsoTaG) should be used to map the glycome of the model organisms. IsoTaG is based on linking metabolically labelled glycans to an IsoTaG-probe that is designed to enrich tagged glycoproteins on a solid support and recode the specific isotope pattern that they display. However, the choanoflagellates could not be provided in sufficient quantities, which eventually led to a change of focus in the project. In the scope of my project I was able to design the next generation of bioortogonal probes for the investigation of glycoproteins that can be used with the IsoTag technology. As an advantage towards the conventional existing IsoTaG probes, the new design is based on individual building blocks that can be combined in a large variety to give a number of different biorthogonal probes that allow a combination of multiple analytical applications. The design is based on a dipeptide core structure, where a biorthogonal handle can be C-terminal introduced and the N-terminus can be equipped with different analytical tools, such as fluorophores, affinity probes, cross-linking or radioactive label. The probes are synthesized via established methods of solid phase peptide synthesis, which significantly simplified the synthetic demand in comparison to the conventional IsoTaG probes and thereby significantly increased the accessibility of analytical biorthogonal probes. As an additional advantage, the new probes are not restricted to a Mass Spectrometry-based application. The freely combinable toolbox-principle allows the synthesis of probes that can be used in a combination of analytical approaches. A probe that contains for instance an isotopic label and a fluorophore allows the combination of a MS analysis with an imaging experiment. In a similar manna, an isolation (pull down) approach could be combined with a subsequent MS analysis. Many other combinations are also possible. The new collection of bioortogonal probes is summarized under the name “Easy tag” platform (provisional patent filed with Stanford licensing office). In addition to establishing a new generation of bioortogonal probes for studying glycoproteins, I investigated of the molecular mechanisms that underlie the pathology of a rare genetic glycosylation disorder called NGLY1 deficiency. This autosomal recessive disorder is caused by a heterozygous inactivating mutation in the ngly1 gene, which leads to a lack of the enzyme responsible for de-N- glycosylating misfolded glycoproteins as part of the ER-associated degradation (ERAD). Patients exhibit a spectrum of severe symptoms, such as developmental delay, hypotonia, seizures that could prior to my work not be linked to the missing enzyme. During my project, I was able to identify a so far unknown function of NGLY1. In a combination of biochemical assays, I demonstrated that NGLY1 activity is crucial for the activation of an important transcription factor of the "cap'n'collar" (CNC) bZIP family; the Nuclear Factor Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1). Nrf1 is important to maintain proteasome homeostasis in cells. It contains multiple N-Glycans and is activated via the ERAD pathway. I was able to demonstrate that the de-N-glycosylation by NGLY1 is crucial for its proper function. In NGLY1 deficient cells, Nrf1 is mis-processed, shows altered subcellular localization and impaired activation. Since Nrf1 is a master regulator in cells and important during embryonic development, we concluded that impaired de- N-glycosylation of Nrf1 in absence of NGLY1 results in an abrogated bounce back response that contributes to the disease symptoms of NGLY1 deficiency. In addition, this mechanistic link lead to a new potential cancer therapy for certain blood cancers such as multiple myeloma that is currently treated with proteasome inhibitors and shows a high rate of resistance mechanism involving Nrf1. In a small targeted library screen, I identified a peptide-based inhibitor of NGLY1. The compound VW139 showed a potentiation of the efficacy of proteasome inhibitors when given as a co-treatment in several MM and leukemia cell lines. The idea was patented with Stanford’s OTL.
Projektbezogene Publikationen (Auswahl)
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“Inhibition of NGLY1 inactivates the transcription factor Nrf1 and potentiates proteasome inhibitor cytotoxicity” ACS Cent. Sci. 2017, 3, 1143-1155
F. M. Tomlin§, U. I. M. Gerling-Driessen§, Y-C. Liu, R. A. Flynn, J. R. Vangala, C. S. Lentz, S Clauder- Muenster, P. Jakob, W. F. Mueller, D. Ordonez, M. Paulsen, N. Matsui, D. Foley, A. Rafalko, T. Suzuki, M. Bogyo, L. M. Steinmetz, S. K. Radhakrishnan, C. R. Bertozzi
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“Agents that inhibit NGLY1 and methods of use thereof” PCT/US2018/015380
Carolyn R. Bertozzi, Frederick M. Tomlin, Ulla I.M. Gerling-Driessen
