Elucidating the role of n-glycanase in… (F32GM161060) – Project Grant to The Leland Stanford Junior…

Abstract: N-glycanase 1 (NGLY1) is a ubiquitously expressed pngase and is the only cytosolic DE-n-glycosidase. Mutations to NGLY1 result in the severe rare genetic disease known as NGLY1 deficiency, characterized by global developmental delay and intellectual disabilities. Despite decades of study, there is limited biochemistry describing NGLY1 activity and no biochemical link between genotype and phenotype in NGLY1 deficiency. Our current understanding of NGLY1 activity links DE-n-glycosylation to cellular homeostasis through proteasome activity. This occurs through DE-n-glycosylation of proteins bound for ER associated degradation (erad) and the activation of a transcription factor, NRF1, by DE-n-glycosylation and nuclear trafficking in response to proteasome insufficiency. Glycosylation is suggested to affect the subcellular trafficking and activation of other transcriptionally active glycoproteins; emerging evidence has established a link between DE-n-glycosylation and nuclear translocation of membrane receptors in the nucleus (mrins) such as PD-L1 and fgfr, which serve as or bind to transcription factors. As a transcriptional regulator, nuclear fgfr is known to act as a global regulator of brain/neuron, muscle/muscle cell, and skeleton/osteoblast and osteoclast development and differentiation. Therefore, i hypothesize that NGLY1 activity is essential for efficient subcellular glycoprotein trafficking, activity, and degradation, and that dysregulation of these processes presents as the NGLY1 deficiency phenotype. Herein i describe a multi-pronged approach consisting of proximity labeling/interactomics (aim 1), and chemoproteomics/fluorescence microscopy (aim 2) to investigate the behavior of NGLY1 substrates in the presence or absence of NGLY1 activity. Furthermore, i will develop am in vitro biochemical model of NGLY1 and erad, measuring the effect of cytosolic protein DE-n-glycosylation on the kinetics of degradation (aim 3). Together, these studies will identify, validate, and characterize novel substrates and modalities relating to the cellular functions of NGLY1 and the etiology of NGLY1 deficiency. Furthermore, these studies will validate NGLY1 as a therapeutic target to modulate the surface presentation of disease-associated membrane receptors for cancer and other diseases. This research will serve as a steppingstone for the candidate to launch his independent career. Herein, a research training plan involving the development of glycobiology research skills, training in scientific communication, cultivation of a professional network, developing group leadership and management skills, and continued training in mentorship and pedagogy, is described in detail. The candidate plans to launch an independent research career based off the blueprint developed here; achieving a molecular-level understanding of complex human rare genetic diseases, with a specific focus on congenital disorders of glycosylation. This research is expected to discover new biology and inform the development of therapeutics for rare disease and common diseases where the etiologies leverage shared pathways.

Elucidating the role of n-glycanase in glycoprotein activation, trafficking, and degradation

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