Current project: Development of next generation artificial bioorthogonal precursors to study levels and interaction partners of GlcNAc-containing glycoconjugates.
Description: Changes in GlcNAc-containing glycoconjugates are associated with a variety of human diseases, such as cancer, diabetes, and neurodegenerative diseases, however the mechanistic details linking altered glycosylation to disease pathology remain poorly understood. Recent efforts in the development of methods to study biomolecules in their native environment have unlocked the door of bioorthogonal chemistry. Functional group modifications around the sugar hydroxyl groups are tolerated by the biosynthetic pathways and transform them into the corresponding sugar-nucleotide donors. Our work is on the development of accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-Acetylglucosamine (GlcNAc) represents a critical link between cellular metabolism and glycoconjugates, such as O-GlcNAc and N-linked glycans, which regulate an important and ubiquitous cell signaling paradigm, as well as substrate function, localization, and stability. Our goal is to design and synthesis of easy-to-use bioorthogonal tools that can be used by any biomedical researcher to track levels and interaction partners of GlcNAc-containing glycoconjugates, and then to use them to understand their roles in human health and diseases. Our work prioritizes approaches that are simple to implement and makes use of “off-the shelf” reagents and procedures to develop and engineer bioorthogonal next-generation artificial metabolic reporters capable of specifically labeling GlcNAc-containing glycoconjugates and then applying them to pathogenic disease (metabolic dysfunction, neurodegeneration, and cancer) treatment.
Cells are able to catabolize a variety of nutrient fuels to generate energy, with carbohydrate and fatty acid oxidation being dominant drivers of ATP production in many cells outside the brain. This type of fuel flexibility is important for constitutively active tissues (e.g. the heart) or regulators of systemic metabolism (e.g. liver, adipose) to maintain optimal function despite natural fluctuations in the circulating nutrient supply. In addition, however, cells can shift their overall fuel preference in response to persistent conditions such as high fat diet, chronic stress or exercise training. Little is known about the mechanisms that drive this type of fuel shifting but fuel inflexibility has been implicated in the pathology of multiple diseases including cardiomyopathy, diabetes and non-alcoholic fatty liver disease (NAFLD). One candidate mechanism is through protein O-GlcNAcylation, which is dynamically responsive to acute nutrient levels, interactive with energy and metabolic signaling, but at a timescale that typically outlasts its triggering conditions. Mice with a genetically driven partial deletion of the O-GlcNAc removal enzyme O-GlcNAcase (OGA) demonstrate a persistent whole-body fuel shift away from fatty acid oxidation while pharmacological OGA depression delays the natural transition from carbohydrate to fatty acid fuel preference in mice moving from their fed active state to their resting and fasting state. These published data suggest that removal of O-GlcNAcylation from some protein targets may be required for the efficient utilization of fatty acid oxidation in metabolically flexible cell types. My research in the Hanover lab aims to investigate this supposition across three mechanistic hypotheses.
- The O-GlcNAc status of transcription factors/co-factors guide their targeting or activity at the promoters of genes that encode for lipid oxidation proteins.
- O-GlcNAc status influences the activity or stability of proteins that control the availability of fatty acids for oxidation.
- Protein O-GlcNAcylation, whether through transcriptional or direct action, manipulates carnitine homeostasis, which is critical for the mitochondrial uptake of fatty acids.
A key question of interest is whether a non-dominant splice isoform of OGA (sOGA), which has been localized at both the lipid droplet membrane and in mitochondria, might play a particularly active or lipid-sensitive role in any of these hypothesized mechanisms.
Significance/Impact – Not only will these studies expand our understanding of protein O-GlcNAcylation as a context-dependent metabolic rheostat but they may shed light on the unknown function of OGA splice regulation, which has been putatively linked to the manifestation of clinical diabetes. Furthermore, insight into the mechanisms behind adaptive fuel preference may aid in the development of therapies to mimic the metabolic benefits of exercise and fasting or to mitigate conditions of toxic lipid accumulation.
Relevant Publications - Lockridge, A & Hanover, J. A Nexus of Lipid and O-GlcNAc Metabolism in Physiology and Disease. Frontiers in Endocrinology, section Molecular and Structural Endocrinology. Front. Endocrinol. 30 August 2022. Doi: 10.3389/fendo.2022.943576
O-GlcNAc modulation impacts DNA damage repair machinery in highly replicative cells
Highly replicative cells accumulate DNA mutations leading to accumulation of point mutations and chromosome abnormalities. Using mouse embryonic stem cells, mouse embryonic fibroblasts and cancer cells, Dr. Cruz aims to understand how O-GlcNAc modulation impacts replication stress, DNA damage machinery and cell fate. Dr. Cruz’s work has focused on how O-GlcNAc acts in cell plasticity and differentiation with the goal of defining metabolic and molecular O-GlcNAc targets that are involved in the response to replicative stress.
Metabolites such as glucose or glucosamine get converted into their corresponding nucleotide sugar UDP-GlcNAc through the hexosamine biosynthetic pathway (HBP) or salvage pathway. These nucleotide sugars can subsequently be used for numerous post translational modifications (PTMs) such as the modification of glycans, lipids, proteins, etc. Since these monosaccharide precursors are often shared by several pathways, selectivity for or against any particular type of modification has been difficult to attain. Functional group modifications around the sugar hydroxyl groups are tolerated by the HBP, allowing for the transformation of these bioorthogonal sugars into their corresponding nucleotide-sugar donors. This provides a unique opportunity to develop and engineer artificial metabolic chemical reporters (MCRs) that allow for monitoring of the PTMs in vivo. My work involves the development of accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates by interrogating the selectivity and labeling partners of these bioorthogonal sugars. The use of bioorthogonal sugars has helped increase our understanding of the cellular glycosylation of lipids and proteins, the imaging of glycans, nucleic acids and cellular organelles, the identification and tracking of active enzymes, and the mechanisms of click and release drug delivery. My project is to design and synthesize bioorthogonal artificial MCRs capable of specifically labeling GlcNAc-containing glycoconjugates and to apply them to the study of pathogenic disease (metabolic dysfunction, neurodegeneration, and cancer), as well as its treatment.