PI: Mitra, Sharmistha
Glycogen is the largest soluble macromolecule in the cell and serves as a critical energy store in tissues. Co-
ordinated actions by multiple enzymes control regular branching, radial spherical growth of the molecule
ensuring the solubility, which is critical for tissue homeostasis. Regulatory enzymes of the glycogenesis
process includes at least three E3 ubiquitin ligases, dedicated to securing glycogen’s spherical architecture.
Absence of any of them leads to a glycogen structure akin to the insoluble starch amylopectin (now called
polyglucosans). This process of forming polyglucosans is called amylopectinosis. Polyglucosans tend to
aggregate producing insoluble deposits called polyglucosan bodies (PB). At the tissue level, precipitated PBs
lead to untreatable pathologies such as fatal cardiomyopathy (in heart), disabling skeletal-myopathy (muscle),
motor neuron loss (central nervous system) and neurodegeneration (brain). How deficiencies of each of these
ubiquitin ligases (or their interacting proteins) and through what substrates and pathways, result in the
derangement of glycogen structure is not known. In the current proposal, the PI proposes to work with Linear
Ubiquitin Chain Assembly Complex (LUBAC) to identify its role in glycogen solubility control. LUBAC is a multi-
protein complex composed of two E3 ubiquitin ligases – RBCK1, HOIP and an adaptor protein SHARPIN.
LUBAC-deficient patients exhibit PBs in different organs, especially skeletal and cardiac muscle resulting in
myopathy and cardiomyopathy with heart failure. This emphasizes LUBAC’s important role in glycogen
metabolism. To date, glycogen metabolism related LUBAC substrate(s) and associated molecular mechanisms
are not known. Utilizing newly created mouse models, cell lines and novel approaches, the proposed work
tests the central hypothesis that LUBAC downregulates the activity of glycogen synthase (GS) and
ubiquitinates longer less-branched glycogen chains to keep the glycogen soluble. Aim 1 of the research uses
two newly created mouse models of LUBAC deficiency to understand Rbck1’s glycogen association.
Furthermore, to understand the mechanisms of amylopectinosis, the PI investigates the influence of glycogen
phosphate, and muscle/brain cell specificity for PB accumulation. In Aim 2, utilizing newly created
overexpression cell lines and mouse models, a detailed mechanistic pathway by which Rbck1 deficiency
results in high GS activity is tested. Additionally, aim 2 by using a novel in-vivo approach, tests the possibility of
Rbck1 directly ubiquitinating long, precipitation-prone glycogen chains. Finally, in aim 3, using novel cell
models and quantitative proteomics, the proposal identifies additional pathways/substrates for GS activation.
Taken together, the proposed aims comprehensively study LUBAC’s function in glycogen solubility with novel
understandings of its role in amylopectinosis. Deeper understandings of LUBAC mediated control of glycogen
metabolism will not only provide new insights towards developing a treatment for a fatal rare condition, but also
have implications in other common disease research such as cancers and Alzheimer’s disease where both
glycogen metabolism and LUBAC are often dysregulated.