The objective of this grant is to elucidate the regulation, downstream signaling, and structural dynamics of the glucan phosphatase laforin. Mammalian cells store readily mobilized energy in the form of glycogen, a water-soluble carbohydrate. Recessive mutations in genes encoding the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin disrupt glycogen metabolism and result in a fatal, neurodegenerative epilepsy called Lafora disease (LD). A hallmark of LD is poorly branched, hyperphosphorylated insoluble carbohydrate accumulations called Lafora bodies (LBs), thought to be the causative agent of LD. We established that malin is a single-subunit E3 ubiquitin ligase that ubiquitinates and triggers the degradation of multiple proteins involved in glycogen metabolism. In addition, we discovered that laforin is the founding member of a unique class of phosphatases that dephosphorylate phospho-glucans. These results allowed us to propose molecular mechanisms that cause LD: 1) loss of malin results in LBs due to an imbalance in glycogen metabolism proteins, i.e. protein levels are not properly maintained;2) loss of laforin results in LBs due to glucan hyperphosphorylation that inhibits glucan branching;3) laforin also acts as a targeting subunit for malin, so that loss of laforin disrupts some malin-directed ubiquitination events. While we have made significant strides in determining the molecular mechanisms of LD, we lack an understanding of how these enzymes are regulated. This proposal will elucidate the deficiencies in our current knowledge. We recently identified novel phosphorylation and ubiquitination events on laforin. Phosphorylation and ubiquitination are post-translational modifications that direct changes in protein concentration, enzymatic activity, protein localization, protein-protein interactions, and structural dynamics.
In Aim 1 we will define the in vivo conditions triggering laforin phosphorylation and characterize the functional consequences. We will utilize 1) overexpression and endogenous protein levels in tissue culture cells to monitor the status of laforin localization and modification, 2) assay laforin function in vitro using purified proteins, and 3) utilize a LD mouse model to verify our results.
In Aim 2, we will utilize a similar strategy to determine the affects of ubiquitination on laforin function. In addition, we will define the role of laforin in malin directed ubiquitination, as we have recently discovered that laforin acts as a targeting protein for malin.
In Aim 3, we will determine how perturbations of the structural components of glucan phosphatases contribute to LD. We will utilize Hydrogen- Deuterium exchange mass spectrometry to define the structural dynamics of laforin and x-ray crystallography to determine the structure of a glucan phosphatase. This proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intercalated events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better understanding of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration.

Public Health Relevance

The focus of our work is to determine how loss of function of either of two proteins leads to the fatal, neurodegenerative epilepsy called Lafora disease. Our proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intertwined events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better comprehension of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS070899-03
Application #
8245575
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Stewart, Randall R
Project Start
2010-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$287,799
Indirect Cost
$83,178
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Meekins, David A; Vander Kooi, Craig W; Gentry, Matthew S (2016) Structural mechanisms of plant glucan phosphatases in starch metabolism. FEBS J 283:2427-47
Emanuelle, Shane; Brewer, M Kathryn; Meekins, David A et al. (2016) Unique carbohydrate binding platforms employed by the glucan phosphatases. Cell Mol Life Sci 73:2765-78
Gentry, Matthew S; Brewer, M Kathryn; Vander Kooi, Craig W (2016) Structural biology of glucan phosphatases from humans to plants. Curr Opin Struct Biol 40:62-69
Raththagala, Madushi; Brewer, M Kathryn; Parker, Matthew W et al. (2015) Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease. Mol Cell 57:261-72
Meekins, David A; Raththagala, Madushi; Auger, Kyle D et al. (2015) Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates. J Biol Chem 290:23361-70
Meekins, David A; Raththagala, Madushi; Husodo, Satrio et al. (2014) Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity. Proc Natl Acad Sci U S A 111:7272-7
Jang, Eun Ryoung; Shi, Ping; Bryant, Jamal et al. (2014) HUWE1 is a molecular link controlling RAF-1 activity supported by the Shoc2 scaffold. Mol Cell Biol 34:3579-93
Brewer, M Kathryn; Husodo, Satrio; Dukhande, Vikas V et al. (2014) Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli. BMC Biochem 15:8
Gentry, Matthew S; Romá-Mateo, Carlos; Sanz, Pascual (2013) Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii. FEBS J 280:525-37
Meekins, David A; Guo, Hou-Fu; Husodo, Satrio et al. (2013) Structure of the Arabidopsis glucan phosphatase like sex four2 reveals a unique mechanism for starch dephosphorylation. Plant Cell 25:2302-14

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