The National Institute of Diabetes & Digestive & Kidney Diseases

NIDDK Home > Laboratories at NIDDK > Intramural Faculty

Dona C. Love and John A. Hanover Current Research

Dona C. Love and John A. Hanover

The following is a brief overview of our research interests, accomplishments and future plans.

Research Focus I: The Hexosamine Signaling Pathway: Key Cellular Sensor of Nutrient Excess deregulated in Diabetes

We have proposed that the Hexosamine Signaling Pathway, terminating in the addition of O-GlcNAc to target proteins, is one of the key cellular responses to nutrient excess (See Figure 1) (1, 2). Levels of the sugar nucleotide UDP-GlcNAc are highly responsive to concentrations of glucose, amino acid, and lipids. UDP-GlcNAc is utililzed by O-GlcNAc transferase to modify target proteins. The large number of O-GlcNAc modified proteins includes transcription factors, nuclear pores, proteasomes and signaling kinases. We recently solved the structure of the TPR domain of OGT (see OGT sructure, right). The TPR domain forms a homodimeric complex that allows interaction with the numerous intracellular substrates of OGT. In addition, alternatively spliced isoforms of OGT are differentially targeted to nucleus and mitochondria (See image, right). We have recently shown that overexpression of the O-GlcNAc Transferase (OGT) in muscle and fat leads to insulin resistance and hyperleptinemia (3). The other enzyme of hexosamine signaling, O-GlcNAcase was recently shown to be a NIDDM susceptibility gene associated with obesity in the Mexican American population (4). We have found that one isoform of this enzyme is associated with lipid droplets while the other is nuclear and cytoplasmic (Kim, E. J., Love DC. and Hanover JA, in press). Interestingly, the polymorphism most closely associated with NIDDM maps to the region of the protein that diverges in these two isoforms (5). Current efforts are aimed at understanding the relationship between O-GlcNAc metabolism and lipid storage and mobilization. Much of this work centers on chemical biology approaches to inhibitor design and optimization. We also recently developed a novel imaging reagent to examine O-GlcNAc removal - a caged GlcNAc fluorogenic substrate (See Right).

To examine the function of hexosamine signaling in a genetically amenable organism, we have examined null alleles of OGT (OGT-1) and the O-GlcNAcase (OGA-1) in Caenorhabditis elegans that are viable and fertile. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity and dauer formation. We demonstrate that the OGT and OGA null mutants exhibit striking metabolic changes manifested in an elevation in trehalose levels and glycogen stores with a concomitant decrease in triglycerides levels. The OGT knockout suppresses dauer larvae formation induced by a temperature sensitive allele of the insulin-like receptor gene daf-2. The OGA knockout enhances dauer formation suggesting the development of insulin resistance in the absence of O-GlcNAcase activity (See Figure at right). Our findings demonstrate that OGT and O-GlcNAcase modulate insulin action in C. elegans and provide a unique genetic model for examining the role of O-GlcNAc in cellular signaling, insulin resistance and obesity (6). This work is carried out in collaboration with Michael Krause.

Future Plans:

1. Genetic analysis of C. elegans insulin-like signaling to determine where insulin signaling is altered by the hexosamine signaling pathway.
2. Use of small molecule inhibitors of the enzymes of O-GlcNAc cycling to block insulin resistance.
3. Use of our recently developed novel imaging reagents to examine O-GlcNAc modification in living cells.

References

1. Lubas, W. A., Frank, D. W., Krause, M. & Hanover, J. A. (1997) J Biol Chem 272, 9316-9324.
2. Hanover, J. A. (2001) FASEB J 15, 1865-1876.
3. McClain, D. A., Lubas, W. A., Cooksey, R. C., Hazel, M., Parker, G. J., Love, D. C. & Hanover, J. A. (2002) Proc Natl Acad Sci U S A 99, 10695-10699.
4. Duggirala, R., Bangero, J., Almasy, L., Dyer, D. T., Williams, K. J., Leach, R. J., O'Connell, P., Stern, M. P. & O'brien, R. M. (1999) Am. J. Hum. Genet. 64, 1127-1140.
5. Lehman, D. M., Fu, D. J., Freeman, A. B., Hunt, K. J., Leach, R. J., Johnson-Pais, T., Hamlington, J., Dyer, T. D., Arya, R., Abboud, H., Goring, H. H., Duggirala, R., Blangero, J., Konrad, R. J. & Stern, M. P. (2005) Diabetes 54, 1214-1221.
6. Hanover, J. A., Forsythe, M. E., Hennessey, P. T., Brodigan, T. M., Love, D. C., Ashwell, G. & Krause, M. (2005) PNAS 102, 11266-71.

Research Focus II: A Role of the Hexosamine Signaling Pathway in Tauopathy: A C. elegans model

The hexosamine signaling pathway terminating in O-GlcNAc addition has been proposed to play a key role in neurodegeneration (1). In these disorders, the proteins accumulating as aggregates such as tau and amyloid precursor protein are heavily modified with O-GlcNAc and are also phosphorylated. To examine the role of O-GlcNAc in tauopathy we have developed a C. elegans model of tauopathy in which the enzymes of hexosamine signaling have been systematically deleted. This strategy is based on previous work demonstrating the utility of C. elegans in modeling the one form of tauopathy, FTDP-17 (2-4). We find that the loss of OGT-1, the O-GlcNAc transferase protects the nematode from human tau-induced neuropathy. This protection is associated with a decrease in the hyperphosphorylation of tau associated with aggregate formation (See Figure 2). This genetically amenable model of tauopathy is being exploited to examine how removal of OGT-1 exerts its protective effect on tau-induced neuropathy. Small molecule inhibitors are being explored as potential therapeutic agents. This work is carried out in collaboration with Michael Krause.

Future Plans:

1. Determine whether the neuroprotective effects of the C. elegans OGT knockout can be mimicked using small molecule inhibitors of OGT.
2. Examination of other neurodegeneration models in the OGT-1 null allele of C. elegans.
3. Use of Novel Imaging reagents to examine O-GlcNAc metabolism in neurons of C. elegans and cultured mammalian neurons (See also Research Focus I).

References



1. Robertson, L. A., Moya, K. L. & Breen, K. C. (2004) J Alzheimers Dis 6, 489-495.
2. Goedert, M. (2003) Proc Natl Acad Sci U S A 100, 9653-9655.
3. Kraemer, B. C., Zhang, B., Leverenz, J. B., Thomas, J. H., Trojanowski, J. Q. & Schellenberg, G. D. (2003) Proc Natl Acad Sci U S A 100, 9980-9985.
4. Lee, V. M., Kenyon, T. K. & Trojanowski, J. Q. (2005) Biochim Biophys Acta 1739, 251-259.

Research Focus III: Calmodulin-Dependent Nuclear Import

We previously identified a Ran-independent nuclear import pathway mediated by calmodulin, the ubiquitous nucleocytoplasmic calcium sensor. This evolutionarily ancient, calcium-dependent import pathway was initially proposed to facilitate uptake of a distinct subset of nuclear proteins during cell activation (1)(See Figure 3). Calmodulin mediated transport is now known to be highly conserved thoughout metazoan evolution. Furthermore, the physiological significance of the Ca⁺²-calmodulin-regulated transport pathway has been solidified by the finding that defects in calmodulin-dependent nuclear import underlie certain forms of human sex reversal (2, 3). The HMG-box architectural transcription factors SRY and SOX9 must enter the nucleus of Sertoli cells and bind tightly to target DNA for proper male gonad development. Thus, SRY acts as the primary trigger for maleness; nuclear transport of SRY isrequired to release this trigger. In a subset of human patients with autosomal sex reversal (Swyer’s syndrome, Campomelic dysplasia) nuclear transport of SRY and SOX9 is hindered. The mutations associated with these defects reside in a conserved calmodulin-binding motif near the amino terminus of SRY and SOX9. Thus, the SOX family of transcription factors appears to use Ca⁺²/calmodulin both as import receptor and molecular switch allowing for nuclear import and DNA binding. A similar autosomal sex reversal phenotype occurs when three insulin-related receptors are ablated in mice suggesting that intracellular signaling cascades may impact the normal functions of SRY and SOX9 (4, 5). The import of nuclear proteins by calmodulin, while functionally redundant with the canonic Ran-dependent pathway, is subject to independent regulation by intracellular Ca+2 mobilization. Perhaps owing to this redundancy, abnormalities in either calmodulin-dependent import or Ran-dependent import emerge as defects in patients with autosomal sex-reversal. These sex-reversal syndromes represent the first direct examples of a defect in a nuclear import pathway leading to human disease. We are currently involved in screens for small molecules to inhibit calmodulin-dependent import.

To study this pathway using a genetically amenable system we have examined calmodulin-dependent import in the yeast S. cerevisae. We have isolated mutants defective in calmodulin-dependent import in yeast and are characterizing the genes responsible. The yeast studies are carried out in collaboration with Will Prinz.

Future Plans:

1. Complementation analysis of yeast mutants defective in Calmodulin-dependent nuclear import.
2. Examine the structure of Calmodulin-architectural transcription factor complexes
3. Focused small molecule screens in yeast and mammalian cells to identify specific inhibitors of Calmodulin-dependent import.
4. Identification of other protein utilizing calmodulin dependent nuclear import.

References



1. Sweitzer, T. D. & Hanover, J. A. (1996) Proc Natl Acad Sci U S A 93, 14574-14579.
2. Argentaro, A., Sim, H., Kelly, S., Preiss, S., Clayton, A., Jans, D. A. & Harley, V. R. (2003) J Biol Chem 278, 33839-33847.
3. Harley, V. R., Layfield, S., Mitchell, C. L., Forwood, J. K., John, A. P., Briggs, L. J., McDowall, S. G. & Jans, D. A. (2003) Proc Natl Acad Sci U S A 100, 7045-7050.
4. Nef, S., Verma-Kurvari, S., Merenmies, J., Vassalli, J. D., Efstratiadis, A., Accili, D. & Parada, L. F. (2003) Nature 426, 291-295.
5. Koopman, P. (2003) Nature 426, 241.

Staff Members

Eun Ju Kim, Ph.D.,

Myung Sun Lee, Ph.D.,

Brooke Lazarus, Ph.D.,

Michele Forsythe, Ph.D.,

Chithra Keembiyehetty, Ph.D.,

Dona C. Love, Ph.D., Staff Scientist

John Hanover, Ph.D.,

Peng Wang, Ph.D.,

Marcy Comley, M.S.,

Osamu Sekine, M.D., Ph.D.,

Gayani Weersinghe, B.S.,

Salil Ghosh, Ph.D.,