Nutrient-Mediated Signaling Unit

Head of the Unit:

Dr. Pascual Sanz Bigorra
Instituto de Biomedicina de Valencia (CSIC)
Jaime Roig, 11
46010-Valencia
Tel. 34-96-3391779
Fax. 34-96-3690800
e-mail: sanz@ibv.csic.es

Summary.

At the moment, the Unit carries out two complementary research lines:


1) Study of the glucose signalling process and its implications in pathophysiology.
2) Study of the molecular bases of the progressive myoclonus epilepsy of the Lafora type.

1) Study of the glucose signalling process and its implications in pathophysiology.

Pancreatic beta cells regulate the synthesis and secretion of insulin in response to the levels of glucose in blood. The components that regulate this glucose signalling event are the glucose transporter GLUT2, which facilitates the entry of the sugar inside the cell, and glucokinase (GK), which phosphorylates glucose to yield glucose-6P. Glucokinase has been described as the “glucose sensor” in pancreatic beta cells. In fact, insulin secretion directly correlates with the enzymatic activity of glucokinase, and alterations in the activity of this enzyme may cause from a particular form of monogenic diabetes (MODY2), to different forms of hyperinsulinism. Recently, a third component which can regulate insulin secretion in response to glucose has been described; it is the AMP-activated protein kinase (AMPK), which acts as a cellular energy sensor. Low levels of glucose in blood activate this kinase, what leads to the inhibition of insulin synthesis and secretion, among other physiological effects. Since the activity of AMPK inversely correlates with the levels of glucose in blood, we are studying whether glucokinase participates in the inhibition of the activity of AMPK in response to glucose. With this aim, we are studying the inactivation of AMPK in response to glucose in cells lacking or not glucokinase. It has also been described that in the presence of glucose the activity of the protein phosphatase 1 (PP1) is increased. For this reason we are also studying whether glucokinase participates in the activation of PP1 in response to glucose. Since inactivation of AMPK correlates with a dephosphorylation of its catalytic subunit, we are studying whether PP1 is involved in the dephosphorylation and inactivation of AMPK in response to glucose.


            In parallel, our group participates in the characterization of new mutations in the glucokinase gene (GCK) that are responsible either of an inactivation of the resulting enzyme, leading to monogenic diabetes MODY2, or to an overactivation of the resulting enzyme, leading to a particular form of hypoglycaemic hyperinsulinism of the infancy (PHHI).


2) Study of the molecular bases of the progressive myoclonus epilepsy of the Lafora type.
Progressive myoclonus epilepsy of the Lafora type (Lafora disease, LD, OMIM 274780) is a rare disease characterized by the presence of neurodegeneration, epilepsy and accumulation of poorly branched polyglycosans in different tissues. This disease leads to the death of the patient around 10 years from the onset. At the moment only a limited knowledge about the molecular bases of the disease is known and there is no available treatment. LD is a recessive autosomic disease and so far two genes have been described to be involved in the pathogenesis of the disease: EPM2A and EPM2B. EPM2A codes for a protein called laforin which has a carbohydrate binding domain (CBD) at the N-terminus and a dual specificity phosphatase (DSP) domain at the C-terminus. EPM2B codes for a protein called malin (with E3-ubiquitin ligase activity), which has a RING finger domain at the N-terminus and six NHL domains at the C-terminus involved in protein-protein interaction. Recently, our group has described that laforin and malin form a functional complex, suggesting that both proteins participate in a common physiological process and justifying why patients with mutation in either EPM2A or EPM2B are neurologically and histologically indistinguishable. One of the functions of this complex is to downregulate the levels of R5/PTG, a protein involved in the upregulation of glycogen synthesis. This function would explain why in the absence of a functional laforin-malin complex, cells would accumulate polyglycosans (a pathological determinant of the disease). In the complex, laforin acts as a targeting subunit of malin, being able to recognize different substrates that eventually will be ubiquitinated by malin and targeted for degradation. In addition to the role that laforin and malin may have on the regulation of glycogen synthesis, these proteins have alternative roles in cellular physiology. For this reason, we are studying the involvement laforin and malin in the regulation of different signalling pathways, such as those involving AMPK. These new regulatory functions of laforin and malin will help us to understand the pathophysiology of the disease and to define possible therapeutic targets.

            Recently, it has been described that laforin and malin are localized around the endoplasmic reticulum (ER) and that, after conditions of proteasome inhibition, these two proteins form aggregates that include ER chaperones, E2-ubiquin ligases and proteasome subunits. Since this manifestation is similar to other conditions of ER-stress, and since it has been described that maintained ER-stress may lead to apoptosis and neurodegeneration (another pathologic determinant of Lafora disease), we are studying whether the process of ER-stress is altered in samples from Lafora disease patients. To confirm this point we are also using cultured cell lines lacking either laforin or malin and animal models of the disease (EPM2A or EPM2B KO mice).

 

Key words.

Glucose signalling, glucose repression, glucose induction, glucokinase, AMP-activated protein kinase, diabetes, yeast, hexokinase PII (Hxk2), protein kinase Snf1.


Protein-protein interaction, two-hybrid analysis, pull-down assays with GST, co-immunoprecipitation methods, protein phosphorylation studies.

 

Selected Papers.

Pedelini, L., Garcia-Gimeno, A., Marina, A., Gomez-Zumaquero, J.M., Rodriguez-Badia, P., Lopez-Enriquez, S., Soriguer, F.C., Cuesta-Muñoz, A.L. and Sanz, P. “Structure-function analysis of alpha5 and alpha13 helixes of human glucokinase: description of two novel activating mutations” Protein Science 14, 2080-2086 (2005).

Solaz-Fuster, M.C., Gimeno-Alcañiz, J.V., Casado, M. & Sanz, P. “TRIP6 tranascriptional co-activator is a novel substrate of AMP-activated protein kinase”. Cellular Signalling. 18, 1702-1712 (2006).

Sanz, P. “Yeast as a model system to study glucose-mediated signalling and response”. Frontiers in Bioscience. 12, 2358-2371 (2007).

Viana, R., Towler, M., Pan, D.A., Carling, D., Viollet, B., Hardie, DG. and Sanz, P. “AMP-activated protein kinase gamma subunits interact with beta subunits via a conserved sequence immediately N-terminal to the Bateman domains”. J. Biol. Chem. 282, 16117-16125 (2007).

Solaz-Fuster, M.C., Gimeno-Alcañiz, J.V., Ros, S., Fernandez-Sanchez, M.E., Garcia-Fojeda, B., Criado, O., Vilchez, D., Domínguez, J., Garcia-Rocha, M., Sanchez-Piris, M., Aguado, C., Knecht, E., Serratosa, J., Guinovart, J.J., Sanz, P.* and Rodríguez de Cordoba, S. “Regulation of glycogen síntesis by the laforin-malin complex is modulated by the AMP-activated protein kinase”. Human Mol. Genet. 17, 667-678 (2008) (*co-senior author).

Viana, R., Aguado, C., Esteban, I., Moreno, D., Viollet, B., Knecht, E., and Sanz, P. “Role of AMP-activated protein kinase in autophagy and proteasome function”. Biochem. Biophys Res. Commun. 369, 964-968 (2008).

Estalella, I., Garcia-Gimeno, M.A., Marina, A., Castaño, L. and Sanz, P. “Biochemical characterization of novel glucokinase mutations isolated from Spanish maturity onset diabetes of the young (MODY2) patients”. J. Human Genetics. 53, 460-466 (2008).

Moreno, D., Knecht, E., Viollet, B.,and Sanz, P. “A769662, a novel activator of AMP-activated protein kinase, inhibits non-proteolytic components of the 26S proteasome by an AMPK-independent mechanism”. FEBS Letters. 582, 2650-2654 (2008).

Riera, A., Ahuatzi, D., Herrero, P., Garcia-Gimeno, M.A., Sanz, P. and Moreno, F. Human pancreatic beta-cell glucokinase: subcellular localization and glucose repression signalling function in the yeast cell”. Biochemical J. 415, 233-239 (2008).

Sanz, P. “AMP-activated protein kinase: structure and regulation”. Current Prot. Pept. Sci. 9, 478-492 (2008).

Vernia, S., Solaz-Fuster, M.C., Gimeno-Alcañiz, J.V., Rubio, T., Garcia-Haro, L., Foretz, M., Rodriguez de Cordoba, S., and Sanz, P. “AMP-activated proteína kinase phosphorylates R5/PTG, the glycogen targeting subunit of the R5/PTG-PP1 holoenzyme and accelerates its downregulation by the laforin-malin complex. J. Biol. Chem. 284, 8247-8255 (2009).

Meissner, T., Marquard, J., Cobo-Vuilleumier, N., Maringa, M., Rodriguez-Bada, P., Garcia-Gimeno, M.A., Castro, M.J., Aledo, J.C., Baixeras, E., Weber, J., Olek, K., Sanz, P., Mayatepek, E. and Cuesta-Muñoz, A.L. Diagnostic difficulties in glucokinase hyperinsulinism. Horm. Metab. Res. 41, 320-326 (2009).

Vernia, S., Rubio, T., Heredia, M., Rodriguez de Cordoba, S. and Sanz, P. “Increased endoplasmic reticulum stress and decreased proteasomal function in Lafora disease models lacking the phosphatase laforin”. PLoS ONE 4, e5907 (2009).

Moreno, D., Viana, R. and Sanz, P. “Two-hybrid analysis identifies PSMD11, a non-ATPase subunit of the proteasome, as a novel interaction partner of AMP-activated protein kinase”. Int. J. Biochem Cell Biol. 41, 2431-2439 (2009).

Barbetti, F., Cobo-Vuilleumier, N., Dionisi-Vici, C., Toni, S., Ciampalini, P., Massa, O., Rodriguez-Bada, P., Colombo, C., Lenzi, L., Garcia-Gimeno, M.A., Bermudez F.J., Rodriguez de Fonseca F., Banin, P., Aledo, J.C., Baixeras, E., Sanz, P. and Cuesta Muñoz, A.L. “Opposite clinical phenotypes of glucokinase disease: description of a novel activating mutation and contiguous inactivating mutations in human glucokinase (GCK) gene”. Molecular Endocrionol. 23, 1983-1989 (2009).