GBE1 interactions with membranes: GBE1 interactions with membranes: Implications in APBDImplications in APBD

Rafael Álvarez, David J. López, Maitane Ibarguren and Pablo V. EscribáUniversity of the Balearic Islands, Palma de Mallorca, Spain.

10th Meeting of the APBDRF. New York City, 2013

Glycogen storage disease typesGlycogen storage disease types

Corpora amylacea in the brain cortex of a 56-yr man. A) PAS-hematoxylin stain; bar = 20 m. B) Hematoxylin-eosin stain; bar = 40 m.

Considerations about etiology and therapyConsiderations about etiology and therapy

1. The late onset of APBD suggests a critical cellular event in the etiology of APBD different from mutations.

2. Decline of DHA and PE with age suggests that GBE1-lipid interactions could be associated with APBD.

3. Nutraceutical supplementation with Triheptanoin links lipids with APBD and suggests that MLT approaches could be used to treat this condition.

4. Some authors suggest that misfolded GBE1 may cause APBD. We believe that misfolding could alter the cell localization of GBE1.

GBE1 (OriGene) bindingGBE1 (OriGene) bindingto model membranesto model membranes

GBE1 in U118 cellsGBE1 in U118 cells

Confocal microscopy of GBE1 in U118 cells. Cells were stained with Hoechst 33342 (blue, nucleus) andwith an anti-GBE1 antibody (red). Bar=10 μm.

GBE1 amino acid sequence GBE1 amino acid sequence WT Glucan (1,4-alpha-) branching enzyme 1 ML Databases

WT Glucan (1,4-alpha-) branching enzyme 1 OriGene Tech. Co.

GBE1 amino acid sequence GBE1 amino acid sequence WT Glucan (1,4-alpha-) branching enzyme 1 Escribá

Y329S Glucan (1,4-alpha-) branching enzyme 1 Escribá

GBE1 IN Sf9 MEMBRANESGBE1 IN Sf9 MEMBRANES

GBE1 in Sf9 cell Cytosol and purificationGBE1 in Sf9 cell Cytosol and purification

GBE1 bionding to model membranesGBE1 bionding to model membranes

APBD: Drug design and screeningAPBD: Drug design and screeningTonset (ºC) Tm (ºC) ΔH

(Kcal/mol)Tonset (ºC) TH (ºC) ΔH (Kcal/mol) Effect on TH

POPE 22.8 ± 0.2 23.6 ± 0.2 4.42 ± 0.03 68.3 ± 0.4 69.1 ± 0.5 0.42 ± 0.03 -----

+ 1 mol% Triheptanoin 21.4 ± 0.4 23.1 ± 0.2 5.90 ± 0.22 61.0 ± 0.9 62.5 ± 0.7 0.50 ± 0.06 ↓7 ºC

+ 1 mol% TGM7 21.4 ± 0.1 22.7 ± 0.2 5.88 ± 0.97 66.6 ± 0.6 68.3 ± 0.6 0.51 ± 0.04 ↓1 ºC

+ 1 mol% TG140 22.3± 0.5 24.4 ± 0.3 4.41 ± 0.05 58.7 ± 1.7 63.8 ± 0.7 0.23 ± 0.06 ↓5 ºC

+ 1 mol% TG141 22.3 ± 0.5 23.5 ± 0.2 5.12 ± 0.12 61.0 ± 1.4 64.7 ± 0.8 0.21 ± 0.10 ↓4 ºC

+ 1 mol% TG160 22.7 ± 0.3 23.8 ± 0.2 10.2 ± 0.19 58.2 ± 0.2 58.6 ± 0.2 1.21 ± 0.24 ↓10 ºC

+ 1 mol% TG161 22.6 ± 0.3 23.5 ± 0.2 4.2 ± 0.10 61.3 ± 1.1 64.5 ± 0.7 0.15 ± 0.05 ↓5 ºC

+ 1 mol% TG180 22.8 ± 0.3 23.8 ± 0.2 5.52 ± 0.19 62.7 ± 0.2 63.6 ± 0.4 0.84 ± 0.05 ↓5 ºC

+ 1 mol% TG181 22.6 ± 0.3 23.3 ± 0.2 4.55 ± 0.09 58.5 ± 4.0 60.8 ± 3.3 0.24 ± 0.11 ↓8 ºC

+ 1 mol% TGM1 22.7 ± 0.2 23.8 ± 0.3 5.78 ± 0.08 64.0 ± 0.6 66.0 ± 0.9 0.55 ± 0.06 ↓3 ºC

+ 1 mol% TG182 22.2 ± 0.2 23.4 ± 0.2 5.52 ± 0.07 52.7 ± 5.2 61.6 ± 1.2 0.36 ± 0.11 ↓8 ºC

+ 1 mol% TGM2 21.5 ± 0.2 23.0 ± 0.2 5.78 ± 0.07 56.0 ± 2.1 62.0 ± 1.1 0.67 ± 0.24 ↓7 ºC

+ 1 mol% TG204 22.2 ± 0.4 23.4 ± 0.2 5.68 ± 0.22 58.9 ± 0.2 63.2 ± 0.4 0.42 ± 0.03 ↓5 ºC

+ 1 mol% TGM4 21.9 ± 0. 2 23.2 ± 0.2 3.55 ± 0.22 60.4 ± 1.5 64.6 ± 0.8 0.37 ± 0.09 ↓5 ºC

+ 1 mol% TG226 22.5 ± 0.2 23.5 ± 0.2 5.73 ± 0.10 59.5 ± 2.3 63.7 ± 1.9 0.40 ± 0.07 ↓5 ºC

+ 1 mol% TGM6 22.0 ± 0.1 23.3 ± 0.2 5.54 ± 0.12 59.5 ± 1.3 64.9 ± 0.9 0.48 ± 0.13 ↓4 ºCTable 1. Enthalpies and transition temperatures of POPE mixed with 1 mol% hydroxylated and non-hydroxylated triacylglycerides

Membrane structure modulation

POPE

15ºC

18ºC

80ºC

LL

H

POPE

POPE + 5 mol% TG 2OHOA

15ºC

18ºC

80ºC

Lamellar-to-hexagonal transition temperature (TH) decreases drastically

POPE + 5 mol% TG 2OHOALamellar-to-hexagonal transition temperature (TH)

decreases drastically

APBD FUTURE RESEARCHAPBD FUTURE RESEARCH

1. Determination of our GBE1 proteins (WT and Y329S) activity.2. Protein production and purification.3. Investigate of WT and Y329S GBM1 with different types of membranes4. Investigate the role of TGMs and other LP drugs on these interactions.5. Get a proof of principle for one oe more molecules.6. (GBE1 structure).