April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Glycolytic Enzyme Modulates the Cellular Distribution and Isomerase Activity of Rpe65
Author Affiliations & Notes
  • P. Brabet
    INSERM, Montpellier, France
  • K. Chekroud
    INSERM, Montpellier, France
  • L. Guillou
    INSERM, Montpellier, France
  • J. Andries
    INSERM, Montpellier, France
  • M. O. Péquignot
    INSERM, Montpellier, France
  • C. P. Hamel
    INSERM, Montpellier, France
  • Footnotes
    Commercial Relationships  P. Brabet, None; K. Chekroud, None; L. Guillou, None; J. Andries, None; M.O. Péquignot, None; C.P. Hamel, None.
  • Footnotes
    Support  Fédération des Aveugles et Handicapés Visuels de France, IRRP, Retina France, SOS Rétinite, Région Languedoc Roussillon, French Ministry for National Education, and INSERM.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3400. doi:
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      P. Brabet, K. Chekroud, L. Guillou, J. Andries, M. O. Péquignot, C. P. Hamel; Glycolytic Enzyme Modulates the Cellular Distribution and Isomerase Activity of Rpe65. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3400.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : we had identified two glycolytic enzymes, aldolase A and pyruvate kinase M2, as proteins interacting with RPE65. These proteins are known to form a complex with RPE65, CRALBP and actin in RPE microsomes (JS Crabb et al. Invest Ophthalmol Vis Sci 2006; 47: E-Abstract 2039). We demonstrated these protein interactions using two methods, the two-hybrid yeast system and GST pull-down. Our next objective was to assess whether these interactions could regulate the cellular distribution and activity of isomerase RPE65.

Methods: : porcine RPE cells were split by differential centrifugation to isolate a subcellular fraction enriched in RPE65, aldolase A, and F-actin. This subcellular fration was incubated with substrates for RPE65 and aldolase A or with cytochalasin D to disrupt actin microfilaments and proteins were analyzed by Western blot. HEK293 cells were transfected, selected with Geneticin for stable expression of fusion proteins, GFP-RPE65 and LRAT-CFP, and then sorted by FACS. HEK293LR cells were incubated with vitamin A and the production of all trans-retinyl esters and 11-cis retinol was followed by reverse HPLC. Other substrates or inhibitors of aldolase A and the cytochalsin D was added to vitamin A. In parallel, the mobility of fusion proteins GFPnRPE65 was studied by FRAP experiments using a confocal microscope Zeiss LSM 5 DUO Laser Scanning.

Results: : Fructose 1, 6 diphosphate (F1,6diP), glyceraldehyde-3 phosphate (G-3P) and dihydroacetone phosphate (DHAP), the substrate and the products of aldolase A, respectively, separate the aldolase A from the particulate fraction, which contains the F-actin, while F6P had no effect. Meanwhile, a significant fraction of RPE65 dissociates from the particulate to the cytosolic fraction in the presence of G-3P and F1,6diP. On the contrary 100 µM Ca2 + promotes the association of RPE65 to the particulate fraction, but it has not abrogated its dissociation in the presence of G-3P and F1,6diP. HEK293LR cells showed both LRAT and RPE65 activities after the addition of vitamin A in culture, and fluorochromes GFP and CFP co-located with the ER-Traker. Incubation of HEK293LR cells with 2 µg / ml cytochalasin D for 1 hour disrupted the actin filaments, causing their aggregation along the cell periphery. Similarly, the green fluorescent RPE65 linked to co-locate these actin aggregates. Photo-bleaching of GFPnRPE65 showed that it was possible to measure its dynamics in living HEK293LR cells subject to various stimuli and metabolic inhibitions.

Conclusions: : We confirmed that the aldolase A and RPE65 may complex in vitro and in the cell and that their interaction could modulate the mobility, cellular distribution and isomerase activity of the RPE65.

Keywords: metabolism • retinoids/retinoid binding proteins • enzymes/enzyme inhibitors 

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