April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Antichaperonic Activity of Oxidized -Synuclein
Author Affiliations & Notes
  • I. G. Surgucheva
    Retinal Biology Lab, VA Medical Center, Kansas City, Missouri
    Neurology, Kansas University Medical Center, Kansas City, Kansas
  • K. Cain
    Retinal Biology Lab, VA Medical Center, Kansas City, Missouri
  • E. Dremina
    Pharmacology, Higuchi Bioscience Center, Lawrence, Kansas
  • V. Sharov
    Pharmacology, Higuchi Bioscience Center, Lawrence, Kansas
  • A. Surguchov
    Retinal Biology Lab, VA Medical Center, Kansas City, Missouri
    Neurology, Kansas University Medical Center, Kansas City, Kansas
  • Footnotes
    Commercial Relationships  I.G. Surgucheva, None; K. Cain, None; E. Dremina, None; V. Sharov, None; A. Surguchov, None.
  • Footnotes
    Support  VA Merit Review Grant and The Glaucoma Foundation Grant
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6446. doi:
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    • Get Citation

      I. G. Surgucheva, K. Cain, E. Dremina, V. Sharov, A. Surguchov; Antichaperonic Activity of Oxidized -Synuclein. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6446.

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

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Purpose: : Intrinsically unstructured proteins expressed in eye tissues are prone to aggregate and form deposits causing eye pathology. We previously described such protein deposits in glial cells of the optic nerve in glaucoma patients and in transgenic animals. In this study we investigate what can cause protein aggregation and formation of inclusions.

Methods: : We used intrinsically unstructured (naturally unfolded) proteins synucleins as model to study the mechanism of protein aggregation. Aggregation was monitored by SDS-electrophoresis in a gradient 4-15% polyacrylamide gel and Western blotting. Immortalized cultures of rat optic nerve A7 astrocytes were incubated with Alexa-488 labeled synucleins and the intracellular localization was analyzed by immunocytochemistry. Post-translational modifications of synucleins were revealed by mass-spectrometry.

Results: : Incubation of the proteins at 370C is accompanied by a slow oligomerization (molecular mass ~65 kDa) and aggregation (molecular mass >200 kDa) of α- and γ-synuclein. The neurotransmitter dopamine (DA) significantly accelerates the level of synucleins aggregation. γ-Synuclein most easily forms high molecular weight aggregates (6% monomers and 94% aggregates after overnight incubation of γ-synuclein versus 22% monomers and 78% aggregates for α-synuclein). To find out if oligomerization of γ-synuclein is associated with its chemical modification, we utilized the method of mass spectrometry. The predominant untreated γ-synuclein species had a mass of 13,330.8, whereas after incubation under oxidative conditions the mass increased to 13,363.8 Da. The mass difference corresponds to the addition of two oxygen atoms to γ-synuclein. Further MS-MS analysis showed that this form of the protein contained oxidized methionine 38 (Met38) and tyrosine 39 (Tyr39) in peptide K.TKEGVM*Y*VGAK.T, which corresponds to delta mass found in undigested protein. High propensity of γ-synuclein may be explained by two structural features: a) the presence of hydrophobic stretch of amino acids in the central part of its molecule and b) localization of two oxygen acceptor amino acids Met38 -Tyr39 in the adjacent positions. Incubation of astrocytes with monomeric or aggregated γ-synuclein caused its internalization and accumulation inside the cells in aggregated form.

Conclusions: : Oxidized γ-synuclein easily aggregates and may serve as a core for the aggregation of other proteins, thus expressing antichaperonic properties. Protein aggregation may be an important step in the etiology of glaucoma and other eye pathology.

Keywords: chaperones • astrocyte • optic nerve 

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