ABSTRACT
BACKGROUND: Isomerization of aspartate to isoaspartate (isoAsp) on aging causes protein damage and malfunction. Protein-L-isoaspartyl methyltransferase (PIMT) performs a neuroprotective role by repairing such residues. A hexapeptide, Val-Tyr-Pro-(isoAsp)-His-Ala (VA6), a substrate of PIMT, is shown to form fibrils, while the normal Asp-containing peptide does not. Considering the role of PIMT against epileptic seizure, the combined effect of PIMT and two antiepileptic drugs (AEDs) (valproic acid and stiripentol) was investigated for anti-fibrillation activity. METHODS: Structural/functional modulations due to the binding of AEDs to PIMT were investigated using biophysical techniques. Thioflavin T (ThT) fluorescence assay and microscopic methods were employed to study fibril formation by VA6. In vitro experiments with PC12 cells were carried out with PIMT/AEDs. RESULTS: ThT assay indicated reduction of fibrillation of VA6 by PIMT. AEDs stabilize PIMT, bind close to the cofactor binding site, possibly exerting allosteric effect, increase the enzymatic activity, and anti-fibrillation efficacy. Furthermore, Aß42, implicated in Alzheimer's disease, undergoes ß-sheet to α-helix transition in presence of PIMT. Studies with PC12 derived neurons showed that PIMT and PIMT/AEDs exerted neuroprotective effect against anti-NGF induced neurotoxicity. This was further validated against neurotoxicity induced by Aß42 in primary rat cortical neurons. CONCLUSIONS: The study provides a new perspective to the role isoAsp in protein fibrillation, PIMT in its prevention and AEDs in enhancing the activity of the enzyme. GENERAL SIGNIFICANCE: IsoAsp, with an additional C atom in the main-chain of polypeptide chain, may make it more susceptible to fibrillation. PIMT alone, or in association with AEDs prevents this.
Subject(s)
Amyloid/metabolism , Isoaspartic Acid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Alzheimer Disease/metabolism , Amyloid/physiology , Amyloid beta-Peptides/metabolism , Animals , Anticonvulsants/pharmacology , Aspartic Acid/metabolism , Benzothiazoles/metabolism , Brain/metabolism , Dioxolanes/pharmacology , Epilepsy/metabolism , Humans , Isoaspartic Acid/physiology , Neurons/metabolism , PC12 Cells , Peptides/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/physiology , Rats , Structure-Activity Relationship , Valproic Acid/pharmacologyABSTRACT
Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-l-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an alpha(v)beta3-integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates alpha(v)beta3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.
Subject(s)
Fibronectins/chemistry , Isoaspartic Acid/physiology , Amino Acid Sequence , Animals , Binding, Competitive , Cell Adhesion , Extracellular Matrix/metabolism , Humans , Integrins/chemistry , Isoaspartic Acid/chemistry , Melanoma, Experimental , Mice , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemistry , Recombinant Proteins/chemistryABSTRACT
Isomerization of L-aspartate and deamidation of L-asparagine in proteins or peptides dominantly give rise to L-isoaspartate by a non-enzymatic reaction via succinimide as a intermediate under physiological conditions. Isoaspartates have been identified in a variety of cellular proteins in vivo as well as pathologically deposited proteins in neurodegenerative brain tissue. We described here that the formation of isoaspartate is enhanced in amyloid-beta (Abeta) peptides in Alzheimer's disease (AD). Specific antibodies recognizing isoaspartate of Abeta revealed that isomerized Abeta peptides were deposited in senile plaques as well as amyloid-bearing vessels. Moreover, it was revealed that Abeta peptides, isomerized at position 7 or 23, were differentially deposited in senile plaques and vascular amyloids in AD brains. In vitro experiments showed that the modification at position 23 greatly enhanced the aggregation of Abeta. Furthermore, systematic proline substitution analyses revealed that the beta-turn structure at positions 22 and 23 of Abeta42 plays a crucial role in the aggregation and neurotoxicity of Abeta peptides. It is suggested that spontaneous isomerization at position 23 induces the conformational change to form a beta-turn at position 23, which plays a pathogenic role in the deposition of Abeta peptides in sporadic AD. Protein L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which converts L-isoaspartyl residues in damaged proteins to normal L-aspartyl residues. PIMT-deficient mice manifested neurodegenerative changes concomitant with the accumulation of L-isoaspartate in the brain. We discuss here the pathological implications of the formation of isoaspartate in damaged proteins during neurodegeneration in model mice and AD.
