Tanaya Chatterjee1, Gaurav Das2, Barun K Chatterjee3, Jesmita Dhar4, Surajit Ghosh2, Pinak Chakrabarti5. 1. Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata 700054, India. Electronic address: tanaya_chatterjee@yahoo.com. 2. Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India; Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India. 3. Department of Physics, Bose Institute, 93/1 A.P.C. Road, Kolkata 700009, India. 4. Bioinformatics Center, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata 700054, India. 5. Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata 700054, India; Bioinformatics Center, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata 700054, India. Electronic address: pinak@jcbose.ac.in.
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, Aβ42, implicated in Alzheimer's disease, undergoes β-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 Aβ42 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.
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, Aβ42, implicated in Alzheimer's disease, undergoes β-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 Aβ42 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.