title: Ser422phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer's Disease.
abstract: Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer's Disease (AD). This cleavage likely occurs between Asp421↓Ser422leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter's proteolytic truncation. Interestingly, it is further noted that Ser422residue present in the P1' position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser422residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser422phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp421↓Ser422]. Corresponding phospho-, dextro-Ser422and dextro-Asp421analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp421↓Ser422site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser422peptide remained completely resistant to the cleavage. Substitution of Asp421by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser422by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser422phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser422or Asp421are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.
authors.affiliation: Interdisciplinary School of Health Sciences, Faculty of Health Science, U Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: Priya
authors.initials: P
authors.lastname: Sandhu
authors.affiliation: Interdisciplinary School of Health Sciences, Faculty of Health Science, U Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: Mansur Mohammad
authors.initials: MM
authors.lastname: Naeem
authors.affiliation: Interdisciplinary School of Health Sciences, Faculty of Health Science, U Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Lu
authors.affiliation: Environmental Health Science and Research Bureau, Environmental and Radiation Health Sciences Directorate, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON K1A 0K9, Canada.
authors.forename: Premkumari
authors.initials: P
authors.lastname: Kumarathasan
authors.affiliation: Interdisciplinary School of Health Sciences, Faculty of Health Science, U Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: James
authors.initials: J
authors.lastname: Gomes
authors.affiliation: Interdisciplinary School of Health Sciences, Faculty of Health Science, U Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada; Chronic Disease Program, Ottawa Hospital Research Institute, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada. Electronic address: abasa2@uottawa.ca.
authors.forename: Ajoy
authors.initials: A
authors.lastname: Basak
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D016875:tau Proteins; D012694:Serine; D053148:Caspase 3
keywords: Alzheimer’s Disease; Caspase-3 cleavage; Caspase-3 inhibitor; Kinases; Peptide digestion; Peptide synthesis; Reverse Phase High Performance Liquid Chromatography; Ser(422) phosphorylation; Tau phospho and non-phospho peptides; Tau protein