title: Role of the carboxy-termini of tubulin on its chaperone-like activity.
abstract: Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.
authors.affiliation: Department of Biochemistry, Centenary Campus, Bose Institute, Calcutta, India.
authors.forename: T
authors.initials: T
authors.lastname: Sarkar
authors.forename: T
authors.initials: T
authors.lastname: Manna
authors.forename: S
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: P
authors.initials: P
authors.lastname: Mahapatra
authors.forename: A
authors.initials: A
authors.lastname: Poddar
authors.forename: S
authors.initials: S
authors.lastname: Roy
authors.forename: J
authors.initials: J
authors.lastname: Pena
authors.forename: R
authors.initials: R
authors.lastname: Solana
authors.forename: R
authors.initials: R
authors.lastname: Tarazona
authors.forename: B
authors.initials: B
authors.lastname: Bhattacharyya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006041:Goats; D015395:Histocompatibility Antigens Class I / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020860:Subtilisin / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015395:Histocompatibility Antigens Class I; D018832:Molecular Chaperones; D014404:Tubulin; D010447:Peptide Hydrolases; D020860:Subtilisin