Synthesis and Characterization of a Peptide Identified as a Functional Element in αA-crystallin

Abstract

Eye lens ␣-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to ␣-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in ␣-crystallin. To investigate the mechanism of the interaction between ␣-crystallin and substrate proteins, we determined the melittin-binding sites in ␣-crystallin by cross-linking studies. Localization of melittin-binding sites in ␣-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of ␣A-crystallin and FSVNLDVK of ␣B-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1-bi(4-anilino) naphthalene-5,5-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPED-LTVK, having the melittin as well as bis-ANS-binding sequence of ␣A-crystallin. We show that this peptide has characteristics similar to that of ␣A-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the ␤3 and ␤4 region present in the ␣-crystallin domain of sHSP 16.5. We hypothesize that the ␣-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…We identified 19-to 20-mer sequences with chaperone function from aA-crystallin and aB-crystallin. 12,13 The individual amino acid, the location, and the chain length of the amino acid sequence are important determinants of the function of a specific peptide. For example, while a 19-mer peptide consisting of a 70 to 88 sequence of aA-crystallin is antiapoptotic, sequences 66 to 80 of a 15-mer peptide cause aggregation and toxicity.…”

“…11 Potential sequences of a-crystallins that exhibit chaperone activity have been identified. 12,13 We previously identified a 19-mer sequence corresponding to beta3 and beta4 regions of aA-crystallin domain, which has similar chaperone activity in vitro to that of aA-crystallin. 12 Further studies have revealed that the 19-mer peptide sequence of aA-crystallin inhibits fibril formation of Ab-amyloid peptides and suppresses the toxic action of Ab-peptide in rat pheochromocytoma (PC12) cells.…”

“…12,13 We previously identified a 19-mer sequence corresponding to beta3 and beta4 regions of aA-crystallin domain, which has similar chaperone activity in vitro to that of aA-crystallin. 12 Further studies have revealed that the 19-mer peptide sequence of aA-crystallin inhibits fibril formation of Ab-amyloid peptides and suppresses the toxic action of Ab-peptide in rat pheochromocytoma (PC12) cells. 11 Similarly, interactive sequences consisting of residues 73 to 92 in aB-crystallin are able to prevent the aggregation of substrate proteins similar to the action of native aB-crystallin.…”

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…This part of Hsp21 is a disordered, methionine-rich domain and has previously been suggested to be substratebinding (Harndahl et al 2001). For other sHsps, the Nterminal region has also previously been shown to be implicated in substrate-binding (Jaya et al 2009;Sharma et al 2000). Residues M1 and K27, and K18, are the only aminegroup-containing amino acid residues out of the 81 amino acid residues in the disordered N-terminal region of wild- Fig.…”

Small Heat‐shock Proteins and Clusterin: Intra‐ and Extracellular Molecular Chaperones with a Common Mechanism of Action and Function?

“…Recent advances in NMR spectroscopy, such as the development of Transverse Relaxation Optimized SpectroscopY (TROSY) methods, may provide a route to determine the structures of these large chaperone aggregates [34]. It would also be valuable to determine the chaperone binding site(s) in clusterin; this might be accomplished via cross-linking studies with bound target peptides and proteins, as has been done for sHsps [8,35]. Furthermore, the hypothesis that subunit exchange is a crucial factor in the activation of clusterin chaperone action could be tested by directly measuring the rate of clusterin subunit exchange and its variation with pH, temperature and the presence of target protein.…”