The critical role of the central hydrophobic core (residues 71–77) of amyloid-forming αA66-80 peptide in α-crystallin aggregation: a systematic proline replacement study

Abstract

Age-related cataract formation is marked by the progressive aggregation of lens proteins. The formation of protein aggregates in the aging lens has been shown to correlate with the progressive accumulation of a range of post-translational crystallin modifications, including oxidation, deamidation, racemization, methylation, acetylation, N- and C-terminal truncations and low molecular weight (LMW) crystallin fragments. We found that an αA-crystallin-derived peptide, αA66-80 (1.8 Kda), is a prominent LMW peptide concentrated in water-insoluble fractions of the aging lens. The peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from lens-soluble fractions. It binds at multiple sites and forms a hydrophobically driven non-covalent complex with α-crystallin to induce α-crystallin aggregation. To define the specific role of the αA66-80 peptide in age-related protein aggregation and cataract formation, it is important to understand the mechanisms by which this peptide acts. We used scanning proline mutagenesis to identify which particular sequences of the peptide drive it to form amyloid-like fibrils and induce α-crystallin aggregation. The secondary structure and the aggregate morphology of the peptides were determined using circular dichroism and transmission electron microscopy, respectively. Peptides were also tested for their ability to induce α-crystallin aggregation. We found that proline replacement of any residue in the sequence FVIFLDV, which corresponds to residues 71-77, led to an absence of both fibril formation and α-crystallin aggregation. The apparently critical role of 71-77 residues in αA66-80 explains their significance in the self-assembly processes of the peptide and further provide insights into the mechanism of peptide-induced aggregation. Our findings may have applications in the design of peptide aggregation inhibitors.

RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH

“…The 70 KFVIF 74 sequence present in the core of the peptide (Fig. 1) is similar to the 16 KLVFF 20 region of β-amyloid peptide, enabling the peptide to form amyloid fibrils (Kannan et al, 2014;Santhoshkumar et al, 2011), as well as generate H 2 O 2 (Raju et al, 2017), when incubated in vitro.…”

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

“…Bis-ANS binding experiments suggest that αA66-80 peptide has hydrophobicity (Santhoshkumar et al, 2011). In a separate study we showed that Pro substitutions that disrupt the beta sheet structure decrease interaction of the peptide with crystallins (Kannan et al, 2014). In a previous study we found that αA66-80 interacts with 70–74, 75–90, 91–103, 93–107, and 164–174 regions in αB-crystallin (Kannan et al, 2013).…”

Amyloid‐like aggregation of designer bolaamphiphilic peptides: Effect of hydrophobic section and hydrophilic heads

“…[20][21][22][23][24][25] Although these peptides were still quite different from each other considering their exact amino acid sequences, a large proportion of them showed a common feature of amphiphilic structure, suggesting the essential role of hydrophobic interaction in the formation of amyloid fibrils. [26][27][28][29][30] Among all these amyloid-like peptides, Aβ [16][17][18][19][20][21][22] with the sequence of KLVFFAE might be the most widely studied one. 31 A lot of work have been done on this peptide, but its bolaamphiphilic feature, ie, 2 hydrophilic heads connected by a hydrophobic section, has not received enough attention.…”