Abstract
Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long-lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N-terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser- and Thr-containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N-terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co-ordinates zinc and that once zinc is co-ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long-lived proteins, for example truncation on the N-terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long-term stability of cellular proteins.
Highlights
Long-lived proteins such as those found in the brain, heart, connective tissue, lung and the lens are subject to many age-related modifications including racemization (Shapira et al, 1988; Hooi & Truscott, 2011), crosslinking (Wang et al, 2014) and isomerization of Asp and Asn (Shapira et al, 1988; Stephenson & Clarke, 1989)
One of the signatures of long-lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N-terminal side of Ser residues, and this is important since cleavage at Ser, and Thr, has been observed in a number of human proteins
The autolysis of Ser- and Thr-containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH
Summary
Long-lived proteins such as those found in the brain, heart, connective tissue, lung and the lens are subject to many age-related modifications including racemization (Shapira et al, 1988; Hooi & Truscott, 2011), crosslinking (Wang et al, 2014) and isomerization of Asp and Asn (Shapira et al, 1988; Stephenson & Clarke, 1989). The Accepted for publication 25 October 2015 mechanisms that underpin some of these processes are still poorly understood One such modification, which is commonly observed in long-lived proteins, is truncation at the N-terminal side of Ser and Thr residues (Lampi et al, 1998; Friedrich et al, 2012, 2000; Sergeant et al, 2003; Santhoshkumar et al, 2008; Vlad et al, 2011; Su et al, 2012). Several cleavage events on the N-terminal side of Ser and Thr have been documented (Lampi et al, 1998; Schey et al, 2000; Santhoshkumar et al, 2008; Su et al, 2012)
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