Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

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.

Keywords: Alzheimer's disease; ageing; aging; lifespan; longevity; neurodegenerative disease; protein chemistry; proteomics.

Figure 1

Zinc‐induced truncation at Ser and the effect of amino acid sequence. One representative HPLC trace from each triplicate is shown following the incubation of PFHSPSY (a,b), PFASPSY (c,d) and PFHAPSY (e,f) at 60°C for 14 days. Racemization of N‐terminal residues following incubation (i.e. D‐ProFHASPY) has been characterized in a previous publication (22). Peptides were incubated in 50 mm Tris, pH 7.4 ± 2 mm ZnCl₂ as indicated. Of interest, cleavage of the C‐terminal Tyr residue was more evident in samples that lacked Zn. Detection at 216 nm.

Figure 2

Time course illustrating the effect of (a) peptide sequence on cleavage and (b) zinc concentration on PFHSPAY cleavage next to Ser. (c) Bar graphs highlighting the effect of sequence following incubation of 12 peptides (Ac‐AASAA, Ac‐AADSAA, Ac‐AAESAA, Ac‐AAPSAA, Ac‐AALSAA, Ac‐AAHSAA, Ac‐AASPAA, Ac‐AASHAA, Ac‐AAHSPAA, Ac‐AA(O‐Me)AA, Ac‐AAHTAA and Ac‐AAHAA) for 21 days. All peptides were incubated in 50 mm Tris, pH 7.4 ± 2 mm ZnCl₂ at 60°C. The position of His was statistically significant with respect to the rate of cleavage: HS vs. SH (P = 0.000096), as was the presence of C‐terminal Pro: HS vs. HSP (P = 0.000090), SP vs. HSP (0.000000014).

Figure 3

Truncation at Ser/Thr residues of peptides. HPLC traces of (a) Ac‐YEVRSDRDY, (b) Ac‐YEVRSDRDY + zinc, (c) Ac‐YPERTIPY and (d) Ac‐YPERTIPY + zinc following 21‐day incubation at 60°C. The rate of cleavage beside Thr (f) in this peptide was slower, but comparable with that next to Ser (e). Peptides were incubated in 50 mM tris pH 7.4, +/− 2 mM ZnCl₂.

Figure 4

NMR titration experiment showing the effect of zinc addition on (a) the aromatic proton region of PFHSPSY and (b) the Ser and His carbonyl regions of PFHSPSY. PFHSPSY was dissolved (2 mg mL⁻¹) in 20 mm deuterated Tris pH 7.4 (in D₂O) and ZnCl₂ (in 20 mm deutrated Tris pH 7.4 / D₂O) was added stepwise from 0.6 to 4 molar equivalents. (c) A mechanism that accounts for zinc‐induced truncation at the N‐terminal side of Ser residues. An analogous mechanism is proposed for Thr.

Figure 5

Representative HPLC traces following incubation of (a) PFHSPSY and (b) PFHSPSY + ZnCl2 at 37°C for 21 days. (c) Time course illustrating the effect of zinc on truncation at Ser 4. PFHSPSY was incubated in 50 mm Tris, pH 7.4 ± 2 mm ZnCl₂ as indicated.

Figure 6

Truncation of recombinant alpha B crystallin at Ser is induced by zinc. (a) Incubation of recombinant (a) alpha B crystallin and (b) alpha B crystallin + zinc at 60°C for 14 days. Alpha B was incubated in 50 mm Tris, pH 7.4 ± 2 mm ZnCl₂ as indicated.