Succinylation Is a Gain-of-Function Modification in Human Lens αB-Crystallin

Abstract

Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All of the major crystallins exhibited Nε-succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC–MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses (age > 60 years) relative to young lenses (age < 30 years). Immunohistochemical analyses revealed that SuccK was present in epithelium and fiber cells. Western blotting and immunoprecipitation experiments revealed that SuccK is particularly prominent in αB-crystallin, and succinylation in vitro revealed that αB-crystallin is more prone to succinylation than αA-crystallin. Mass spectrometric analyses showed succinylation at K72, K90, K92, K166, K175, and potentially K174 in human lens aB-crystallin. We detected succinylation at K72, K82, K90, K92, K103, K121, K150, K166, K175, and potentially K174 by mass spectrometry in mildly succinylated αB-crystallin. Mild succinylation improved the chaperone activity of αB-crystallin along with minor perturbation in tertiary and quaternary structure of the protein. These observations imply that succinylation is beneficial to αB-crystallin by improving its chaperone activity with only mild conformational alterations.

Structure, Biosynthesis, and Biological Activity of Succinylated Forms of Bacteriocin BacSp222

“…This explanation is supported by literature data: suc-CoA solutions are stable for about 2 h [41]. The lysine modification reaction is not specific for BacSp222, as equivalent reactions have been described for a variety of proteins [20,42,43]; however, the site-specificity of modification is BacSp222 specific. The yield of succinylation of BacSp222 by suc-CoA was also dependent on the pH of the reaction.…”

“…Many authors emphasize that succinylation is an essential post-translational modification that alters e.g., the structure of proteins [2,3,15,35]; unfortunately, there are almost no experimental findings in favour of such a conclusion. Certain reports on in vitro succinylation of bovine serum albumin or glutamate dehydrogenase suggest such a possibility [4]; however, the only study evaluating the influence of physiological succinylation on the structure of a functional protein was focused on αB-crystallin from the human lens [20]. As shown by the results, the modification does not alter the secondary structure of crystallin but mildly perturbates its tertiary and quaternary structure, consequently leading to increased chaperone activity of this protein.…”

“…These studies are primarily focused on evaluation of the effects of in vitro succinylation on such proteins as bovine serum albumin, γ-globulin, lysozyme, and glutamate dehydrogenase [8,18,19]. The influence of physiological succinylation on the structure and activity of a functional protein was most extensively studied in the case of αB-crystallin from the human lens [20]. The present work reports investigation of the structure, biological activity, and mechanism of succinylation of bacteriocin BacSp222.…”

Small Heat Shock Proteins in Retinal Diseases

“…Glycation is a major modification of the human lens proteins HspB4 and HspB5 ( Swamy et al, 1992 ; Cherian and Abraham, 1995 ), and this modification can either improve or reduce the function of these proteins, depending on which carbonyl compound initiates the glycation ( Nagaraj et al, 2012a ). Various acyl modifications, such as acetylation, succinylation, malonylation, and propionylation, have also been reported in the human lenses proteins HspB4 and HspB5 ( Nagaraj et al, 2012b ; Nahomi et al, 2013b ; Nandi et al, 2019a ; Nahomi et al, 2020 ). These modifications (acetylation and succinylation) have also been shown to improve the chaperone-like activity of HspB5 ( Nahomi et al, 2013a ; Nahomi et al, 2013b ; Nandi et al, 2019a ; Nandi et al, 2019b ).…”

Glycation-mediated inter-protein cross-linking is promoted by chaperone–client complexes of α-crystallin: Implications for lens aging and presbyopia

“…Therefore, it is tempting to speculate that chaperone activity is beneficial in early life but could have a negative connotation later in life. The loss and gain of chaperone activity as a result of chemical modifications (46)(47)(48)(49)(50) could also dictate the kinetics of AGE-mediated crosslinking of α-crystallin-client protein complexes. The inter-protein association through disulfide bonds, which has been well documented in human lenses, could also predispose them to AGE-mediated crosslinking, similar to a-crystallin-client complexes.…”