In situ maleimide bridging of disulfides and a new approach to protein PEGylation.
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ABSTRACT:
SUBMITTER: Schumacher FF
PROVIDER: S-EPMC3039925 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Conjugation with poly(ethylene glycol) (PEG) or PEGylation is a widely used tool to overcome the shortcomings of native proteins, such as poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity. However, PEGylation is often accompanied by an unwanted detrimental effect on bioactivity, particularly, resulting from the amphiphilic nature of PEG. This is especially true for PEGylated proteins with large binding targets. Pegasys, a PEGylated interferon alpha-2a (IFN-α2a) bearing a 40 kDa branched PEG, is a typical example that displays only 7% in vitro activity of the unmodified IFN-α2a. In this work, by employing IFN-α2a as a model protein, we demonstrated that a protein conjugated with zwitterionic polymers (or zwitterlation) could significantly mitigate the antiproliferative bioactivity loss in vitro after polymer conjugation. The retained antiproliferative activity of zwitterlated IFN-α2a is 4.4-fold higher than that of the PEGylated IFN-α2a with the same polymer molecular weight, or 3-fold higher than that of the PEGylated IFN-α2a with a similar hydrodynamic size. It is hypothesized that nonspecific interactions between zwitterionic polymers and IFN-α2a/IFN-α2a receptors can be mitigated due to the super-hydrophilic nature of zwitterionic polymers. This, in turn, reduces the 'nonspecific blocking' between IFN-α2a and IFN-α2a receptors. In addition, we demonstrated that zwitterlated IFN-α2a showed a prolonged circulation time and a mitigated accelerated blood clearance after repeated injections in rats.
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Project description:Experiments were carried out to detect cysteine residues on human Keap1 protein that may be sensors of oxidative stress that gives rise to changes in the GSH/GSSG redox couple. Human Keap1 protein, at a final concentration of 6 microM, was incubated for two hours in aqueous buffer containing 0.010 M GSH, pH 8, in an argon atmosphere. Subsequently, excess iodoacetamide and trypsin were added to generate a peptide map effected by LCMS analysis. Peptides containing all 27 carboxamidomethylated cysteines were identified. Replacement of GSH by 0.010 M GSSG yielded a map in which 13 of the original carboxamidomethylated peptides were unperturbed, while other caboxamidomethylated cysteine-containing peptides were undetected, and a number of new cysteine-containing peptide peaks were observed. By mass analysis, and in some cases, by isolation, reduction, carboxamidomethylation, and reanalysis, these were identified as S-glutathionylated (Type 1) or Cys-Cys (Type 2) disulfides. Such peptides derived from the N-terminal, dimerization, central linker, Kelch repeat and C-terminal domains of Keap1. Experiments were carried out in which Keap1 was incubated similarly but in the presence of various GSH/GSSG ratios between 100 and 1 ([GSH + GSSG] = 0.010 M), with subsequent caraboxamidomethylation and trypsinolysis to determine differences in sensitivities of the different cysteines to the type 1 and type 2 modifications. Cysteines most sensitive to S-glutathionylation include Cys77, Cys297, Cys319, Cys368, and Cys434, while cysteine disulfides most readily formed are Cys23-Cys38 and Cys257-Cys297. The most reducing conditions at which these modifications are at GSH/GSSG = 10, which computes to an oxidation potential of E h = -268.5 mV, a physiologically relevant value. Under somewhat more oxidizing, but still physiologically relevant, conditions, GSH/GSSG = 1 ( E h = -231.1 mV), a Cys319-Cys319 disulfide is detected far from the dimerization domain of the Keap1 homodimer. The potential impact on protein structure of the glutathionylation of Cys434 and Cys368, the two modified residues in the Kelch repeat domain, was analyzed by docking and energy minimizations of glutathione residues attached to the Kelch repeat domain, whose coordinates are known. The energy minimizations indicated marked alterations in structure with a substantial constriction of Neh2 binding domain of the Keap1 Kelch repeat domain. This alteration appears to be enforced by an extended hydrogen-bonding network between residues on the glutathione moiety attached to Cys434 and amino acid side chains that have been shown to be essential for repression of Nrf2 by Keap1. The modifications of Keap1 detected in the present study are discussed in the context of previous work of others who have examined the sensitivity of cysteines on Keap1 to electrophile assault.
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