Destabilizing single chain major histocompatibility complex class I protein for repurposed enterokinase proteolysis

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• High-throughput screening of stabilizing peptides for peptide-major histocompatibility complex class I (pMHC-I) molecules is lacking.
• Current methods rely on in silico tools, often without experimental biophysical characterization.
• Existing labeling- and antibody-based assays for MHC-I are time-consuming.

Purpose of the Study:

• To develop a rapid, high-throughput assay for screening stabilizing peptides for pMHC-I molecules.
• To characterize peptide-MHC-I stability as a predictor of immunogenicity.
• To explore the use of enterokinase for protein instability-based assays.

Main Methods:

• Development of the EASY MHC-I (EZ MHC-I) assay based on de novo protein fragmentation.
• Utilizing the inherent instability of empty single-chain MHC-I proteins.
• Employing repurposed enterokinase to fragment unstable MHC-I unless stabilized by a peptide.

Main Results:

• The EZ MHC-I assay enables peptide antigen screening in seconds per peptide.
• Assay demonstrates that protein stability is a superior indicator of immunogenicity compared to classical affinity.
• Identified a novel application of enterokinase to target destabilized MHC-I proteins lacking the standard cleavage motif.

Conclusions:

• The EZ MHC-I assay offers a rapid and efficient method for peptide screening.
• Protein stability is a critical factor in MHC-I immunogenicity.
• Enterokinase can be repurposed for novel protein instability-based assays beyond MHC-I.