Systematic Analysis of C-Termini of Small Open Reading Frame-Encoded Peptides in Human Cancer Cell Lines

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Protein C-termini regulate biological functions through structures and modifications.
• Small open reading frame-encoded peptides (SEPs) are a novel class of gene products with diverse biological activities.
• The C-termini of SEPs influence their function, but their polymorphism is not well understood.

Purpose of the Study:

• To systematically investigate the polymorphism of C-termini in SEPs.
• To identify and characterize novel C-terminal peptides from SEPs.
• To explore the mechanisms generating SEP C-terminal diversity.

Main Methods:

• C-terminal proteomics was employed to identify C-terminal peptides from proteins in human cancer cell lines.
• Bioinformatics analysis was used to predict the origin and processing of identified SEP C-termini.
• Immunoblotting was performed to validate the processing of specific SEPs.

Main Results:

• 3636 C-terminal peptides were identified from 2168 proteins, including 272 from 267 SEPs.
• Over 85% of identified SEP C-terminal peptides were novel, suggesting extensive post-translational processing.
• Protease cleavage by KLK, MMP, and CAT families was predicted as a major source of new SEP C-termini.
• Alternative splicing or protein trimming may also contribute to SEP C-terminal diversity.
• Experimental validation confirmed the in vivo processing of some SEPs, with cleavage occurring within functional domains.

Conclusions:

• This study significantly expands the known sequence information for SEPs.
• It provides crucial experimental evidence for the in vivo processing of SEPs.
• The findings lay the groundwork for future functional analyses of SEP processing and its impact on biological activity.