Degradation of proteins upon storage at near-neutral pH: indications of a proteolytic/gelatinolytic activity associated with aggregates.

Background: The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.
Methods: Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.
Results: Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of 'gelatinolytic' activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.
Conclusions: Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.
General Significance: The study suggests that a bi-directional cause-effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.