Strategies for the Assessment of Protein Aggregates in Pharmaceutical Biotech Product Development

Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics

Sti1 is a noncompetitive inhibitor of the Hsp90 ATPase. Binding prevents the N-terminal dimerization reaction during the ATPase cycle

The molecular chaperone Hsp90 is known to be involved in the activation of key regulatory proteins such as kinases, steroid hormone receptors, and transcription factors in an ATP-dependent manner. During the chaperone cycle, Hsp90 has been found associated with the partner protein Hop/Sti1, which seems to be required for the progression of the cycle. However, little is known about its specific function. Here we have investigated the interaction of Sti1 from Saccharomyces cerevisiae with Hsp90 and its influence on the ATPase activity. We show that the inhibitory mechanism of Sti1 on the ATPase activity of Hsp90 is non-competitive. Sti1 binds to the N- and C-terminal part of Hsp90 and prevents the N-terminal dimerization reaction that is required for efficient ATP hydrolysis. The first 24 amino acids of Hsp90, a region shown previously to be important for the association of the N-terminal domains and stimulation of ATP hydrolysis, seems to be important for this interaction

The Charged Linker Region Is an Important Regulator of Hsp90 Function*

Hsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins