The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nM. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.
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.
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. Hsp903 is an ATP-dependent molecular chaperone present in the cytosol of eubacteria and eucaryotes. It regulates the maturation and activation of numerous proteins involved in signal transduction, cell cycle control, hormone signaling, and transcription (1-3). Recent crystal structures of HtpG from Escherichia coli and Hsp90 from Saccharomyces cerevisiae show that the overall structural organization of the proteins is highly conserved (4 -6). Both prokaryotic and eukaryotic Hsp90 proteins consist of an N-terminal ATP binding domain, a middle domain involved in client protein binding, and a C-terminal dimerization domain. During the ATPase cycle, large conformational changes in the Hsp90 dimer lead to the transient dimerization of the N-terminal domains and their association with the middle domains (7-12). Despite the conservation of the basic molecular architecture and the ATPase mechanism, there are major differences between Hsp90 from prokaryotes and eukaryotes. The most striking difference is the emergence of a large set of co-chaperones in eucaryotes that bind to Hsp90 and seem to modulate and expand its properties (13). Furthermore, in contrast to procaryotes, Hsp90 is an essential protein in eucaryotes (14, 15) and it had been shown in S. cerevisiae that ATP hydrolysis by Hsp90 is required for sustaining its essential function (16 -18). Finally, eukaryotic Hsp90 contains additional structural elements compared with prokaryotic Hsp90; that is, a long charged linker between the N terminus and the middle...
Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance.Cytotoxic CD8 T cells have an essential role in cellular immunity in that they destroy infected or malignantly transformed cells. They are activated by the recognition of complexes of major histocompatibility complex (MHC) 4 class I and antigenic peptides present on the surface of antigen-presenting cells (APC). Conventionally, the antigenic peptides presented by MHC class I are derived from endogenous cytosolic antigens. In specialized situations, MHC class I molecules additionally present peptides derived from exogenous antigens. This noncanonical MHC class I presentation, which is referred to as cross-presentation, requires that the exogenous antigen is internalized by APCs, subsequently enzymatically processed into peptides, and channeled into the MHC class I loading pathway (1, 2). Cross-presentation is crucial for the generation of CD8 T cell responses against antigens that are not endogenously produced by APCs, such as tumor antigens and pathogen-derived proteins. In an applied setting, cross-presentation is the required pathway for the generation of protein-based vaccines that are intended to stimulate antigen-specific CD8 responses. Critical parameters that define the efficacy of a vaccine are the amount of delivered antigen and the context in which the antigen is presented to the T cells. As the physiological capacity of APCs to cross-present antigen is generally low (3), there is significant interest to de...
PurposeFollowing two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial.MethodsWe undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches.ResultsSoluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein.ConclusionsWe propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.
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