The conserved heat shock protein (HSP) families are a group of molecular chaperones expressed in all prokaryotic and eukaryotic cells. HSP are the most abundant consti-tutively expressed proteins in cells. The expression of many HSP is also induced during cellular stress responses such as those caused by exposure to toxins, glucose starvation, anoxia, irradiation and heat (Hassen et al., 2005; Maytin, 1992; Spence et al., 1990; Zimmerman et al., 1991). The major functions ascribed to HSP are in the assembly and disassembly of proteins, the prevention of aggregation and their ability to bind the hydrophobic regions of nascent polypeptide chains (Gething and Sambrook, 1992; Richarme and Kohiyama, 1993). As discussed above, this latter function has generated immense interest amongst immunologists, as HSP-derived from tumor cells have been shown to confer tumor specific immunity (Richarme and Kohiyama, 1993; Srivastava, 2002a). This has led to the identification of several HSP with immunological activity. Whilst the two most widely used are gp96 and Hsp70, it is also clear that Hsp90, Hsp65 and calreticulin can also elicit anti-tumor immune responses (Anthony et al., 1999; Basu and Srivastava, 1999; Udono and Srivastava, 1994). The association of HSP with tumor specific cellular peptides explains the induction of protective tumor specific immunity and the generation of CTL specific for the tumor cells. Following internalization of HSP-peptide complexes by professional antigen presenting cells (pAPC) such as Dendritic cells (DC), the tumor specific peptides are released from the HSP and gain access to the MHC class-I pathway in a process termed cross-presentation (Li et al., 2002). This observation has been expanded from tumor-derived proteins to antigens derived from infectious agents and minor histocompatibility antigens (Arnold et al., 1995; Ciupitu et al., 1998).
Hence, the ability to bind and chaperone antigenic peptides to the immune system is thought to be a critical aspect of HSP activity for the elicitation of adaptive tumor specific responses. For the rational design of HSP based immunotherapeutics, it is important to understand how specific peptide substrates are bound and how much bound antigenic peptide is required to elicit protective responses.
The role of heat shock proteins in the elicitation of immune responses 177 Peptide Binding Properties of GP96
For gp96, Sastry and colleagues utilized a photoreactive azido tagged peptide from vesicular stomatitis virus capsid combined with specific proteolysis to map the substrate-binding site to amino acids 624-630. This site is situated adjacent to a proposed dimerization domain and tri-dimensional model of peptide binding was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. This gp96-peptide model predicts that the peptide ligand is held in a groove formed by a-helices and lies on a hydrophobic surface consisting of anti-parallel ^-sheets (Linderoth et al., 2001). The same group has also reported that gp96 can exist as higher order structures or aggregates from dimers to octamers and retain the potential to bind antigenic peptides (Linderoth et al., 2001). Whilst this model is extremely interesting, it remains speculative due the use of chemical cross-linking to bind the peptide substrates to gp96. This is because the experimental conditions required to generate a more physiological or non-covalent form of peptide binding to gp96 have not been defined. There is also a lack of corroborative crystal structure data on gp96-peptide complexes.
These are two areas where studies on Hsp70/peptide interactions are significantly advanced in comparison to gp96. There is a 60% amino acid sequence identity between eukaryotic Hsp70, and a 40% identity between eukaryotic and the E. coli Hsp70-equivalent DnaK (Robert et al., 2001). Hsp70 contains an N-terminal ATPase subunit (44kD) and a C-terminal peptide binding domain (27kD). The structural features of the Hsp70 substrate binding and the crystal structure of the bacterial Hsp70 homologue DnaK, have been resolved. Analysis of DnaK has demonstrated the presence of a hydrophobic binding channel (Zhu et al., 1996). Access of nascent hydrophobic polypeptide chains to this domain is controlled by ATP binding and hydrolysis. Binding of ATP to the nucleotide-binding domain induces a confor-mational change that results in the opening of a helical lid allowing peptides access to the substrate binding domain. Following hydrolysis of ATP to ADP, the helical lid is closed, preventing access to the cavity and resulting in slow 'on' and 'off' rates for bound substrates (Boice and Hightower, 1997; Popp et al., 2005).
The nature of the polypeptides, which preferentially bind to Hsp70, has been studied. Gething and colleagues tested a large panel (Srivastava, 2002b) of synthesized peptides for their binding activity. They found that the peptides which bound with the highest affinities were made up of at least 7 residues and included large hydrophobic and basic amino acids with few or none acidic residues. By comparing several HspP70 family members (Hsc70, DnaK and BiP), they were also able to demonstrate significant differences in the peptide binding specificities of these 3 closely related proteins, perhaps reflecting some notable differences in the sequence of their peptide binding domains. These are important considerations for the design of linkers and antigenic epitopes for generation of peptide/HSP complexes (Fourie et al., 1994). Hence, NRLLLTG or HWDFAWPWGS linker sequences are often fused to the amino or carboxy terminals of antigenic peptides to facilitate HSP binding in studies of Hsp70 (Castellino et al., 2000; Javid et al., 2004).
The importance of peptide binding for Hsp70 immune activity was formally demonstrated using peptide-binding mutants of Mycobacterial Hsp70. The design of these mutants was based on earlier studies of DnaK (Mayer et al., 2000) that demonstrated the importance of a conserved Valine residue in the peptide-binding domain. By substituting Valine 310 for Phenylalanine in Mycobacterial Hsp70, we were able to severely impair peptide binding and also compromised the ability of the HSP to induce virus specific T-lymphocytes, even in the presence of excess viral peptides. Moreover, by utilizing biophysical approaches such as fluorescence anisotropy, we have also shown that as little as 120pM antigenic peptide when delivered to Dendritic Cells in association with Hsp70 is sufficient to elicit T-lymphocyte responses (MacAry et al., 2004).
For HSP mediated cross-presentation to occur, professional antigen presenting cells of the immune system, such as immature dendritic cells (iDC) need to bind and internalizes HSP-peptide complexes (Singh-Jasuja et al., 2000; Wassenberg et al., 1999). A further requirement is for the DC to be modified and/or stimulated by the HSP in a way that facilitates their maturation into cells that can process and present the HSP chaperoned peptides efficiently (Kuppner et al., 2001). These cells must also be capable of migrating to local lymphoid tissues to expose themselves to circulating T-cells and be able to form direct physical interactions with other immune cells to allow for the formation of immunological synapses (Benvenuti et al., 2004; Kupiec-Weglinski et al., 1988).
While the specificity of HSP can be attributed to peptide occupancy of the substrate-binding domain, the effectiveness of HSP also relies on their pro-inflammatory activity. However, the ability of HSP to directly stimulate inflammatory responses in DC remains controversial with a large divergence in the stimulatory activity reported between the different HSP expressed by cells and in the species from which the HSP is derived (Tsan and Gao, 2004). An additional complicating factor has been the absence of suitable controls and safeguards for contaminating endotoxin activity with several studies intimating that this is responsible for the pro-inflammatory effects observed (Gao and Tsan, 2003; Reed et al., 2003). It is also clear that in studies where poor pro-inflammatory activity has been observed, the HSP are still found to be proficient in the induction of peptide specific CTL (Bausinger et al., 2002).
Controversy aside, It has been shown that different HSP can activate innate immune cells both in vivo and in vitro (Srivastava, 2002b). However, the route of activation of innate immune cells has been poorly defined. Specific HSP binding to the cell surface of both murine and human dendritic cells and monocytes has been demonstrated and a number of potential HSP receptors described, including CD91, SR-A, CD14, CD40, TLR4 and Lox-1 (Asea et al., 2000; Berwin et al., 2003; Binder et al., 2000; Delneste et al., 2002; Vabulas et al., 2002b; Wang et al., 2001). However, a clear consensus has not emerged regarding the role that these individual receptors play in uptake and/or pro-inflammatory signaling for the different species of HSP.
The Van Eden laboratory has proposed that some HSP can play an important role in regulating immune responses by inducing a regulatory phenotype in T-lymphocytes which helps to control potentially damaging autoimmune responses (Wendling et al., 2000). The potentiation of immune responses to chaperoned peptides may reflect a balance between the generation of IL-10 secreting regulatory T-cells specific for HSP epitopes versus effector cells specific for the chaperoned peptide (van Eden et al., 2003). In a previous study, a role for Mycobacterial Hsp70 in the DC mediated cross-presentation of viral peptides derived from Influenza A (Inf A) and human Cytomegalovirus (huCMV) was shown to be dependent on the induction of an intracellular calcium signaling cascade within the DC, and not on the direct stimulation of the cells to produce cytokines and chemokines. However, the provision of a second signal mediated by effector T-cell/DC interactions, greatly augmented the HSP effect on the DC resulting in the increased secretion of pro-inflammatory cytokines and chemokines (MacAry et al., 2004). Hence, Mycobacterial Hsp70 can be said to have a 'licensing' effect on human DC by lowering their threshold of activation, thus making them more responsive to additional pro-inflammatory signals. Given that the immune system will usually encounter HSP in association with the release of proteins from damaged tissues caused by pathological events (necrosis), this represents a powerful model, which potentially explains many of the conflicting observations in the Mycobacterial Hsp70 literature. The extension of this model to other species of HSP requires further investigation.
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