The ability of members of the hsp70 family to bind to peptides in vivo and in vitro suggests that they may be involved in the processing of antigens for binding to Major Histocompatibility (MHC) class I and/or class n molecules. The aims of this thesis have been to provide evidence for the involvement of hsp70s in antigen processing and to characterise the binding of peptides by hspTOs by structural and functional studies. Firstly, the peptide-binding domains of two hsp70s, hsp70hom and PBP74, were expressed in isolation from the rest of the molecule for structure determination. Both of these hsp70s were implicated in antigen processing: hsp70hom in the class I pathway, due to its cytoplasmic localisation and constitutive expression, and the presence of its gene in the MHC; and PBP74 in the class n pathway because published work indicated that it was localised to endosomes and that antibodies against it inhibited antigen processing. The expression and purification of both peptide-binding domains was very successful, and one dimensional NMR experiments indicated that they were folded. However, it was not possible to determine their structures by NMR spectroscopy or X-ray crystallography because they aggregated in solution at high concentrations. Instead, the structure of the C-terminal region of hsp70hom, which includes its peptidebinding domain, was modelled based on the known structure of the equivalent portion of dnaK, the hsp70 of E.coli. The structure of hsp70hom is predicted to be very similar to that of dnaK, and modelling studies suggest that it is likely to bind peptides in a closely related fashion. The modelling of complexes between hsp70hom and two peptides suggest that the peptide-binding groove is very versatile, accounting for the broad peptide-binding specificity of hsp70s. The interactions of hsp70hom and PB74 with peptides were investigated using plate binding assays and isothermattitration calorimetry. A biotinylated peptide bound to the peptide-binding domain of hsp70hom, immobilised in plastic wells, with a Kd of <25 μM, which is within the range of Kds reported for other hsp70-peptide complexes (0.1-100 μM). In solution, isothermal titration calorimetry showed that the binding of peptides to the peptide-binding domains of hsp70hom and PBP74 was likely to be entropically rather than enthalpically driven, and, therefore, the interactions involved are likely to be predominantly hydrophobic. Secondly, PBP74, an hsp70 thought to be involved in the class II antigen processing pathway in endosomes, was localised by immunofluorescence microscopy. It was shown to be a mitochondrial protein, and is, therefore, unlikely to be involved in antigen processing. The presence of other members of the hsp70 family in lysosomes purified from a B cell line by Percoll density gradient centrifugation was investigated using antibodies that reacted with many Afferent members of the hsp70 family. No hsp70s were detected in these late endocytic compartments, even after heat shock or serum starvation. However, the presence of an hsp70 in endosomes, or of a member of this family not detected by the antibodies used, in lysosomes, cannot be ruled out. A third approach investigated the induction of the three hsp70 genes found in the MHC by four cytokines. The hsp70-l and hsp70-2 genes are induced at the mRNA level by IFN-γ and IL- 1, while TNF induces hsp70-2 alone. This data supports a role for the heat-inducible hsp70 in MHC class I antigen processing, as it appears to be coregulated with known members of this antigen processing pathway. The expression of hsp70hom was unaffected by any of the four cytokines examined. In addition, the mitochondrial hsp70 (which is not encoded in the MHC) appears to be induced by IFN-γ at the protein level. The research presented in this thesis provides a greater understanding of the peptide-binding properties of two hsp70s. Further work is necessary to show conclusively whether any of the hsp70s is involved in antigen processing.

Indiana University-Purdue University Indianapolis (IUPUI)

Indiana University-Purdue University Indianapolis (IUPUI) / Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene. To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta 1 and alpha V. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta 1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alpha V at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.