Chaperonin 10 (Cpn10) is a mitochondrial protein with protein folding function. There is substantial evidence that extracellular Cpn10 regulates the immune response. Prior research has shown that Cpn10 binds to T cells, inhibits LPS-induced RAW264.7 macrophage cell- and healthy donor peripheral blood mononuclear cell (PBMC)-activation, and downregulates lipopolysaccharide (LPS)-induced membrane distribution of the MHC II molecule on dendritic cells (DC). Recent Phase IIa rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS) clinical trials demonstrate improved disease amelioration with Cpn10. Despite compelling evidence of the anti-inflammatory properties of Cpn10, the precise mechanisms of action are unknown. The principal aim was to characterise the modulation of inflammation by Cpn10 and in the process create a bioassay that would allow for the reliable assessment of batch-to-batch variability of Cpn10 preparations. For this purpose, a Cpn10 bioassay was performed in the RAW264.7 cell line and expanded to DC and T cell lines. Furthermore, the analysis of gene expression in healthy donor PBMC was performed, as a mixed cell population experiment, to reflect possible involvement of cell-to-cell communication pathways. Initial data showed that Cpn10 reduced LPS-induced tumour necrosis factor ?? (TNF??) expression in RAW264.7 cells. However, the Cpn10 preparation was shown to contain trace lipid contaminants, which induced cellular tolerance, resulting in the observed reduction in TNF??. Experiments with a second batch of Cpn10 showed no reduction of LPS-induced TNF?? in the RAW264.7 cells, seen with the primary batch of Cpn10 and previously reported characterisation of Cpn10. The Cpn10 bioassay conducted in DC and T cell lines was shown to have the potential to decrease toll-like receptor 9 (TLR9) expression, suggesting that Cpn10 may attenuate immune responses by downregulating receptor recognition of bacterial components. The Cpn10 bioassay conducted in LPS-stimulated PBMCs revealed that Cpn10 downregulates gene expression of Th1 related genes including the polarising cytokines IL-7, IL-12B and IL-23A and Th2 related genes including the transcriptions factors GATA3, GFI1 and CEBPB. The downregulation of these genes may play an immuno-modulatory role, having improved efficacy of Cpn10 in T cell mediated autoimmune diseases, with possible therapeutic implications in Th2 mediated diseases such as asthma. The research carried out in this thesis provides insight into the success of Cpn10 in the RA, MS and psoriasis clinical trials. These results have also supported previously published data and provide additional insight into the mechanism of action of Cpn10. In addition, a Cpn10 bioassay has been established using healthy donor PBMCs stimulated with LPS and results show a reduced expression of Th1 and Th2 associated genes. The findings that in mixed cell populations, Cpn10 downregulates not only genes involved in Th1 polarisation mainly at the signal 3 level, but is also capable of downregulating Th2 polarising genes at the signal 1 level of TCR mediated transcription factors, are of particular interest. Ultimately, research from this project has confirmed the anti-inflammatory action of Cpn10 and given useful insight into how Cpn10 acts to modulate the inflammatory response.

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.