Method development for the analysis of protein interactions

Abstract: Biological processes take place through interactions between macromolecules, such as nucleic acids and proteins. It is, therefore, fundamental to understand the functions of proteins and how they form complexes in order to carry out their role. Importantly, including spatial information in the analyses of protein complexes allows us to account for cell or tissue heterogeneity, highlighting the importance of in situ studies in biologically and clinically relevant material.To that end, methods for the analysis of protein complexes in situ have been developed, such as in situ proximity ligation assay (PLA) and proximity-dependent initiation of hybridisation chain reaction (proxHCR). Both methods depend on antibodies for target recognition and utilise oligonucleotide systems in order to generate reporter signals with fluorescence readout. While in situ PLA employs rolling circle amplification, proxHCR is an enzyme-free method that takes advantage of DNA hybridisation properties. Both methods, however, yield a polymerised reporter signal of protein complex formation.To further the use of proxHCR, we optimised the design of the oligonucleotide system as well as the experimental procedure, so as to increase the robustness and versatility of the assay. In addition, we developed a novel method, MolBoolean, that simultaneously reports the levels of free proteins as well as protein complexes. In this way, we address limitations of earlier methods and provide the opportunity to obtain a more comprehensive picture of biological processes. Our methods provide a means to circumvent the resolution limits of light microscopy by utilising molecular tricks so that protein binding events, that occur below the resolving power of conventional instrumentation, are made visible.  The methods presented in the present doctoral thesis provide powerful tools in the analysis of protein interactions and have applications in cell biology studies as well as in diagnostics. Part of this thesis was the examination of the function of 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor, potentially linked to vitamin D3. We investigated the expression and subcellular localisation of 1,25D3-MARRS in an array of cell lines and employed siRNA-mediated depletion to examine effects on cellular processes in androgen-independent prostate cancer cell models. Our data suggest that 1,25D3-MARRS supports cell proliferation and might have a role in cell migration. Additionally, we observe an effect on the regulation of intracellular vitamin D3 levels. With this study, we contribute to the understanding of the role of 1,25D3-MARRS in prostate cancer cells, that could potentially prove of value in the adaptation of therapeutic strategy for prostate carcinoma.