Method development for the analysis of protein interactions

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

Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3) : Effects on proliferation, migration, and genes in control of active vitamin D

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels

Optimization of proximity-dependent initiation of hybridization chain reaction for improved performance

Proximity based detection methods are invaluable tools in the field of molecular biology, increasing selectivity and allowing for analysis of protein interactions. ProxHCR utilizes pairs of antibodies labelled with oligonucleotides to probe for proximal binding and to initiate a hybridization chain reaction (HCR) to generate an amplified detection signal. As HCR is based upon hybridization of DNA hairpins, the performance is dependent on salt concentrations and temperature. Herein we have redesigned the proxHCR system to increase the performance and to reduce dependency on temperature and salt concentrations. The new oligonucleotides provide an increased signal when performed at physiological salt concentrations and in room temperature

A method for Boolean analysis of protein interactions at a molecular level

Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research