Research using animal model systems has been instrumental in delivering improved therapies for breast cancer, as well as in generating new insights into the mechanisms that underpin development of the disease. A large number of different models are now available, reflecting different types and stages of the disease; choosing which one to use depends on the specific research question(s) to be investigated. Based on presentations and discussions from leading experts who attended a recent workshop focused on in vivo models of breast cancer, this article provides a perspective on the many varied uses of these models in breast cancer research, their strengths, associated challenges and future directions. Among the questions discussed were: how well do models represent the different stages of human disease; how can we model the involvement of the human immune system and microenvironment in breast cancer; what are the appropriate models of metastatic disease; can we use models to carry out preclinical drug trials and identify pathways responsible for drug resistance; and what are the limitations of patient-derived xenograft models? We briefly outline the areas where the existing breast cancer models require improvement in light of the increased understanding of the disease process, reflecting the drive towards more personalised therapies and identification of mechanisms of drug resistance.
A proteomics-based approach is described that combines gel electrophoresis and MS in order to identify protein interactions and the nature of the interaction interface with high-sample throughput and sensitivity. Results for protein antigens of the influenza virus have demonstrated that the approach can be successfully employed to detect determinants within the hemagglutinin antigen of two divergent type A forms of the virus in present circulation. The determinants are localised to residues 206-224 following tryptic digestion of the hemagglutinin antigen. Specific peptide-antibody complexes formed after treatment of gel-recovered antigen are shown to be able to be preserved on the MALDI target array as has been previously demonstrated in this laboratory for whole virus. The approach has broad applicability for the analysis of a wide array of protein complexes with identification of the interaction interface in a single step with high-sample throughput and at low sample levels.
The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.
The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.
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