Profiling protein-protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia.

B-cell-lymphoma-2 (BCL2) homology-3 (BH3) mimetics are inhibitors of protein-protein interactions (PPIs) that saturate anti-apoptotic proteins in the BCL2 family to induce apoptosis in cancer cells. Despite the success of the BH3-mimetic ABT-199 for the treatment of haematological malignancies, only a fraction of patients respond to the drug and most patients eventually develop resistance to it. Here we show that the efficacy of ABT-199 can be predicted by profiling the rewired status of the PPI network of the BCL2 family via single-molecule pull-down and co-immunoprecipitation to quantify more than 20 types of PPI from a total of only 1.

The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab.

G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported.

Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy.

Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant.

Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme.