Over the past decade, it became evident that proteins perform critical functions as components of specialized macromolecular complexes. Here, we discuss a recent study by Wan and colleagues, which highlights the significance of protein complexes by studying their conservation in organisms separated by up to a billion years of evolution.
Human mutations in the cardiac transcription factor gene TBX5 cause congenital heart disease (CHD), although the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the nucleosome remodeling and deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD mis-sense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD.
In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability.
The integration of proteomic methods to virology has facilitated a significant breadth of biological insight into mechanisms of virus replication, antiviral host responses and viral subversion of host defenses. Throughout the course of infection, these cellular mechanisms rely heavily on the formation of temporally and spatially regulated virus-host protein-protein interactions. Reviewed here are proteomic-based approaches that have been used to characterize this dynamic virus-host interplay. Specifically discussed are the contribution of integrative mass spectrometry, antibody-based affinity purification of protein complexes, cross-linking and protein array techniques for elucidating complex networks of virus-host protein associations during infection with a diverse range of RNA and DNA viruses. The benefits and limitations of applying proteomic methods to virology are explored, and the contribution of these approaches to important biological discoveries and to inspiring new tractable avenues for the design of antiviral therapeutics is highlighted.