TM, Greco, Guise AJ, and Cristea IM. “Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.”. Methods Mol Biol (Forthcoming). Web. PubMedAbstract

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. 

TM, Greco, and Cristea IM. “The Biochemical Evolution of Protein Complexes.”. Trends Biochem Sci. 41.1 (2016): , 41, 1, 4-6. Web. PubMedAbstract

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. 

L, Waldron, et al.The Cardiac TBX5 Interactome Reveals a Chromatin Remodeling Network Essential for Cardiac Septation.”. Dev Cell 36.3 (2016): , 36, 3, 262-75. Web. PubMedAbstract

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. 

KK, Lum, and Cristea IM. “Proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection.”. Expert Rev Proteomics 13.3 (2016): , 13, 3, 325-40. Web. PubMedAbstract

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. 

Song, R, et al.Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion.”. MBio 71 (2016). Web. PubMedAbstract

nfection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion.


Neurons are highly polarized cells with long axonal processes that connect to distant targets. PNS axons that innervate peripheral tissues are exposed to various situations that follow infection, inflammation, and damage of the tissue. After viral infection in the periphery, axons represent potential front-line barriers to PNS infection and damage. Indeed, most viral infections do not spread to the PNS, yet the mechanisms responsible are not well studied. We devised an experimental system to study how axons respond to inflammatory cytokines that would be produced by infected tissues. We found that axons respond differentially to type I and type II interferons. The response to type I interferon (IFN-β) is a rapid axon-only response. The response to type II interferon (IFN-γ) involves long-distance signaling to the PNS cell body. These responses to two interferons erect an efficient and rapid barrier to PNS infection.