How do the cells of an organism, all of which have exactly the same genetic code, adopt such different fates, morphologies and functions? And how do they then respond to the signals and stresses around them in order to make up a living, growing, healthy organism that can adapt to its environment?
Seminal work in the field of Epigenetics has taught us that the answer to the first question lies in the fact that our genome is subject to epigenetic regulation, which ensures its stability and determines when and where genes produce their transcript and protein products. And the answer to the second question lies largely within the fact that these proteins, which then go on to execute most of the cell’s functions, are themselves subject to regulatory mechanisms which determine when, where and how a protein will function. Our lab combines these two fascinating biological questions to understand how genome-regulating proteins are themselves regulated during development.
We employ biochemistry, cell and molecular biology, genomic and epigenetic approaches and mouse model systems to understand the mechanisms that modulate the function of epigenetic regulators, how these mechanisms are perturbed in disease and how they may be targeted for therapeutic effect. We have a particular interest in protein post-translational modifications (PTMs). These are small chemical changes that happen on proteins as a result of cell signalling changes and can quickly alter the activity, stability and sub-cellular localisation of these proteins, as well as their affinity for other molecules. As a result, PTMs add an enormous degree of sophistication to biological systems, beyond what can be achieved by gene regulation.
Our favourite PTM is citrullination, the conversion of an arginine residue to the non-coded amino acid citrulline. Exciting developments in this classically under-explored field have shown that citrullination and the enzymes that catalyse it, the peptidylarginine deiminases (PADIs or PADs), regulate many aspects of cell physiology, while their deregulation contributes to the development of pathologies such as autoimmunity, neurodegeneration and cancer. Understanding the mechanisms that control PADIs and other epigenetic regulators in response to developmental cues and cellular stresses can offer valuable insights into human health, which can be exploited towards therapeutic benefit in a variety of disease conditions.
Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to a lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >10 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, a cell-active inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_11, a cell-active PADI4 activator. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts.
Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.
During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from knock-out () mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for in the specification of the mesodermal lineage and reports the associated transcriptome, providing a platform for understanding the physiological functions of in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.