A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates.

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.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

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 Padi4 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 Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Intrinsic function of the peptidylarginine deiminase PADI4 is dispensable for normal haematopoiesis.

Peptidylarginine deiminases (PADIs) are strongly associated with the development of autoimmunity, neurodegeneration and cancer but their physiological roles are ill-defined. The nuclear deiminase PADI4 regulates pluripotency in the mammalian pre-implantation embryo but its function in tissue development is unknown. PADI4 is primarily expressed in the bone marrow, as part of a self-renewal-associated gene signature. It has been shown to regulate the proliferation of multipotent haematopoietic progenitors and proposed to impact on the differentiation of haematopoietic stem cells (HSCs), suggesting that it controls haematopoietic development or regeneration. Using conditional in vivo models of steady state and acute Padi4 ablation, we examined the role of PADI4 in the development and function of the haematopoietic system. We found that PADI4 loss does not significantly affect HSC self-renewal or differentiation potential upon injury or serial transplantation, nor does it lead to HSC exhaustion or premature ageing. Thus PADI4 is dispensable for cell-autonomous HSC maintenance, differentiation and haematopoietic regeneration. This work represents the first study of PADI4 in tissue development and indicates that pharmacological PADI4 inhibition may be tolerated without adverse effects.

Citrullination Alters the Antiviral and Immunomodulatory Activities of the Human Cathelicidin LL-37 During Rhinovirus Infection.

Human rhinoviruses (HRV) are the most common cause of viral respiratory tract infections. While normally mild and self-limiting in healthy adults, HRV infections are associated with bronchiolitis in infants, pneumonia in immunocompromised patients, and exacerbations of asthma and COPD. The human cathelicidin LL-37 is a host defense peptide (HDP) with broad immunomodulatory and antimicrobial activities that has direct antiviral effects against HRV. However, LL-37 is known to be susceptible to the enzymatic activity of peptidyl arginine deiminases (PAD), and exposure of the peptide to these enzymes results in the conversion of positively charged arginines to neutral citrullines (citrullination). Here, we demonstrate that citrullination of LL-37 reduced its direct antiviral activity against HRV. Furthermore, while the anti-rhinovirus activity of LL-37 results in dampened epithelial cell inflammatory responses, citrullination of the peptide, and a loss in antiviral activity, ameliorates this effect. This study also demonstrates that HRV infection upregulates PAD2 protein expression, and increases levels of protein citrullination, including histone H3, in human bronchial epithelial cells. Increased PADI gene expression and HDP citrullination during infection may represent a novel viral evasion mechanism, likely applicable to a wide range of pathogens, and should therefore be considered in the design of therapeutic peptide derivatives.

Citrullination regulates pluripotency and histone H1 binding to chromatin.

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma.

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.

Temporal dissection of p53 function in vitro and in vivo.

To investigate the functions of the p53 tumor suppressor, we created a new knock-in gene replacement mouse model in which the endogenous Trp53 gene is substituted by one encoding p53ER(TAM), a p53 fusion protein whose function is completely dependent on ectopic provision of 4-hydroxytamoxifen. We show here that both tissues in vivo and cells in vitro derived from such mice can be rapidly toggled between wild-type and p53 knockout states. Using this rapid perturbation model, we define the kinetics, dependence, persistence and reversibility of p53-mediated responses to DNA damage in tissues in vivo and to activation of the Ras oncoprotein and stress in vitro. This is the first example to our knowledge of a new class of genetic model that allows the specific, rapid and reversible perturbation of the function of a single endogenous gene in vivo.