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Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor alpha target gene activation.

Mon, 06/15/2015 - 12:00pm

Cofactors for estrogen receptor alpha (ERalpha) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERalpha-positive cells with 17beta-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERalpha at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERalpha after E2 stimulation and that inhibition of either PAD2 or ERalpha strongly suppresses E2-induced H3R26 citrullination and ERalpha recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERalpha, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.

Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.

Mon, 06/15/2015 - 12:00pm

Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.

D-amino acid based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy.

Mon, 06/15/2015 - 12:00pm

The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Since D-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of D-amino acid analogs of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.

Mon, 06/15/2015 - 12:00pm

BACKGROUND: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.

METHODS: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.

RESULTS: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 x 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45alpha, and Ki67.

CONCLUSION: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest.

Mon, 06/15/2015 - 12:00pm

Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.

Design, synthesis, and kinetic characterization of protein N-terminal acetyltransferase inhibitors.

Mon, 06/15/2015 - 12:00pm

The N-termini of 80-90% of human proteins are acetylated by the N-terminal acetyltransferases (NATs), NatA-NatF. The major NAT complex, NatA, and particularly the catalytic subunit hNaa10 (ARD1) has been implicated in cancer development. For example, knockdown of hNaa10 results in cancer cell death and the arrest of cell proliferation. It also sensitized cancer cells to drug-induced cytotoxicity. Human NatE has a distinct substrate specificity and is essential for normal chromosome segregation. Thus, NAT inhibitors may potentially be valuable anticancer therapeutics, either directly or as adjuvants. Herein, we report the design and synthesis of the first inhibitors targeting these enzymes. Using the substrate specificity of the enzymes as a guide, we synthesized three bisubstrate analogues that potently and selectively inhibit the NatA complex (CoA-Ac-SES4; IC50 = 15.1 muM), hNaa10, the catalytic subunit of NatA (CoA-Ac-EEE4; Ki = 1.6 muM), and NatE/hNaa50 (CoA-Ac-MLG7; Ki* = 8 nM); CoA-Ac-EEE4 is a reversible competitive inhibitor of hNaa10, and CoA-Ac-MLG7 is a slow tight binding inhibitor of hNaa50. Our demonstration that it is possible to develop NAT selective inhibitors should assist future efforts to develop NAT inhibitors with more drug-like properties that can be used to chemically interrogate in vivo NAT function.

The protein arginine deiminases: Structure, function, inhibition, and disease.

Mon, 06/15/2015 - 12:00pm

The post-translational modification of histones has significant effects on overall chromatin function. One such modification is citrullination, which is catalyzed by the protein arginine deiminases (PADs), a unique family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins. Overexpression and/or increased PAD activity is observed in several diseases, including rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, lupus, Parkinson's disease, and cancer. This review discusses the important structural and mechanistic characteristics of the PADs, as well as recent investigations into the role of the PADs in increasing disease severity in RA and colitis and the importance of PAD activity in mediating neutrophil extracellular trap formation through chromatin decondensation. Lastly, efforts to develop PAD inhibitors with excellent potency, selectivity and in vivo efficacy are discussed, highlighting the most promising inhibitors. (c) 2012 Wiley Periodicals, Inc. Biopolymers 99: 155-163, 2013.

NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.

Mon, 06/15/2015 - 12:00pm

The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti-citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor-alpha (TNF-alpha) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus.

Mon, 06/15/2015 - 12:00pm

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.

Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5.

Mon, 06/15/2015 - 12:00pm

Protein arginine methyltransferases (PRMTs) have emerged as attractive therapeutic targets for heart disease and cancers. PRMT5 is a particularly interesting target because it is overexpressed in blood, breast, colon, and stomach cancers and promotes cell survival in the face of DNA damaging agents. As the only known member of the PRMT enzyme family to catalyze the formation of mono- and symmetrically dimethylated arginine residues, PRMT5 is also mechanistically unique. As a part of a program to characterize the mechanisms and regulation of the PRMTs and develop chemical probes targeting these enzymes, we characterized the substrate specificity, processivity, and kinetic mechanism of bacterially expressed Caenorhabditis elegans PRMT5 (cPRMT5). In this report, we demonstrate that distal positively charged residues contribute to substrate binding in a synergistic fashion. Additionally, we show that cPRMT5 catalyzes symmetric dimethylation in a distributive fashion. Finally, the results of initial velocity, product, and dead-end inhibition studies indicate that cPRMT5 uses a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes. In total, these studies will guide PRMT5 inhibitor development and lay the foundation for studying how the activity of this medically relevant enzyme is regulated.

Targeting the arginine phosphatase YwlE with a catalytic redox-based inhibitor.

Mon, 06/15/2015 - 12:00pm

Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) family, was shown to efficiently target phosphoarginine. YwlE shares several sequence motifs with this family including the C(X)4 CR(S/T) motif that is crucial for catalysis and redox regulation of the enzyme. Herein we confirm that Cys9 and Cys14 play important roles in YwlE catalysis and regulation. On the basis of these observations, we designed and synthesized a YwlE inhibitor, denoted cyc-SeCN-amidine, that irreversibly inhibits YwlE (kinact/KI = 310 M(-1) min(-1)) by inducing disulfide bond formation between the two active site cysteine residues. Interestingly, inactivation appears to be catalytic, since the compound is neither destroyed nor altered after enzyme inhibition. Although the exact mechanism of disulfide induction remains elusive, we propose several potential mechanisms accounting for the cyc-SeCN-amidine mediated inhibition of YwlE. These findings could stimulate the design of similar selenium-based compounds targeting other redox-sensitive enzymes.

Automethylation of protein arginine methyltransferase 8 (PRMT8) regulates activity by impeding S-adenosylmethionine sensitivity.

Mon, 06/15/2015 - 12:00pm

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet.

Chemical and biological methods to detect post-translational modifications of arginine.

Mon, 06/15/2015 - 12:00pm

Post-translational modifications (PTMs) of protein embedded arginines are increasingly being recognized as playing an important role in both prokaryotic and eukaryotic biology, and it is now clear that these PTMs modulate a number of cellular processes including DNA binding, gene transcription, protein-protein interactions, immune system activation, and proteolysis. There are currently four known enzymatic PTMs of arginine (i.e., citrullination, methylation, phosphorylation, and ADP-ribosylation), and two non-enzymatic PTMs [i.e., carbonylation, advanced glycation end-products (AGEs)]. Enzymatic modification of arginine is tightly controlled during normal cellular function, and can be drastically altered in response to various second messengers and in different disease states. Non-enzymatic arginine modifications are associated with a loss of metabolite regulation during normal human aging. This abnormally large number of modifications to a single amino acid creates a diverse set of structural perturbations that can lead to altered biological responses. While the biological role of methylation has been the most extensively characterized of the arginine PTMs, recent advances have shown that the once obscure modification known as citrullination is involved in the onset and progression of inflammatory diseases and cancer. This review will highlight the reported arginine PTMs and their methods of detection, with a focus on new chemical methods to detect protein citrullination.

(c) 2013 Wiley Periodicals, Inc. Biopolymers 101: 133-143, 2014.

Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation.

Mon, 06/15/2015 - 12:00pm

Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1-4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.

Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation

Mon, 06/15/2015 - 12:00pm

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki's ranging from 6 to 50 muM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.

Mon, 06/15/2015 - 12:00pm

RATIONALE: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-alpha-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation.

OBJECTIVE: To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis.

METHODS AND RESULTS: Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-alpha in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-alpha expression.

CONCLUSIONS: Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

Using unnatural amino acid mutagenesis to probe the regulation of PRMT1.

Mon, 06/15/2015 - 12:00pm

Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-l-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-l-phenylalanine (pBpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.

A FluoPol-ABPP PAD2 high-throughput screen identifies the first calcium site inhibitor targeting the PADs.

Mon, 06/15/2015 - 12:00pm

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERalpha target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 muM), to preferentially bind the apoenzyme.

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway.

Mon, 06/15/2015 - 12:00pm

PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.