Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

High-valent iron(IV)−oxo complexes are of great significance as reactive intermediates implicated in diverse chemical and biological systems. The aqueous iron(IV)−oxo complex (Fe aq IV O 2+ ) is the simplest but one of the most powerful ferryl ion species, which possesses a high-spin state, high reduction potential, and long lifetime. It has been well documented that Fe aq IV O 2+ reacts with organic compounds through various pathways (hydrogen-atom, hydride, oxygen-atom, and electron transfer as well as electrophilic addition) at moderate reaction rates and show selective reactivity toward inorganic ions prevailing in natural water, which single out Fe aq IV O 2+ as a superior candidate for oxidative water treatment. This review provides state-of-the-art knowledge on the chemical properties and oxidation mechanism and kinetics of Fe aq IV O 2+ , with special attention to the similarities and differences to two representative free radicals (hydroxyl radical and sulfate radical). Moreover, the prospective role of Fe aq IV O 2+ in Fe aq 2+ activation-initiated advanced oxidation processes (AOPs) has been intensively investigated over the past 20 years, which has significantly challenged the conventional recognition that free radicals dominated in these AOPs. The latest progress in identifying the contribution of Fe aq IV O 2+ in Fe aq 2+ -based AOPs is thereby reviewed, highlighting controversies on the nature of the reactive oxidants formed in several Fe aq 2+ activated peroxide and oxyacid processes. Finally, future perspectives for advancing the evaluation of Fe aq IV O 2+ reactivity from an engineering viewpoint are proposed.

Gene expression is regulated by a number of interrelated posttranslational modifications of histones, including citrullination. For example, peptidylarginine deminase 4 (PAD4) converts peptidyl arginine to citrulline in histone H3 and can repress gene expression. However, regulation of gene expression through citrullination of non-histone proteins is less well defined. Herein, we identify a tumor suppressor protein, inhibitor of growth 4 (ING4), as a novel non-histone substrate of PAD4. ING4 is known to bind p53 via its nuclear localization signal (NLS) region and to enhance transcriptional activity of p53. We show that PAD4 preferentially citrullinates ING4 in the same NLS region and thereby disrupts the interaction between ING4 and p53. A citrulline-mimicking Arg-NLS-Gln ING4 mutant, which has all Arg residues in the NLS mutated to Gln, loses its affinity for p53, can no longer promote p53 acetylation, and results in repression of downstream p21 expression. In addition, we found that citrullination leads to increased susceptibility of ING4 to degradation, likely impacting p53-independent pathways as well. These findings elucidate an interaction between posttranslational citrullination, acetylation, and methylation and highlight an unusual mechanism whereby citrullination of a non-histone protein impacts gene regulation.Protein arginine deiminases (PADs 2 or PADIs) catalyze the posttranslational modification of peptidyl arginine and, more slowly, peptidyl monomethylarginine to form peptidyl citrulline. This citrullination reaction is a hydrolytic deimination that eliminates the positive charge of the arginine side chain, can lead to changes in inter-and intraprotein interactions, and can even cause localized protein unfolding (1, 2). The human PAD4 isoform is calcium-regulated, nuclear-targeted, and is of particular interest because of its role in gene regulation as a catalyst of histone modification and chromatin remodeling (3). PAD4 expression is up-regulated in a variety of human malignant cancers, and PAD4-specific inhibitors can kill a selection of cancerous cells lines including HL-60, MCF7, and HT-29 (4). Accordingly, PAD4 has been suggested as a possible target for anticancer therapeutics (5).Some PAD4-interacting non-histone proteins have also been identified, including the tumor suppressor p53 (6). PAD4 binds p53 and inhibits p53-regulated cell cycle arrest and apoptosis. Through this interaction, PAD4 gets recruited to gene promoters that are targeted by p53 and, in the case of p21, can downregulate expression. Reciprocally, p53 can regulate the expression of PAD4 through a p53-response element located in a PAD4 gene intron (7). These findings suggest one mechanism whereby PAD4 can promote cancerous growth through its interactions with p53, although other unidentified pathways may also contribute.PAD4 also acts through citrullination of non-histone proteins, such as p300 (8). The p300 protein, histone methyltransferase coactivator-associated arginine methyltransferase-1 (CARM1) and glucocorticoid rec...

It is generally recognized that hepatic fibrogenesis is an end result of increased extracellular matrix (ECM) production from the activation and proliferation of hepatic stellate cells (HSCs). An in-depth understanding of the mechanisms of HSC necroptosis might provide a new therapeutic strategy for prevention and treatment of hepatic fibrosis. In this study, we attempted to investigate the effect of curcumol on necroptosis in HSCs, and further to explore the molecular mechanisms. We found that curcumol ameliorated the carbon tetrachloride (CCl4)-induced mice liver fibrosis and suppressed HSC proliferation and activation, which was associated with regulating HSC necroptosis through increasing the phosphorylation of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3). Moreover, curcumol promoted the migration of RIPK1 and RIPK3 into necrosome in HSCs. RIPK3 depletion impaired the anti-fibrotic effect of curcumol. Importantly, we showed that curcumol-induced RIPK3 up-regulation significantly increased mitochondrial reactive oxygen species (ROS) production and mitochondrial depolarization. ROS scavenger, N-acetyl-L-cysteine (NAC) impaired RIPK3-mediated necroptosis. In addition, our study also identified that the activation of c-Jun N-terminal kinase1/2 (JNK1/2) was regulated by RIPK3, which mediated curcumol-induced ROS production. Down-regulation of RIPK3 expression, using siRIPK3, markedly abrogated JNK1/2 expression. The use of specific JNK1/2 inhibitor (SP600125) resulted in the suppression of curcumol-induced ROS production and mitochondrial depolarization, which in turn, contributed to the inhibition of curcumol-triggered necroptosis. In summary, our study results reveal the molecular mechanism of curcumol-induced HSC necroptosis, and suggest a potential clinical use of curcumol-targeted RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling for the treatment of hepatic fibrosis.