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The AlkB family of nucleic acid demethylases is currently of intense chemical, biological, and medical interest because of its critical roles in several key cellular processes, including epigenetic gene regulation, RNA metabolism, and DNA repair. Emerging evidence suggests that dysregulation of AlkB demethylases may underlie the pathogenesis of several human diseases, particularly obesity, diabetes, and cancer. Hence there is strong interest in developing selective inhibitors for these enzymes to facilitate their mechanistic and functional studies and to validate their therapeutic potential. Herein we review the remarkable advances made over the past 20 years in AlkB demethylase inhibition research. We discuss the rational design of reported inhibitors, their mode-of-binding, selectivity, cellular activity, and therapeutic opportunities. We further discuss unexplored structural elements of the AlkB subfamilies and propose potential strategies to enable subfamily selectivity. It is hoped that this perspective will inspire novel inhibitor design and advance drug discovery research in this field.

The human AlkB homolog family (ALKBH) of proteins play a critical role in some types of cancer. However, the expression and function of the lysine demethylase ALKBH4 in cancer are poorly understood. Here, we examined the expression and function of ALKBH4 in non-small-cell lung cancer (NSCLC) and found that ALKBH4 was highly expressed in NSCLC, as compared to that in adjacent normal lung tissues. ALKBH4 knockdown significantly induced the downregulation of NSCLC cell proliferation via cell cycle arrest at the G1 phase of in vivo tumour growth. ALKBH4 knockdown downregulated E2F transcription factor 1 (E2F1) and its target gene expression in NSCLC cells. ALKBH4 and E2F1 expression was significantly correlated in NSCLC clinical specimens. Moreover, patients with high ALKBH4 expression showed a poor prognosis, suggesting that ALKBH4 plays a pivotal tumour-promoting role in NSCLC.

Summary Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing., Graphical Abstract, Highlights • 6mA deposition and erasure by mammalian Mettl4 and Alkbh4, respectively • Mettl4-deficient mice display craniofacial dysmorphism • 6mA triggers proteolysis of its cognate sensor proteins ASXL1 and MPND • Adversarial 6mA network architecture preserves Polycomb silencing, Kweon et al. reveal the molecular infrastructure of a mammalian DNA N6-methyladenine (6mA) methylation, erasure, and sensing system. Deposition of 6mA by the Mettl4 methyltransferase triggers the proteolytic destruction of its cognate sensor proteins, the histone H2A deubiquitinases ASXL1 and MPND, thus preserving Polycomb gene silencing.

During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly.

The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins., National Institutes of Health (U.S.) (Grant P01 CA26731), National Institutes of Health (U.S.) (Grant R37 CA080024), National Institutes of Health (U.S.) (Grant P30 ES002109)

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.

ALKBH4, an AlkB homologue in the 2-oxoglutarate and Fe2+ dependent hydroxylase family, has previously been shown to regulate the level of monomethylated lysine-84 in actin and thereby indirectly influences the ability of non-muscular myosin II to bind actin filaments. ALKBH4 modulates fundamental processes including cytokinesis and cell motility, and its depletion is lethal during early preimplantation embryo stage. The aim of this study was to investigate the effect of ALKBH4 deficiency in a physiological context, using inducible Alkbh4 knockout mice. Here, we report that ALKBH4 is essential for the development of spermatocytes during the prophase of meiosis, and that ALKBH4 depletion leads to insufficient establishment of the synaptonemal complex. We also show that ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia and primary spermatocytes.

Regulation of actomyosin dynamics by post-transcriptional modifications in cytoplasmic actin is still poorly understood. Here we demonstrate that dioxygenase ALKBH4-mediated demethylation of a monomethylated site in actin (K84me1) regulates actin–myosin interaction and actomyosin-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring and midbody via association with methylated actin. ALKBH4-mediated regulation of actomyosin dynamics is completely dependent on its catalytic activity. Disorganization of cleavage furrow components and multinucleation associated with ALKBH4 deficiency can all be restored by reconstitution with wild-type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction., The division of a single eukaryotic cell into two requires actomyosin-dependent contraction. Here the authors show that lysine methylation of actin inhibits contractility during cytokinesis by blocking its association with myosin, and this modification is reversed at the contractile ring by the demethylase ALKBH4.

The Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from E. coli is a demethylase which repairs alkyl lesions in DNA, as well as RNA, through a direct reversal mechanism. Humans possess nine AlkB homologs (ALKBH1-8 and FTO). ALKBH2 and ALKBH3 display demethylase activities corresponding to that of AlkB, and both ALKBH8 and FTO are RNA modification enzymes. The biochemical functions of the rest of the homologs are still unknown. To increase our knowledge on the functions of ALKBH4 and ALKBH7 we have here performed yeast two-hybrid screens to identify interaction partners of the two proteins. While no high-confidence hits were detected in the case of ALKBH7, several proteins associated with chromatin and/or involved in transcription were found to interact with ALKBH4. For all interaction partners, the regions mediating binding to ALKBH4 comprised domains previously reported to be involved in interaction with DNA or chromatin. Furthermore, some of these partners showed nuclear co-localization with ALKBH4. However, the global gene expression pattern was only marginally altered upon ALKBH4 over-expression, and larger effects were observed in the case of ALKBH7. Although the molecular function of both proteins remains to be revealed, our findings suggest a role for ALKBH4 in regulation of gene expression or chromatin state. © 2012 Bjørnstad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV–visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a gmax of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.