Human and Arabidopsis alpha‐ketoglutarate‐dependent dioxygenase homolog proteins—New players in important regulatory processes

Abstract

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N 6 methyladenine (N 6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N 6 meA demethylase influencing plant susceptibility to viral infections via m 6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N 6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of Abbreviations: (AlkB; FOXM1, Forkhead box protein M1; FTO, FaT mass and Obesity-associated; GWAS, genome-wide-associated studies; HIF-1alpha, hypoxia inducible factor 1; HNSCC, head and neck squamous cell carcinoma; IRX3, Iroquois-class homeodomain protein 3; mcm5U, 5-methoxycarbonylmethyluridine; MMS, mthyl methanosulphonate; N6meA, N6 methyladenine; NANOG, homeobox protein NANOG; PCNA, proliferating cell nuclear antigen; SNP, single nucleotide polymorphism; TBK1, TANK-binding kinase 1.Michał Marcinkowski and Tomaš Pilžys contributed equally to this study. Tomasz J. Sarnowski and Elzbieta Grzesiuk contributed equally to this study.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

“…Even more potential phosphorylation sites were identified in the PhosphoSitePlus [ 30 ] database that integrates both low- and high-throughput data sources. Among them was S256 as the highest occurrence, followed by T4, S260, T6, S184, S229, S173, Y199, T32, S55, Y106, Y108, T150, Y185, Y220, S355 and S458 [ 24 ]. The majority of these sites are located in the N-terminal catalytic subunit of FTO.…”

“…Despite the plentitude of studies on the involvement of FTO in metabolic regulation, relatively few data concerning the relationship between FTO structure and function have been published. Several phosphorylated amino acids have been identified in FTO [ 24 ], and their phosphorylation status shown to affect the protein fate. Tai and co-workers reported that threonine phosphorylation by protein kinase Cβ increased the FTO lifetime [ 25 ], while Faulds and co-workers showed that in mice phosphorylation of specific serines in the N-terminal domain—S249 and S253—led to FTO degradation [ 26 ].…”

Functional Characterization of a Putative RNA Demethylase ALKBH6 in Arabidopsis Growth and Abiotic Stress Responses

“…Although the writer proteins that install these m 6 A marks have been identified, the eraser proteins targeting only the RRACH motif have been identified, including the human ALKBH5 and FTO proteins [33,34] and plant ALKBH9B and ALKBH10B proteins [35,36]. The function and RNA target of ALKBH6 are unknown in plants as well as in animals [32]. Previous studies have demonstrated that ALKBH6 in humans is localized mainly in the nucleus and also in cytoplasm [30,61], and that ALKBH6 possesses a positively charged surface, suggesting its possible interaction with nucleic acids [62].…”

“…The AlkB homolog (ALKBH) proteins, which are members of the alpha-ketoglutarate (αKG) and Fe(II)-dependent dioxygenase superfamily and can remove alkyl and methyl groups from DNAs, have been suggested to function as RNA demethylases [26,27]. Mammals have nine different ALKBH family members: ALKBH1 to ALKBH8 and the fat mass and obesity-associated (FTO) protein [28][29][30][31][32]. Among them, human ALKBH5 and FTO protein have been demonstrated to function as m 6 A demethylases, which are involved in obesity, diabetes, and hypoxia response [33,34].…”

“…In plants, the role of eraser proteins has been determined in only a few cases. Thirteen Arabidopsis ALKBH family members have been identified by bioinformatics analysis [30,32]. Among them, the role of ALKBH9B and ALKBH10B has been demonstrated; ALKBH10B was identified as an mRNA m 6 A eraser, influencing floral transition [35], and ALKBH9B was shown to affect methylation of alfalfa mosaic virus RNA, mediating systemic infection [36].…”