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  • Fmoc-Glu(OtBu)-OPfp A key step in the ADAR reaction

    2024-05-23

    A key step in the ADAR reaction is the formation of the adenosine covalent hydrate with a tetrahedral center at C6. The nucleoside analog 8-azanebularine (box in Fig. 6), with its relatively high propensity to form a covalent hydrate, is useful as an adenosine replacement in ADAR2 substrates to trap protein–RNA complexes [121,150,166]. Since the 8-azanebularine covalent hydrate is similar in structure to the adenosine deamination transition state, high affinity complexes are formed by the binding of ADAR2 to RNAs bearing this analog [121,150]. Furthermore, once formed in the ADAR2 active site, the 8-azanebularine hydrate cannot proceed forward along the reaction pathway since it lacks a C6 amino group [166]. Indeed, stabilization of human ADAR2 deaminase domain–RNA complexes by replacing the reactive adenosine with 8-azanebularine enabled trapping and crystallization of the complex in the “flipped out” conformation (Fig. 5) [121]. It remains to be seen whether a similar approach can stabilize ADAR1–RNA complexes.
    Substrate Specificity Editing reactions catalyzed by ADARs are not strictly sequence specific but do show preferences for adenosines at certain locations in RNA substrates. In addition, ADARs can bind most duplex RNAs beyond a certain length (~15bp). It is now known that the opposite base, the flanking sequence, and the local secondary structure each play an important role in determining ADAR editing efficiency. Below we discuss each of these factors in detail.
    Regulation RNA editing levels differ with developmental stage, pathological or carcinogenic conditions, and in a tissue-specific manner [94,179–184]. Some of these changes correlate with changes in ADAR expression levels, but not all do, indicating that other mechanisms must exist for regulation of ADAR activity [185–189]. Indeed, ADARs have been shown to be regulated by posttranscriptional RNA processing (i.e., splicing and auto editing of ADAR pre-mRNA), by posttranslational modifications (e.g., phosphorylation, ubiquitination, and SUMOylation), by protein–protein interactions, by colocalization with substrate RNAs, by Fmoc-Glu(OtBu)-OPfp with substrate RNAs from both proteins and RNAs, as well as by changes in ADAR expression levels. In this section we discuss different mechanisms for Fmoc-Glu(OtBu)-OPfp regulation of ADAR activity.
    Consequence of A-to-I Editing
    A-to-I Editing and Human Diseases
    Acknowledgments
    Introduction Chronic and excessive ethanol consumption is associated with various biochemical and physiological changes in CNS. Some of these changes are pertaining to alteration of specific neurotransmitter systems (Chandler et al., 1997) and signaling pathways (Hoek and Kholodenko, 1998). Besides GABA, glutamate, dopamine, and noradrenaline, ethanol acts on purinergic signaling changing P2X receptor function (Franke and Illes, 2006) and also the adenosine levels (Mailliard and Diamond, 2004). Purinergic signaling involves the role of nucleotides and nucleosides in CNS. After released, ATP is catabolized to adenosine via ectonucleotidase pathway, such as nucleotide pyrophosphatase/phosphodiesterases (NPP), nucleoside triphosphate diphosphohydrolases (NTPDases), and 5′-nucleotidase, or it can be released from any cell when the intracellular concentration rises (Fredholm, 2002, Yegutkin, 2008). Extracellular adenosine acts as a neuromodulator in the CNS (Ralevic and Burnstock, 1998, Burnstock, 2006) and can mediate different cellular functions by operating G-protein-coupled receptors (A1, A2A, A2B, A3), which can inhibit (A1 and A3) or facilitate (A2A and A2B) neuronal communication (Burnstock, 2007). Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme in the purine catabolic pathway that catalyses the conversion of adenosine and deoxyadenosine into inosine and deoxyinosine, respectively. Since this enzyme is located on the membrane surface of many cells, are considered as ecto-enzymes (Franco et al., 1997, Kanbak et al., 2008). Adenosine is involved in several acute and chronic effects of ethanol (Dar et al., 1983, Newton and Messing, 2006). In mammalian brains, adenosine deaminase activity is located mainly in the cytosol, but the presence of ecto-adenosine deaminase has been also established on the surface of synaptosomes and neurons by activity assays and immunohistochemistry (Ruiz et al., 2000).