**4. Integration of different signals by 2-ODDs: HIF prolyl hydroxylases and TET dioxygenases**

As mentioned above, the unusual catalytic mechanism of 2-ODDs requires, besides the substrate undergoing the specific modification, the cooperation of four different co-substrates, namely O2, 2-oxoglutarate, Fe2+, and AsA. From an evolutionary point of view, it is unlikely that such a complex mechanism developed just by chance. Unexpectedly, further studies on an apparently unrelated subject brought novel information about 2-ODD-mediated catalysis in general, and more specifically about the role of AsA. Starting from the observation that low-oxygen (hypoxic) conditions induce nuclear expression of a novel Hypoxia Inducible Factor (HIF) orchestrating downstream cellular responses [25], an interesting oxygen-sensing system was identified in human cells (**Figure 2**). The oxygen level is critical to activate a prolyl-hydroxylase, different from the collagen-related one. When two prolines in the HIF protein are modified to hydroxyprolines (hypro) by a highly specific HIF-prolyl hydroxylase (HPH), the HIF protein binds another factor known as the Von Hippel Lindau protein (pVHL), targeting the complex to ubiquitination and degradation. On the other hand, if oxygen is not available, the hydroxylation cannot take place, and the unhydroxylated HIF moves to the nucleus, where it acts as a transcriptional regulator of several genes involved in the hypoxic response [26]. The three main

*The Function of Ascorbic Acid through Occam's Razor: What We Know, What We Presume… DOI: http://dx.doi.org/10.5772/intechopen.109434*

**Figure 2.**

*The mechanism of oxygen sensing mediated by the Hypoxia-Inducible Factor 1α. Left: in the presence of oxygen, the HIF Prolyl Hydroxylase (HPH) catalyzes the conversion of two critical proline (pro) residues into hydroxyproline (hypro). Hydroxylated HIF1α binds the von Hippel–Lindau protein (pVHL) and the resulting complex is ubiquitinated and degraded in the proteasome. Right: If oxygen is not available, no hydroxylation of HIF1α occurs, and the transcription factor moves to the nucleus, activating transcriptional responses to hypoxia.*

investigators (Semenza, Kaelin, and Ratcliffe) who deciphered this elegant mechanism received the 2019 Nobel prize in Physiology or Medicine.

Although at the very beginning the HIF mechanism was mainly considered an oxygen-sensing mechanism, some studies tried to assess whether the remaining co-substrates also modulate HIF hydroxylation and the consequent hypoxic response. It was immediately clear that both AsA and Fe2+ cooperate with oxygen in the regulation of the response [27]. High AsA, by favoring the hydroxylation reaction, targets the HIF factor to degradation thus preventing the transcriptional cascade [28]. A new group of oxygen-sensing thiol dioxygenases, operating with a mechanism functionally very similar to the one observed in the HIF signaling module, has been recently characterized [29].

If in the past some doubts had been expressed about the specificity of AsA in the reaction catalyzed by collagen prolyl hydroxylase (see above), in the case of HIF prolyl hydroxylases sound experimental data confirm beyond any reasonable doubt that different reducing agents (such as glutathione, or dithiothreitol) cannot take over [30, 31]. Therefore, AsA is not just a generic reducing agent required to keep iron in the reduced state [20]; on the contrary, it appears tailored to the needs of HIF prolyl hydroxylase (and, by extension, of many other 2-ODDs), in conjunction with the other co-substrates.

At first sight, the four co-substrates may seem an odd combination of unrelated molecules, but a careful examination suggests that three out of the four co-substrates bring relevant molecular information regarding energy metabolism: 2-oxoglutarate, as a key intermediate of the Krebs cycle; oxygen, as the obvious respiratory substrate; iron, not only for its involvement in respiration but also for oxygen transport (hemoglobin) [32, 33]. In this perspective, the contribution of AsA to the potential signaling-related content of 2-ODDs appears less clear and will be further discussed below (Section 8). The fascinating hypothesis that the co-substrates can coordinate in parallel all the many different 2-ODDs operating in a cell [34] needs further experimental support, but at least provides a tentative explanation to the surprising complexity of the 2-ODD catalytic mechanism.

Another striking discovery stressing the outstanding importance of dioxygenases in the regulation of cellular responses was the identification of another group of

2-ODDs involved in gene expression. DNA methylation is a widespread mechanism for the epigenetic regulation of gene expression. The hydroxylase activity catalyzed by Ten Eleven Translocation 1 (TET1) converts methylcytosine into hydroxymethylcytosine [35]. The same enzyme also catalyzes further steps toward demethylation and the consequent modulation of gene expression [36]. It has been demonstrated that TET activity is strictly AsA-dependent [37], opening new and unexpected perspectives into our understanding of AsA biological function. Epigenetic modulation is essential to implement the developmental program of any organism. The involvement of AsA in the regulation of gene expression has been widely investigated, in relation to stem cell and cancer research [38–41].

Besides TET hydroxylases, epigenetic regulation by AsA occurs by means of histone demethylases characterized by the Jumonji domain [42]. In this case, the methylation of lysine residues in histone proteins is removed by a specific dioxygenase, thus modulating gene expression.
