Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger

Abstract

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (Np n Ns), the best studied of which is diadenosine tetraphosphate (Ap 4 A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap 4 A and Ap 4 N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the “friend” and “foe” scenarios. The “friend (or alarmone) hypothesis” suggests that Ap n N act as bona fide secondary messengers mediating responses to stress. In contrast, the “foe” hypothesis proposes that Ap n N and other Np n N are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for Ap n N in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to Ap n N. Finally, areas for future research are discussed with possible but unproven roles for intracellular Ap n N to encourage further research into the signaling networks that are regulated by these nucleotides.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

“…Diadenosine tetraphosphate (Ap 4 A), which was shown in several bacteria to function as an stress induced second messenger ( Lee et al, 1983 ; Pálfi et al, 1991 ; Kimura et al, 2017 ; Ferguson et al, 2020 ), could be detected in cell extracts from both archaeal model organisms ( Figures 3A,B ). For both species, levels of Ap 4 A were higher during exponential growth.…”

“…The observed differences between exponential and stationary growth phases as well as the high amounts of Ap 4 A specifically produced by exponentially growing S. acidocaldarius cells imply a general physiological relevance of this di-nucleotide. Noteworthy, bioinformatical identification of any Ap 4 A synthesizing enzyme in S. acidocaldarius and H. volcanii is particularly complicated as a broad variety of aminoacyl-tRNA synthetases and also other enzymes like, for example, DNA and RNA ligases, are known to be capable of forming this di-nucleotide ( Fraga and Fontes, 2011 ; Ferguson et al, 2020 ).…”

Deletion of the yqeK gene leads to the accumulation of Ap4A and reduced biofilm formation in Streptococcus mutans

“…Cas proteins are involved in DNA repair (Serbanescu et al., 2015), and Ap4A directly inhibits the initial phase of DNA replication, while elongation is not affected in a cell‐free replication system comprising mouse cell nuclei and cytoplasmic factors (Marriott et al., 2015). This evidence suggests that when cells are under stress, Ap4A may be involved in inhibiting the start of DNA replication to preserve the genomic integrity during recovery (Ferguson et al., 2020).…”

“…Streptococcus mutans needs to adapt to the environment of the oral cavity (Lemos et al., 2005), and for this reason, it produces dinucleoside polyphosphates, among which, the most studied is diadenosine‐5′,5′′′‐ P 1, P 4‐tetraphosphate (Ap4A; Ferguson et al., 2020). Ap4A is a dinucleotide metabolite that consists of two adenosines joined in the 5′−5′ linkage by four phosphates (Ji et al., 2019).…”

Data_Sheet_1.PDF

“…• Actual function as second messenger currently unclear • In bacteria, both are the degradation product of 3 ,5 -c-di-GMP and 3 ,5 -c-di-AMP, respectively (Rao et al, 2010;Stelitano et al, 2013) • Both have the capability to bind to some targets which are regularly binding the respective, unhydrolyzed c-di-NMP (Smith et al, 2012;Stelitano et al, 2013;Bowman et al, 2016;Kuipers et al, 2016) • As a nanoRNA, both have the potential to affect gene transcription on the level of transcription initiation (Goldman et al, 2011;Nickels and Dove, 2011) Ap 4 A In a multitude of uni-and multicellular species (Plesner and Ottesen, 1980;Flodgaard and Klenow, 1982;Garrison and Barnes, 1984;McLennan and Prescott, 1984) • Potential stress signaling related alarmone (Brevet et al, 1985;Baltzinger et al, 1986;Coste et al, 1987;Garrison et al, 1989) • In metazoa: regulatory effects on the cardiovascular and immune system and neuronal signal transduction (Vahlensieck et al, 1999;Miras-Portugal et al, 2003;Lee et al, 2004) Synthesizing and hydrolyzing enzymes present in various phyla (Ferguson et al, 2020) • Potential stress signaling related alarmone (Lee et al, 1983;Pálfi et al, 1991;Kimura et al, 2017) • Cellular development (Nishimura et al, 1997;Kimura et al, 2017) • Biofilm formation (Monds et al, 2010) Oligo-nucleotide-based signaling molecules cOA According to current knowledge absent in eukaryotes In species utilizing a type III CRISPR system (Kazlauskiene et al, 2017;Niewoehner et al, 2017) • Produced upon presence of invader RNA (Kazlauskiene et al, 2017;Niewoehner et al, 2017) • Activate effectors leading t...…”