Abstract
The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Highlights
The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells
Ap3A is cleaved into AMP and ADP by the tumor suppressor protein fragile histidine triad (FHIT)[8], whereas Nudix type motif 2 (NUDT2) hydrolyzes Ap4A to AMP and ATP9
As it has already been reported that Ubiquitin-like modifier-activating enzyme 1 (UBA1) is able to synthesize Ap4A in vitro[7], we investigated if Ap3A and Ap4A can substitute for ATP in the Ub cascade
Summary
The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. The most widely studied family members are diadenosine polyphosphates (ApnAs), like diadenosine triphosphate Ap3A and diadenosine tetraphosphate Ap4A2,3 As their cellular concentrations increase in response to extracellular cues such as pH, temperature and oxidative stress from a nanomolar to a lower micromolar range, ApnAs have been considered to be alarmones that signal cellular stress[2,4,5]. Ap3A is cleaved into AMP and ADP by the tumor suppressor protein fragile histidine triad (FHIT)[8], whereas Nudix (nucleoside diphosphate linked to X) type motif 2 (NUDT2) hydrolyzes Ap4A to AMP and ATP9 The malfunction of these enzymes results in severe consequences for the affected cells or organisms. The absence of NUDT2, on the other hand, causes downregulation of the immune response[12]
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