ARTD10/PARP10 Induces ADP-Ribosylation of GAPDH and Recruits GAPDH into Cytosolic Membrane-Free Cell Bodies When Overexpressed in Mammalian Cells

Abstract

Protein ADP-ribosylation is a reversible post-translational modification of cellular proteins that is catalysed by enzymes that transfer one (mono) or several (poly) units of ADP-ribose from β-NAD + to a specific amino acid of the target protein. The most studied member of the ADP-ribosyltransferase family is PARP1 (also known as ADP-ribosyltransferase diphtheria toxin-like 1, ARTD1), which is directly activated by DNA strand breaks and is involved in DNA damage repair, chromatin remodelling and transcriptional regulation. Much less is known about the further 16 members of this family. Among these, ARTD10/PARP10 has been previously characterised as a mono-ADP-ribosyltransferase with a role in cell proliferation and in NF-kB signalling. In the present study, we identified the glycolytic enzyme GAPDH as an interactor and a novel cellular target for ARTD10/PARP10. Moreover, we detected the co-localisation of GAPDH and ARTD10/PARP10 in well-defined cytosolic bodies, which we show here to be membrane-free, rounded structures using immunogold labelling and electron microscopy. Using the cognitive binding module macro domain to visualise ADP-ribosylated proteins by immunofluorescence microscopy in cells over-expressing the ARTD10/PARP10 enzyme, we show that the staining of the ARTD10/PARP10-dependent cytosolic bodies was lost when the cells were treated with compounds that inhibit ARTD10/PARP10, either by directly inhibiting the enzyme or by reducing the cellular NAD + levels. In parallel, the same treatment affected the co-localisation of GAPDH and ARTD10/PARP10, as GAPDH disappeared from the cytosolic cell bodies, which indicates that its presence there depends on the catalytic activity of ARTD10/PARP10. In line with this, in cells over-expressing the ARTD10/PARP10 catalytic domain alone, which we show here to form stress granules, GAPDH was recruited into stress granules. These data identify ARTD10/PARP10 as the enzyme that modifies and recruits GAPDH into cytosolic structures.

Regulation of Glucose Metabolism by NAD+ and ADP-Ribosylation

“…Nonetheless, potential functions of ADP-ribosylation in the cytoplasm are rather poorly described. The best-studied ARTDs localizing to the cytoplasm are ARTD10, ARTD12, ARTD15 and the tankyrases (TNKs) [95,96,97,98,99,100]. ARTD10 and the TNKs have been linked to cytoplasmic metabolic processes, while the other cytoplasmic ARTDs have thus far not been directly associated with cell/carbohydrate metabolism.…”

“…ARTD10 and the TNKs have been linked to cytoplasmic metabolic processes, while the other cytoplasmic ARTDs have thus far not been directly associated with cell/carbohydrate metabolism. ARTD12 localizes to the Golgi and is involved in protein sorting and trafficking [95,96], while ARTD15 localizes to the ER and has been linked to the unfolded protein response [97,98]. For more detailed information about the function of other (cytoplasmic) ARTs, the reader is referred to other reviews [101].…”

The ADP-Ribosyl-Transferases Diphtheria Toxin-Like (ARTDs) Family: An Overview

“…We have recently provided evidence that ARTD10 interacts with and mono-ADP-ribosylates the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that links metabolic activity to various cellular processes, including cell survival and proliferation [160]. The co-localization of GAPDH and ARTD10 occurs in well-defined cytosolic cell bodies, which we have shown to be rounded membrane-free structures.…”

“…Inhibition of ARTD10 activity resulted in the release of GAPDH from these cytosolic cell bodies, indicating that the catalytic activity of ARTD10 is required to recruit GAPDH, but it is not required to induce the formation of these cell bodies. Moreover, the dehydrogenase activity of GAPDH appears not to be regulated by its ADP-ribosylation, thus leading to hypothesis that ADP-ribosylation is important for the recruitment of GAPDH into cell bodies [160].…”

System-wide identification and prioritization of enzyme substrates by thermal analysis (SIESTA)

“…In total, 58 proteins were identified as potential PARP10 substrates ( Fig. 4a, Supplementary Data 12), some of which already known, such as ILF2, ILF3, IPO4 and PUM1 42 as well as GAPDH 43 . Melting curves for some of the putative substrates are shown in Fig.…”