ADP-ribosylation of mitochondrial proteins is mediated by Neuralized-like protein 4 (NEURL4)

Abstract

ADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to amino acid side-chains of target proteins either as mono-ADP-ribose (MARylation or MAR) or poly-ADP-ribose chains (PARylation or PAR) by a class of enzymes called ADP-ribosyltransferases (ARTs). Although ADP-ribosylation is best known for its nuclear roles, ADP-ribosylation of extra nuclear proteins is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins, in particular, has been widely reported, even though the extent to which ADP-ribosylation of specific proteins regulates mitochondrial functions is unclear and the exact nature of mitochondrial ART enzymes is debated.Here, we have identified Neuralized-like protein 4 (NEURL4) as a mitochondrial ART enzyme and profiled the NEURL4-dependent ADP-ribosylome in mitochondrial extracts from Hela cells by LC-MS/MS, using isobaric tandem mass tag (TMT) labeling for relative quantification. Comparison of WT and NEURL4-KO cells generated by CRISPR/Cas9 genome editing revealed that most ART activity associated with mitochondria is lost in absence of NEURL4. Putative NEURL4 targets include numerous mitochondrial proteins previously shown to be ADP-ribosylated. In particular, we show that NEURL4 enzymatic activity is required for the regulation of mtDNA integrity via poly-ADP-ribosylation of mitochondrial specific Ligase III (mtLIG3), the rate-limiting enzyme for mitochondrial DNA (mtDNA) Base Excision Repair (BER).Collectively, our studies reveal that NEURL4 acts as the main mitochondrial ART enzyme under physiological conditions and provide novel insights in the regulation of mitochondria homeostasis through ADP-ribosylation.

ADP-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control

“…Finally, a new enzyme with supposed PARylating activity has been associated with mitochondria: Neuralized-like protein 4 (NEURL4) possesses an H-Y-E-like ART domain in the C-terminal domain, in addition to six Neuralized Homology Repeat (NHR) domains, functioning as protein–protein interaction scaffolds [ 201 , 202 , 203 ]. The PARylating activity associated with mitochondria is lost in cells lacking NEURL4.…”

Adp-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control

“…Finally, a new enzyme with supposed PARylating activity has been associated with mitochondria: Neuralized-like protein 4 (NEURL4) possesses an H-Y-E like ART domain in the C-terminal domain, in addition to six Neuralized Homology Repeat (NHR) domains, functioning as protein-protein interaction scaffolds [199][200][201]. PARylating activity associated with mitochondria is lost in cells lacking NEURL4.…”

“…One main protein target for PARylation is mitochondrial Ligase III (mtLIG3), required for mtDNA stability. Furthermore, LRRC9-ART has been characterized at expression level, and is predicted to interact with ZFP36L2, an RNA binding protein that controls cell cycle and is involved in pancreatic cancer [200,201]. Also new macrodomain proteins have been identified, such as C12ORF4, a cytoplasmic protein predicted to have eraser activity, involved in mast cell degranulation [201].…”

Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight

“…To date, besides ARTD1, SIRT4, ARH3, PARG and MacroD1 are also proposed to be involved in the regulation of ADP-ribosylation in mitochondria by catalyzing either its synthesis or removal [28,29,[187][188][189][190][191][192][193]. In addition to the potential contribution of those four enzymes (reviewed in detail elsewhere [39]), a recent study proposed NEURL4 as a new ADP-ribosylation catalyzing enzyme within mitochondria [16,17].…”

“…In addition to these enzymes, recent studies have proposed that other protein families, including NEURL4-like enzymes and certain leucine-rich repeat-containing enzymes, are able to catalyze ADP-ribosylation as well [16,17]. In recent years, protein ADP-ribosylation has emerged as a complex and dynamic post-translational modification that either directly affects the modified proteins or leads to modification-dependent protein complex formation thus serving as a signaling molecule [18].…”