Functional Localization of Two Poly(ADP-Ribose)-Degrading Enzymes to the Mitochondrial Matrix

Abstract

Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific "knock-down" of the NAD content which is readily detectable by PAR formation.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

“…Altering Mitochondrial NAD ϩ Levels Evokes a Metabolic Shift from Oxidative Phosphorylation to Glycolysis-293mito-PARP cells were previously found to display increased medium acidification compared with control 293 cells (29). Unexpectedly, when culturing 293AtNDT2 cells, we also observed an accelerated color shift of the pH indicator phenol red, indicating that medium acidification occurred faster in these cells compared with controls and prompted us to monitor lactate concentration in the cell culture medium.…”

“…Modulation of Mitochondrial NAD ϩ Levels-In a previous study, it was noted that 293mitoPARP cells have a constitutively lower NAD ϩ content, because of the mitochondrial NAD ϩ consumption introduced by the mitoPARP construct (29). 293mitoPARP cells exhibited a higher lactate production rate and lowered mitochondrial membrane potential but did not have any obvious deficiencies.…”

“…For example, pharmacological inhibitors are likely to affect other cellular NAD ϩ pools as well and may induce acute responses independent of long term alterations of the mitochondrial NAD content (28). Using a transgenic cell-based approach, we have previously shown that a diminished mitochondrial NAD ϩ pool affects mitochondrial metabolism and leads to accelerate lactate production (29).…”

“…To determine whether overexpression of these transporters in human cells had an influence on mitochondrial NAD ϩ contents, we made use of the previously established PARAPLAY system (23,30). This system is based on the accumulation of immunodetectable PAR mediated by mitoPARP, a mitochondrially targeted fusion protein consisting of an enhanced GFP (EGFP) portion and the catalytic domain of PARP1, which converts mitochondrial NAD ϩ to PAR in a concentration-dependent manner (23,29). In HEK293 cells (subsequently referred to as 293 cells) transiently expressing mitoPARP, the PAR signal is readily detectable (Fig.…”

Poly(ADP-ribose) polymerases as modulators of mitochondrial activity

“…However, these studies were based on the extraction of PARylated proteins using a PAR antibody and given the existing discrepancies between proteins identified by the different studies [29,30]; additional, non-immunological methods are required to verify these findings. Importantly, when PARP-1 was overexpressed in mitochondria, in vitro, mitochondrial PARylation increased, which was accompanied by decreased mitochondrial output and unaltered glycolytic flux [33], highlighting a possible functional consequence of mitochondrial PARylation. Taken together, PAR-degrading activity appears to be present in mitochondria, while well-defined PARP activity or presence is missing.…”