Abstract
NAD+ is one of the most crucial molecules in the body, playing a vital role in energy metabolism and cellular signaling. NAD+ exists in oxidized (NAD+) and reduced (NADH) as well as in its phosphorylated forms (NADP+ and NADPH). These states are constantly interchanging, and recent developments of genetically encoded tools have improved the detection and monitoring of NAD+ levels in cells, thereby substantially advancing the field of NAD+ research. NAD+ can also be used as a substrate for post-translational modifications i.e. ADP-ribosylation, catalyzed by the enzyme family of ADP-ribosyltransferases. Among these, PARP9 has recently emerged as an interesting player in the innate immune response pathway. Although PARP9 has been found to act pro-tumorigenic through its role as a transcriptional repressor of IRF1, the general role of PARP9 remains poorly explored. This thesis aims to further elucidate the role of PARP9 in cellular homeostasis by focusing on (i) its impact on autophagy; (ii) its influence on ROS metabolism; and (iii) its effect on cell proliferation. In our first study, we found that PARP9 acts as a transcriptional activator of the autophagy receptor p62, since PARP9 knockdown led to a decrease in the specific autophagy receptor p62. Reduction of p62 due to loss of PARP9 resulted in an increased levels of peroxisomes, mitochondria and protein aggregates within the cell. This increase in peroxisomes subsequently raised ROS levels, leading to oxidative stress related modification of RNA and chromatin. The observed dysregulation of cellular homeostasis likely reduced proliferation and accumulation of cells in the G2 phase. In our second study, we discovered that two antibodies which were raised against ADP-ribose to detect ADP-ribosylation cross-reacted with formaldehyde-fixed NADH and NADPH. Notably, the reduction of mitochondrial NADPH-producing enzymes significantly impacted the observed heterogenous immunofluorescence signals intensities, confirming their crucial role in maintaining proper NADPH levels. Similarly, reducing cytoplasmic NADPH-modulating enzymes affects the mitochondrial NADPH pool, underscoring the interconnected nature of cellular compartments in regulating NADPH. Interestingly, the expression levels of p62 showed a strong correlation with the mitochondria-associated NADPH signal, and knockdown of p62 reduced the signal, highlighting its potential role as a regulator in the homeostasis of NADH/NADPH levels. Taken together, our findings indicate that PARP9 regulates transcriptional activation of p62 as well as p62-regulated selective autophagy and thus plays a critical role in cellular homeostasis. In addition, we provide evidence that certain antibodies can detect NADH and NADPH, making them a valuable addition to the toolkit for studying these metabolites in cells. Furthermore, our study showed that the specific autophagy receptor p62 plays a role in maintaining mitochondrial NADH/NADPH levels.
Muskalla, Lukas