Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress

Abstract

Protein–protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein–protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein–protein interactions. We show that proteasome-mediated degradation of Polβ is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein–protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polβ in the cytosol via interaction between Polβ and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polβ with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polβ to the nuclear compartment and regulates the stability of Polβ via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polβ/NQO1 complex, enhancing the interaction of Polβ with XRCC1. Our results reveal that somatic mutations such as T304I in Polβ impact critical protein–protein interactions, altering the stability and sub-cellular localization of Polβ and providing mechanistic insight into how key protein–protein interactions regulate cellular responses to stress.

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…At least four different ligation states (with small molecules bound) are relevant to understand the intracellular stability and enzymatic activity of NQO1 as well as its effects on the function and stability of its macromolecular partners: i) NQO1apo, which has no ligand bound; ii) NQO1holo, which contains a molecule of oxidized FAD per NQO1 monomer; iii) NQO1holo-red , containing the FAD cofactor reduced upon interaction with NAD(P)H and hydride transfer; and iv) NQO1dic, a ternary complex of NQO1holo with the inhibitor dicoumarol bound. These different ligation states interact differently with NQO1 protein partners [78,117,127]. High-resolution structural models for these complexes are, to the best of our knowledge, not reported.…”

“…Indeed, NQO1 overexpression in mice is known to enhance glycolytic and mitochondrial respiration activities and enhance metabolic flexibility, mimicking the beneficial effects of caloric restriction [124]. It is worth noting that the proteasomal protein degradation machinery may operate through rather similar mechanisms in the cytosol and the nucleus [125,126], and thus, the well-known chaperone role of NQO1 for different protein partners (see Table A2), may indeed operate in the cytosol thus increasing the levels of cytosolic proteins amenable to import to other organelles (such as nucleus or mitochondria; [74,127]) and plausibly by stabilizing these proteins upon import of both the partner and NQO1 in these organelles. Noteworthy, although more rarely described, the presence of NQO1 in other subcellular locations such as cytoskeleton may explain other roles of the multifunctional NQO1 protein.…”

“…However, it is worth commenting on some studies carried out under less stringent (i.e. aerobic) conditions, particularly because a wealth of work support that generally, this state may strongly interact with NQO1 protein partners [117,127]. Using biochemical in vitro assays with NADP(H), it has been reported that NQO1holo-red influences the conformation of the CTD affecting the interaction with antibodies raised against it [118] and apparently increasing the thermodynamic stability of this domain [80,118].…”

“…Binding of dicoumarol (or NAD + ) causes small structural rearrangements in the protein structure [75,78,81,131,134] (Figure 6A). Therefore, from NQO1 structural point of view, it is not straightforward to understand how NQO1dic prevents binding to or largely decrease binding affinity for macromolecular partners with subsequent effects on the stability of these partners [78,127]. An alternative explanation has emerged from studies on NQO1 protein dynamics by HDXMS [129].…”

Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

“…January 17, 2021 by guest http://jvi.asm.org/ Downloaded from S1-RBD samples with the hAPN and purified SARS-CoV S-trimer, S1-RBD samples with the hACE2 were measured by surface plasmon resonance (OpenSPR, Nicoyalife), as described previously(42)(43)(44). Briefly, the hAPN or hACE2(200µl, 5µg) was immobilized on the OpenSPR™ COOH Sensor Chip (Nicoya # SEN-AU-100-12-COOH).…”