Cellular redox sensor HSCARG negatively regulates the translesion synthesis pathway and exacerbates mammary tumorigenesis

Abstract

The translesion synthesis (TLS) pathway is a double-edged sword in terms of genome integrity. Deficiency in TLS leads to generation of DNA double strand break (DSB) during replication stress, while excessive activation of the TLS pathway increases the risk of point mutation. Here we demonstrate that HSCARG, a cellular redox sensor, directly interacts with the key protein PCNA in the TLS pathway. HSCARG enhances the interaction between PCNA and the deubiquitinase complex USP1/UAF1 and inhibits the monoubiquitination of PCNA, thereby impairing the recruitment of Y-family polymerases and increasing cell sensitivity to stimuli that trigger replication fork blockades. In response to oxidative stress, disaggregation of HSCARG dimers into monomers and the nuclear transport of HSCARG activate the regulatory function of HSCARG in the TLS pathway. Moreover, HSCARG, which is highly expressed in breast carcinoma, promotes the accumulation of DSBs and mutations. HSCARG knockout PyMT transgenic mice exhibit delayed mammary tumorigenesis compared with that in HSCARG wild-type or heterozygous PyMT mice. Taken together, these findings expand our understanding of TLS regulatory mechanisms and establish a link between the cellular redox status and the DNA damage response (DDR).

Structure and functions of cellular redox sensor HSCARG/NMRAL1, a linkage among redox status, innate immunity, DNA damage response, and cancer

“…In contrast, mutant K41S retains the capability of homodimerization because serine, unlike alanine, is able to form similar hydrogen bond with NADPH as lysine [ 12 ]. In comparison to the wild-type protein, mutants R37A and Y81A, but not mutant K41S, exhibit enhanced interactions with their binding partners, indicating that formation of asymmetrical dimers inactivates the regulatory functions of HSCARG [ 13 , 14 ].…”

“…Accordingly, enhancement of interaction between HSCARG and ASS was observed in DHEA-treated cells, proving that the ability of HSCARG to impair arginine recycling and NO synthesis is activated after dissociation with NADPH [ 14 ]. The conclusion was reinforced by the fact that HSCARG NADPH-unbound mutants R37A and Y81A, which exists only as monomer, exhibit increased interaction with ASS and more efficient inhibitory effects on p47phox protein level in comparison to the wild-type protein [ 12 , 13 ]. Considering both NADPH oxidase-catalyzed ROS production and NOS-catalyzed NO production are couple with oxidation of NADPH into NADP + , one can speculate that HSCARG might regulate NADPH homeostasis via feedback inhibition of NADPH-consuming pathways and promote cell resistance against oxidative stress.…”

“…Overexpression of HSCARG partly rescues UV-induced decrease of chromatin-associated USP1 level. As a result, upregulation of HSCARG or nuclear enrichment of HSCARG caused by high dosage of SNAP treatment block the accumulation of POLH at replication stalling sites in response to UV radiation, which leads to collapse of replication forks and generation of DSBs [ 13 ].…”

“…Consistently, deletion of HSCARG in female polyomavirus middle T antigen (PyMT) transgenic mice delays the genesis of breast carcinoma. Interestingly, in newly developed tumors from PyMT mice, the upregulated HSCARG protein accumulate in the nucleus, which is most likely due to oxidative stress in the microenvironment [ 13 ]. This observation reinforces the conclusion that negative regulation of DNA damage response in the nucleus is, at least in part, responsible for the oncogenic effects of HSCARG ( Fig.…”

“…On the one hand, upon dissociation from NADPH, HSCARG transforms from functionally inactive homodimer to active monomer. Indeed, it was observed that treatment of cells with moderate amount of SNAP (50 μM) and DHEA (100 μM) activated the interaction between HSCARG and ASS and elevated the inhibitory effect of HSCARG on ROS production [ 13 , 14 ]. On the other hand, over-enrichment of HSCARG in the nucleus under violent and persistent oxidative stress attenuates its functions in the cytoplasm.…”