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

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).

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase-dependent manner and regulates the DNA damage response.

Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (γH2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G2-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.

Deubiquitinase OTUB2 exacerbates the progression of colorectal cancer by promoting PKM2 activity and glycolysis.

Aberrant regulation of ubiquitination often leads to metabolic reprogramming in tumor cells. However, the underlying mechanisms are not fully understood. Here we demonstrate that OTUB2, an OTU deubiquitinase, is upregulated in colorectal cancer (CRC) and exacerbates the progression of CRC through modulating the aerobic glycolysis. Mechanistically, OTUB2 directly interacts with pyruvate kinase M2 (PKM2) and inhibits its ubiquitination by blocking the interaction between PKM2 and its ubiquitin E3 ligase Parkin, thereby enhancing PKM2 activity and promoting glycolysis. In response to glucose starvation stress, the effect of OTUB2 on PKM2 is enhanced, which confers metabolic advantage to CRC cells. Moreover, OTUB2 depletion reduces glucose consumption, lactate production, and cellular ATP production. OTUB2-knockout CRC cells exhibit attenuated proliferation and migration, as well as an elevated level of apoptosis and increased sensitivity to chemotherapy drugs. Furthermore, in vivo assays show that knockout of OTUB2 inhibits tumor growth in mice. Taken together, these findings reveal the critical role of OTUB2 in the regulation of glycolysis and illustrate the molecular mechanism underlying its role as a negative regulator of PKM2 ubiquitination in CRC, establishing a bridge between OTUB2-regulated PKM2 ubiquitination and altered metabolic patterns in CRC and suggesting that OTUB2 is a promising target for CRC treatment.

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

NmrA-like proteins are NAD(P) (H) interacting molecules whose structures are similar to that of short-chain dehydrogenases. In this review, we focus on an NADP(H) sensor, HSCARG (also named NMRAL1), which is a NmrA-like protein that is widely present in mammals, and provide a comprehensive overview of the current knowledge of its structure and physiological functions. HSCARG selectively binds to the reduced form of type II coenzyme NADPH via its Rossmann fold domain. In response to reduction of intracellular NADPH concentration, HSCARG transforms from homodimer to monomer and exhibits enhanced interactions with its binding partners. In the cytoplasm, HSCARG negatively regulates innate immunity through impairing the activities of NF-κB and RLR pathways. Besides, HSCARG regulates redox homeostasis via suppression of ROS and NO generation. Intensive and persistent oxidative stress leads to translocation of HSCARG from the cytoplasm to the nucleus, where it regulates the DNA damage response. Taken together, HSCARG functions as a linkage between cellular redox status and other signaling pathways and fine-tunes cellular response to redox changes.

A20/TNFAIP3 Regulates the DNA Damage Response and Mediates Tumor Cell Resistance to DNA-Damaging Therapy.

A competent DNA damage response (DDR) helps prevent cancer, but once cancer has arisen, DDR can blunt the efficacy of chemotherapy and radiotherapy that cause lethal DNA breakage in cancer cells. Thus, blocking DDR may improve the efficacy of these modalities. Here, we report a new DDR mechanism that interfaces with inflammatory signaling and might be blocked to improve anticancer outcomes. Specifically, we report that the ubiquitin-editing enzyme A20/TNFAIP3 binds and inhibits the E3 ubiquitin ligase RNF168, which is responsible for regulating histone H2A turnover critical for proper DNA repair. A20 induced after DNA damage disrupted RNF168-H2A interaction in a manner independent of its enzymatic activity. Furthermore, it inhibited accumulation of RNF168 and downstream repair protein 53BP1 during DNA repair. A20 was also required for disassembly of RNF168 and 53BP1 from damage sites after repair. Conversely, A20 deletion increased the efficiency of error-prone nonhomologous DNA end-joining and decreased error-free DNA homologous recombination, destablizing the genome and increasing sensitivity to DNA damage. In clinical specimens of invasive breast carcinoma, A20 was widely overexpressed, consistent with its candidacy as a therapeutic target. Taken together, our findings suggest that A20 is critical for proper functioning of the DDR in cancer cells and it establishes a new link between this NFκB-regulated ubiquitin-editing enzyme and the DDR pathway.Significance: This study identifies the ubiquitin-editing enzyme A20 as a key factor in mediating cancer cell resistance to DNA-damaging therapy, with implications for blocking its function to leverage the efficacy of chemotherapy and radiotherapy. Cancer Res; 78(4); 1069-82. ©2017 AACR.