Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A DDB2 . The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A DDB2 complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A DDB2 ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.E3 ubiquitin ligase ͉ monoubiquitinated histone H2A ͉ nucleotide excision repair

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4 DCAF1 ). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1-and CUL4-associated factor 1 (DCAF1 The viral protein R (Vpr) 2 is one of four HIV-1 accessory proteins (Nef, Vif, Vpr, and Vpu), that regulate virus infectivity, primarily through interactions with host proteins (1). Vpr is highly conserved in HIV-1, HIV-2, and the simian immunodeficiency viruses (2-4). Several biological roles for Vpr during viral infection of cells have been described, including facilitation of nuclear translocation of preintegration complexes (5-7), modulation of mutation frequency (8, 9), induction of cell cycle arrest in the G 2 /M phase (10 -15), and stimulation of host cell apoptosis (16 -18). More recently, Vpr has been postulated to enhance infection by mediating the degradation of unknown cellular defense factors (1, 19).To date, three of the four HIV-1 accessory proteins, including Vpr, have been found to interact with cullin-RING finger E3 ubiquitin ligases (CRLs). E3 ligases are multisubunit complexes that include a cullin (CUL), a RING H2 finger protein (RBX1), an adaptor, and a substrate recognition subunit (20). More specifically, Vpr interacts with the CRL4 DCAF1 E3 ubiquitin ligase, assembled with cullin 4A (CUL4A), RBX1, DDB1 (DNA damage-binding protein 1), and DCAF1 (DDB1-and CUL4-associated factor 1) (1, 21). The substrate recognition subunit of this CRL4, DCAF1, previously known as Vpr-binding protein (VprBP), was originally identified via co-precipitation with Vpr (22). At the present time, substantial evidence suggests that Vpr usurps CRL4 DCAF1 E3 ubiquitin ligases to ubiquitinate (ubiquitylate) and degrade unknown cellular proteins required for cell cycle progression (23-31). In fact, Vpr was reported to bind and modulate the activity of cell cycle-related proteins, such as CDC25 (32), WEE1 kinase (33), and SAP145 (34, 35). In addition, Vpr activates ATM and Rad3-related checkpoint kinase (ATR)-dependent DNA damage signaling pathways, including phosphorylation of the histone 2A variant-X and BRCA1 (36, 37). However, whether Vpr interactions with these proposed host cellular factors result in their degradation via CRL4 DCAF1-Vpr E3 ubiquitin ligases had not been established.One potential target of Vpr is uracil-DNA glycosylase-2 (UNG2), which removes uracil lesions from single-stranded and double-stranded DNA in the base excision repair pathway. Initially identified in a yeast two-hybrid screen (38), UNG2 has been implicated as a Vpr-dependent substrate of the CRL4 E3 ubiquitin ligase (39,40).However, a number of studies investigating the roles of UNG2 in HIV replication resulted in opposing views regarding HIV biology. For example, some studies found that virion-associated UNG2 modulates the innate ...

By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatinembedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A DDB2 . We previously showed that DDB1-CUL4A DDB2 ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A DDB2 and DDB1-CUL4B DDB2 . To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B DDB2 E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B DDB2 is more efficient than DDB1-CUL4A DDB2 in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B DDB2 E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER. [Cancer Res 2008;68(13):5014-22]

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.