Arsenite Binds to the RING Finger Domain of FANCL E3 Ubiquitin Ligase and Inhibits DNA Interstrand Crosslink Repair

Abstract

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in FA-mediated DNA repair. Here, we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5′,8-trimethylpsoralen plus UVA irradiation. Furthermore, clonogenic survival assay results showed that arsenite coexposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may compromise genomic stability via perturbation of the Fanconi anemia pathway, thereby conferring its carcinogenic effect.

Arsenite Binds to ZNF598 to Perturb Ribosome-Associated Protein Quality Control

“…In light of the previous observations about the effects of As(III) binding on modulating the E3 ubiquitin ligase activities of other RING finger proteins, [20][21][22]25 we reasoned that the decreases in ubiquitination of RPS10 and RPS20 may be attributed to the interaction between As(III) and ZNF598 and the ensuing loss of its E3 ubiquitin ligase activity. To test this, we first examined the interaction between As(III) and ZNF598.…”

“…16 To this end, we performed a quantitative proteomic experiment, which relies on metabolic labeling using SILAC and immunoprecipitation of tryptic peptides containing diglycine remnant-modified lysine(s), and LC-MS/MS analysis, to identify proteins in GM00637 human skin fibroblasts exhibiting substantial alterations in their ubiquitination levels after a 24 h exposure to 5.0 μM NaAsO 2 (Figure 1A). 25 We found that the ubiquitination levels of lysine 138 and 139 (K138 and K139) in RPS10 and K8 in RPS20 were decreased to 60% and 52% of the levels observed in control untreated cells, respectively (Figure 1B). Representative MS and MS/MS results for monitoring the ubiquitination of K8 in RPS20 are shown in Figure 2, and the MS/MS for monitoring the ubiquitinations of K138 and K139 in RPS10 are shown in Figure S1.…”

“…As 3+ Binds to RING Finger Motif of ZNF598 and Induces Proteotoxic Stress. As 3+ can interact with zinc finger proteins, [20][21][22]25,27,28 especially with high affinities to C 3 H and C 4 types of zinc fingers. 18 In this study, we provided two lines of evidence to support the notion that arsenite can bind to the RING finger domain of ZNF598 by displacing its bound Zn 2+ ions.…”

“…SILAC labeling of GM00637 cells in light or heavy RPMI 1640 medium, As 3+ exposure, immunoprecipitation of ubiquitin remnant peptides, and LC−MS/MS analysis of the enriched peptides were described previously. 25 Fluorescence Microscopy. Wild-type pcDNA4/TO-GFP-ZNF598 plasmid (0.5 μg) was transfected into 1 × 10 5 HEK293T cells seeded on cover glasses placed in a 24-well plate.…”

Arsenite Targets the RING Finger Domain of Rbx1 E3 Ubiquitin Ligase to Inhibit Proteasome-Mediated Degradation of Nrf2

“…6 Since most RING finger domains harbor a C3H- and a C4-type zinc finger, 7–9 our recently published work demonstrated the interactions between arsenite and the RING finger domains of proteins, including several E3 ligases, such as FANCL and RNF20-RNF40, which ultimately led to the inhibition of the repair of DNA interstrand cross-link lesions and double strand breaks, respectively. 10,11 Our previous work also elucidated the interaction between arsenite and the zinc finger motifs of Tip60 histone acetyltransferase and ten-eleven translocation (Tet) family proteins, which perturbs histone and DNA epigenetic marks. 12,13…”