NEIL3-mediated proteasomal degradation facilitates the repair of cisplatin-induced DNA damage in human cells

Abstract

Anti-neoplastic effect of DNA cross-linking agents such as cisplatin, mitomycin C, and psoralen is attributed to their ability to induce DNA interstrand cross-links (ICLs), which block replication, transcription, and linear repair pathways by preventing DNA strand separation and trigger apoptosis. It is generally agreed that the Fanconi anemia (FA) pathway orchestrates the removal of ICLs by the combined actions of various DNA repair pathways. Recently, attention has been focused on the ability of the NEIL3-initiated base excision repair pathway to resolve psoralen- and abasic site-induced ICLs in an FA-independent manner. Intriguingly, overexpression of NEIL3 is associated with chemo-resistance and poor prognosis in many solid tumors. Here, using loss- and gain-of-function approaches, we demonstrate that NEIL3 confers resistance to cisplatin and participates in the removal of cisplatin-DNA adducts. Proteomic studies reveal that the NEIL3 protein interacts with the 26S proteasome in a cisplatin-dependent manner. NEIL3 mediates proteasomal degradation of WRNIP1, a protein involved in the early step of ICL repair. We propose that NEIL3 participates in the repair of ICL-stalled replication fork by recruitment of the proteasome to ensure a timely transition from lesion recognition to repair via the degradation of early-step vanguard proteins.

Figure 1

NEIL3 is involved in the repair of cisplatin-induced DNA lesions and protects human cells from cisplatin-induced apoptosis. (a) Western blot of Flag IP samples purified from HeLa S3 cells overexpressing NEIL3 protein (NEIL3-FH). Ponceau S staining was used as a loading control. Note that the membrane was cropped to remove irrelevant parts. (b) Measurement of apoptosis by flow cytometry of Annexin V and Propidium Iodide (PI) stained cells incubated overnight in the presence of different doses of cisplatin (CDDP). Data shown are the mean ± SD (n = 4). *p <0.05; **p <0.01; ***p <0.001. (c) Dot blot with an antibody recognizing cisplatin-modified DNA to measure the accumulation of cisplatin–DNA adducts after overnight exposure to cisplatin (15 µg/mL). SYBR Gold staining was used as a loading control. (d) Characterization of the HeLa S3 NEIL3-/- cell lines constructed using CRISPR-Cas9 genome editing technique. The disruption of the NEIL3 gene in clones #1–1 and #2–6 was confirmed by RT-qPCR. Data shown are the mean ± SD (n = 5). ****p <0.0001. (e, f) Same as in “b” and “c” panels except that HeLa S3 NEIL3^-/- cells were used. See Materials and Methods for additional details.

Figure 2

26S proteasome plays a role in the NEIL3-mediated removal of cisplatin–DNA adducts. (a) Schematic outline of the mass-spectrometry analysis of Flag IP samples performed before and after 30 min treatment of HeLa S3 NEIL3-FH cells with 15 µg/mL cisplatin. (b) Venn diagrams representing the results of mass-spectrometry: “only ctrl” denotes proteins found in control non-treated cells; “cis < ” and “cis > ” indicate proteins that increase or decrease, respectively, after cisplatin treatment; “cis = ” refers to proteins that do not change after the treatment; “only cis” designates proteins that are detected only after the treatment. (c) Western blot of Flag IP samples purified from chromatin fractions of NEIL3-FH cells treated with cisplatin (CDDP, 15 µg/mL) for the indicated period of time. Note that the membrane was cropped to remove irrelevant parts. (d) Measurement of apoptosis by flow cytometry of Annexin V and Propidium Iodide (PI) stained cells pretreated with 0.5 µM CFZ followed by overnight incubation with different doses of CDDP. Data shown are the mean ± SD (n = 3). **p <0.01; ***p <0.001. (e) Dot blot with an antibody recognizing cisplatin-modified DNA to measure the accumulation of cisplatin–DNA adducts after the pretreatment with 0.5 µM CFZ followed by overnight exposure to CDDP (15 µg/mL). SYBR Gold staining was used as a loading control. See Materials and Methods for additional details.

Figure 3

WRNIP1 interaction with the 26S proteasome is stabilized by the proteasome inhibitors. (a) Proteins identified by mass spectrometry in the NEIL3-Flag/HA protein complexes analyzed before and after 3 h treatment with either 15 µg/mL CDDP or 0.5 μM CFZ, or a combination of both. (b) Western blot of Flag IP samples purified from NEIL3-FH cells treated with CDDP, CFZ, or both drugs. (c) Western blot of Flag IP samples purified from NEIL3-FH cells treated with different doses of MG-132, a reversible proteasome inhibitor. (d) Western blot of Flag IP samples purified from NEIL3-FH cells pre-treated with 0.5 μM CFZ followed by 15 µg/mL CDDP treatment for different time points demonstrating the dynamics of interaction between NEIL3, WRNIP1 and 26S proteasome. Note that membranes were cropped to remove overexposed parts. IP experiments were normalized by Flag/HA tagged protein used as a bait and quantified using ImageJ software. Ratios for each protein are indicated below the given blot. See Materials and Methods for additional details.

Figure 4

WRNIP1 is degraded in the ubiquitin-proteasome pathway. (a) Schematic outline of the experiment shown in panel “b”. (b) Western blot of Flag IP samples purified from PSMB2-FH cells treated with MG-132 as depicted in panel “a”. (c) Western blot of Flag IP samples purified from HeLa S3 PSMB2-FH cells treated with broad ubiquitylation inhibitor TAK-243 and carfilzomib. (d) Effect of NEIL3 knockdown on WRNIP1 degradation. Left, Western blot of Flag IP samples purified from PSMB2-FH cells treated with cisplatin, CFZ, and NEIL3 siRNA; Right, confirmation of NEIL3 knockdown by RT-qPCR. Data shown are the mean ± SD (n = 3). **p <0.01. Note that the membranes were cropped to remove overexposed parts. CF, chromatin fraction; SF, soluble fraction that contains cytoplasmic and nuclear proteins; FT, flow-through; W1, first wash of the beads. IP experiments were normalized by Flag/HA tagged protein used as a bait and quantified using ImageJ software. Ratios for each protein are indicated below the given blot. See Materials and Methods for additional details.

Figure 5

A model of the NEIL3-dependent proteasomal degradation of the WRNIP1 protein upon cisplatin-induced ICLs.