XPA is susceptible to proteolytic cleavage by cathepsin L during lysis of quiescent cells

Abstract

The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3-4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.

Keywords: Cathepsin; Keratinocyte; Nucleotide excision repair; Proteolysis; Quiescence; UV radiation; XPA.

Figure 1.. A proteolytically cleaved form of XPA is generated during lysis of quiescent cells and fails to interact with the NER factor TFIIH.

(A) Cell lysates were prepared from sub-confluent HaCaT cells (proliferating) and cells grown to confluence and then maintained for 2 days in low serum (quiescent). Lysates were analyzed by western blotting with four different anti-XPA antibodies (#1, Santa Cruz sc-28353; #2, Abcam ab65963; #3, Cell Signaling #14607; and #4, Santa Cruz sc-853). (B) XPA protein expression was detected in lysates prepared from wild-type (WT) HaCaT cells and a derivative line in which XPA was knocked out using CRISPR/Cas9 (XPA-KO). (C) Cell lysates prepared from cells after growth for the indicated periods of time were analyzed by immunoblotting for XPA. (D) HaCaT cell pellets were resuspended in lysis buffer and then protease inhibitor cocktail (PIC) was added to the lysis solution at the indicated time points. Lysates were analyzed by immunoblotting for XPA. (E) Cells were analyzed as in (D) except SDS was added at the indicated time points. (F) MBP-XPA was incubated in TLB in the absence or presence of confluent HaCaT cells and PIC and then examined by Coomassie staining or western blotting. (G) HaCaT cells were lysed in TLB containing MBP-XPA in the absence or presence of PIC. Anti-MBP magnetic beads were used to isolate the MBP-XPA from the cell lysates, which were then analyzed by western blotting with antibodies against the indicated proteins.

Figure 2.. Cathepsin L is expressed at high levels in quiescent cells and is responsible for XPA cleavage but does not impact UVB survival or photoproduct removal from DNA.

(A) Confluent HaCaT cells were lysed in TLB containing the indicated protease inhibitor. (B) Lysates from proliferating and quiescent cells were analyzed by western blotting with antibodies against cathepsin B and L (CTSB and CTSL). (C) Lysates from HaCaT cells grown to confluence (Figure 2) were probed for CTSB and CTSL. (D) HaCaT cells were lysed in the presence of the indicated specific cathepsin inhibitors. (E) Quiescent HaCaT cells were treated with 10 μM CTSLi 30 min prior to cell lysis (Pre-) or at the time of cell lysis (Post-). (F) MBP-XPA was incubated with recombinant cathepsin L or confluent HaCaT cells in the absence or presence of SDS or cathepsin inhibitor (CTSLi). (G) Quiescent HaCaT cells were treated for 24 hr with 10 μM CTSLi before exposure to the indicated fluence of UVB radiation. MTT assays were performed 3 days later, and the graph shows results from 3 independent experiments (average and SEM). (H) Quiescent HaCaT cells were pre-treated with CTSLi for 30 min before exposure to 150 J/m2 UVB. Genomic DNA was purified from cells at the indicated time points and analyzed by DNA immunoblotting with an anti-(6–4)PP antibody. Results are the average and SEM from two independent experiments.