We screened a collection of LF mutants, which were generated by error-prone PCR, for a mutant that was defective at killing RAW 264.7 cells (data not shown). One of the identified mutants contained two substitution mutations, K518E and E682G (). Amino acid K518 is within a patch of amino acids that was previously implicated in binding MAPKKs [22
]. Amino acid E682 is within an α-helix that also contains the amino acids that form the 686
metalloprotease motif () [23
]. We performed a limited tryptic digest to assess whether the mutations altered the tertiary structure of LF. Purified wild-type LF or LF-K518E/E682G was incubated with various concentrations of trypsin and the mixtures were then subjected to SDS-PAGE. Differences between the patterns of tryptic fragments were observed for LF-K518E/E682G and wild-type LF and the mutant appeared to be somewhat more sensitive to trypsin (). This suggested that while the mutations altered the tertiary structure of the protein, they did not cause it to become grossly misfolded and destabilized. Since we were interested in characterizing a mutant with altered catalytic properties, rather than identifying amino acids that might bind substrates directly, we decided to study this mutant further.
Figure 1 An LF double mutant, LF-K518E/E682G. (A) Structure of the catalytic domain of LF. Amino acids 518 and 682 are shown in red. Residues of the HExxH motif are depicted in green. An optimized peptide substrate is shown in blue. The model was created using (more ...)
We first assessed the severity of the cytotoxicity defect caused by the mutations. PA and various concentrations of either wild-type LF or LF-K518E/E682G were incubated with RAW 264.7 cells for 4 h and cell viability was estimated using the MTS assay, which measures mitochondrial function. Whereas the concentration of LF required to kill 50% of the cells (EC50) was estimated to be 4 × 10−11 M, LF-K518E/E682G did not cause enough cell death under these conditions for an accurate EC50 to be determined (). Increasing the time of toxin exposure from 4 h to 24 h did not markedly decrease the EC50 for wild-type LF or decrease the viability of cells exposed to the mutant (data not shown). The reduced ability of the LF mutant to kill RAW 264.7 cells was tested further using a trypan blue exclusion assay (). Cells were left untreated (black bars) or were exposed to a mixture of 10−8 M PA and 10−8 M wild-type LF (white bars) or mutant LF (grey bars) for either 4 h or 24 h and the fraction of cells that excluded trypan blue under each condition was determined. Similar to what was observed using the MTS assay, this assay indicated that LF-K518E/E682G was less cytotoxic than wild-type LF; increasing the time of toxin incubation from 4 h to 24 h did not lead to an increased level of cell death ().
Figure 2 LF-K518E/E682G is defective at killing RAW 264.7 cells and inducing the Nlrp1b inflammasome. (A) PA and various concentrations of wild-type LF (σ) or LF-K518E/E682G (○) were incubated with RAW 264.7 cells and viability was assessed after (more ...)
To confirm that LF-K518E/E682G is defective at activating Nlrp1b, we used an independent approach that takes advantage of a recently developed heterologous expression system [24
]. HT1080 human fibroblasts were transfected with plasmids encoding murine Nlrp1b, pro-caspase-1 and pro-IL-1β and after ~24 h the cells were treated with combinations of PA, LF and LF-K518E/E682G. PA and LF activated the inflammasome as determined by the loss of pro-IL-1β in the cytosol and the appearance of IL-1β in the cell supernatants (). A lower level of IL-1β was found in the supernatants of cells treated with LF-K518E/E682G, suggesting that the mutant was defective at activating the inflammasome. LF-K518E/E682G entered cells and was catalytically active, however, since it cleaved MAPKK1 ().
Since it is unclear whether cleavage of MAPKKs by LF causes pyroptosis of RAW 264.7 cells, we attempted to correlate cyotoxicity with downregulation of the MAPK pathways. RAW 264.7 cells were treated with PA and either wild-type LF or LF-K518E/E682G and then the cells were stimulated with lipopolysaccharide to activate the signaling pathways. Cellular lysates were prepared and probed for phosphorylated ERK, p38, and JNK by Western blotting (). Exposure of cells to PA and increasing concentrations of wild-type LF for 1 h resulted in decreased phosphorylation of the three MAPKs. Interestingly, increasing the LF concentration from 10−11 M to 10−10 M had a considerable effect on cell viability, but relatively minor effects on the phosphorylation of the MAPKs (compare Figs. and ). LF-K518E/E682G decreased phosphorylation of ERK almost as effectively as wild-type LF, but did not decrease phosphorylation of p38 or JNK below the level observed in cells treated with lipopolysaccharide alone. Thus, while wild-type LF interfered with signaling in all three MAPK pathways, LF-K518E/E682G selectively downregulated the ERK pathway.
Figure 3 LF-K518E/E682G inhibits the phosphorylation of ERK, but not of p38 or JNK. RAW 264.7 cells were treated with 10−8 M PA and the indicated concentrations of either wild-type LF or LF-K518E/E682G for 1 h and then treated with lipopolysaccharide for (more ...)
To examine why the mutant demonstrated increased specificity towards downregulating the ERK pathway, we next compared the abilities of wild-type LF and LF-K518E/E682G to cleave MAPKKs (). MAPKK1 and MAPKK2, which phosphorylate ERK, were both cleaved by wild-type LF as assessed by Western blotting. At the highest concentration of LF tested (10−8 M), ~50% of MAPKK1 and ~60% of MAPKK2 was cleaved after 1 h. Treatment of cells with PA and 10−8 M LF-K518E/E682G resulted in ~50% of MAPKK1 and ~20% of MAPKK2 being cleaved. Since the mutant was able to downregulate the ERK pathway almost as efficiently as wild-type LF, these results suggest that MAPKK1 is primarily responsible for ERK activation under these conditions.
Figure 4 LF-K518E/E682G has a reduced ability to cleave some MAPKKs. RAW 264.7 cells were treated with 10−8 M PA and the indicated concentrations of either wild-type LF or LF-K518E/E682G for 1 h. Cellular lysates were prepared and probed for phosphorylated (more ...)
We next sought to determine the cause of the mutant’s deficiency at downregulating p38 by examining the cleavage of MAPKK3 and MAPKK6. LF-K518E/E682G was modestly defective at cleaving MAPKK3 compared to wild-type LF, but was considerably more defective at cleaving MAPKK6. The inability of the mutant to prevent phosphorylation of p38 () indicates that the level of MAPPK3/6 that remained in the cell was sufficient to support maximal p38 phosphorylation.
We next probed cellular lysates for MAPKK4 and MAPKK7, which phosphorylate JNK. LF-K518E/E682G cleaved similar amounts of MAPKK4 as did wild-type LF. Neither wild-type LF nor the mutant cleaved appreciable amounts of MAPKK7 after 1 h of toxin treatment. Thus, wild-type LF and LF-K518E/E682G exhibited similar activities towards MAPKK4 and 7, but only wild-type LF reduced the level of phosphorylation of JNK to ~50% as compared to the control. There is no evident explanation for these results; the difference in JNK phosphorylation observed might be due to an indirect effect of intoxication.
Since downregulation of the ERK pathway has been shown to be sufficient to cause apoptosis in MALME-3M cells [20
], we next compared the activities of wild-type LF and LF-K518E/E682G in a cytotoxicity assay using this melanoma cell line. PA and either wild-type or mutant LF were incubated with MALME-3M cells for 72 h and viability was estimated using the MTS assay () [20
]. The EC50
for wild-type LF was determined to be 2 × 10−13
M and the EC50
for LF-K518E/E682G was only ~3-fold higher at 7 × 10−13
M. These results indicate that LF-K518E/E682G is markedly more defective compared to wild-type LF at killing the murine macrophage cells than the melanoma cells. We next assessed the phosphorylation of ERK in MALME-3M cells treated with either wild-type or mutant LF and found that LF-K518E/E682G downregulated the ERK pathway almost as effectively as wild-type LF did (). This is consistent with previous work that indicated the requirement of ERK signaling for survival of these cells and suggests that different types of cells are killed by LF as a result of the cleavage of distinct substrates.
Figure 5 LF-K518E/E682G causes death of melanoma cells. (A) PA and various concentrations of wild-type LF (σ) or LF-K518E/E682G (○) were incubated with MALME-3M cells and viability was assessed after 72 h using the MTS assay. Values represent the (more ...)
To summarize, we have isolated an LF mutant that is impaired in its ability to activate the Nlrp1b inflammasome, but remains able to cause apoptosis in a melanoma cell line. LF-K518E/E682G activity prevented phosphorylation of ERK, but did not prevent phosphorylation of JNK or p38. This observation serves to explain why the mutant retains its ability to kill the melanoma cells, as it has been shown previously that inhibition of the ERK pathway is sufficient to induce apoptosis. It is unclear why the mutant is defective at causing pyroptosis, but it is presumably because LF-K518E/E682G has a diminished capacity to cleave a substrate that is involved in the activation of Nlrp1b.