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Protein disulfide isomerase as a target in leukemia
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Figure 2. SK053 binds to protein disulfide isomerase. (A) HL-60 cells were incubated with 100 mM SK-BIO and lysed. Proteins binding SK-BIO were precipitated with avidin-coated beads and eluted with buffers E1 and E2 as described in the Methods section. Next, the samples were separated by SDS-PAGE. A band obtained with buffer E1 (black rectangle) was excised from silver-stained gel and analyzed by mass spectrometry. An inactive biotinylated SK053 analog (SK-IN) was used as a neg- ative control. A representative result of a series of experiments is presented. (B) Recombinant human (rhu) PDI was incubated with a 10× molar excess of SK-BIO for 10-120 min at 37°C (left) or preincubated for 1 h with a 10× molar excess of SK053 followed by 1 h incubation with a 10× molar excess of SK-BIO (right). Next, proteins were separated by SDS-PAGE followed by western blotting (WB) using anti-biotin monoclonal antibody. Membranes stained with Ponceau red served as load- ing controls. A representative result of a series of experiments is presented. (C) Reduced (red) and oxidized (ox) rhuPDI were incubated with SK-BIO followed by immunoblotting for biotin. Gels stained with Coomasie blue served as loading controls. SK-IN was used as a negative control. A representative result of a series of experiments is presented. (D) The lysates of HL-60 cells were incubated with SK-BIO followed by biotin immunoprecipitation and western blotting. Biotin detection in total cell lysates (input) was used as a loading control. Lysate incubated with protein G agarose beads in the absence of antibodies was used as a negative control (No Ab). A representative result of a series of experiments is presented. IP: immunoprecipitation; TXN: thioredoxin.
detection of biotin revealed covalent binding of SK-BIO to recombinant human (rhu) PDI (Figure 2B). Oxidation of rhuPDI, which decreases the number of sulfhydryl groups, reduced binding of SK-BIO to PDI indicating a thiol-dependent interaction (Figure 2C). Moreover, in a competition assay, pre-incubation of rhuPDI with SK053 prevented binding of SK-BIO, indicating the same binding site for both compounds (Figure 2B).
Immunoprecipitation of PDI and thioredoxin from the lysates of HL-60 cells cultured with SK-BIO revealed the presence of biotinylated PDI but not thioredoxin (Figure 2D), indicating that in HL-60 cells PDI is a preferential tar- get for the compound. Further mass spectrometry revealed that SK053 binds to the catalytic cysteines local- ized in the fourth thioredoxin-like domain of PDI (Online Supplementary Figure S9E). Furthermore, the molecular dynamics simulation followed by covalent docking of SK053 to the fourth thioredoxin-like domain of human PDI (pdb|4ekz) indicated that the binding of the truncated form of SK053 (a molecule without the leaving group, Online Supplementary Figure S8D) is equally likely to occur by forming a covalent bond with either sulfur from Cys397 or Cys400 (Online Supplementary Figure S10, Online Supplementary Movie 1). Next, we sought to deter- mine whether binding of SK053 translates into inhibition of PDI activity. A turbidimetric assay of PDI-mediated insulin reduction revealed that SK053 efficiently blocks the enzymatic activity of rhuPDI with an IC50 value of
9.98 mM (Figure 3A,B). To further investigate whether SK053 can inhibit PDI activity in cells, we transfected HeLa cervical cancer cells with a plasmid encoding an endoplasmic reticulum-tuned fluorescent redox-respon- sive probe (roGFPiE)12 and measured the response of this probe using fluorescence lifetime imaging in a time-corre- lated single-photon counting mode. The fluorescent life- time of the probe declined in SK053-treated HeLa cells, reflecting the reductive shift in the dithiol-disulfide steady state and indicating SK053-mediated inhibition of PDI activity in the endoplasmic reticulum of living cells (Figure 3C).
Inhibition of protein disulfide isomerase increases C/EBPa levels and inhibits the growth of acute myeloid leukemia cells
Recently, it was shown that PDI interacts with the stem loop region of mRNA for C/EBPa thereby blocking its translation.13 Considering that C/EBPa is a crucial tran- scription factor driving myeloid cells differentiation14,15 we investigated the effects of SK053 on C/EBPa levels in AML cells. Incubation of HL-60 cells with SK053 increased the nuclear levels of the 42 kDa C/EBPa isoform (Figure 4A) and induced CEBPA mRNA expression (Figure 4C). Additionally, the oncogenic p30 kDa C/EBPa levels were strongly suppressed in cells incubated with SK053 (Figure 4B). Accordingly, SK053 decreased the levels of SOX4 mRNA (Figure 1G), a direct target of C/EBPa, the expres-
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