UBL3 Interacts with Alpha-Synuclein in Cells and the Interaction Is Downregulated by the EGFR Pathway Inhibitor Osimertinib

Abstract

Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as α-synucleinopathies. Alpha-synuclein (α-syn) has been widely studied for its involvement in α-synucleinopathies. However, it is still unknown whether UBL3 interacts with α-syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and α-syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with α-syn by the Gaussia princeps based split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with α-syn in cells and is significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for α-synucleinopathies in the future.

Keywords: EGFR pathway inhibitor; UBL3; downregulate; drug screen; interaction; osimertinib; α-synuclein; α-synucleinopathies.

Figure 1

Expression of p-S-129 α-syn was upregulated in the substantia nigra of Ubl3^−/− mice brain. (A) Representative images of immunocytochemistry staining of p-S-129 α-syn in the substantia nigra of WT and Ubl3^−/− mice. Scale bars: 10 μm. The red boxes represent the selection zones of the magnified pictures below. (B) Quantification comparison of the immunohistochemical expression of p-S-129 α-syn in the substantia nigra between WT and Ubl3^−/− mice. Histograms represent the mean + SD (the number of mice in each group was three). A Student’s t test was performed. *: p-value < 0.05; WT: wild type; Ubl3^−/−: Ubl3 knock out.

Figure 2

UBL3 interacted with α-syn in cells. (A) Schematic representation of the Gaussia princeps based split luciferase complementation assay system. Whether there is an interaction between UBL3 and α-syn is still unknown (Red question mark). (B) Schematic of split Gluc-tagged proteins, NGluc-UBL3, NGluc-UBL3∆5, and α-syn-CGluc, both containing an IKSS sequence, which allows successfully expressed fragments and their interacting complexes to be secreted into the CM. (C) Immunoblotting of CM and CL from transfected HEK293 cells by anti-UBL3 polyclonal antibody and anti-α-syn antibody. (D) Luminescence of the CM (left) and CL (right) from transfected HEK293 cells. The luminescence ± SD in triplicate experiments is shown. (E) Schematic representation of 3xFlag-UBL3, 3xFlag-UBL3∆5, and 6xMYC-α-syn for co-immunoprecipitation (Co-IP). (F) Co-immunoprecipitated 3xFlag-UBL3 and 3xFlag-UBL3∆5 interact with 6xMYC-α-syn. The input lanes are 1% of the sample prior to Co-IP and the Co-IP lanes are 20% of the Co-IP products. IKSS: immunoglobulin kappa secretory signal; CM: cell culture medium; CL: cell lysate.

Figure 3

The interaction level in cells was upregulated by the treatment of MPP⁺. (A) Luminescence of CM from transfected HEK293 cells, which were treated with 50, 100, 300, 500, and 600 μM of MPP⁺ for 48 h. (B) The cell viability of transfected HEK293 cells was treated with different concentrations of MPP⁺ for 48 h. (C) The ratios of luminescence divided by cell viability were calculated in triplicate. The luminescence ± SD, cell viability ± SD, and ratio ± SD in triplicate are shown. One-way ANOVA and Dunnett’s post hoc test were performed. ns (non-significant) p > 0.05, * p < 0.05. FC: fold change; DMSO: dimethyl sulfoxide.

Figure 4

Drug screening results of 32 drugs at concentrations of 1 μM and 10 μM. (A) The screen result under a concentration of 1 μM. (B) The screen result under a concentration of 10 μM. The volcano plot showed the fold-change (x-axis) versus the significance (y-axis) of 32 drugs. The significance (non-adjusted p-value) and the fold-change are converted to −Log10(p-value) and Log2(fold-change), respectively. The vertical and horizontal dotted lines show the cut-off of fold-change ± 1.25, and p-value = 0.05, respectively. The luminescence of CM from transfected HEK293 cells was upregulated by >1.25-fold with a p-value < 0.05 (upper-right, dots colored red) and the luminescence of CM from transfected HEK293 cells was downregulated by < −1.25-fold with p-value < 0.05 (upper-left, dots colored blue). The luminescence ± SD in triplicate experiments is shown. One-way ANOVA and Dunnett’s post hoc test were performed. p < 0.05 was considered statistically significant.

Figure 5

The interaction between UBL3 and α-syn in cells was regulated by the treatment of clinical drugs. (A) The cell viability of transfected HEK293 cells treated with selected drugs at concentrations of 1 and 10 μM for 48 h, including docetaxel, erlotinib, gemcitabine, methylcobalamin, osimertinib, pemetrexed, and sulfasalazine. (B) The ratio of luminescence divided by cell viability was calculated in triplicate. The cell viability ± SD and the ratio ± SD in triplicate experiments are shown. One-way ANOVA and Dunnett’s post hoc test were performed. ns (non-significant) p > 0.05; * p < 0.05.