UBL3 Interaction with α-Synuclein Is Downregulated by Silencing MGST3

Abstract

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that plays a crucial role in sorting proteins into small extracellular vesicles. Aggregations of alpha-synuclein (α-syn) are associated with the pathology of neurodegenerative diseases such as Parkinson's disease. Recently, the interaction between UBL3 and α-syn was discovered, with potential implications in clearing excess α-syn from neurons and its role in disease spread. However, the regulator that can mediate the interaction between UBL3 and α-syn remains unclear. In this study, using the split gaussian luciferase complementation assay and RNA interference technology, we identified that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1, and ATG3 were involved in downregulating the interaction between UBL3 and α-syn. Notably, silencing MGST3 had the most significant impact. Immunocytochemistry staining confirmed the impact of MGST3 silencing on the co-localization of UBL3 and α-syn in cells. MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We induced oxidative stress with hydrogen peroxide, observing its effect on the UBL3-α-syn interaction, and showing that 800 µM of H₂O₂ downregulated this interaction. In conclusion, silencing MGST3 downregulates the interaction between UBL3 and α-syn.

Keywords: hydrogen peroxide; microsomal glutathione s-transferase 3; oxidative stress; ubiquitin-like 3; α-synuclein.

Figure 1

Effect of siRNA on the interaction of UBL3 with α-syn using split-luciferase complementation assay. (A) Luminescence of culture medium of HEK293 cells co-transfected with siRNA, NGluc-UBL3, and α-syn-Cgluc. Two siRNAs were available for each candidate protein. The groups were compared with the UBL3 and α-syn co-transfected group, and statistically analyzed. (B) Fold change log2 of luminescence. Silencing MGST3 resulted in luminescence fold change log2 < −1. The luminescence ± S.D. in three independent experiments as shown. ns: non-significant; *: p < 0.05; **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. NC siRNA: negative-control siRNA, QSOX1: quiescin sulfhydryl oxidase 1, QSOX2: quiescin sulfhydryl oxidase 2, HTATIP2: HIV-1 Tat interactive protein 2, UBE3C: ubiquitin protein ligase E3C, COPS5: COP9 signalosome subunit 5, MGST3: microsomal glutathione S-transferase 3, NSF: N-ethylmaleimide sensitive factor, HECTD1: HECT domain E3 ubiquitin protein ligase 1, SAE1: SUMO1 activating enzyme subunit 1, ATG3: autophagy related 3.

Figure 2

MGST3 was knocked down in HEK293 cells transfected with MGST3 siRNA. NC siRNA: negative-control siRNA.

Figure 3

Effect of silencing MGST3 on co-localization of UBL3 and α-syn. (A) Representative images of HEK293 cells showing the effect of silencing MGST3 on the co-localization of UBL3 and α-syn. Green represents UBL3, Red represents α-syn, Blue represents cell nucleus and yellow shows the co-localization of UBL3 and α-syn. Scale bars, 10 and 2 μm. (B) Quantitative analysis of the effect of silencing MGST3 on co-localization of Flag-UBL3 with MYC-α-syn. (n = 13). ns: non-significant; **: p < 0.01, ***: p < 0.001.

Figure 4

Effect of H₂O₂ on the interaction between UBL3 and α-syn. (A) Luminescence of culture medium from HEK293 cells co-transfected with NGluc-UBL3 and α-syn-CGluc at different concentrations of H₂O₂. (B) Effect of different concentrations of H₂O₂ on the viability of HEK293 cells co-transfected with NGluc-UBL3 and α-syn-CGluc. (C) The ratio of luminescence to cell viability in culture medium from HEK293 cells co-transfected with NGluc-UBL3 and α-syn-CGluc treated with different concentrations of H₂O₂. These groups were compared to the group treated with 0 μM H₂O₂, and statistically analyzed. (D) The ratio of luminescence to cell viability of culture medium from HEK293 cells transfected with Gluc treated with 800 μM H₂O₂. The luminescence ± S.D., cell viability ± S.D., and ratio ± S.D. in three independent experiments as shown. ns: non-significant, ***: p < 0.001.

Figure 5

Effect of silencing MGST3 on the interaction between UBL3 and α-syn with 800 μM H₂O₂. (A) Luminescence of culture medium from HEK293 cells co-transfected NGluc-UBL3 with α-syn-Cgluc. (B) Viability of the HEK 293 cells. (C) The ratio of luminescence to cell viability of HEK293 cell culture medium co-transfected with Ngluc-UBL3 and α-syn-Cgluc. The luminescence ± S.D., cell viability ± S.D. and ratio ± S.D. in three independent experiments as shown. NC siRNA: negative-control siRNA, ns: non-significant, * p < 0.05, ** p < 0.01.