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siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Published on: May 24, 2014
Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking
Andrey S Selyunin1,2,3, Lakesla R Iles4, Geoffrey Bartholomeusz4
1Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX.
Abstract:
Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors.
Insights
Shiga toxin 2 (STx2) uses a specific trafficking pathway involving UNC50 and GBF1 to reach the cytosol. Understanding this mechanism is key to developing new therapies against STx2 toxicity.
Area of Science:
- Cell Biology
- Molecular Biology
- Toxicology
Background:
- Shiga toxins (STx1 and STx2) are potent protein toxins that enter cells via retrograde transport to the cytosol.
- Early endosome-to-Golgi transport is a critical step for STx1 and STx2 to evade lysosomal degradation.
- The precise trafficking mechanism of the more potent STx2 remains incompletely understood, hindering therapeutic development.
Purpose of the Study:
- To identify host factors essential for the early endosome-to-Golgi trafficking of Shiga toxin 2 (STx2).
- To elucidate the molecular mechanism by which STx2 utilizes this specific trafficking route.
Main Methods:
- Genome-wide siRNA screen in HeLa cells to identify genes required for STx2 toxicity.
- Validation of endosome/Golgi-localized host factors specifically involved in STx2 trafficking.
- Investigating the role of identified host factors, such as UNC50 and GBF1, in toxin transport and degradation.
Main Results:
- A genome-wide screen identified distinct sets of host genes required for STx1 and STx2 toxicity.
- Depletion of UNC50 specifically blocked early endosome-to-Golgi trafficking of STx2, leading to its lysosomal degradation.
- UNC50 was found to recruit GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi apparatus.
Conclusions:
- UNC50 and GBF1 are critical host factors for the intracellular trafficking of Shiga toxin 2 (STx2).
- Targeting the UNC50-GBF1 interaction offers a potential therapeutic strategy to inhibit STx2.
- This study provides novel insights into STx2 trafficking, paving the way for developing effective toxin-trafficking inhibitors.
