Monitoring Stub1-Mediated Pexophagy

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality ...

Academia Sinica

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.

Profiling carbohydrate-receptor interaction with recombinant innate immunity receptor-Fc fusion proteins.

Monodansylpentane (MDH) is a fluorophore that displays selective labeling of lipid droplets (LDs). The dye preferentially segregates into the neutral lipid cores of LDs and emits blue fluorescence, compatible with the simultaneous use of green and red fluorescent reporters in multi-color live-cell imaging. MDH can be used for visualizing LDs not only in cell cultures, but also in fixed tissues such as the fat body and ovaries from Drosophila. MDH is therefore a versatile marker for LDs in fluorescence microscopy.

Staining of Lipid Droplets with Monodansylpentane.

Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.

Article	Year
Profiling carbohydrate-receptor interaction with recombinant innate immunity receptor-Fc fusion proteins. The Journal of biological chemistry\| PubMed ID: 19837675	2009
Staining of Lipid Droplets with Monodansylpentane. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28155158	2017
Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes. Nature communications\| PubMed ID: 33077711	2020

Bo-Hua Chen

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