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Published on: June 13, 2021
Purification and biochemical characterization of Msi3, an essential Hsp110 molecular chaperone in Candida albicans
Ying Wang1, Hongtao Li1, Cancan Sun1
1Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Abstract:
Hsp110s are unique and essential molecular chaperones in the eukaryotic cytosol. They play important roles in maintaining cellular protein homeostasis. Candida albicans is the most prevalent yeast opportunistic pathogen that causes fungal infections in humans. As the only Hsp110 in Candida albicans, Msi3 is essential for the growth and infection of Candida albicans. In this study, we have expressed and purified Msi3 in nucleotide-free state and carried out biochemical analyses. Sse1 is the major Hsp110 in budding yeast S. cerevisiae and the best characterized Hsp110. Msi3 can substitute Sse1 in complementing the temperature-sensitive phenotype of S. cerevisiae carrying a deletion of SSE1 gene although Msi3 shares only 63.4% sequence identity with Sse1. Consistent with this functional similarity, the purified Msi3 protein shares many similar biochemical activities with Sse1 including binding ATP with high affinity, changing conformation upon ATP binding, stimulating the nucleotide-exchange for Hsp70, preventing protein aggregation, and assisting Hsp70 in refolding denatured luciferase. These biochemical characterizations suggested that Msi3 can be used as a model for studying the molecular mechanisms of Hsp110s.
Insights
The fungal protein Msi3, essential for Candida albicans growth and infection, functions similarly to yeast Hsp110 (heat shock protein 110), aiding in protein homeostasis and refolding.
Area of Science:
- Molecular Biology
- Biochemistry
- Mycology
Background:
- Heat shock proteins 110 (Hsp110s) are crucial eukaryotic molecular chaperones maintaining protein homeostasis.
- Msi3 is the sole Hsp110 in Candida albicans, an opportunistic fungal pathogen, and is vital for its growth and virulence.
- Sse1 is the primary Hsp110 in Saccharomyces cerevisiae and is well-characterized.
Purpose of the Study:
- To express and purify the Candida albicans Hsp110, Msi3, in a nucleotide-free state.
- To biochemically characterize Msi3 and compare its functions to the well-studied Sse1 from S. cerevisiae.
- To evaluate Msi3's potential as a model for studying Hsp110 molecular mechanisms.
Main Methods:
- Expression and purification of nucleotide-free Msi3.
- Biochemical assays to assess ATP binding, conformational changes, and Hsp70 interaction.
- Functional complementation assay in S. cerevisiae lacking SSE1.
Main Results:
- Msi3 successfully substituted for Sse1 in S. cerevisiae, despite only 63.4% sequence identity.
- Purified Msi3 exhibited biochemical activities similar to Sse1, including high-affinity ATP binding and ATP-dependent conformational changes.
- Msi3 demonstrated the ability to stimulate Hsp70 nucleotide exchange, prevent protein aggregation, and assist Hsp70 in refolding denatured proteins.
Conclusions:
- Msi3 shares significant functional and biochemical similarities with Sse1, indicating conserved Hsp110 mechanisms.
- Msi3 can functionally complement the loss of Sse1 in S. cerevisiae.
- Msi3 serves as a valuable model for elucidating the molecular mechanisms of Hsp110 chaperones.
