Journal article
Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus
G3 : genes - genomes - genetics, Vol.16(2), jkaf295
02/04/2026
DOI: 10.1093/g3journal/jkaf295
PMCID: PMC12869073
PMID: 41351542
Abstract
Aspergillosis caused by Aspergillus fumigatus is a clinical issue of such severity that the World Health Organization has designated this organism as 1 of the 4 most critical fungi to study. Progress in A. fumigatus has been limited by the availability of genetic tools with which to study this filamentous fungus. Currently available means of altering the dosage of genes and gene products include construction of disruption mutants as well as regulated promoters. These are powerful techniques but somewhat limited for the analysis of essential genes. Here we describe a new method that permits regulated proteolysis of any A. fumigatus protein that can be made as a fusion protein to the well-described Green Fluorescent Protein (GFP) of Aequorea victoria. A GFP fusion protein of interest can be targeted for degradation using a single-chain antibody called a nanobody that recognizes GFP (GFPNb). This GFPNb is in turn fused to an E3 ligase protein called Rnf4 from rat that efficiently ubiquitinates target proteins. A fusion gene was constructed under control of a doxycycline-inducible promoter that produced a GFPNb-Rnf4 fusion protein in A. fumigatus. Here we show that production of this GFPNb-Rnf4 protein led to the rapid proteolysis of a variety of GFP fusion proteins. Additionally, we found that some GFP fusion proteins triggered a corresponding genomic response when their degradation was induced while others were simply degraded. These studies provide a new means to directly regulate protein levels in A. fumigatus and generate new alleles of genes exposing the underlying regulatory circuitry.
Details
- Title: Subtitle
- Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus
- Creators
- Sanjoy Paul - University of IowaW Scott Moye-Rowley - University of Iowa
- Resource Type
- Journal article
- Publication Details
- G3 : genes - genomes - genetics, Vol.16(2), jkaf295
- DOI
- 10.1093/g3journal/jkaf295
- PMID
- 41351542
- PMCID
- PMC12869073
- NLM abbreviation
- G3 (Bethesda)
- ISSN
- 2160-1836
- eISSN
- 2160-1836
- Publisher
- Oxford University Press
- Grant note
- NIH: R01AI143198, R21AI190747
Funding for this work was provided by National Institutes of Health grants R01AI143198 and R21AI190747.
- Language
- English
- Electronic publication date
- 12/06/2025
- Date published
- 02/04/2026
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9985090731002771
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