Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS.

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Nuclear import receptors (NIRs) are crucial for mitigating the mislocalization and aggregation of RNA-binding proteins (RBPs), such as FUS and TDP-43.
• These aggregated RBPs are implicated in the pathogenesis of neurodegenerative diseases.
• Mutations in the nuclear localization signal (NLS) of RBPs can impair NIR binding and reduce their therapeutic efficacy.

Purpose of the Study:

• To define the key features governing the anti-aggregation activity of NIRs and their cognate NLSs.
• To understand how binding affinity and NLS location influence NIR-mediated disaggregation of RBPs.
• To explore the therapeutic potential of engineered RBPs that leverage NIR interactions.

Main Methods:

• Investigated the relationship between NIR-NLS binding affinity and NIR disaggregation activity.
• Analyzed the impact of NLS positioning relative to the aggregation-prone domain on NIR efficacy.
• Developed and tested a designed FUS chimera (FUS_IBB) incorporating the importin β binding (IBB) domain for in vitro solubilization, cellular nuclear translocation, and gene expression modulation.

Main Results:

• High binding affinity between NIRs and NLSs is critical for potent disaggregation activity.
• Optimal NLS location relative to the aggregating domain significantly enhances NIR disaggregation efficacy.
• The FUS_IBB chimera demonstrated successful in vitro solubilization, nuclear import in cells, and downregulation of endogenous FUS expression.

Conclusions:

• Guiding the specific recognition between NLSs and NIRs is a promising strategy for developing novel therapeutics against neurodegenerative diseases.
• Engineered RBPs, like the FUS_IBB chimera, can replace aggregation-prone endogenous proteins, offering a potential therapeutic avenue.