C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Nuclear import receptors (NIRs) regulate RNA-binding protein (RBP) transport and phase transitions via nuclear localization signals (NLSs).
• C9orf72-associated neurodegeneration involves toxic arginine-rich poly-dipeptides causing nucleocytoplasmic transport deficits.
• The precise mechanism by which these poly-dipeptides impair NIR function remains unclear.

Purpose of the Study:

• To elucidate the molecular basis of arginine-rich poly-dipeptides' toxicity on NIRs' role in RBP phase transition modification.
• To investigate the interaction between C9orf72-derived poly-dipeptides and NIRs.

Main Methods:

• Isothermal titration calorimetry (ITC) to quantify binding affinity.
• Size-exclusion chromatography (SEC) to assess complex formation.
• Nuclear magnetic resonance (NMR) spectroscopy to identify binding sites.

Main Results:

• Proline:arginine (PR) poly-dipeptides bind karyopherin-β2 (Kapβ2), a key NIR, with a 1:1 stoichiometry.
• NMR data indicate that PR poly-dipeptides bind to the NLS-binding site on Kapβ2.
• This binding competitively inhibits Kapβ2's interaction with NLS-containing proteins.

Conclusions:

• Arginine-rich poly-dipeptides directly interfere with NIRs, specifically Kapβ2, by targeting the NLS binding site.
• This molecular interaction explains the observed nucleocytoplasmic transport deficits in C9orf72-related disorders.
• The findings provide mechanistic insights into the disruption of RBP phase transitions in neurodegeneration.