NF-YA enters cells through cell penetrating peptides

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Cell-penetrating peptides (CPPs) are known for their ability to translocate cell membranes and facilitate molecular delivery.
• NF-Y is a transcription factor complex composed of two Histone Fold Domain (HFD) subunits and the sequence-specific NF-YA subunit.
• NF-YA contains alpha-helices rich in basic residues, a common characteristic of CPPs.

Purpose of the Study:

• To investigate whether the NF-YA subunit possesses intrinsic cell-penetrating capabilities.
• To identify the specific regions within NF-YA responsible for cell entry.
• To assess the functional consequences of NF-YA internalization within cells.

Main Methods:

• Mutagenesis studies to identify functional domains within NF-YA.
• Cellular uptake assays to determine NF-YA internalization efficiency.
• Protein trimerization and DNA binding assays to assess functional activity.
• Inhibition of differentiation assays in C2C12 myoblasts.

Main Results:

• NF-YA efficiently enters cells at nanomolar concentrations without the need for carrier peptides.
• Mutagenesis identified at least two distinct CPPs within NF-YA (A1 and A2), overlapping with nuclear localization signals (NLS).
• Internalized NF-YA retains its ability to trimerize with HFD subunits and bind the CCAAT DNA motif.
• Transfection of NF-YA into C2C12 cells inhibited myoblast differentiation.

Conclusions:

• The NF-YA subunit possesses inherent cell-penetrating peptide (CPP) activity, a novel and unexpected property.
• These CPPs are located in regions overlapping with known nuclear localization signals.
• Internalized NF-YA is functional, capable of forming the transcription factor complex and influencing cellular processes like differentiation.