Abstract
Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein–protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3’ end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin®XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.
Highlights
Neurofibromatosis Type 1 (NF1) occurs in ~1:3500 births due to pathogenic variants in the NF1 gene that encodes the protein neurofibromin
We showed that 14-3-3σ is up-regulated when NF1 is deleted in HEK293 cells and that restoration of neurofibromin expression leads to decreased/normalization of expression [19]. 14-3-3 proteins have been implicated in other Rasopathy phenotypes, including cardio-facio-cutaneous syndrome [20] and Noonan syndrome [21]
Given the importance of NF1 protein–protein interactions (PPIs), we developed and describe a novel tandem affinity purification tag linked to full-length murine Nf1 cDNA
Summary
Neurofibromatosis Type 1 (NF1) occurs in ~1:3500 births due to pathogenic variants in the NF1 gene that encodes the protein neurofibromin. FAF2 protein directly interacts with and negatively regulates neurofibromin by promoting its ubiquitin-dependent proteolysis. Legius syndrome and NF1 have phenotypic overlap (including CALMs and freckling); no pathogenic SPRED1 mutations have been reported in individuals with Lisch nodules, neurofibromas, or optic pathway gliomas (OPGs). Ras signaling when transfected into NF1 null HEK293 cells We utilized this tagged NF1 cDNA to affinity purify binding partners. This led to identification of 21 proteins with a high confidence of truly interacting with neurofibromin. One of the binding partners we identified was neurofibromin itself, indicating that it oligomerizes As this has several implications, we validated this interaction through reverse immunoprecipitation with a halo-tagged full-length human NF1 cDNA (hNF1). Further characterization of neurofibromin oligomerization and putative PPIs will help define neurofibromin function and potential therapeutic targets
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