Conformation of neurofilament heavy chain effect on neurofilament-neurofilament association and bundling.

Abstract

Mammalian neurofilaments (NFs) are composed of Heavy (NF-H), Medium (NF-M), and Light (NF-L) subunits, named according to their molecular mass, along with α-internexin or peripherin (in central or peripheral nervous systems, respectively). Phosphorylated NF subunits assemble linearly, with C-terminal regions extending from the filament “backbone.” Phosphorylation of C-terminal “side arms” of NF-H promotes NF-NF interactions, providing stability to axons. Transport of NFs from the perikaryon into the axon and NF-NF interactions are dependent upon regulation of multiple kinases. The complete sequence of phosphorylation events does not occur until NFs have entered the axon. Sidearm phosphorylation fosters lateral extension away from the NF backbone. By contrast, sidearm conformation prior to phosphorylation remains unresolved. Scrutiny of the NF-H side-arm sequence revealed 4 proline-proline (PP) motifs; these motifs allow tremendous rotation of their peptide chains from a closed, folded-back hairpin (cis) to a fully extended, linear confirmation (trans). Notably, one PP motif is located within each of the consensus sequences for the NF kinases. We hypothesize that phosphorylation-induced alterations in the configuration of these PP motifs are critical for NF transport, NF-NF association within axons. Mutated NFs constructs with changed PP motifs were expressed (with or without PP motifs /Phosphorylated or unphosphorylated) and incubated with NFs medium chain then trapped using a his-tag column to demonstrate bundling. Our initial data were inconclusive. A different experimental approach, such as Localized Surface Plasma Resonance (LSPR) is necessary to analyze the role of PP motifs in NFs association. We predict that constructs lacking PP motifs will present lower association affinity to NF-M (even when phosphorylated. Understanding the mechanisms that allow NF-NF associations within neurites will help guide targeted treatments for NF-based neurodegenerative disorders.