ABSTRACT
Neurofilaments (NF) are important determinants of the shape and size of nerve cells. The oxidation of NF, relevant to aging, neurodegenerative disorders, and axonal (Wallerian) degeneration, has not been studied. In this investigation, we have combined biochemical and ultrastructural methods to study the metal-catalyzed oxidation (MCO) of bovine NF using an ascorbate/Fe+3/O2 system. The oxidation of NF proteins was documented by increases in carbonyl content, which were time- and concentration-dependent. Polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses revealed the fragmentation of oxidized NF proteins, predominantly NF-H and NF-M. Electron microscopy (EM) showed that oxidized NF formed dense aggregates and bundles of laterally aggregated filaments. Finally, we also demonstrated that oxidized NF proteins were more susceptible to calpain proteolysis. In view of the growing evidence supporting increased oxidative stress on the nervous system in aging and the report of Cu/Zn superoxide dismutase mutation in familial motor neuron disease, oxidative injury of NF may be relevant to cell atrophy and degeneration of nerve cells and to the formation of abnormal cytoskeletal structures.
Subject(s)
Ferric Compounds/pharmacology , Intermediate Filaments/metabolism , Neurofilament Proteins/metabolism , Spinal Cord/metabolism , Animals , Ascorbic Acid/pharmacology , Calpain/metabolism , Cattle , Cell Fractionation , Chlorides , Electrophoresis, Polyacrylamide Gel , Guanidine , Guanidines/pharmacology , Intermediate Filaments/drug effects , Intermediate Filaments/ultrastructure , Microscopy, Electron , Molecular Weight , Neurofilament Proteins/isolation & purification , Neurofilament Proteins/ultrastructure , Oxidation-Reduction , Solubility , Spinal Cord/ultrastructure , UltracentrifugationABSTRACT
The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium-binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200-kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160-kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain-induced breakdown products of native and dephosphorylated 160-kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68-kDa neurofilament protein. Calmodulin binding to the purified individual 160- and 200-kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium-activated proteins calpain and calmodulin.
Subject(s)
Calmodulin/metabolism , Calpain/pharmacology , Neurofilament Proteins/metabolism , Peptide Hydrolases/metabolism , Animals , Molecular Weight , Neurofilament Proteins/chemistry , PhosphorylationABSTRACT
The in vitro degradation of individual neurofilament proteins by calpain and the effects of calmodulin on this proteolysis were studied. Two major results are reported. First, in the presence of calcium, calmodulin binds to the 200-kD neurofilament protein, but only weakly associates with the 150-kD neurofilament protein. The 70-kD neurofilament protein shows no specific calmodulin-binding. Second, calmodulin inhibits the calpain-mediated degradation of the 200-kD neurofilament protein, but does not alter the hydrolysis of the 150-kD and 70-kD neurofilament proteins. In addition, calmodulin is able to bind to the 200-kD neurofilament protein in the presence of other neurofilament subunits, indicating that calmodulin may play a role in the regulation of the metabolism of the 200-kD neurofilament protein in vivo.
