We have previously shown that there is a deficiency in the structural
protein, nonerythroid
alpha spectrin (alphaIISp), in cells from patients with
Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alphaIISp and correlates with a decreased level of repair of
DNA interstrand cross-links and
chromosomal instability in FA cells. An important factor in the stability of alphaIISp is its susceptibility to cleavage by the
protease,
mu-calpain. We hypothesized that an increased level of
mu-calpain cleavage of alphaIISp in FA cells leads to an increased level of breakdown of alphaIISp and that knocking down expression of
mu-calpain in FA cells should restore levels of alphaIISp and correct a number of the phenotypic defects observed. The results showed that there is increased
mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alphaIISp in these FA cells.
Protein interaction studies indicated that FANCA and FANCG bind directly to
mu-calpain. We hypothesize that this binding may lead to inhibition of
mu-calpain activity in normal cells. Knocking down
mu-calpain by
siRNA in FA-A cells restored levels of alphaIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the
chromosomal instability observed after exposure to
a DNA interstrand cross-linking agent. These studies indicate that FA
proteins may play an important role in maintaining the stability of alphaIISp in the cell by regulating its cleavage by
mu-calpain. Thus, by reducing the level of breakdown of alphaIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.