Knockdown of μ-Calpain in Fanconi Anemia, FA-A, Cells by siRNA Restores αII Spectrin Levels and Corrects Chromosomal Instability and Defective DNA Interstrand Cross-Link Repair

We have previously shown that there is a deficiency in the structural protein, nonerythroid α spectrin (αIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of αIISp and correlates with a decreased level of repair of DNA...

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Published in:Biochemistry (Easton) 2010-07, Vol.49 (26), p.5570-5581
Main Authors: Zhang, Pan, Sridharan, Deepa, Lambert, Muriel W
Format: Article
Language:English
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Summary:We have previously shown that there is a deficiency in the structural protein, nonerythroid α spectrin (αIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of αIISp 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 αIISp is its susceptibility to cleavage by the protease, μ-calpain. We hypothesized that an increased level of μ-calpain cleavage of αIISp in FA cells leads to an increased level of breakdown of αIISp and that knocking down expression of μ-calpain in FA cells should restore levels of αIISp and correct a number of the phenotypic defects observed. The results showed that there is increased μ-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in αIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to μ-calpain. We hypothesize that this binding may lead to inhibition of μ-calpain activity in normal cells. Knocking down μ-calpain by siRNA in FA-A cells restored levels of αIISp 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 αIISp in the cell by regulating its cleavage by μ-calpain. Thus, by reducing the level of breakdown of αIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi100656j