Knockdown of mu-calpain in Fanconi anemia, FA-A, cells by siRNA restores alphaII 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 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.
Zhang
Pan
P
Department of Pathology and Laboratory Medicine, New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Sridharan
Deepa
D
Lambert
Muriel W
MW
D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D043171:Chromosomal Instability / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D005199:Fanconi Anemia / Q000473:pathology; D006801:Humans; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology*; D013049:Spectrin / Q000378:metabolism*
D034741:RNA, Small Interfering; D013049:Spectrin; D002154:Calpain; C449002:mu-calpain