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

Pan Zhang, Deepa Sridharan, Muriel Lambert

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)5570-5581
Number of pages12
JournalBiochemistry
Volume49
Issue number26
DOIs
StatePublished - Jul 6 2010

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Fanconi Anemia
Spectrin
Chromosomal Instability
Calpain
Small Interfering RNA
Repair
DNA
Fanconi Anemia Complementation Group Proteins
DNA Repair
Defects
Gastrin-Secreting Cells
Proteins
Peptide Hydrolases

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "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",
abstract = "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.",
author = "Pan Zhang and Deepa Sridharan and Muriel Lambert",
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T1 - 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

AU - Zhang, Pan

AU - Sridharan, Deepa

AU - Lambert, Muriel

PY - 2010/7/6

Y1 - 2010/7/6

N2 - 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.

AB - 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.

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SN - 0006-2960

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