Dataset Information

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.

ABSTRACT: 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.

SUBMITTER: Zhang P

PROVIDER: S-EPMC2899890 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Publications

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.

Zhang Pan P Sridharan Deepa D Lambert Muriel W MW

Biochemistry 20100701 26

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 hypoth ...[more]

PMID: 20518497

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Project description:Calpain-catalyzed proteolysis of II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.

Dataset Information

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.

Publications

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.

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