Inactivation of the SMN Complex by Oxidative Stress

Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148, USA.
Molecular cell (Impact Factor: 14.02). 07/2008; 31(2):244-54. DOI: 10.1016/j.molcel.2008.06.004
Source: PubMed

ABSTRACT The SMN complex is essential for the biogenesis of small nuclear ribonucleoproteins (snRNPs), the major constituents of the spliceosome. Deficiency in functional SMN protein causes spinal muscular atrophy, a common motor neuron degenerative disease of severity commensurate with SMN levels and, correspondingly, snRNP assembly decreases. We developed a high-throughput screen for snRNP assembly modifiers and discovered that reactive oxygen species (ROS) inhibit SMN-complex activity in a dose-dependent manner. ROS-generating compounds, e.g., the environmental toxins menadione and beta-lapachone (in vivo IC(50) = 0.45 muM) also cause intermolecular disulfide crosslinking of SMN. Both the oxidative inactivation and SMN crosslinking can be reversed by reductants. We identified two cysteines that form SMN-SMN disulfide crosslinks, defining specific contact points in oligomeric SMN. Thus, the SMN complex is a redox-sensitive assemblyosome and an ROS target, suggesting that it may play a role in oxidative stress pathophysiology, which is associated with many degenerative diseases.

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Available from: Elizabeth Ottinger, Feb 03, 2014
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    • "Their data indicate that SMN knockdown alone is not critical to cell viability. However, when SMN knockdown cells are under stress, such as oxidative stress [48], their survival rate may significantly decrease [47]. In parallel, SOD3 (extracellular superoxide dismutase, EC-SOD) was shown to be the predominant form in extracellular fluids. "
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    Evidence-based Complementary and Alternative Medicine 03/2014; 2014(3):280204. DOI:10.1155/2014/280204 · 1.88 Impact Factor
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    • "Unlike sulfenic acids that can be reduced by major cellular reductants, sulfinic and sulfonic acids cannot. Oxidation of Cys residues in proteins can lead to diverse functional consequences, such as inhibition or activation of enzymatic activities, inhibition of binding activities [13], [15]–[16]. To maintain the intracellular thiol-disulfide redox status under reducing conditions ([-SH]>[-S-S-]), living cells possess two major systems, the thioredoxin (Trx) and glutaredoxin (Grx) pathways [17]. "
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    ABSTRACT: In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.
    PLoS ONE 10/2013; 8(10):e75751. DOI:10.1371/journal.pone.0075751 · 3.23 Impact Factor
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    • "The splicing changes that we uncovered in SMN2 transgene transcripts during late stage SMA were not described in these studies, although in other studies, SMN2 splicing was shown to decrease in type I mice at P1 (Jodelka et al. 2010; Ruggiu et al. 2012). Dying mice likely suffer from hypoxia; hypoxic stress induces reactive oxygen species generation, which inactivates the SMN complex (Wan et al. 2008). This complex plays fundamental roles in assembling snRNPs, which are required for splicing (Burghes and Beattie 2009), and low SMN levels result in decreased SMN2 exon 7 splicing through a feedback loop (Jodelka et al. 2010; Ruggiu et al. 2012). "
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