Biochemical Characterization of Glutaredoxins from Chlamydomonas reinhardtii Reveals the Unique Properties of a Chloroplastic CGFS-type Glutaredoxin

Abstract

Aspirin and other nonsteroidal anti-inflammatory drugs inhibit cell proliferation and induce apoptosis in various cancer cell lines, which is considered to be an important mechanism for their anti-tumor activity and prevention of carcinogenesis. However, the molecular mechanisms through which these compounds induce apoptosis are not well understood. Here we have found that aspirin treatment of the mouse Neuro 2a cells impaired the proteasome function and caused severe mitochondrial abnormalities. Treatment with aspirin lead to a dose-and time-dependent decrease in proteasome activity and an increase in the accumulation of ubiquitylated proteins in the cells, which correlated with its effect on cell death. Aspirin exposure also resulted in an increase in the half-life of pd1EGFP, a model substrate of proteasome, as well as various intracellular substrates like Bax, IB-␣, p53, and p27 kip1 . Aspirin-induced proteasomal malfunction might be responsible, at least in part, for the downregulation of NF-B activity and neurite outgrowth. Finally, we have shown that aspirin treatment caused changes in the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and -3, which could be because of the proteasomal dysfunction.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

“…DHA and a k cat of 0.23 Ϯ 0.01 s Ϫ1 (Table 2). These values are in the same order as those obtained for PtGrxS12 and CrGrx1 (6,27). The apparent K m value for GSH was determined by varying the amount of GSH in the DHA assay.…”

“…Importantly, GrxC5, GrxS14, and GrxS16 could exist as both apo-and holoforms, which exhibit different functionalities. From the literature on plant Grxs, it is likely that all proteins, except GrxS16, possess the capacity to deglutathionylate protein substrates, making them potential central components regulating many plastidial metabolic pathways as several glutathionylated proteins have been identified in chloroplasts (6,54,55). However, this reaction is likely to be preferentially carried out by class I Grxs as only GrxC5 and -S12 have the capacity to efficiently regenerate target proteins, which uses only one cysteine for catalysis, such as PrxIIE and MsrB1 (14,48).…”

“…However, this reaction is likely to be preferentially carried out by class I Grxs as only GrxC5 and -S12 have the capacity to efficiently regenerate target proteins, which uses only one cysteine for catalysis, such as PrxIIE and MsrB1 (14,48). 6 Another major difference is that the reductase activity of GrxC5 and -S12 is dependent on GSH, whereas by analogy to the work achieved with C. reinhardtii Grx3, GrxS14 and presumably GrxS16 may be reduced by ferredoxin-thioredoxin reductase but not glutathione (6).…”

“…Although Trxs have been reported to catalyze deglutathionylation in some organisms (16), this reaction is generally considered to be specifically catalyzed by Grxs (17). Protein deglutathionylation can proceed via a monothiol or a dithiol pathway depending on the involvement of one or two cysteines in the catalytic mechanism (6,18,19).…”

“…This variation correlates with the type of Grx considered and depends on their redox potential and catalytic mechanism. Indeed, whereas classical Grxs have midpoint redox potentials between Ϫ170 and Ϫ230 mV (pH 7.0) (7,8), Chlamydomonas Grx3 forms during its catalytic cycle an atypical intramolecular disulfide between the first active site cysteine and a second cysteine located in the C-terminal part of the protein (6). The more negative redox potential (Ϫ323 mV at pH 7.9) observed for this protein most likely explains its ability to receive electrons from ferredoxin:thioredoxin reductase but not from glutathione (6).…”