Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development.

We studied the effect of reducing the levels of the mitochondrial electron carrier cytochrome c (CYTc) in Arabidopsis thaliana. Plants with CYTc deficiency have delayed growth and development, and reach flowering several days later than the wild-type but with the same number of leaves. CYTc-deficient plants accumulate starch and glucose during the day, and contain lower levels of active gibberellins (GA) and higher levels of DELLA proteins, involved in GA signaling. GA treatment abolishes the developmental delay and reduces glucose accumulation in CYTc-deficient plants, which also show a lower raise in ATP levels in response to glucose. Treatment of wild-type plants with inhibitors of mitochondrial energy production limits plant growth and increases the levels of DELLA proteins, thus mimicking the effects of CYTc deficiency. In addition, an increase in the amount of CYTc decreases DELLA protein levels and expedites growth, and this depends on active GA synthesis. We conclude that CYTc levels impinge on the activity of the GA pathway, most likely through changes in mitochondrial energy production. In this way, hormone-dependent growth would be coupled to the activity of components of the mitochondrial respiratory chain.

Redox state-dependent aggregation of mitochondria induced by cytochrome c.

Cytochrome c is known to play central role in apoptosis. Here, it is shown that ferricytochrome c, but not ferrocytochrome c is able to directly induce the aggregation of rat liver mitochondria, similar to the effect caused by magnesium ions at high concentrations. The aggregation was revealed by a decrease in light dispersion of mitochondrial suspension and it was confirmed by the optical microscopy. In the medium containing NADH and cytochrome c, mitochondrial aggregation was initiated only after exhaustion of NADH leading to oxidation of cytochrome c. The aggregation induced by 30 µM ferricytochrome c, but not by 5 mM MgCl(2), was completely inhibited by 30-100 µM ferricyanide, thus indicating that ferricyanide-cytochrome c specific interaction prevents mitochondrial aggregation. After completion of the aggregation caused by ferricytochrome c, this effect cannot be readily reversed by subsequent reduction of cytochrome c. The aggregation induced by ferricytochrome c and/or magnesium ions explains masking of the external NADH-oxidase activity of mitochondria in vitro reported in the literature. This new cytochrome c redox state-dependent phenomenon might also be involved in more complex mechanisms controlling aggregation (clustering) of mitochondria in vivo under the influence of pro-apoptotic factors and requires further study.

Bluetongue virus infection: activation of the MAP kinase-dependent pathway is required for apoptosis.

Bluetongue virus (BTV) is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. Based on information that members of the mitogen-activated protein kinase family (MAPKs) are mediators of apoptosis, we have examined in detail the MAPK-dependent apoptosis in BTV infection. Previously, we have shown that apoptosis in BTV infection requires the participation of mitochondrial apoptotic pathways. In addition, we demonstrated that NF-κB is activated and that its inhibition substantially reduces cellular apoptosis. For the first time, here we demonstrated the activation of MAPKs after BTV infection. Moreover, by pre-treatment with MAPK inhibitors, c-Jun N-terminal kinases (JNKs) and p38 MAPK, but not extracellular signal-related kinase (ERK), significantly decreased the induction of apoptosis. JNK and p38 activation regulated the cytochrome c released from mitochondria and caspase 3 activation. These results strengthen the understanding of BTV infection and contribute to our previous data confirming that BTV infection induces robust apoptosis in mammalian cells and is likely to play a primary role in BTV pathophysiology.

Nitrocytochrome c: synthesis, purification, and functional studies.

Posttranslational protein tyrosine oxidation, to yield 3-nitrotyrosine, is a biologically relevant protein modification related with acute and chronic inflammation and degenerative processes. It is usually associated with a decrease or loss in protein function. However, in some proteins, tyrosine nitration results in an increase or gain in protein function. Nitration of cytochrome c by biological oxidants in vitro can be achieved via different mechanisms, which include reactions with peroxynitrite, nitrite plus hydrogen peroxide, and nitric oxide plus hydrogen peroxide, and result in a loss in its electron transport capacity and in a higher peroxidatic activity. This chapter describes the methodology for studying chemical and biological properties of nitrocytochrome c. In particular, we report methods to synthesize tyrosine-nitrated cytochrome c, purify cytochrome c mononitrated species, map the sites of tyrosine nitration, and investigate the functional consequences of nitrated cytochrome c on mitochondrial electron transport properties, peroxidatic activity, and apoptosome assembly.

A cyclic chimeric interferon-alpha2b peptide induces apoptosis in tumor cells.

Interferons alpha (IFNsalpha) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsalpha have been widely employed for the pharmacological treatment of different types of cancer, the therapeutic efficacy occasionally can be diminished by the appearance of side effects, neutralizing antibodies or tumor resistance. In the search of mimetic peptides of the IFN-alpha2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-alpha2b binding to its receptor and exerts an IFN-like antiproliferative activity. In order to study the mechanism of growth inhibition of the cyclic chimera, we evaluated its ability to induce cell cycle arrest or apoptosis in WISH cells. We found that the chimeric peptide did not cause a cell cycle arrest, although the entire IFN-alpha2b molecule did modify cell cycle by increasing the number of S-phase cells. In spite of this difference, both molecules were able to induce apoptosis through the activation of caspases 8 and 9, indicating the involvement of death receptor and mitochondrial pathways. In addition, both peptidic derivative and IFN-alpha2b altered the expression of Bcl-2 family proteins and induced the release of cytochrome C to cytosol, supporting the participation of mitochondrial pathway in the induction of apoptosis. In conclusion, we demonstrated that the chimeric cyclic peptide behaved as a potent inducer of apoptosis and it could be a potentially useful agent for the treatment of certain malignancies.