Cytochrome C

Desulfovibrio vulgaris Hildenborough cytochrome c 3 contains four hemes in a low-spin state with bis-histidinyl coordination. Highspin forms of cytochrome c 3 can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.

Mitochondrial encephalomyopathies, arising from deficiencies of the electron transport chain (ETC) give rise to a wide clinical spectrum of presentation and are often progressive in nature. The aetiology of mitochondrial encephalomyopathies have yet to be fully elucidated, however, a successive loss of ETC function may contribute to the progressive nature of these disorders. The possibility arises that as a consequence of a primary impairment of ETC activity, secondary damage to the ETC may occur. In order to investigate this hypothesis, we established a model of cytochrome oxidase (Complex IV) deficiency in cultured human astrocytoma 1321N cells. Potassium cyanide (KCN, 1 mM) resulted in a sustained 50% (p < 0.01) loss of complex IV. At 24 h activities of the other ETC complexes were unaffected. However, at 72 h significant loss of succinate-cytochrome c reductase (complex II-III) activity expressed as a ratio to the mitochondrial marker, citrate synthase was observed. (KCN treated; 0.065 ± 0.011 vs controls; 0.118 ± 0.017 mean ± SEM, n = 8, p < 0.05). These results provide a possible mechanism for the progressive nature of ETC defects and why in some patients multiple patterns of ETC deficiencies can be demonstrated.

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of diheme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c 551. Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a ''charge hopping'' mechanism may be operative in the process of intra-molecular electron transfer.

Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this minireview we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein interactions, using a Debye-Hiickel-based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface-charge/ E" relationships. This approach shows that a number of anions, most of which are of biological relevance, namely C1-, HPO:-, HCO;, NO;, SO:-, ClO;, citrate'-and oxalate2-,. bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities. The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence-related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. Specific binding of this large set of anions bearing different charges allows the electrostatic effect on E" due to neutralization of net positive protein surface charge(s) to be monitored. 'H NMR indeed indicates the absence of significant salt-induced structural perturbations, hence the above change in ED is predominantly electrostatic in origin. A systematic study of protein surface-chargelE" relationships using this approach is unprecedented. Values of 15 -25 mV (extrapolated at zero ionic strength) are obtained for the decrease in E" due to neutralization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or chemical modification of surface lysines. The effects of the anion-induced decrease of net positive charge on ED persist also at a relatively high ionic strength and add to the general effects related to the charge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electron transfer between cytochromes c and redox partners could also involve salt-induced changes in the driving force.

Molecular motion of metal-free and metal-substituted cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal-free cytochrome c is around 10 ns. We find that the anisotropy of fluorescence of metal-free cytochrome c when bound to mitochondria does not decay, but when bound to phospholipids has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of zinc cytochrome c, as measured by triplet-triplet absorption and phosphorescence emission is z 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane-bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 ps and the slow component with relaxation time of z 6 ms.

Some 35 years ago, I had the good fortune to be accepted into Ray Wu’s lab as a graduate student. Ray was already well known for his pioneering studies in DNA sequencing, and his lab was quietly humming with the excitement of the new approaches being developed for DNA labeling, sequencing and synthesis. Looking back, I am somewhat surprised that Ray accepted me into his lab-I was not a chemistry or biochemistry student, and I was transferring from another Department where I had become disillusioned with the prospects for completing a thesis project (of my own naïve design). I’d like to briefly recount the story of how I came to work for Ray, as I think it sheds some light on his personality, in particular the importance that he placed on the support and teaching of young, inexperienced students. If he saw potential in someone, he would do whatever it took to provide the opportunities and resources for that person to develop as a scientist. John Stiles, a fellow graduate student, and I had come up with what was, in 1973, a technically challenging but novel approach to the detection of a specific mRNA. The idea was based on the recent completion of a short stretch of the DNA sequence of the yeast iso-cytochrome c gene, which had been determined by the remarkable strategy of sequencing the protein products of a series of frameshift mutants (in Fred Sherman’s lab, in nearby Rochester, N.Y.). We felt that it should be possible to chemically synthesize a DNA probe that would allow us to detect the gene and its mRNA by hybridization. But how could we actually do this? The genetic tools for the project were available in Fred Sherman’s lab, but the chemical synthesis of DNA oligonucleotides was still a significant undertaking back then. Looking around at the Cornell faculty of the time, it seemed to us that Prof. Wu’s laboratory was the best and probably the only place that this project could be carried out. But would the famous Prof. Wu listen to a couple of unknown graduate students pushing a wild plan? It was with some trepidation that John and I went to see Prof. Wu, and nervously tried to explain the idea we had come up with. Fortunately, Ray was indeed intrigued by the idea. Although our proposal must have seemed somewhat obvious to him, Ray smiled and nodded and said that it sounded “very interesting” (only later would I learn what high praise this was coming from Ray Wu!). Eventually, Ray agreed to help us carry out our plan, and to arrange the necessary collabora

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k a increased 20-fold when the concentration of urea was raised from 0 M to 6 M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the k a value reached its maximum (k a,max ) in all urea concentrations. Interestingly, the k a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe-S(Met80) bond disruption and for ligand binding to heme iron.

The work presented here was initiated to explore the mechanisms underlying vinorelbine resistance in two previously established murine leukemia P388 cell lines (N.63 and N2.5). IC50 measurements demonstrated that the vinorelbine-resistant cell line N.63 was sensitive to both vinblastine and vinflunine. In addition, vinorelbine-resistant cell line N2.5 retained sensitivity to vinflunine. We used flow cytometry with propidium iodide to measure G2/M arrest in response to drug treatment. Annexin V labeling was used as a marker of apoptosis and JC-1 dye labeling as a marker of mitochondrial membrane depolarization to explore differential responses that might help explain the absence of cross resistance to vinflunine. At equipotent (10X IC50) doses, after 8 h of drug treatment, vinflunine induced G2/M arrest in a significantly larger fraction of vinorelbine- resistant cells compared to vinorelbine. At the same drug doses, at 16 h after initiation of drug treatment, vinflunine induced a statistically significant greater apoptotic response and mitochondrial depolarization. The mitochondrial depolarization at 16 h was confirmed by Western blotting that showed release of cytochrome c. Comparison of apoptotic and mitochondrial depolarization responses in vinorelbine-resistant cells upon exposure to vinorelbine, vinblastine and vinflunine demonstrated the following pattern of drug activity: vinflunine > vinblastine > vinorelbine, confirming the importance of a antimitotic-induced mitochondria-mediated pathways in these P388 cell lines. We conclude that vinflunine may be preferred for treatment of specific cancers compared to other vinca alkaloids due to its enhanced effects on apoptotic pathways that follow G2/M arrest.

In this study, a-bisabolol, a sesquiterpene alcohol present in natural essential oil, was found to have a strong time-and dosedependent cytotoxic effect on human and rat glioma cells. After 24 h of treatment with 2.5-3.5 lM a-bisabolol, the viability of these cells was reduced by 50% with respect to untreated cells. Furthermore, the viability of normal rat glial cells was not affected by treatment with a-bisabolol at the same concentrations as above. Glioma cells treated with high concentration of a-bisabolol (10 lM) resulted in a 100% cell death. Judging from hypo-G1 accumulation, poly(ADP-ribose) polymerase cleavage, and DNA ladder formation, the cytotoxicity triggered by a-bisabolol resulted from apoptosis induction. Moreover, the dissipation of mitochondrialinner transmembrane potential and the release of cytochrome c from mitochondria indicated that, in these glioma cells, apoptosis occurred through an intrinsic pathway. As pointed out by the experimental results, a-bisabolol may be considered a novel compound able to inhibit glioma cell growth and survival.

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/ nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we have studied the influence of differently substituted benzofurans on the CRTAase activity to study the effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl) on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase. Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin (DAMC).

Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis. We show for the first time that paclitaxel acts directly on mitochondria isolated from human cancer cells. In isolated yeast mitochondria, paclitaxel (15 μm) induced an 18% increase in the ...

Our previous work suggests that cone photoreceptor inner segment (CIS) mitochondria demand and produce more ATP than rods. The CISs utilize two complimentary strategies to increase ATP production: increase the absolute number of mitochondria and their cristae surface membrane area. In this treatise, we ask: How are crista junctions formed and regulated? Once formed, are there physical mechanisms that constrain their diameter? How are the constrictions in cristae regulated and is this key for cytochrome c release during apoptosis? What are their differences in rod and cone susceptibility to apoptotic cell death during calcium overload and oxidative stress?

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA Gly genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in diVerent cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining diVerent strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15 N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV-visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.

Superoxide radical plays an important role in cell signaling. However, certain events can result in a large increase in superoxide concentration which has been linked to, among other conditions, inflammation, neurodegenerative diseases, and cancer. Consequently, in vivo detection of superoxide is of great interest. Previously, due to brittleness, instability, or size, superoxide sensors have been limited in their ability for

Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the inXuence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a signiWcant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G signiWcantly accelerated hydroperoxideinduced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro-and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H 2 O 2 ) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus -18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H 2 O 2 via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.

This work characterized the binding of an RNA aptamer recognizing hen egg white lysozyme, as well as a literature-reported single-stranded DNA analog of sequence identical to the original RNA aptamer, using fluorescence anisotropy, isothermal titration calorimetry (ITC) and analytical ultracentrifugation. The polyanionic DNA aptamer analog is selective for lysozyme even over cationic cytochrome c and has been reported to be successfully used in biosensing applications. The association however, is predominantly of electrostatic character, strongly salt-sensitive and entropically-driven, in contrast to previously described enthalpically-driven antibody-lysozyme and DNA aptamer-VEGF interactions. With a moderate selectivity for their target, high salt-sensitivity along with fast association and dissociation behavior, these molecules might serve as pseudo-affinity ligands for biomolecular separations.

En esta revisión se analizan las principales moléculas que participan en la supervivencia y la muerte por apoptosis de las células de los folículos antrales y del cuerpo lúteo (CL). Las gonadotropinas, los estrógenos y factores de crecimiento como IGF-I y VEGF, deben estar presentes en el ambiente folicular para que el folículo se mantenga sano. Mientras que la progesterona, LH y los factores de crecimiento mencionados, son vitales para las células del CL. En la apoptosis de las células ováricas, igual que en otros tipos celulares, participan una gran variedad de moléculas, dentro de las que destacan: Fas/FasL y otros integrantes de la familia del factor de necrosis tumoral (TNF) y sus ligandos, miembros proapoptóticos de la familia de proteínas Bcl-2 (Bax, Bok, Bim, Bad y Bid), citocromo c, Factor Inductor de Apoptosis (AIF), Factor activador de la apoptosis (Apaf-1), p53, caspasas de iniciación (1, 8 y 9), caspasas efectoras (3, 6 y 7), Smac/Diablo y proteínas inhibidoras de la Apoptosis (IAPs). Un ambiente folicular con bajos niveles de factores de sobrevivencia, incrementa la expresión de Fas y su interacción con el ligando FasL en las células foliculares, provocándoles la muerte por apoptosis, sin embargo, este ambiente inadecuado también puede ocasionar la liberación de proteínas proapoptóticas desde la mitocondria. La apoptosis en las células lúteas es inducida por la PGF2-a, la cual incrementa la expresión de miembros proapoptóticos tales como Bax y de receptores de muerte (RTNF-α α α α α)

The interaction of metalloproteins such as cytochromes with oxides is of interest for a number of reasons, including molecular catalysis of environmentally important mineral-solution electron transfer reactions (e.g., dehalogenations) and photovoltaic applications. Iron reduction by bacteria, thought to be cytochrome mediated, is of interest for geochemical and environmental remediation reasons. As a baseline for understanding cytochrome interaction with ferric oxide surfaces, we report on the interaction of mitochondrial cytochrome c (Mcc), a well-studied protein, with hematite (
-Fe2O3) surfaces. Mcc sorbs strongly to hematite from aqueous solution in a narrow pH range corresponding to opposite charge on Mcc and hematite (between pH 8.5 and 10, Mcc is positively charged and hematite surfaces are negatively charged). Cyclic voltammetry of Mcc
using hematite electrodes gives redox potentials characteristic of Mcc in a native conformational state, with no evidence for unfolding on the hematite surface. Atomic force microscopy imaging is consistent with a loosely attached adsorbate that is easily deformed by the AFM tip. In phosphate-containing solution, Mcc adhers to the surface more strongly. These results establish hematite as a viable material for electrochemical
and spectroscopic characterization of cytochrome–mineral interaction.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers show or acquire resistance to TRAIL. In this study we report that TRAIL activates a TGF-β-activated kinase 1→mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6→p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the `death-inducing signaling complex' (DISC), activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis (IAP) antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and activation of the apoptosome was dispensable. Even after MOMP, however, mitochondrial-generated reactive oxygen species (ROS) activated a secondary signaling pathway, involving c-Jun Nterminal kinases (JNKs), that similarly upregulated MCL-1 expression and partially rescued some cells from death. Thus, stress kinases activated at distinct steps, before and after mitochondrial injury, mediate TRAIL resistance through maintenance of MCL-1 expression.

We have shown previously that the pathways leading to Fas-mediated apoptosis in prostatic carcinoma cell lines are intact, because apoptosis can be triggered either by Fas ligation alone in the Fas-sensitive cell lines PC3 and ALVA31 or by rendering the Fas-resistant cell lines DU145 and JCA1 Fas-sensitive by combined treatment with anti-Fas monoclonal antibody and cycloheximide (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997). In this study, we demonstrate that two of the early events after Fas ligation are the release of cytochrome c from the mitochondria and activation of caspase-9. We also found that Bid is processed after Fas ligation and thus might activate the mitochondria-dependent apoptotic cascade. In a cell-free system, cytochrome c induced caspase-3-like activity in cytoplasmic extracts from all four cell lines studied, although differences in the level of enzymatic activity were observed. Western blot analysis revealed that caspase-7 is activated by cytochrome c...

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release.

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-⑀-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities. Cytochrome c has been identified as an important participant in apoptosis. In living cells, cytochrome c is present in the intermembrane space of mitochondria, where it plays an indispensable role in respiration. During apoptosis, however, cytochrome c translocates to the cytosol by mechanisms that are still unclear (reviewed in Refs. 1 and 2). In the cytosol, cytochrome c binds to Apaf-1 in a dATP/ATP-dependent manner (3), precipitating the oligomerization of Apaf-1 (4-8). The ensuing recruitment and activation of caspase-9 results in activation of further caspases, such as caspase-3, which orchestrate the final packaging of the apoptotic cell (see, e.g., Refs. 9-11). Cytochromes c are encoded by nuclear genes and translated in the cytosol. The resulting apocytochromes c are subsequently translocated through the outer mitochondrial mem-* This work was supported by American Cancer Society Grant DB-97 (to D. D. N.

BID, a pro-apoptotic Bcl-2 family member, promotes cytochrome c release during apoptosis initiated by CD95L or TNF. Activation of caspase-8 in the latter pathways results in cleavage of BID, translocation of activated BID to mitochondria, followed by redistribution of cytochrome c to the cytosol. However, it is unclear whether BID participates in cytochrome c release in other (non-death receptor) cell death pathways. Here, we show that BID is cleaved in response to multiple death-inducing stimuli (staurosporine, UV radiation, cycloheximide, etoposide). However BID cleavage in these contexts was blocked by Bcl-2, suggesting that proteolysis of BID occurred distal to cytochrome c release. Furthermore, addition of cytochrome c to Jurkat post-nuclear extracts triggered breakdown of BID at Asp-59 which was catalysed by caspase-3 rather than caspase-8. We provide evidence that caspase-3 catalysed cleavage of BID represents a feedback loop for the amplification of mitochondrial cytochrome c release during cytotoxic drug and UV radiation-induced apoptosis. Cell Death and Differentiation (2000) 7, 556 ± 565.

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.

Haplophytin-A (10-methoxy-2,2-dimethyl-2,6-dihydro-pyrano[3,2-c]quinolin-5-one), a novel quinoline alkaloid, was isolated from the Haplophyllum acutifolium. In this study, we investigated the effect of haplophytin-A on the apoptotic activity and the molecular mechanism of action in human promyelocytic leukemia HL-60 cells. Treatment with haplophytin-A (50 M) induced classical features of apoptosis, such as, DNA fragmentation, DNA ladder formation, and the externalization of annexin-Vtargeted phosphatidylserine residues in HL-60 cells. In addition, haplophytin-A triggered the activations of caspase-8, -9, and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, haplophytin-A caused the loss of mitochondrial membrane potential ( « m ) and the release of cytochrome c and Smac/DIABLO to the cytosol, and modulated the expression levels of Bcl-2 family proteins. We further demonstrated that knockdown of caspase-8 using its siRNA inhibited the mitochondrial translocation of tBid, the activations of caspase-9 and caspase-3, and subsequent DNA fragmentation by haplophytin-A. Furthermore, haplophytin-A-induced the formation of death-inducing signaling complex (DISC) and then activated caspase-8 in HL-60 cells. During haplophytin-A-induced apoptosis, caspase-8-stimulated tBid provide a link between the death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway. Taken together, these results suggest that the novel compound haplophytin-A play therapeutical role for leukemia via the potent apoptotic activity through the extrinsic pathway, involving the intrinsic pathway.

Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization. These events are not induced by mitochondria-independent apoptotic stimuli,

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (⌬m) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the ⌬m drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.

Apigenin, a plant flavone, potentially activates wild-type p53 and induces apoptosis in cancer cells. We conducted detailed studies to understand its mechanism of action. Exposure of human prostate cancer 22Rv1 cells, harboring wild-type p53, to growth-suppressive concentrations (10-80 μM) of apigenin resulted in the stabilization of p53 by phosphorylation on critical serine sites, p14 ARFmediated downregulation of MDM2 protein, inhibition of NF-κB/p65 transcriptional activity, and induction of p21/WAF-1 in a dose-and time-dependent manner. Apigenin at these doses resulted in ROS generation, which was accompanied by rapid glutathione depletion, disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. Interestingly, we observed accumulation of a p53 fraction to the mitochondria, which was rapid and occurred between 1 and 3 h after apigenin treatment. All these effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine, p53 inhibitor pifithrin-α, and enzyme catalase. Apigenin-mediated p53 activation and apoptosis were further attenuated by p53 antisense oligonucleotide treatment. Exposure of cells to apigenin led to a decrease in the levels of Bcl-XL and Bcl-2 and increase in Bax, triggering caspase activation. Treatment with the caspase inhibitors Z-VAD-FMK and DEVD-CHO partially rescued these cells from apigenin-induced apoptosis. In vivo, apigenin administration demonstrated p53-mediated induction of apoptosis in 22Rv1 tumors. These results indicate that apigenin-induced apoptosis in 22Rv1 cells is initiated by a ROS-dependent disruption of the mitochondrial membrane potential through transcriptional-dependent and -independent p53 pathways.

Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardioli...