Eukaryotic Cell Death Research Articles

A Proteomic Survey of Chlamydomonas reinhardtii Mitochondria Sheds New Light on the Metabolic Plasticity of the Organelle and on the Nature of the -Proteobacterial Mitochondrial Ancestor

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Having a swell time – mitochondrial morphology and plant cell death programmes

Cell death is a vital process in multi-cellular eukaryotes. Rather than being a contradiction in terms, this statement highlights the importance of limited and localized cell killing to the health and normal development of complex organisms. The main focus of this article is the role of mitochondrial morphological changes during cell death programmes, and the conserved role of mitochondrial permeability transition (increased permeability of either the outer or inner membrane) as an early mechanistic event preceding cell death in both plant and non-plant eukaryotes. A second focus of this article is a review of the terminology and fundamental paradigms underpinning cell death research. Because of the importance of the process of cell death, there has been an enormous quantity of research performed to try to understand the underlying biological mechanisms. One result of such a large and varied research effort, and a result that is perhaps particularly evident to investigators coming into the field anew, is that some of the basic tenets of cell death research appear to have become confused. In this short article, I make an attempt to clarify the subject, focussing on the role of mitochondria, and the difficulties in comprehensibility arising from the sometimes-erroneous, or at least unnecessarily confusing use of specific terminology; there are several key terms in the cell death literature that appear interchangeable when they are not, or are interchanged when they should not be.

Characterisation of cDNAs of key genes involved in apoptosis in common carp ( Cyprinus carpio L.)

Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Many of the cell deaths that occur during normal embryological development and during normal tissue turnover display the morphological hallmarks of apoptosis. In the last 20 or so years a better biochemical picture of how the process occurs has been produced, at least in higher vertebrates. It is now widely accepted that many of the proteolytic cleavages occurring during apoptosis are mediated by caspases, the activation of which, in turn, has been found to be tightly regulated. The current study focuses on the sequencing and analysis of key genes involved in the apoptotic process, based on sequence similarity to known apoptosis genes from genetic models such as zebrafish ( Danio rerio) and other vertebrates. The present study identifies key components of the apoptotic process in common carp ( Cyprinus carpio L.), which in turn can be used, for example, to monitor the fate of the cellular components of the immune system after an immune challenge.

Marine Biofilm Bacteria Evade Eukaryotic Predation by Targeted Chemical Defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

A role for actin in regulating apoptosis/programmed cell death: evidence spanning yeast, plants and animals

Achieving an understanding of how apoptosis/PCD (programmed cell death) is integrated within cellular responses to environmental and intracellular signals is a daunting task. From the sensation of a stimulus to the point of no return, a programme of cell death must engage specific pro-death components, whose effects can in turn be enhanced or repressed by downstream regulatory factors. In recent years, considerable progress has been made in our understanding of how components involved in these processes function. We now know that some of the factors involved in PCD networks have ancient origins that pre-date multicellularity and, indeed, eukaryotes themselves. A subject attracting much attention is the role that the actin cytoskeleton, itself a cellular component with ancient origins, plays in cell death regulation. Actin, a key cellular component, has an established role as a cellular sensor, with reorganization and alterations in actin dynamics being a well known consequence of signalling. A range of studies have revealed that actin also plays a key role in apoptosis/PCD regulation. Evidence implicating actin as a regulator of eukaryotic cell death has emerged from studies from the Animal, Plant and Fungal Kingdoms. Here we review recent data that provide evidence for an active, functional role for actin in determining whether PCD is triggered and executed, and discuss these findings within the context of regulation of actin dynamics.

Molecular control of bacterial death and lysis.

Although the phenomenon of bacterial cell death and lysis has been studied for over 100 years, the contribution of these important processes to bacterial physiology and development has only recently been recognized. Contemporary study of cell death and lysis in a number of different bacteria has revealed that these processes, once thought of as being passive and unregulated, are actually governed by highly complex regulatory systems. An emerging paradigm in this field suggests that, analogous to programmed cell death in eukaryotes, regulated cell death and lysis in bacteria play an important role in both developmental processes, such as competence and biofilm development, and the elimination of damaged cells, such as those irreversibly injured by environmental or antibiotic stress. Further study in this exciting field of bacterial research may provide new insight into the potential evolutionary link between control of cell death in bacteria and programmed cell death (apoptosis) in eukaryotes.

EXOG, a novel paralog of Endonuclease G in higher eukaryotes

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed ‘yeast Endonuclease G (EndoG)’, is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5′–3′ exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog ‘EXOG’, a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5′–3′ exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

BackgroundDNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.ResultsTo determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant.ConclusionThese results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Methylation Profiling of Apoptotic Genes in Leukemia Cell Lines and Patients.

^equal contribution Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Apoptosis is executed via 2 main pathways that lead to the activation of caspases: the death receptor (DR) pathway and the mitochondrial pathway. Defects in apoptotic mechanisms play an important role in enabling neoplastic cells to survive by ignoring death inducing signals leading to protection from the lack of survival factors. DNA methylation is a reversible mechanism of gene inactivation that might be responsible for impaired apoptosis in leukemia cells. Methylation profiling of apoptotic genes can identify possible gene targets for epigenetic therapy using demethylating agents like 5-aza-deoxycytidine and azacytidine. It can also be used for classification and prognosis. We performed a comprehensive DNA methylation analysis of apoptosis pathway genes with CpG islands in promoter/exon 1 regions in 5 leukemic cell lines (TF1, K562, Raji, HL60 and Jurkat), 20 patients with acute myelogenous leukemia (AML), 20 patients with acute lymphoblastic leukemia (ALL), and 6 control samples (normal peripheral blood cells). We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Our analysis covered a total of 25 genes: 11 in the intrinsic pathway (BAX, BAK, HRK, CYC1, DIABLO, ATM, APAF1, MAP3K5, CHEK2,PAK1, and BNIP 3), 4 in the extrinsic pathway (TRADD,PYCARD, CARD 10 and BID), 5 genes of the Caspase family (Caspase 2, 3, 6, 7, 9) in addition to 4 of the programmed cell death genes (PDCD 2, 4, 6, and 11). By using a 15% cutoff level to call a gene methylated, methylation was observed in 7 genes in at least one cell line: BNIP3 (Raji), PYCARD (K562), HRK (Tf1, Raji and Jurkat), CARD10 (Raji and HL-60), BID (Raji), PAK1 (Raji) and Casp 6 (Raji). We then performed methylation analysis of 4 genes (BNIP3, PYCARD, PAK1 and CARD 10) in AML and ALL patient samples. Methylation frequencies over a 15% cutoff are summarized in Table 1. We conclude that DNA methylation-associated epigenetic silencing is likely to play a role in modulating apoptosis in a subset of AML and ALL patients, with PYCARD being the most frequent target.Table 1GenesMethylation in AML patientsMethylation in ALL patientsPYCARD6/204/20BNIP31/203/20CARD100/202/20PAK10/201/20

Flagella Facilitate Escape ofSalmonellafrom Oncotic Macrophages

The intracellular parasite Salmonella enterica serovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival of Salmonella in phagocytes is well understood, little has been documented about the exit of intracellular Salmonella from host cells. Here we report that in a population of infected macrophages Salmonella induces "oncosis," an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMphis) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMphis filled with bacilli remains impermeable, and intracellular Salmonella bacilli move vigorously using flagella. Eventually, intracellular Salmonella bacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy whereby Salmonella escapes from host macrophages.

Bacteriophage-encoded toxins: the ?-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells

The bacteriophage-encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage lambda-holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of lambda-holin-induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle-specific fluorescence immunocytochemistry indicated the presence of the lambda-holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria-specific fluorochrome JC-1 demonstrated a loss of mitochondrial transmembrane potential in response to lambda-holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP-ribose) polymerase (PARP) in an apoptosis-specific manner. These findings suggest that the lambda-holin protein mediates a caspase-independent non-apoptotic mode of cell death.

ER Stress Induces the Expression of Jpk, which Inhibits Cell Cycle Progression in F9 Teratocarcinoma Cell

Jopock (Jpk), a transacting factor associated with the position-specific regulatory element of murine Hoxa-7, has shown to induce cell death in both prokaryotic and eukaryotic cells when introduced and overexpressed. Since Jpk protein harbors a transmembrane domain (TM) and a putative endoplasmic reticulum (ER) -retention signal at the N terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP). Both N-term- (Jpk-EGFP) and C-term-fused Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscope after staining the cells with ER- and Mito-Tracker. Through deletion analysis TM turned out to be important for ER localization of Jpk. When flow cytometric analysis was performed, both cells expressing Jpk-EGFP and EGFP-Jpk led cell cycle arrest and subsequent apoptotic cell death. In order to see whether Jpk is expressed during ER stress-mediated apoptosis, F9 cells were treated with DTT, an ER stress inducer. In the presence of 4 mM of DTT, about 50% of cells died strongly expressing Jpk (sevenfold) as well as Grp78, a molecular chaperone, and CHOP-10, a well-known apoptotic protein. When cells were transfected with both pEGFP-Jpk and pJpk-EGFP, cell cycle progression was interrupted compared to those of control cells. In summary, excess ER stress upregulated the expression of Jpk, which seemed to inhibit the cell cycle progression. These results altogether suggest that Jpk could be a useful cell death-triggering molecule applicable for cancer therapy as well as a useful target molecule for the treatment of certain neurodegenerative diseases caused by ER stress.

Ultraviolet irradiation-induced apoptosis does not trigger nuclear fragmentation but translocation of chromatin from nucleus into cytoplasm in the microglial cell-line, BV-2

Chromatin condensation, decrease of nuclear volume, and nuclear fragmentation are key features of apoptosis (programmed cell death) in many eukaryotic cells. How chromatin is redistributed in a continuously shrinking nucleus is an intriguing question. To evaluate this interesting spatial problem, we studied the ultrastructural distribution of chromatin in distinct stages of apoptosis using the microglial cell-line, BV-2, as a model and UV irradiation as a trigger of apoptosis. During apoptosis, condensed chromatin accumulated initially at the nuclear periphery and, subsequently, occupied almost the entire nucleus. Surprisingly, nuclei did not fragmentize, but apoptotic cells showed condensed chromatin in the nucleus as well as in the nucleus-attached cytoplasm. During apoptosis, the nuclear envelope dilated and decreased in extension by formation of numerous electron lucent vesicles, which accumulated in the cytoplasm. Furthermore, we observed in BV-2 cells well-known apoptotic features, like increased caspase-3/7 activity and annexin V labeling, as well as a sequence of cell morphological alterations, including cell shrinkage, zeiosis, and formation of apoptotic bodies. Thus, our findings suggest that UV-induced chromatin degradation is not restricted to the nucleus but may also take place in the cytoplasm in BV-2 cells.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

Simian Virus 40 Late Proteins Possess Lytic Properties That Render Them Capable of Permeabilizing Cellular Membranes

Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

Mycoplasma alligatorisInfection Promotes CD95 (FasR) Expression and Apoptosis of Primary Cardiac Fibroblasts

Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2'-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.

The actin cytoskeleton, RAS–cAMP signaling and mitochondrial ROS in yeast apoptosis

The release of reactive oxygen species (ROS) by mitochondria instigates the pathways of programmed cell death in eukaryotic cells. Gourlay and Ayscough present intriguing experimental evidence that mutations in the genes encoding the regulatory proteins End3p and Sla1p, which influence actin dynamics in budding yeast, lead to a loss of mitochondrial membrane potential, resulting in ROS production and apoptosis. This effect can be suppressed by downregulation of the RAS-cAMP signaling pathway, thus establishing the existence of a new and complex regulatory network.

Metacaspases of Trypanosoma cruzi: Possible candidates for programmed cell death mediators

The genome of Trypanosoma cruzi, the Protozoan parasite causing the American Trypanosomiasis, Chagas disease, contains two genes, TcMCA3 and TcMCA5, with homology to those encoding metacaspases, distantly related to the caspases involved in programmed cell death (PCD) in higher eukaryotes. TcMCA3 is present in the CL Brener clone at 16 copies per haploid genome, arrayed in two tandems located in chromosomes of 0.54 and 0.98 Mbp. TcMCA5, on the other hand, is present as a single copy gene. The proteins encoded were expressed in Escherichia coli BL21 [DE3] cells, and used to generate antibodies, which allowed demonstrating that TcMCA3 is expressed in the four major developmental stages of the parasite, whereas TcMCA5 is expressed only in the epimastigote form. Moreover, recombinant TcMCA3, but not TcMCA5, was recognized by most sera from chronic Chagasic patients, showing that the protein is expressed during natural infections. All attempts to show processing and enzyme activity in the recombinant proteins have been unsuccesful so far; however, indirect evidence suggests that the metacaspases might be involved in PCD of the parasite. (1) Immunofluorescence experiments showed that both proteins change their subcellular localization during fresh human serum (FHS)-induced PCD migrating into the nucleus. (2) Epimastigotes over-expressing TcMCA5 were more sensitive to FHS-induced PCD than the controls. (3) PCD was paralelled by an increase in peptidase activity against Z-YVAD-AFC, a typical caspase substrate, and the apoptotic nuclei cells were labeled in vivo with the pan-caspase fluorescent inhibitor SR-VAD-FMK. Further experiments will be required to complete the characterization of these proteins and elucidate their role in the parasite.