Andrey Kropotov

Processed caspase-2 can induce mitochondria-mediated apoptosis independently of its enzymatic activity

The mechanism by which caspase‐2 executes apoptosis remains obscure. Recent findings indicate that caspase‐2 is activated early in response to DNA‐damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase‐2 stimulates mitochondrial release of cytochrome c and Smac/DIABLO, but not apoptosis‐inducing factor (AIF). This event occurs independently of several Bcl‐2 family proteins, including Bax, Bak and Bcl‐2, and inactivation experiments reveal that the proteolytic activity of caspase‐2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase‐2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase‐2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity.

Caspase-2 Permeabilizes the Outer Mitochondrial Membrane and Disrupts the Binding of Cytochrome c to Anionic Phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.

Constitutive expression of the human peroxiredoxin V gene contributes to protection of the genome from oxidative DNA lesions and to suppression of transcription of noncoding DNA

Peroxiredoxins belong to a family of antioxidant proteins that neutralize reactive oxygen species. One member of this family, peroxiredoxin I (PRDX1), suppresses DNA oxidation. Peroxiredoxin V (PRDX5) has been cloned as a transcriptional corepressor, as a peroxisomal/mitochondrial antioxidant protein, and as an inhibitor of p53‐dependent apoptosis. Promoters of mammalian PRDX5 genes contain clusters of antioxidant response elements, which can bind the transcription factor NRF2. However, we found that expression of the human PRDX5 gene in situ was not stimulated by the oxidative agent menadione. Silencing of the NRF2 gene in the absence of oxidative stress by specific siRNA did not decrease PRDX5 protein concentration. We also constructed clones of human lung epithelial cells A549 with siRNA‐mediated knockdown of the PRDX5 gene. This led to a significant increase in 8‐oxoguanine formation in cell DNA. In the PRDX5 knockdown clone, an increase in transcripts containing sequences of alpha‐satellite and satellite III DNAs was also detected, suggesting that this protein may be required for silencing of heterochromatin. Together, these results suggest that constitutively expressed PRDX5 gene plays an important role in protecting the genome against oxidation and may also be involved in the control of transcription of noncoding DNA.

Mitochondrial targeting of human peroxiredoxin V protein and regulation of PRDX5 gene expression by nuclear transcription factors controlling biogenesis of mitochondria.

Peroxiredoxin V (PRDX5) is a member of the family of mammalian proteins that neutralize reactive oxygen species. The PRDX5 gene is constitutively expressed at a high level in many human tissues, but functional elements of its promoter responsible for a high basal activity in the absence of oxidative stress have still not been identified. Among predicted binding sites for transcription factors in the human PRDX5 promoter are binding sites for nuclear respiratory factor 1 (NFR‐1) and nuclear respiratory factor 2 (also called GABPA), which regulate the biogenesis of mitochondria. We constructed luciferase reporter gene plasmids containing stepwise deletions of the PRDX5 promoter and examined their activities in transient transfections. Our results suggest that basal PRDX5 promoter activity mostly depends on NFR‐1 and GABPA sites. The latter, in the PRDX5 promoter, were conserved in the six mammalian genomes analyzed (human, chimpanzee, cow, mouse, rat and dog) and a fraction of human PRDX5 associates with the mitochondrial matrix. We also found that the N‐terminal 50 amino acids of the full‐length human PRDX5 (24 kDa) translated from its first AUG codon targets this protein exclusively to mitochondria. However, the short form of PRDX5 (17 kDa), translated from its second AUG codon, has cytoplasmic and nuclear localization, which is also typical for endogenously expressed protein. Together, our results indicate that high basal expression of the PRDX5 gene is coordinated with the expression of nuclear genes encoding mitochondrial proteins and that the PRDX5 protein might play a major role in permanent defense against reactive oxygen species produced by mitochondria.

Dynamics of γH2AX formation and elimination in mammalian cells after X-irradiation

Phosphorylation of the replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γH2AX can be used as an effective marker for DSB repair and DNA damage response. In this study, we examined a bystander effect (BE) in locally irradiated embryonic human fibroblasts. Using fluorescence microscopy, we found that BE could be observed 1 h after X-ray irradiation (IR) and was completely eliminated 24 h after IR. Using immunohistochemistry and immunoblotting, we also studied kinetics of γH2AX formation and elimination in Syrian hamster and mouse tissues after whole body IR of animals. Analysis of hamster tissues at different times after IR at the dose 5 Gy showed that γH2AX-associated fluorescence in heart was decreased slowly with about a half level remaining 24 h after IR; at the same time, in brain, the level of γH2AX was about 3 times increased over the control level, and in liver, γH2AX level decreased to control values. We also report that in mouse heart the level of γH2AX measured by immunoblotting is lower than in brain, kidney and liver at different times after IR at the dose 3 Gy. Our observations indicate that there are significant variations in dynamics of γH2AX formation and elimination between non-proliferating mammalian tissues. These variations in γH2AX dynamics in indicated organs partially correlated with the expression level of the major kinase genes involved in H2AX phosphorylation (ATM and DNA-PK).

Phosphorylation of histone H2AX in human lymphocytes as a possible marker of effective cellular response to ionizing radiation

DNA double-strand breaks (DSBs) that occur in cells after ionizing radiation (IR) or chemical agents are the most dangerous lesions in eukaryotic cells resulting in cell death or chromosomal aberrations and cancer. DSB repair is very important for maintenance of genome stability. One of the earliest cellular responses to DSBs is phosphorylation at 139 serine of core variant histone H2AX in megabase chromatin domains around DSB (γ-H2AX), which amplifies the signal and makes it possible to identify even a very few DSBs in a genome. Here, using immunofluorescent and Western blotting techniques, we studied the dynamics of γ-H2AX formation in human lymphocytes of various individuals irradiated ex vivo. We found that the dynamics of γ-H2AX formation in lymphocytes differ between individuals but had similar kinetics and statistically is independent of people’s age.

Transcription factor GABP/NRF-2 controlling biogenesis of mitochondria regulates basal expression of peroxiredoxin V but the mitochondrial function of peroxiredoxin V is dispensable in the dog

Peroxiredoxins (PRDXs) represent a conserved family of six antioxidant proteins which are widely expressed in different organisms. Human PRDX5 is detected in the cytosol and nucleus and can also target peroxisomes and mitochondria. However, it remains unknown if mitochondrial localization of PRDX5 is essential for its functions. Here we studied whether the known regulator of mitochondrial biogenesis, transcription factor GABP/NRF-2, is required for the basal expression of the human PRDX5 gene and what the significance is of the mitochondrial targeting of the PRDX5 protein. It was found that mutation-mediated inactivation of all potential binding sites for GAPB in the PRDX5 promoter lead to ∼80% inhibition of its basal activity in a reporter gene assay. Co-transfection of plasmids expressing GABP-alpha and GABP-beta stimulated activity of the non-mutated PRDX5 promoter but had no effect on the mutated promoter, suggesting that basal expression of the human PRDX5 gene is regulated by GABP. We found that the dog c-Myc-tagged PRDX5 did not target the mitochondria of human cells. Endogenously expressed PRDX5 also showed no association with mitochondria in the dog cells. It appears, therefore, that during evolution the dog PRDX5 gene lost its upstream ATG codon and mitochondrial targeting signal without major functional consequences.

Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity

Disturbances at the childhood age increase risk of appearance of cardiovascular disease decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable source of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles understanding on the triggers and mechanisms are the cross-species comparative data on life-cycle and heart aerobic capacity. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capacity in adults. Basing on these data, we suggested that the neonatal infectious gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathologic changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of atrial septum (ostuim primum), we have established that a gradual increase of intensity of infestation with Cryptosoridium parvum oocysts causes sharp changes corresponding to “all-or-nothing” response. At a weak infestation the atrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of α- and β- myosin were absent. At the intermediate and severe infestation, in the atrial septum the foramen ovale was visualized and there were observed the cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity of β-chain. Thus, by disturbing the frequency-strength ratios and causing the outflow of resources from the formed heart, the neonatal cryptosporidiosis produces pathological changes of the organ molecular and anatomical structure. Our results can be interest to evolutionary biologists and physicians, as they show the importance of knowledge of evolutionary-comparative investigations for search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of atrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

Remodeling of rat cardiomyocytes after neonatal cryptosporidiosis. I. Change of ratio of isoforms of myosin heavy chains

Diseases of the human cardiovascular system are the main cause of death in developed countries. Therefore, searching for new risk factors thereof is of particular interest. Upon comparing epidemiological data with data of transcriptome of cardiomyocytes and comparative physiology of vertebrate ontogenesis, we have come to the conclusion that one such factor may be gastroenteritis. This disease includes at once several stimuli able to cause functional and metabolic alterations in the heart: tachycardia, hormonal and ionic misbalance, and outflow of resources from the cardiovascular system. Using the model of rat neonatal gastroenteritis caused by the widespread human and animal enteropathogen Cryptosporidium parvum (Apicomplexa, Sporozoa), we studied the change of expression of α- and β-myosin heavy chains after the developed cryptosporidiosis. Online data obtained by methods of immunocytochemistry, quantitative morphometry, and polymerase chain reaction not only have confirmed our suggestion, but also have shown that moderate 4-day-long cryptosporidiosis is sufficient for producing a significant (1.7- to 4.5-fold) shift in the ratio of myosin isoforms toward the β-isoform beta at the level of mRNA and at the level of protein (2.5–6 times). The reciprocity of the changes, as well as their clear similarity at the level of mRNA and of protein, indicates that the cryptosporidial gastroenteritis involves all the main chains of a complex network of regulation of expression of the myosin heavy chains. A shift of the ratio of myosin isoforms toward the β-isoform that has an ATPase activity several times lower than the α-isoform is the commonly accepted indicator of human heart failure; therefore, the cryptosporidial gastroenteritis can be considered a novel risk factor for decrease of the heart’s contractile ability. Our data may be of interest for clinical and preventive medicine.

The threshold response of neonatal rat cardiomyocytes to gradual enhancement of cryptosporidial invasion

One common cause of excessive cardiac loading in children is infectious gastroenteritis that produces malabsorption and tachyarrythmia. Our recent studies have shown that neonatal cryptosporidial gastroenteritis causes the long-term pathology of cardiomyocytes. In the present work, we studied how cryptosporidiosis of different degrees of severity is reflected on the heart anatomy and polyploidization and remodeling of cardiomyocytes of neonatal rats, as well as on the expression of their gene encoding factor sensitive to hypoxia (HIF-1α). By methods of Real-Time PCR, cytometry, immunocytochemistry, image analysis, and visual study of interatrial septum, we have established that gradual enhancement of infestation by cryptosporidiosis is accompanied by threshold changes in heart. With weak infection, the interatrial septum preserves integrity, while changes in cardiomyocytes are absent. With moderate and strong infection, the changes are expressed approximately equally: in the interatrial septum, foramen ovale appears and the heart becomes atrophied and elongated, while cardiomyocytes lose protein and become thinned and hyperpolyploid. In addition, an excessive level of mRNA of gene HIF-1α appears in the myocardium. It is important to note that the threshold response to the gradual increase of the action is a criterion of triggering of the developmental programming of cardiovascular diseases, as such response is based on irreversible disturbance of the anatomy of the organ and failure of cell differentiation. In our case, the disturbance of the heart is the patent foramen ovale in the interatrial septum, while the disturbance of differentiation is hyperpolyploidization of cardiomyocytes. Our data may be of interest to physicians, as they show for the first time a connection between gastroenteritis, disturbance of the integrity of the interatrial septum, and pathological alterations in cardiomyocytes.