Master of Science / Division of Biology / Rollie J. Clem / Caspases are a conserved family of cysteine proteases that play important roles in apoptosis and innate immunity as well as other cellular processes. Eleven caspase genes have been annotated in the mosquito Aedes aegypti. Amongst these, previous studies have demonstrated functional roles for AeDronc, CASPS7 and CASPS8 in the Ae. aegypti apoptosis pathway, while CASPS18 and CASPS19 have also been functionally characterized. A previous study from our research group showed that AeIAP1 has preferential binding for CASPS7 compared to CASPS8. In this study, it was confirmed that AeIAP1 has a higher capacity to inhibit CASPS7 than CASPS8. Furthermore, five of the remaining Ae. aegypti caspases, namely CASPS15, CASPS16, CASPS17, CASPS20 and CASPS21, were characterized. An attempt was made to classify these caspases as initiator or effector caspases, based on factors such as the length of their prodomain, sequence similarity to known Drosophila initiator and effector caspases, and their substrate specificity. The functions of these caspases in apoptosis was examined in the Ae. aegypti cell line Aag2, by using RNA interference to reduce their expression and test the effect on apoptosis. Recombinant CASPS16, 17, 20 and 21 were produced in bacteria and the abilities of these recombinant proteins to cleave different caspase substrates were examined. From the resulting data, it was concluded that CASPS17 and CASPS21 are likely to be effector caspases since they preferred a effector caspase substrate. When considering the prodomain length, CASPS17 has a short prodomain, but CASPS21 has a long prodomain, which is normally associated with initiator caspases. CASPS20 did not show preference for any specific substrate and has a short prodomain. Since it did not have a specific preference of substrate, it is likely to be an effector caspase based on prodomain length. CASPS16 showed a slightly higher preference for the initiator caspase substrate WEHD, and has a long prodomain. Based on these results, CASPS16 is likely an initiator caspase. To examine the potential roles of CASPS15, 16, 17, 20 and 21 in apoptosis, their expression in Aag2 cells was knocked down using RNA interference. Successful knockdown was verified by qRT-PCR. After silencing specific caspases, the cells were exposed to two different apoptotic stimuli, ultraviolet radiation (UV) or the RNA synthesis inhibitor actinomycin D (ActD). Following apoptotic treatment, apoptosis was measured by two methods; caspase activity was measured using an effector caspase substrate, and phosphatidyl serine exposure on the outer leaflet of the plasma membrane, which occurs in apoptotic cells, was measured by Annexin V staining and flow cytometry. In cells where CASPS15, 16, 17, 20 or 21 had been knocked down and the cells were then treated with UV or ActD, it was observed that effector caspase activity and Annexin V staining were both significantly lower than in UV- or ActD-treated cells that had received control double-stranded RNA. Together these results suggest that all of these caspases are involved in apoptosis in Aag2 cells. This study serves as a starting point for further research on Ae. aegypti caspases and their roles in specific cellular processes.

No description available.

Apoptosis is executed by caspase-mediated cleavage of various proteins. Elucidating the consequence of substrate cleavage provides us with insight into cell death and other biological processes. In this study, we applied the diagonal gel approach, a proteomic strategy, to identify substrates of the inflammatory caspase, caspase-1 and the cell death executioner caspase, caspase-3. Our results showed significant overlap between the substrates cleaved by both caspase-1 and -3. Such substrates are implicated in common cellular functions, including maintenance of the cytoskeleton, folding of proteins, translation, glycolysis, bioenergetics, signaling and trafficking. An important finding is that many glycolysis enzymes were targeted specifically by caspase-1. Processing of these glycolysis enzymes by caspase-1 was confirmed by cleaving in vitro transcribed and translated substrates with recombinant caspase-1. We have focused our further analysis on certain glycolysis enzymes. We have characterized the caspase-1 cleavage site in GAPDH. Point mutation of the Aspartic acid at position 189 to Alanine (D189A) in GAPDH blocked its cleavage by caspase-1. In vivo, in a mice model of septic shock, characterized by hyperactivation of caspase-1, we observed depletion of the full-length forms of these glycolysis enzymes in the diaphragm muscle. Further studies in caspase-1 deficient mice will confirm whether this depletion, in caspase-1 proficient mice, was due to caspase-1 processing of the glycolysis enzymes. This provides a direct link between caspase-1 activation and inhibition of glycolysis, which might have important implications on loss of muscle contractility in septic shock.

Cardiac hypertrophy is an adaptive response in which the heart grows to normalize output during times of increased demand. This increase in size originates from the growth of cardiomyocytes rather than cellular division. Many cellular modifications observed during hypertrophy are reminiscent of apoptosis; caspase proteases, traditionally known for their role in apoptosis, have recently been implicated in non-apoptotic settings including cardiac differentiation. Studies have reported caspase-3 inhibition limits the heart`s ability to undergo pathological hypertrophy in vivo. Data presented here indicate that inhibition of caspase-3 and caspase-8 minimizes hypertrophic growth in primary cardiomyocytes. Phenylephrine induced an increase in cell size, which was attenuated upon addition of caspase inhibitors. These data suggest these proteins may be involved in hypertrophic growth of cardiomyocytes. Furthermore, results suggest that increased caspase activity may not be directly responsible for this effect. Rather, subcellular localization of caspase proteases may contribute to the effects seen during hypertrophy.

Several genes involved in the regulation of apoptosis can influence longevity. Although observations in several different systems imply that apoptosis and aging are closely linked, the relationship between the two remains largely unknown. In this study, Drosophila melanogaster was used as a model organism to explore the relationship between aging and apoptosis regulation. Apoptosis was investigated using two apoptotic hallmarks: caspase activity and DNA fragmentation. The results showed that apoptosis occured in adult flies at all ages and the changes in apoptosis associated with aging were linked to physiological age and were tissue-specific. During normal fly aging, apoptosis increased gradually within the muscle and was activated in the fat of old flies. However, neither of the two apoptotic signs were shown in the nervous system. The analysis of the apoptotic response to starvation and oxidative stress suggested that the increased apoptosis in muscle resulted from the accumulation of oxidative damage associated with aging. Once the presence of apoptosis during Drosophila aging was confirmed, the expression of anti-apoptotic genes was manipulated in specific tissues to examine the impact of a localized alternation of apoptosis on aging. The overexpression of anti-apoptotic genes in muscle extended Drosophila mean and maximum life span up to 99% and 65%, respectively. This extension was mediated by apoptosis inhibition using the detection of caspase activity and DNA fragmentation. In addition, the long-lived animals exhibited increased resistance to oxidative stress and preserved flight ability. Overall this study establishes that muscle apoptosis limits life span and participates in sarcopenia. This finding may have applications in the development of interventions to improve the life quality of elderly human. / Thesis (Ph.D, Biology) -- Queen's University, 2008-10-27 00:24:02.204

The heart has the remarkable ability to adjust in response to varying stress stimuli and myocardium enlargement, referred to as cardiac hypertrophy, is a common form of stress adaptation. Divergent forms of hypertrophy can occur depending on the type and duration of the insult. The beneficial physiological form of hypertrophy is reversible and leads to improved cardiac function, while the pathological form is a maladaptive process that often transitions to heart failure. As a result of the prominence of cardiac disease, investigations into methods of reducing this detrimental form of cardiac remodeling are sought. Interestingly, pathological cardiac hypertrophy shares common features with the regulated form of cell death referred to as apoptosis. Here, we describe an essential role for apoptotic caspase-dependent signaling in the induction of pathological cardiac hypertrophy. Initially, we discovered that primary cardiomyocytes treated with hypertrophy agonists display transient activation of intrinsic-mediated apoptotic-signaling, including caspase 9 and caspase 3 activity. The necessity of functional caspase activation in hypertrophic signaling was shown by both in vitro and in vivo methods. We further investigated caspase cleavage targets histone deacetylase 3 (HDAC3) and gelsolin (GSN). HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Caspase-mediated GSN cleavage occurred at latter stages, coincident with the cytoskeletal alterations that occur during this process. We demonstrated the requirement of GSN and its caspase-mediated processing by use of GSN expressing adenoviruses (AdVs). Use of a non-cleavable GSN-AdV provided evidence for not only the requirement of GSN in the hypertrophic response, but also for caspase mediated GSN cleavage. This body of work implicates caspase pathways and their targets as inductive signaling cues for pathological cardiac hypertrophy. These observations suggest that inhibitors that mute or suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.

Cardiac hypertrophy is an adaptive response in which the heart grows to normalize output during times of increased demand. This increase in size originates from the growth of cardiomyocytes rather than cellular division. Many cellular modifications observed during hypertrophy are reminiscent of apoptosis; caspase proteases, traditionally known for their role in apoptosis, have recently been implicated in non-apoptotic settings including cardiac differentiation. Studies have reported caspase-3 inhibition limits the heart`s ability to undergo pathological hypertrophy in vivo. Data presented here indicate that inhibition of caspase-3 and caspase-8 minimizes hypertrophic growth in primary cardiomyocytes. Phenylephrine induced an increase in cell size, which was attenuated upon addition of caspase inhibitors. These data suggest these proteins may be involved in hypertrophic growth of cardiomyocytes. Furthermore, results suggest that increased caspase activity may not be directly responsible for this effect. Rather, subcellular localization of caspase proteases may contribute to the effects seen during hypertrophy.

The design and development of fluorescent reporters focussed on highly sensitive, specific, and selective imaging of cancer targets is described. These novel optical molecular probes were synthesised with the aim of creating bio-imaging breakthroughs that will aid the clinical analysis of cancer. A specific target of the project was to develop fluorescent reporters for Caspases; intracellular endopeptidases that play an essential role in apoptosis. Lack of activation of the ‘Caspase Cascade’ causes uncontrolled proliferation of cells and has been deemed a ‘Hallmark of Cancer’. In particular, low Caspases-3/7 activities have been associated with a range of cancers, thus molecular detection of Caspases-3/7 activities could therefore lead to advances in oncology. A 14-member FRET library, based upon Caspases-3/7 specific peptide sequences, was initially developed. The cleavage rates and KM values were evaluated for Caspases-3/7, along with the cleavage rates for Cathepsin B, to determine the peptide with the greatest affinity and specificity for Caspase-3. Also developed was a set of internally quenched activity based molecular reporters constructed by attaching fluorophores to a tribranched dendron through the Caspase specific peptide, developed from the FRET Library. The KM values of the dendron probes with Caspase-3 were also evaluated. Furthermore, the dendron reporters were attached to cell penetrating peptides to enable delivery to intracellular Caspase and allow in situ detection of activated Caspase-3 within live cells. In addition, a new labelling moiety was developed enabling dual detection of reporters through fluorescence and MRI imaging. To achieve this, a perfluoro tag (C8F17) was tethered to a Cy5 dye to enable dual detection. The dual 19F-MRI/Cy5 dye was conjugated onto to a cell penetrating peptide to enable in vivo detection of the probe by 19F-MRI and fluorescent imaging.

NF-kB, découvert en 1986 dans des lymphocytes B mature, est un groupe de facteurs de transcription impliqué dans plusieurs processus cellulaires différents tels les réponses immunitaires et inflammatoires, les processus du développement et de la croissance cellulaire ainsi que l'apoptose. L'activité de NF-kB est régulée par IkB ; lors de la phosphorylation de ce dernier, NF-kB est relocalisé vers le noyau. L'apoptose, une mort programmée identifiée en 1972, est caractérisée par une fragmentation de l'ADN et la formation de corps apoptotiques. Les mécanismes contrôlant ce phénomène sont très complexes. Un de ces mécanismes nécessite des protéases à cystéine aspartate-dépendantes que l'on nomme caspases. En utilisant le modèle cellulaire neuronal Neuro 2A, l'objectif de cette étude a été de déterminer si un stress oxydatif (H₂O₂) et un stress thermique (40 à 43°C) pouvaient induire l'apoptose par les mêmes mécanismes et si NF-kB avait un rôle dans la réponse cellulaire à ces deux stress. Une condensation de la chromatine, une caractéristique de l'apoptose, a été observée lors d'un stress oxydatif comme lors d'un stress thermique dans les Neuro 2A. Bien qu'une diminution de la polarité des membranes mitochondriale n'a été observée que pour le choc thermique, l'activation de l'apoptose serait induite au moins par la voie mitochondriale puisqu'il y a eu activation de la caspase 9 dans les deux stress. L'implication de la voie des récepteurs de mort est moins évidente; une activité de la caspase 8 est détectée dans les deux stress mais n'est que peu ou pas augmentée par rapport à l'absence de stress. La présence de H₂O₂ (0 à 25 µM) ne stimule pas l'activité de la caspase 3, mais active significativement l'activité de la caspase 6. Au contraire, durant le choc thermique (42 à 43°C), l'induction de la caspase 3 est très significative, soit environ près de dix fois celle des cellules témoins (37°C), et l'activité de la caspase 6 ne varie pas. La présence de l'inhibiteur Bay 11-7082 (inhibiteur de la phosphorylation de IkB, 4 µM) a peu d'influence sur l'activité des caspases 3 et 6 lors du stress oxydatif mais multiplie l'activité de la caspase 3 d'environ vingt fois lors d'un stress thermique. En conclusion, nos résultats ont permis de montrer l'implication de la voie mitochondriale (caspase 9) dans la mort apoptotique induite par H₂O₂ et le choc thermique et cela, dans la lignée neuronale Neuro 2A. La caspase effectrice 6 serait activée dans le stress oxydatif mais non thermique. Les résultats pour la caspase 6 dans le choc thermique demeurent à être vérifiés en analysant l'activité de cette caspase plus tardivement. En effet, la caspase 6 est située en aval de la caspase 3 dans les mécanismes apoptotiques. L'utilisation de Bay 11-7082 dans le choc thermique montre que NF-kB aurait un effet inhibiteur sur l'activation de la caspase 3. Le Bay 11-7082 en combinaison avec le choc thermique pourrait trouver application dans le traitement de tumeurs neuronales.

L'acroléine est un aldéhyde α,β-insaturé, très électrophile, auquel l'être humain est exposé dans plusieurs situations. Ce composé toxique est un polluant environnemental qui est principalement présent dans les produits de la combustion de la matière organique tels que la fumée de cigarette, les gaz d'échappement et les vapeurs des huiles de cuisson. De façon endogène, l'acroléine est produite lors d'un stress oxydatif et d'une inflammation ce qui suggère qu'elle est impliquée dans plusieurs pathologies telles que les maladies neurodégénératives, respiratoires et l'insuffisance rénale. L'acroléine réagit rapidement avec plusieurs sites nucléophiles de la cellule et forme ainsi des adduits au niveau de l'ADN et des protéines et favorise la peroxydation des lipides. La réaction de l'acroléine avec le glutathion ainsi que les cystéines de plusieurs enzymes résulte en une perturbation de la balance d'oxydoréduction de la cellule ce qui perturbe plusieurs voies de signalisation cellulaire dont la voie apoptotique. L'implication de l'acroléine dans la mort cellulaire a été démontrée dans les études précédentes sans pour autant détailler les mécanismes moléculaires dans les cellules pulmonaires A549. Le but de ce travail est d'étudier l'implication du réticulum endoplasmique (RE) dans la transduction du signal apoptotique induit par l'acroléine (25-200 µM) dans les cellules d'adénocarcinome humain A549. Notre étude montre que l'acroléine (25-200 µM) induit la réponse apoptotique des cellules A549. Plus précisément, l'acroléine cause un stress du RE et induit ainsi la réponse aux protéines à conformations anormales (UPR) qui se traduit par la phosphorylation des kinases PERK et IRE1α ainsi que par le clivage de l'ATF6α. Cependant l'activation de la réponse UPR n'a pas induit l'expression de Bip. De plus, la réponse UPR, induite par un traitement de 2h avec l'acroléine, n'est pas en mesure d'exercer son effet de survie ce qui résulte en l'initiation de la voie pro-apoptogène. Nos résultats indiquent que l'apoptose induite par l'acroléine implique l'induction du facteur de transcription CHOP, l'augmentation du taux de calcium libre intracellulaire qui est responsable de l'activation des calpaïnes qui à leur tour sont impliquées dans l'activation des caspases 12 et 4. Nous avons également montré que le chélateur de calcium BAPTA-AM ainsi que l'inhibiteur des calpaïnes protègent les cellules A549 contre l'apoptose induite par l'acroléine. Toutefois, les inhibiteurs des caspases 12 et 4 n'ont pas montré un effet significatif. Dans le cas de la caspase 4, son inhibition cause une inhibition partielle (non significative) de l'apoptose. Ceci peut suggérer l'implication d'autres voies dans la transduction du signal apoptotique induit par l'acroléine dans les cellules pulmonaires A549. Nos observations suggèrent que le processus apoptotique induit par l'acroléine peut être médié par la voie du RE mais n'implique pas nécessairement l'activation des caspases 12 et 4. Cette étude révèle de nouveaux aspects sur les mécanismes de toxicité de l'acroléine. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Acroléine, Apoptose, Caspase, Réticulum endoplasmique.