Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo

Skeletal Muscle Differentiation Evokes Endogenous XIAP to Restrict the Apoptotic Pathway

Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms

Anti-apoptotic effect of HCV core gene of genotype 3a in Huh-7 cell line

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) Core protein regulates multiple signaling pathways and alters cellular genes expression responsible for HCV induced pathogenesis leading to hepatocellular carcinoma (HCC). Prevalence of HCV genotype 3a associated HCC is higher in Pakistan as compare to the rest of world; however the molecular mechanism behind this is still unclear. This study has been designed to evaluate the effect of HCV core 3a on apoptosis and cell proliferation which are involved in HCC</p> <p>Methodology</p> <p>We examined the in vitro effect of HCV Core protein of genotype 3a and 1a on cellular genes involved in apoptosis by Real time PCR in liver cell line (Huh-7). We analyzed the effect of HCV core of genotype 1a and 3a on cell proliferation by MTT assay and on phosphrylation of Akt by western blotting in Huh-7 cells.</p> <p>Results</p> <p>The HCV 3a Core down regulates the gene expression of Caspases (3, 8, 9 and 10), Cyto C and p53 which are involved in apoptosis. Moreover, HCV 3a Core gene showed stronger effect in regulating protein level of p-Akt as compared to HCV 1a Core accompanied by enhanced cell proliferation in Huh-7 cell line.</p> <p>Conclusion</p> <p>From the current study it has been concluded that reduced expression of cellular genes involved in apoptosis, increased p-Akt (cell survival gene) and enhanced cell proliferation in response to HCV 3a core confirms anti apoptotic effect of HCV 3a Core gene in Huh-7 that may lead to HCC.</p

Regulation of Apoptotic Pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to Survive Thermal Stress and Bleaching

Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like) cloned in this study. In corals exposed to thermal stress (32 or 34°C), caspase activity and the expression levels of the StyBcl-2-like gene increased over time (6–48 h) and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34°C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32°C, and recovered back symbionts when placed at 24°C. These results point to a two-stage response in corals that withstand thermal stress: (i) the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii) acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival

A comprehensive characterization of the caspase gene family in insects from the order Lepidoptera

<p>Abstract</p> <p>Background</p> <p>The cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species.</p> <p>Results</p> <p>In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their <it>Drosophila </it>counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species.</p> <p>Conclusions</p> <p>The basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.</p

The Apoptosome: Emerging Insights and New Potential Targets for Drug Design

Apoptosis plays a crucial role in tissue homeostasis, development and many diseases. The relevance of Apaf1, the molecular core of apoptosome, has been underlined in mitochondria-dependent apoptosis, which according to a growing body of evidence, is involved in various pathologies where the equilibrium of life-and-death is dysregulated, such as heart attack, stroke, liver failure, cancer and autoimmune diseases. Consequently, great interest has emerged in devising therapeutic strategies for regulating the key molecules involved in the life-and-death decision. Here we review recent progress in apoptosis-based pharmacological therapies and, in particular, we point out a possible role of the apoptosome as an emerging and promising pharmacological target

Lactational coumestrol exposure increases ovarian apoptosis in adult rats

This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81–84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135–140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health