Molecular determinants involved in activation of caspase 7

During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7's IDC for its activation and proteolytic activity.     

Seasonal Drivers of the Epidemiology of Arthropod-Borne Viruses in Australia

Arthropod-borne viruses are a major cause of emerging disease with significant public health and economic impacts. However, the factors that determine their activity and seasonality are not well understood. In Australia, a network of sentinel cattle herds is used to monitor the distribution of several such viruses and to define virus-free regions. Herein, we utilize these serological data to describe the seasonality, and its drivers, of three economically important animal arboviruses: bluetongue virus, Akabane virus and bovine ephemeral fever virus. Through epidemiological time-series analyses of sero-surveillance data of 180 sentinel herds between 2004–2012, we compared seasonal parameters across latitudes, ranging from the tropical north (−10°S) to the more temperate south (−40°S). This analysis revealed marked differences in seasonality between distinct geographic regions and climates: seasonality was most pronounced in southern regions and gradually decreased as latitude decreased toward the Equator. Further, we show that both the timing of epidemics and the average number of seroconversions have a strong geographical component, which likely reflect patterns of vector abundance through co-varying climatic factors, especially temperature and rainfall. Notably, despite their differences in biology, including insect vector species, all three viruses exhibited very similar seasonality. By revealing the factors that shape spatial and temporal distributions, our study provides a more complete understanding of arbovirus seasonality that will enable better risk predictions.     

Growth inhibition of several marine diatom species induced by the shading effect and allelopathic activity of marennine, a blue-green polyphenolic pigment of the diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia, a tychopelagic marine diatom distributed world-wide, characteristically settles in oyster-ponds. This diatom synthesizes and releases into the external medium a blue-green polyphenolic pigment called "marennine" (Mn), which is responsible for oyster greening. During the bloom of H. ostrearia in ponds, mainly occurring in autumn along the French Atlantic coast (Marennes-Oléron), a decrease in the abundance of other microalgae, including several diatom species, is observed. In this article, we investigate in vitro the influence of the intra- and extracellular forms of the pigment (IMn and EMn) on the development of diatoms usually coexisting with H. ostrearia in oyster-ponds: Skeletonema costatum, Nitzschia closterium, Haslea crucigera and Entomoneis pseudoduplex. We show that there is no significant difference between IMn and EMn in their ability to reduce, in a dose-dependent manner, the growth of these benthic diatoms. S. costatum, N. closterium and H. crucigera are significantly more sensitive to the inhibitory effect of marennine than E. pseudoduplex. In comparison, H. ostrearia tolerates much higher concentrations of its pigment and can even be stimulated at low marennine concentration. Using an experimental set-up to study independently the inhibitory effect of marennine due to light attenuation, we demonstrate that all the species tested are also inhibited in a dose-dependent manner by a shading effect, without a significant difference between IMn and EMn. Taken together, these results suggest that S. costatum, N. closterium and H. crucigera are inhibited by a cumulative effect of chemical mediation and light attenuation induced by marennine. E. pseudoduplex is inhibited solely by the shading effect. Finally, H. ostrearia is inhibited by the shading effect and stimulated by chemical mediation. The in vitro experimental data presented herein are discussed in relation to an ecophysiological function of marennine, leading us to assume that the form of the pigment released into the external medium could act as an allelochemical at the level of the pond as a whole. Allelopathy may therefore play an important role in interspecific competition and contribute to H. ostrearia bloom maintenance.     

Trypomastigotes and potential flea vectors of the endemic rodents and the introduced Rattus rattus in the rainforests of Madagascar

Transmission of parasites and diseases may be one of the mechanisms for the displacement of native and endemic rodents of Madagascar (subfamily Nesomyinae) by the introduced Rattus rattus (subfamily Murinae). We studied the occurrence of trypomastigotes in rodents at several rainforest sites on the island. Examination of blood smears showed Trypanosoma lewisi-like trypomastigotes in 11.5% of the R. rattus (n = 52). Trypomastigotes differing in morphology from those of T. lewisi were detected in 4% of the endemic rodent Nesomys rufus (n = 23). In contrast to the relatively heavy infections found in R. rattus, only a few trypomastigotes were found in the infected N. rufus. Trypomastigotes were not found in other nesomyine rodents including Eliurus minor (n = 18), E. tanala (n = 15), E. grandidieri (n = 12), E. majori (n = 9) or E. webbi (n = 9). Of potential vectors of trypomastigotes, six endemic species of fleas were identified from the rodents.     

Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.     

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

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Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.