Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP-DEVD-YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.
Two aspartic proteinases, plasmepsins 1 and 11, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin 1. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of LysllOP to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin 1. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin 11. Plasmepsin I had 5-10-fold lower kc.,,/K,n values than plasmepsin I1 for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin 11. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of I? falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin I1 gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.
DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector PET 3a for expression in E. coli. The resultant product was insoluble but accumulated at -20 mg/l of cell culture. Following solubilisation with urea, the zymogen was refolded and, after purification by ion-exchange chromatography, was autoactivated to generate mature Plasmepsin II. The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of -35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors.
BackgroundWnt/β-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between ‘core pathway’ members, to the induction of nuclear transcription from genes flanked by β-catenin/TCF transcription factor binding sites. Wnt/β-catenin signaling is also regulated by a much larger set of ‘non-core regulators’. However the relationship between ‘non-core regulators’ is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues.MethodsMammalian cells engineered to have a partially-active level of Wnt/β-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP).Results141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, ‘core pathway’ components (Eg. β-catenin, GSK-3, Dsh) were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists.ConclusionsThe ‘functional connectome’ identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0475-1) contains supplementary material, which is available to authorized users.
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