A novel mutation in the N-terminal domain of Drosophila BubR1 affects the spindle assembly checkpoint function of BubR1.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that ensures accurate segregation of chromosomes into two daughter cells. BubR1, a key component of the SAC, also plays a role in the mitotic timing since depletion of BubR1 leads to accelerated mitosis. We previously found that mutation of the KEN1-box domain of Drosophila BubR1 (bubR1-KEN1 mutant) affects the binding of BubR1 to Cdc20, the activating co-factor of the APC/C, and does not accelerate the mitotic timing despite resulting in a defective SAC, which was unlike what was reported in mammalian cells. Here, we show that a mutation in a novel Drosophila short sequence (bubR1-KAN mutant) leads to an accelerated mitotic timing as well as SAC failure. Moreover, our data indicate that the level of Fzy, the Drosophila homolog of Cdc20, recruited to kinetochores is diminished in bubR1-KEN1 mutant cells and further diminished in bubR1-KAN mutant cells. Altogether, our data show that this newly identified Drosophila BubR1 KAN motif is required for a functional SAC and suggest that it may play an important role on Cdc20/Fzy kinetochore recruitment.

The monothiol single-domain glutaredoxin is conserved in the highly reduced mitochondria of Giardia intestinalis.

The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.

Oxidative stress and protease dysfunction in the yeast model of Friedreich ataxia.

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.

Labelling of four distinct trophozoite falcipains of Plasmodium falciparum by a cystatin-derived probe.

Trophozoite cysteine protease (TCP) activity, isolated from Plasmodium falciparum soluble 100,000 g extracts, displayed native falcipain-1 kinetic parameters towards peptidyl substrates. The labelling of either isolated TCP or soluble 100,000 g extracts by a cystatin-derived probe (biotinyl-Leu-Val-Gly-CHN2) revealed a single band of ca. 30 kDa by SDS-PAGE, which was resolved into four spots displaying isoelectric points (pI) from 4.7 to 5.3 after two-dimensional separation. The molecular mass and pI correspond to those of falcipain-3, falcipain-2, falcipain-2' and falcipain-1, respectively. The two central spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as falcipain-2 and falcipain-2'. This activity-based probe represents a potential tool for profiling active falcipains in parasites.

Isolation and characterization of Psalmopeotoxin I and II: two novel antimalarial peptides from the venom of the tarantula Psalmopoeus cambridgei.

Two novel peptides that inhibit the intra-erythrocyte stage of Plasmodium falciparum in vitro were identified in the venom of the Trinidad chevron tarantula, Psalmopoeus cambridgei. Psalmopeotoxin I (PcFK1) is a 33-residue peptide and Psalmopeotoxin II (PcFK2) has 28-amino acid residues; both have three disulfide bridges and belong to the Inhibitor Cystine Knot superfamily. The cDNAs encoding both peptides were cloned, and nucleotide sequence analysis showed that the peptides are synthesized with typical signal peptides and pro-sequences that are cleaved at a basic doublet before secretion of the mature peptides. The IC(5O) of PcFK1 for inhibiting P. falciparum growth was 1.59+/-1.15 microM and that of PcFK2 was 1.15+/-0.95 microM. PcFK1 was adsorbed strongly to uninfected erythrocytes, but PcFK2 was not. Neither peptide has significant hemolytic activity at 10 microM. Electrophysiological recordings in isolated frog and mouse neuromuscular preparations revealed that the peptides (at up to 9.3 microM) do not affect neuromuscular transmission or quantal transmitter release. PcFK1 and PcFK2 do not affect the growth or viability of human epithelial cells, nor do they have any antifungal or antibacterial activity at 20 microM. Thus, PcFK1 and PcFK2 seem to interact specifically with infected erythrocytes. They could therefore be promising tools for antimalaria research and be the basis for the rational development of antimalarial drugs.