Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.     

The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features

Inspection of the genome of the ligninolytic basidiomycete Phanerochaete chrysosporium revealed an unusual peroxidase_like sequence. The corresponding full length cDNA was sequenced and an archetypal secretion signal predicted. The deduced mature protein (NoP, novel peroxidase) contains 295 aa residues and is therefore considerably shorter than other Class II (fungal) peroxidases, such as lignin peroxidases and manganese peroxidases. Comparative modeling of NoP was conducted using the crystal structures of Coprinus cinereus and Arthromyces ramosus peroxidases as templates. The model was validated by molecular dynamics and showed several novel structural features. In particular, NoP has only three disulfide bridges and tryptophan replaces the distal phenylalanine within the heme pocket.     

mPER1-mediated nuclear export of mCRY1/2 is an important element in establishing circadian rhythm

Receptor-mediated nucleocytoplasmic transport of clock proteins is an important, conserved element of the core mechanism for circadian rhythmicity. A systematic analysis of the nuclear export characteristics for the different murine period (mPER) and cryptochrome (mCRY) proteins using Xenopus oocytes as an experimental system demonstrates that all three mPER proteins, but neither mCRY1 nor mCRY2, are exported if injected individually. However, nuclear injection of heterodimeric complexes that contain combinations of mPER and mCRY proteins shows that mPER1 serves as an export adaptor for mCRY1 and mCRY2. Functional analysis of dominant-negative mPER1 variants designed either to sequester mPER3 to the cytoplasm or to inhibit nuclear export of mCRY1/2 in synchronized, stably transfected fibroblasts suggests that mPER1-mediated export of mCRY1/2 defines an important new element of the core clock machinery in vertebrates.     

Distribution of Hepatotoxic Cyanobacterial Blooms in Belgium and Luxembourg

A survey of the distribution of cyanobacterial blooms in the southern part of Belgium, in Luxembourg as well as in bordering northeastern France was carried out for 4 years (1997, 1999–2001). In the 64 cyanobacterial bloom samples collected, Microcystis as well as Planktothrix were the most frequently encountered dominant bloom formers, followed by Anabaena, Woronichinia, and Aphanizomenon. The relative frequency of (co-)dominant genera was highly correlated to the geology of the catchments. Microcystins were found in 53% of the analysed blooms and their presence was mainly assigned to Microcystis dominance. The highest microcystin concentration of 2231 μg g⁻¹ seston DW was recorded in a sample dominated by Woronichinia naegeliana. Among the 6 investigated microcystin variants, MC-LR was the most frequently detected whereas MC-LY was never revealed.     

Habits of magnetosome crystals in coccoid magnetotactic bacteria

High-resolution transmission electron microscopy and electron holography were used to study the habits of exceptionally large magnetite crystals in coccoid magnetotactic bacteria. In addition to the crystal habits, the crystallographic positioning of successive crystals in the magnetosome chain appears to be under strict biological control.     

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.     

Olfactory and lens placode formation is controlled by the hedgehog-interacting protein (Xhip) in Xenopus

The integration of multiple signaling pathways is a key issue in several aspects of embryonic development. In this context, extracellular inhibitors of secreted growth factors play an important role, which is to antagonize specifically the activity of the corresponding signaling molecule. We provide evidence that the Hedgehog-interacting protein (Hip) from Xenopus, previously described as a Hedgehog-specific antagonist in the mouse, interferes with Wnt-8 and eFgf/Fgf-8 signaling pathways as well. To address the function of Hip during early embryonic development, we performed gain- and loss-of-function studies in the frog. Overexpression of Xhip or mHip1 resulted in a dramatic increase of retinal structures and larger olfactory placodes primarily at the expense of other brain tissues. Furthermore, loss of Xhip function resulted in a suppression of olfactory and lens placode formation. Therefore, the localized expression of Xhip may counteract certain overlapping signaling activities, which inhibit the induction of distinct sensory placodes.     

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.