Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.     

Stability of tyrosine sulfate in acidic solutions

Tyrosine O-sulfation is a posttranslational modification of secretory and membrane proteins transported through the Golgi apparatus, which is widespread among higher eukaryotes. O-Sulfated tyrosines are not immediately identified during sequencing of peptides and proteins, because the sulfate ester is acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions. Little is known about the hydrolysis at mildly acidic solutions, which are used during several protein purification and analysis procedures. We have examined the stability of tyrosine sulfate using sulfated gastrin-17, caerulein, and drosulfokinin as models for tyrosine O-sulfated peptides. The peptides were incubated in acidic solutions in a pH range of 1 to 3 at different temperatures and time spans. Only marginal hydrolysis of gastrin-17 was observed in triflouroacetic acid at room temperature or below. Comparison of the acid hydrolysis of the three peptides showed that hydrolysis rate depends mainly on the primary amino acid composition of the peptide. The activation energy (E(a)) for the hydrolysis of sulfated gastrin-17 was found to be E(a)=98.7+/-5 kJ mol(-1). This study serves as a general reference for handling tyrosine sulfated peptides in aqueous acidic solutions. We conclude that tyrosine sulfate is more stable under normal protein purification conditions than previously assumed.     

Structural models for the KCNQ1 voltage-gated potassium channel

Mutations in the human voltage-gated potassium channel KCNQ1 are associated with predisposition to deafness and various cardiac arrhythmia syndromes including congenital long QT syndrome, familial atrial fibrillation, and sudden infant death syndrome. In this work 3-D structural models were developed for both the open and closed states of human KCNQ1 to facilitate structurally based hypotheses regarding mutation-phenotype relationships. The KCNQ1 open state was modeled using Rosetta in conjunction with Molecular Operating Environment software, and is based primarily on the recently determined open state structure of rat Kv1.2 (Long, S. B., et al. (2005) Science 309, 897-903). The closed state model for KCNQ1 was developed based on the crystal structures of bacterial potassium channels and the closed state model for Kv1.2 of Yarov-Yarovoy et al. ((2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7292-7207). Using the new models for KCNQ1, we generated a database for the location and predicted residue-residue interactions for more than 85 disease-linked sites in both open and closed states. These data can be used to generate structure-based hypotheses for disease phenotypes associated with each mutation. The potential utility of these models and the database is exemplified by the surprising observation that four of the five known mutations in KCNQ1 that are associated with gain-of-function KCNQ1 defects are predicted to share a common interface in the open state structure between the S1 segment of the voltage sensor in one subunit and both the S5 segment and top of the pore helix from another subunit. This interface evidently plays an important role in channel gating.     

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.     

Strategies for High-Performance Resource-Efficient Compression of Neural Spike Recordings

Brain-machine interfaces (BMIs) based on extracellular recordings with microelectrodes provide means of observing the activities of neurons that orchestrate fundamental brain function, and are therefore powerful tools for exploring the function of the brain. Due to physical restrictions and risks for post-surgical complications, wired BMIs are not suitable for long-term studies in freely behaving animals. Wireless BMIs ideally solve these problems, but they call for low-complexity techniques for data compression that ensure maximum utilization of the wireless link and energy resources, as well as minimum heat dissipation in the surrounding tissues. In this paper, we analyze the performances of various system architectures that involve spike detection, spike alignment and spike compression. Performance is analyzed in terms of spike reconstruction and spike sorting performance after wireless transmission of the compressed spike waveforms. Compression is performed with transform coding, using five different compression bases, one of which we pay special attention to. That basis is a fixed basis derived, by singular value decomposition, from a large assembly of experimentally obtained spike waveforms, and therefore represents a generic basis specially suitable for compressing spike waveforms. Our results show that a compression factor of 99.8%, compared to transmitting the raw acquired data, can be achieved using the fixed generic compression basis without compromising performance in spike reconstruction and spike sorting. Besides illustrating the relative performances of various system architectures and compression bases, our findings show that compression of spikes with a fixed generic compression basis derived from spike data provides better performance than compression with downsampling or the Haar basis, given that no optimization procedures are implemented for compression coefficients, and the performance is similar to that obtained when the optimal SVD based basis is used.     

Oligosaccharides implicated in recognition are predicted to have relatively ordered structures

Fucosylated O- and N-linked glycans are essential recognition molecules in plants and animals. To understand how they impart their functions, through interactions with proteins, requires a detailed analysis of structure and dynamics, but this is presently lacking. In this study, the three-dimensional structure and dynamics of three fucosylated oligosaccharides are investigated using a combination of high field (800 MHz) nuclear magnetic resonance and long (50 ns) molecular dynamics simulations in explicit water. Predictions from dynamics simulations were in agreement with nuclear Overhauser cross-peak intensities. Similarly, a theory of weak alignment in neutral media resulted in reasonable predictions of residual dipolar couplings for the trisaccharide fucosyllactose. However, for larger penta- and hexasaccharides (LNF-1 and LND-1), the anisotropic component of the alignment was underestimated, attributed to shape irregularities of the fucosyl branches on an otherwise linear core, being more pronounced in a singly branched than a doubly branched oligosaccharide. Simulations, confirmed by experiment, predicted fucosylated molecules that are restricted to librations about a single average conformation. This restriction is partly due to microscopic water interactions, which act to stabilize intramolecular hydrogen bonds and maintain tight and ordered conformations; a view not forthcoming from simpler, nonaqueous simulations. Such a conclusion is crucial for understanding how these molecules interact with proteins and impart their recognition properties.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.     

Purified E255L Mutant SERCA1a and Purified PfATP6 Are Sensitive to SERCA-type Inhibitors but Insensitive to Artemisinins

The antimalarial drugs artemisinins have been described as inhibiting Ca²⁺-ATPase activity of PfATP6 (Plasmodium falciparum ATP6) after expression in Xenopus oocytes. Mutation of an amino acid residue in mammalian SERCA1 (Glu²⁵⁵) to the equivalent one predicted in PfATP6 (Leu) was reported to induce sensitivity to artemisinin in the oocyte system. However, in the present experiments, we found that artemisinin did not inhibit mammalian SERCA1a E255L either when expressed in COS cells or after purification of the mutant expressed in Saccharomyces cerevisiae. Moreover, we found that PfATP6 after expression and purification from S. cerevisiae was insensitive to artemisinin and significantly less sensitive to thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone than rabbit SERCA1 but retained higher sensitivity to cyclopiazonic acid, another type of SERCA1 inhibitor. Although mammalian SERCA and purified PfATP6 appear to have different pharmacological profiles, their insensitivity to artemisinins suggests that the mechanism of action of this class of drugs on the calcium metabolism in the intact cell is complex and cannot be ascribed to direct inhibition of PfATP6. Furthermore, the successful purification of PfATP6 affords the opportunity to develop new antimalarials by screening for inhibitors against PfATP6.