Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC

Caspases play a central role in apoptosis, but their activity is under the control of caspase-inhibiting proteins. A characteristic of caspase-inhibiting proteins is direct caspase binding. It is yet unknown how the localization of caspase-inhibiting proteins is regulated and whether there are upstream signals controlling their function. Here we report that the function of ARC is regulated by protein kinase CK2. ARC at threonine 149 is phosphorylated by CK2. This phosphorylation targets ARC to mitochondria. ARC is able to bind to caspase-8 only when it is localized to mitochondria but not to the cytoplasm. Our results reveal a molecular mechanism by which a caspase-inhibiting protein requires phosphorylation in order to prevent apoptosis.     

Aha,another regulator for hsp90 chaperones

A large number of key regulators controlling homeostasis and cell fate are chaperoned by the Hsp90 folding machine. In this issue of Molecular Cell, report the discovery of a new stress-regulated cochaperone, Aha1, which accelerates the dynamics of this machine.     

Grb2 is a key mediator of helicobacter pylori CagA protein activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.     

Dendritic polyglycerol: a new versatile biocompatible-material

Polyglycerol represents the first hyperbranched polymer that can be prepared in a controlled synthesis. It is characterized by the combination of a stable, biocompatible polyether scaffold, high-end group functionality and a compact, well-defined dendrimer-like architecture. These characteristics can be used to generate new materials properties and for biomedical applications to molecularly amplify or multiply effects or to create extremely high local concentrations of drugs, molecular labels, or probe moieties. Therefore, dendritic polyglycerols are expected to lead to new strategies for 'molecular medicine'. In this brief summary, the current state of the art in polyglycerol research is given, focusing on applications in life sciences.     

Model-based analysis of elementary Ca<Superscript>2+</Superscript> release events in skinned mammalian skeletal muscle fibres

Using high temporally and spatially resolved confocal laser scanning microscopy, we have recently demonstrated the existence of elementary Ca(2+) release events (ECRE) in chemically and mechanically skinned fibres from adult mammalian skeletal muscle. Here, we present a first approach to the analysis of mammalian ECRE with a spatio-temporal mathematical model of Ca(2+) ion distribution in skinned muscle fibre preparations. The differential equations for the main processes, including sarcoplasmic reticulum Ca(2+) handling, are solved in a 2-D cylindrical geometry by the method of explicit finite differences. By calculating the various spatio-temporal ion concentrations as well as the theoretical fluorescence signals for confocal microscopy, corrected for the point spread function, the model output can be directly correlated with the experimental data. Thus, the basic features of mammalian ECRE were successfully reproduced with our model. In particular, under our model assumptions a considerable depletion of luminal free calcium is predicted even for short spark-like ECRE. For a full understanding of the molecular and sub-cellular events responsible for EC coupling it is vitally important to combine the experimental and modelling approaches to elucidate the contribution of mammalian ECRE to the global Ca(2+) release and its alteration under various physiological and also pathophysiological conditions.     

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.     

A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step of eukaryotic isoprenoid biosynthesis, and is the main target of cholesterol-lowering drugs. The classical form of the enzyme is a transmembrane-protein anchored to the endoplasmic reticulum. However, during the last years several lines of evidence pointed to the existence of a second isoform of HMGCR localized in peroxisomes, where mevalonate is converted further to farnesyl diphosphate. This finding is relevant for our understanding of the complex regulation and compartmentalization of the cholesterogenic pathway. Here we review experimental evidence suggesting that the peroxisomal activity might be due to a second HMGCR gene in mammals. We then present a comprehensive analysis of completely sequenced eukaryotic genomes, as well as the human and mouse genome drafts. Our results provide evidence for a large number of independent duplications of HMGCR in all eukaryotic kingdoms, but not for a second gene in mammals. We conclude that the peroxisomal HMGCR activity in mammals is due to alternative targeting of the ER enzyme to peroxisomes by an as yet uncharacterized mechanism.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.     

Radiation-induced chromosome aberrations: insights gained from biophysical modeling

Enzymatic misrepair of ionizing-radiation-induced DNA damage can produce large-scale rearrangements of the genome, such as translocations and dicentrics. These and other chromosome exchange aberrations can cause major phenotypic alterations, including cell death, mutation and neoplasia. Exchange formation requires that two (or more) genomic loci come together spatially. Consequently, the surprisingly rich aberration spectra uncovered by recently developed techniques, when combined with biophysically based computer modeling, help characterize large-scale chromatin architecture in the interphase nucleus. Most results are consistent with a picture whereby chromosomes are mainly confined to territories, chromatin motion is limited, and interchromosomal interactions involve mainly territory surfaces. Aberration spectra and modeling also help characterize DNA repair/misrepair mechanisms. Quantitative results for mammalian cells are best described by a breakage-and-reunion model, suggesting that the dominant recombinational mechanism during the G(0)/G(1) phase of the cell cycle is non-homologous end-joining of radiogenic DNA double strand breaks. In turn, better mechanistic and quantitative understanding of aberration formation gives new insights into health-related applications.