Op18/stathmin caps a kinked protofilament-like tubulin tetramer

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended alpha-helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C-terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned alpha/beta-tubulin heterodimers. The appearance of the complex was that of a kinked protofilament-like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N-terminal part of the molecule is to 'cap' tubulin subunits to ensure the specificity of the complex and (ii) the complete C-terminal alpha-helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.     

Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells

This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for ¹⁵N- and ¹³C/¹⁵N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a ¹H–¹⁵N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.     

Mutagenesis at a specific position in a DNA sequence

Predefined changes in a known DNA sequence were introduced by a general method. Oligodeoxyribonucleotides complementary to positions 582 to 593 of the viral DNA strand of the bacteriophage phiX174 am3 mutant (pGTATCCTACAAA), and to the wild type sequence in this region (pGTATCCTACAAA), were synthesized and used as specific mutagens. Each of these oligonucleotides was incorporated into a complete circular complementary strand when used as primer on a genetically heterologous viral strand template, by the combined action of subtilisin-treated Escherichia coli DNA polymerase I and T4 DNA ligase. Incomplete duplexes were removed or were inactivated by nuclease S1 and the products were used to transfect spheroplasts of E. coli. Both oligonucleotides induced specific mutations at high efficiency when used with heterologous template (15% mutants among progeny phage). The am phages isolated by this procedure are phenotypically gene E mutants, and contain A at position 587 of the viral strand. They thus appear identical with am3 and provide evidence that the change G leads to A at position 587 is sufficient to produce a defective E function. Since the template for the induction of am mutants carried another genetic marker (sB1), the strains carrying the induced mutations have the new genotype am3 sB1. It should be possible to introduce the am3 mutation into any known mutant strain of phi174 using this same oligonucleotide. Both possible transition mutations were induced in these experiments. In principle, the method could also induce transversions, insertions, and deletions. The method should be applicable to other circular DNAs of similar size, for example recombinant DNA plasmids.     

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking

Background

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.

Methodology/Principal Findings

We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.

Conclusions/Significance

Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.     

Blood Flow Changes Coincide with Cellular Rearrangements during Blood Vessel Pruning in Zebrafish Embryos

After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y-shaped structure. Subsequently, endothelial cells in parts of the previously perfused branch rearrange from a multicellular into a unicellular tube, followed by blood vessel detachment. This process is accompanied by endothelial cell death. Finally, we show that differences in blood flow between neighboring vessels are important for the completion of the pruning process. Our data suggest that flow induced changes in tubular architecture ensure proper blood vessel pruning.     

Perspectives of biomolecular NMR in drug discovery: the blessing and curse of versatility

The versatility of NMR and its broad applicability to several stages in the drug discovery process is well known and generally considered one of the major strengths of NMR (Pellecchia et al., Nature Rev Drug Discov 1:211–219, 2002; Stockman and Dalvit, Prog Nucl Magn Reson Spectrosc 41:187–231, 2002; Lepre et al., Comb Chem High throughput screen 5:583–590, 2002; Wyss et al., Curr Opin Drug Discov Devel 5:630–647, 2002; Jahnke and Widmer, Cell Mol Life Sci 61:580–599, 2004; Huth et al., Methods Enzymol 394:549–571, 2005b; Klages et al., Mol Biosyst 2:318–332, 2006; Takeuchi and Wagner, Curr Opin Struct Biol 16:109–117, 2006; Zartler and Shapiro, Curr Pharm Des 12:3963–3972, 2006). Indeed, NMR is the only biophysical technique which can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. NMR should therefore be ideally suited and widely requested as a tool for drug discovery research, and numerous examples of drug discovery projects which have substantially benefited from NMR contributions or were even driven by NMR have been described in the literature. However, not all pharmaceutical companies have rigorously implemented NMR as integral tool of their research processes. Some companies invest with limited resources, and others do not use biomolecular NMR at all. This discrepancy in assessing the value of a technology is striking, and calls for clarification—under which circumstances can NMR provide added value to the drug discovery process? What kind of contributions can NMR make, and how is it implemented and integrated for maximum impact? This perspectives article suggests key areas of impact for NMR, and a model of integrating NMR with other technologies to realize synergies and maximize their value for drug discovery.     

Infections with human rhinovirus induce the formation of distinct functional membrane domains.

The plasma membrane contains distinct domains that are characterized by a high concentration of sphingolipids and cholesterol. These membrane microdomains also referred to as rafts, seem to be intimately involved in transmembranous signaling and often initiate interactions of pathogens and the host cell membranes. Here, we investigated the further reorganization of membrane rafts in cultured epithelial cells and ex vivo isolated nasal cells after infection with rhinoviruses. We demonstrate the formation of ceramide-enriched membrane platforms and large glycosphingolipid-enriched membrane domains and the co-localization of fluorochrome-labeled rhinoviruses with these membrane domains during attachment and uptake of human rhinovirus. Destruction of glycosphingolipid-enriched membrane domains blocked infection of human cells with rhinovirus. Furthermore, our studies indicate that the activation of the acid sphingomyelinase (ASM) is intrigued in the formation of ceramide- or GM1- enriched membrane platforms. Inhibition of the ASM reduces the number of ceramide-enriched platforms and glycosphingolipid-enriched membrane domains. These data reveal a critical role of the ASM for the formation of membrane platforms and infection of human cells with rhinoviruses.     

Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib

Current structural understanding of kinases is largely based on x-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. ABL kinase is an important drug target in the treatment of chronic myelogenous leukemia. Here, we present the first characterization of ABL kinase in complex with three clinical inhibitors (imatinib, nilotinib, and dasatinib) by modern solution NMR techniques. Structural and dynamical results were derived from complete backbone resonance assignments, experimental residual dipolar couplings, and (15)N relaxation data. Residual dipolar coupling data on the imatinib and nilotinib complexes show that the activation loop adopts the inactive conformation, whereas the dasatinib complex preserves the active conformation, which does not support contrary predictions based upon molecular modeling. Nanosecond as well as microsecond dynamics can be detected for certain residues in the activation loop in the inactive and active conformation complexes.     

The Cohesin loading factor NIPBL recruits histone deacetylases to mediate local chromatin modifications

Cornelia de Lange Syndrome (CdLS) is a rare congenital malformation disorder. About half of the patients with CdLS carry mutations in the NIPBL gene encoding the NIPBL protein, a subunit of the Cohesin loading complex. Recent studies show association of Cohesin with chromatin-remodeling complexes, either by establishing cohesion or by recruiting Cohesin to specific chromosome locations. In yeast two-hybrid assays, we identified an interaction of NIPBL with the histone deacetylases -1 and -3. These interactions were confirmed in mammalian cells by coimmunoprecipitation and a critical region for interaction was defined to a stretch of 163 amino acids of a highly conserved region of NIPBL, which is mutated in patients with CdLS. Utilizing reporter gene assays, we could show that NIPBL fused to the GAL4-DNA-binding domain (GAL4-DBD) represses promoter activity via the recruitment of histone deacetylases. Interestingly, this effect is dramatically reduced by both NIPBL missense mutations identified in CdLS and by chemical inhibition of the histone deacetylases. Our data are the first to indicate a molecular and functional connection of NIPBL with chromatin-remodeling processes via the direct interaction with histone deacetylases.     

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California

Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.