Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.     

Lysophosphatidic acid-induced platelet shape change proceeds via Rho/Rho kinase-mediated myosin light-chain and moesin phosphorylation

Platelet activation plays an important role in arterial thrombotic disorders. Here we show that the serum-borne phospholipid lysophosphatidic acid (LPA) activates the GTPase Rho and its target Rho-kinase to induce myosin light-chain (MLC) and moesin phosphorylation, leading to platelet shape change. MLC phosphorylation, moesin phosphorylation, and shape change were blocked by preincubating platelets with C3 transferase from Clostridium botulinum and Y-27632-specific inhibitors of Rho and Rho kinase, respectively. LPA did not increase the cytosolic Ca(2+) concentration during shape change. Our results suggest that LPA via Rho-Rho kinase induces MLC and moesin phosphorylation leading to shape change in the absence of an increase in the cytosolic Ca(2+) concentration. Rho/Rho kinase inhibition could be a therapeutic strategy to prevent pathologic platelet activation during arterial thrombotic disorders.     

Towards objectivity in research evaluation using bibliometric indicators – A protocol for incorporating complexity

Publications are thought to be an integrative indicator best suited to measure the multifaceted nature of scientific performance. Therefore, indicators based on the publication record (citation analysis) are the primary tool for rapid evaluation of scientific performance. Nevertheless, it has to be questioned whether the indicators really do measure what they are intended to measure because people adjust to the indicator value system by optimizing their indicator rather than their performance. Thus, no matter how sophisticated an indicator may be, it will never be proof against manipulation. A literature review identifies the most critical problems of citation analysis: database-related problems, inflated citation records, bias in citation rates and crediting of multi-author papers. We present a step-by-step protocol to address these problems. By applying this protocol, reviewers can avoid most of the pitfalls associated with the pure numbers of indicators and achieve a fast but fair evaluation of a scientist's performance. We as ecologists should accept complexity not only in our research but also in our research evaluation and should encourage scientists of other disciplines to do so as well.     

Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii

Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was ¹³C-labeled by incubation in buffer containing [U-¹³C₆]glucose. Subsequently, these ¹³C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. ¹³C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-¹³C₆]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.     

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.     

Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase.

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.     

The far side of auxin signaling: fundamental cellular activities and their contribution to a defined growth response in plants

Recent years have provided us with spectacular insights into the biology of the plant hormone auxin, leaving the impression of a highly versatile molecule involved in virtually every aspect of plant development. A combination of genetics, biochemistry, and cell biology has established auxin signaling pathways, leading to the identification of two distinct modes of auxin perception and downstream regulatory cascades. Major targets of these signaling modules are components of the polar auxin transport machinery, mediating directional distribution of the phytohormone throughout the plant body, and decisively affecting plant development. Alterations in auxin transport, metabolism, or signaling that occur as a result of intrinsic as well as environmental stimuli, control adjustments in morphogenetic programs, giving rise to defined growth responses attributed to the activity of the phytohormone. Some of the results obtained from the analysis of auxin, however, do not fit coherently into a picture of highly specific signaling events, but rather suggest mutual interactions between auxin and fundamental cellular pathways, like the control of intracellular protein sorting or translation. Crosstalk between auxin and these basic determinants of cellular activity and how they might shape auxin effects in the control of morphogenesis are the subject of this review.     

A tobacco homolog of DCN1 is involved in pollen development and embryogenesis

Key Message

We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth.

Abstract

Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called DEFECTIVE IN CULLIN NEDDYLATION 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.     

Sphingosine 1-phosphate dynamically regulates myosin light chain phosphatase activity in human endothelial cells

Sphingosine 1-phosphate (S1P) is known to induce reorganization of the actin cytoskeleton through activation of the GTPase Rho. We have investigated the dynamic behavior of Rho/Rho kinase-regulated myosin light chain (MLC) phosphatase activity and MLC phosphorylation in Human Umbilical Vein Endothelial Cells (HUVEC) stimulated with S1P. Immediately (30-60 s) after S1P stimulation, MLC phosphatase activity dropped and MLC phosphorylation increased in a Rho/Rho kinase-dependent manner. Shortly thereafter (2 min), MLC phosphatase increased above baseline and MLC phosphorylation correspondingly decreased to near control values. At this time point, formation of actin ruffles and Rac activity assays indicated activation of Rac. Finally, between 5 and 15 min, MLC phosphatase dropped to a plateau below baseline. In parallel, MLC phosphorylation became constantly elevated above control values. These findings indicate that S1P is able to induce dynamic cycles of MLC phosphatase deactivation and activation. This novel feature of S1P could contribute to its chemotactic and angiogenic activity.