PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destinati

Involvement of ras p2I in Neurotrophin-induced Response of Sensory, but Not Sympathetic Neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations

Ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors

Kinetic and Structural Analysis of the Mg2+ -binding Site of the Guanine Nucleotide-binding Protein p21 H-ras.

The coordination and binding of the Mg2+ ion in the nucleotide−binding site of p21 have been investigated using site−directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30− or 16−fold on substituting Ser−17 or Asp−57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr−35 is only complexed to Mg2+ in the GTP conformation and both Asp−57 and Ser−17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine−5'−(beta,gamma−imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide− binding site compared to wild−type protein. The mutant proteins show reduced GTPase rates, which are not stimulated by the GTPase−activating protein GAP. p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21−induced survival of primary neuron

Ras-effector interactions, the problem of specificity

AbstractRas plays the role of a molecular switch in many cellular signalling pathways. The Raf-kinase has been identified as the direct target molecule of Ras in mammalian cells. However, in recent reports other proteins have been characterised as putative Ras effectors which have neither a functional nor a structural relationship to each other. In addition it has been shown that also other members of the Ras family like Rap and R-Ras can interact with some of these proteins. To address the problem of specificity and of biological relevance of the interactions, they have to be carefully quantified and the cellular localisation of the proteins involved taken into account

The Rap–RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues

The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb

Perception of biological motion from size-invariant body representations

The visual recognition of action is one of the socially most important and computationally demanding capacities of the human visual system. It combines visual shape recognition with complex non-rigid motion perception. Action presented as a point-light animation is a striking visual experience for anyone who sees it for the first time. Information about the shape and posture of the human body is sparse in point-light animations, but it is essential for action recognition. In the posturo-temporal filter model of biological motion perception posture information is picked up by visual neurons tuned to the form of the human body before body motion is calculated. We tested whether point-light stimuli are processed through posture recognition of the human body form by using a typical feature of form recognition, namely size invariance. We constructed a point-light stimulus that can only be perceived through a size-invariant mechanism. This stimulus changes rapidly in size from one image to the next. It thus disrupts continuity of early visuo-spatial properties but maintains continuity of the body posture representation. Despite this massive manipulation at the visuo-spatial level, size-changing point-light figures are spontaneously recognized by naive observers, and support discrimination of human body motion

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. Results Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands. Conclusion We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.Published versio

Das Verbot langfristiger Lieferverträge im deutschen Erdgasmarkt:die wettbewerbliche Bedeutung der Vertragsaufhebung vor dem Hintergrund eines zweivertraglichen Netzzugangssystems (Entry/Exit-System)

2005 erlies das Bundeskartellamt ein Verbot für langfristige Erdgaslieferverträge zwischen Importunternehmen und nachgelagerten Versorgungsunternehmen in der Hoffnung, dadurch mehr Kunden für neue Gasanbieter freizustellen und so den Wettbewerb nachhaltig zu fördern. Die Arbeit hinterfragt die wettbewerbliche Wirkung des Vertragsverbots vor dem Hintergrund des aktuellen Erdgasnetzzugangssystems. Es wird herausgestellt, dass ein inhärenter Monopoleffekt dem etablierten Versorger erlaubt, seine durchschnittlichen Transportkosten unter die Transportkosten der Konkurrenz zu senken. Auf diese Weise kann er - entgegen den Erwartungen des Kartellamts - Marktneulinge vom Marktzutritt abhalten und sein bestehendes Kundenportfolio langfristig auch ohne entsprechende Verträge an sich binden. Diese Schlussfolgerungen werden durch die Entwicklungen im Erdgasmarkt in den vergangenen ca. zwei Jahren bestätigt

How to name atoms in phosphates, polyphosphates, their derivatives and mimics, and transition state analogues for enzyme-catalysed phosphoryl transfer reactions (IUPAC Recommendations 2016)

Procedures are proposed for the naming of individual atoms, P, O, F, N, and S in phosphate esters, amidates, thiophosphates, polyphosphates, their mimics, and analogues of transition states for enzyme-catalyzed phosphoryl transfer reactions. Their purpose is to enable scientists in very different fields, e.g. biochemistry, biophysics, chemistry, computational chemistry, crystallography, and molecular biology, to share standard protocols for the labelling of individual atoms in complex molecules. This will facilitate clear and unambiguous descriptions of structural results, as well as scientific intercommunication concerning them. At the present time, perusal of the Protein Data Bank (PDB) and other sources shows that there is a limited degree of commonality in nomenclature, but a large measure of irregularity in more complex structures. The recommendations described here adhere to established practice as closely as possible, in particular to IUPAC and IUBMB recommendations and to "best practice" in the PDB, especially to its atom labelling of amino acids, and particularly to Cahn-Ingold-Prelog rules for stereochemical nomenclature. They are designed to work in complex enzyme sites for binding phosphates but also to have utility for non-enzymatic systems. Above all, the recommendations are designed to be easy to comprehend and user-friendly