The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signalling axis

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis

Unraveling the molecular pharmacology of 4-MEC and 4-MePPP- Insights into their mode of action

Aufgrund des weltweit steigenden Mißbrauchs von Designerdrogen ist ein detailliertes Verständnis über die pharmakologischen Wirkmechanismen dieser Substanzen von essentieller Bedeutung. In meiner Arbeit beschreibe ich das neuropharmakologische Profil zweier Mephedron Analoga: 4-Methylethcathinon (4-MEC) and 4'-Methyl-α- pyrrolidinopropiophenone (4-MePPP). Um deren Wirkung auf bekannte Monoamintransporter, Transporter (SERT) sowie den Dopamine Transporter (DAT) zu untersuchen, wurden sowohl in vitro als auch in vivo Methoden verwendet. In vitro Experimente umfassten die Präperation von Synaptosomen aus der Ratte (Rattus Norvegicus), die Verwendung von Krallenfrosch-Oozyten (Xenopus Leavis) als auch heterologe Zellexpressionssysteme. Ebenso wurden die Effekte der zwei Mephedron Analoga auf neurophysiologischer Ebene mittels in vivo Mikrodialyse als auch auf verhaltensbiologischer Ebene in Ratten untersucht. Diese Arbeit beschreibt die individuellen pharmakologischen Profile von 4-MEC und 4-MePPP sowie die molekularen Mechanismen die der Interaktion dieser Substanzen mit ihren Targets zugrunde liegen. Eine besondere Problematik der heutigen Designerdrogen liegt in der immer kürzer werdenen Zeitspanne zwischen Synthetisierung und Verkauf. Als Reaktion auf die Illegalisierung von Mephedron 2010, überschwemmen jetzt 4-MEC und 4- MePPP die Drogenmärkte. Diese Arbeit beschreibt das einzigartige „Hybrid“-Profil von 4-MEC: den Dopamintransport zu blockieren und die Freisetzung von Serotonin zu verstärken; ebenso erzeugt 4-MEC einen SERT-mediierten Einwärtsstrom. In vivo Experimente zeigen daß 4-MEC zu einem erhöten Serotoningehalt in Gehirnen von Ratten führt. 4-MePPP hingegen ihibiert zwar auch den Dopamintransport, zeigt jedoch eine viel geringere Wirksamkeit gegenüber SERT. Dabei kommt es bei Applikation von 4- MePPP zu einem erhöhten Dopamingehalt in Rattengehirnen jedoch ohne den Serotoninspiegel zu beeinflussen. Ebenso wurde eine Erhöhung der motorischen sowie stereotypischen Lokomotion in Ratten gemessen. Unterschiedliche experimentelle Erkenntnisse, die aus den pharmakologischen Profile von 4-MEC und 4-MePPP abgeleitet wurden, sowie die Tatsache, dass beide strukturell von Mephedron abstammen, verlangten nach neuen Erklärungsmodellen. Mittels 12 „Computational docking“ konnten die unterschiedlichen Wirkmechanismen der beiden Mephedron Analoga aufgeklärt werden. Die Anwesenheit einer Pyrrolidin- Ringstruktur in 4-MePPP verhindert eine physische Assoziation mit SERT. Diese Ergebnisse unterstreichen die Bedeutung der Struktur-Funktionsbeziehung auf die pharmakologischen Profile von psychoaktiven Substanzen. Die Bedeutung eines umfassenden Verständnisses über die genauen Mechanismen der zahlreichen psychoaktiven Substanzen spiegelt sich vor allem in der Dringlichkeit therapeutischer Maßnahmen innerhalb der Suchtprävention. Erst wenn wir die Wirkmechanismen verstehen werden, ist es möglich, präventive Maßnahmen zu ergreifen.The growing abuse of psychoactive designer drugs worldwide requires scientific intervention to elucidate their pharmacological properties, physiological effects. It is important to bring forward crucial information about their abuse potential leading to awareness and effective ban on their use. My thesis focuses on two mephedrone analogues, 4-MEC and 4-MePPP, to investigate their neuropharmacological profile. I used a spectrum of techniques including in vitro models of rat brain synaptosomes, HEK293 cells and oocytes from Xenopus laevis to reveal their mode of action on their targets, the monoamine neurotransmitter transporters, the serotonin transporter (SERT) and the dopamine transporter (DAT). The work also looks at their effects in the rat brain by using in vivo microdialysis and monitors their effects on behavioral traits like motor locomotion and stereotypical locomotion. The study sheds light to a distinctive pharmacological profile of 4-MEC and 4-MePPP and at the molecular mechanism of interaction between the drugs and their targets by using the tools of computational biology. The two drugs under investigation are popular drugs of abuse, which have flooded the market since the ban of mephedrone in 2010. The study reveals 4-MEC to possess an unique “hybrid” profile wherein it is effectively able to block the DAT and produce substrate mediated efflux at the SERT. It also produces SERT mediated inward current. In vivo studies revealed its potential to aggravate 5-HT neurotransmitter levels in the rat brain. 4-MePPP displays a different profile by blocking DAT effectively however showing less potency at the SERT. In the rat brain the drug was able to elevate the levels of dopamine without affecting the levels of serotonin. It also increases motor and stereotypical locomotion in rats. Their diverse profile captivated our imagination to seek an answer as to why they have different modes of action, which is striking considering their common ancestral lineage from mephedrone. Computational docking solved this puzzle and disclosed the reason behind 4-MePPPs inactivity at the SERT. This was due to its structural constraint, namely its bulky pyrrolidine ring that could not accommodate itself into the binding pocket of SERT. The study reflects on the importance of structure function relationship of drugs and their profound influence on their pharmacological profile. My thesis puts forward a comprehensive understanding of the mode of action of 4-MEC and 4-MePPP on the monoamine neurotransmitter transporters. The study utilizes various tools and techniques at the molecular, cellular and at the systemic level to understand the biological potential of the drugs. This can be further extrapolated on to other drugs, which still remain a mystery regarding their mode of action as shown by at the later stage of my thesis. In conclusion, my thesis celebrates the importance of research in deciphering the mechanism of action of drugs of abuse by using an array of scientific methods. The work puts forward a model wherein drugs of abuse can be evaluated not only to reveal their biological influence and the molecular mechanism behind them but also point to a therapeutic potential towards a cure for drug addiction.submitted by Kusimika SahaZusamenfassung in deutscher SpracheMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)171435

Mechanical GPCR Activation by Traction Forces Exerted on Receptor N -Glycans

International audienceCells are sensitive to chemical stimulation which is converted into intracellular biochemical signals by the activation of specific receptors. Mechanical stimulations can also induce biochemical responses via the activation of various mechano-sensors. Although principally appreciated for their chemosensory function, G-protein-coupled receptors (GPCRs) may participate in mechano-transduction. They are indirectly activated by the paracrine release of chemical compounds secreted in response to mechanical stimuli, but they might additionally behave as mechano-sensors that are directly stimulated by mechanical forces. Although several studies are consistent with this latter hypothesis, the molecular mechanisms of a potential direct mechanical activation of GPCRs have remained elusive until recently. In particular, investigating the activation of the catecholamine β2-adrenergic receptor by a pathogen revealed that traction forces directly exerted on the N-terminus of the receptor via N-glycan chains activate specific signaling pathways. These findings open new perspectives in GPCR biology and pharmacology since most GPCRs express N-glycan chains in their N-terminus, which might similarly be involved in the interaction with cell-surface glycan-specific lectins in the context of cell-to-cell mechanical signaling

Refinement of the Central Steps of Substrate Transport by the Aspartate Transporter GltPh: Elucidating the Role of the Na2 Sodium Binding Site

<div><p>Glutamate homeostasis in the brain is maintained by glutamate transporter mediated accumulation. Impaired transport is associated with several neurological disorders, including stroke and amyotrophic lateral sclerosis. Crystal structures of the homolog transporter GltPh from <i>Pyrococcus horikoshii</i> revealed large structural changes. Substrate uptake at the atomic level and the mechanism of ion gradient conversion into directional transport remained enigmatic. We observed in repeated simulations that two local structural changes regulated transport. The first change led to formation of the transient Na2 sodium binding site, triggered by side chain rotation of T308. The second change destabilized cytoplasmic ionic interactions. We found that sodium binding to the transiently formed Na2 site energized substrate uptake through reshaping of the energy hypersurface. Uptake experiments in reconstituted proteoliposomes confirmed the proposed mechanism. We reproduced the results in the human glutamate transporter EAAT3 indicating a conserved mechanics from archaea to humans.</p></div

Sialic acid mediated mechanical activation of β2 adrenergic receptors by bacterial pili

International audienceAbstract Meningococcus utilizes β-arrestin selective activation of endothelial cell β 2 adrenergic receptor (β 2 AR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that β 2 AR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on β 2 AR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host

The Na2 site.

<p>(a) Comparison of the Na2 site in the outward-facing conformation before and (b) after rotation of T308. The HP1 re-entrance loop is highlighted in yellow, the HP2 loop in purple. Local changes allowed for an optimal coordination of the bound sodium. (c) Time evolution of the side chain dihedral angle of T308. (d) Level of hydration of T308 measured by the number of water molecules within 0.6 nm from the Cα atom.</p

Substrate uptake by human EAAT3.

<p>Uptake of [³H] L-glutamate into HEK293 cells expressing the human EAAT3 transporter that carrying the same mutations (A, S, V, and W) at the corresponding residue T364. The data are from three independent experiments; error bars show s.e.m.</p

Effects of T308 mutations on substrate uptake.

<p>(a) Uptake of [³H] L-aspartate into GltPh containing proteoliposomes. We could not observe aspartate uptake for the T308W mutation. (b) [³H] L-aspartate uptake into proteoliposomes as a function of [³H] L-aspartate concentration and (c) [³H] L-aspartate uptake into proteoliposomes as a function of sodium concentration. The data are from three independent experiments; error bars show s.e.m.</p

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here, we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions