Sonderforschungsbreich 35

Transmembrane Transporters
in Health and Disease

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Colloquia in Transmembrane Transport:


Uncovering clinically relevant mutations in membrane transporters by genetic analysis linked to the determination of erythrocyte membrane protein expression

The human red blood cell (RBC) membrane contains more than 300 integral membrane proteins, many with high relevance to disease conditions or pharmacological interventions. We have developed a flow cytometry method for the quantitative determination of the RBC membrane expression levels of selected membrane transporters, and examined their molecular genetic background, as well as their potential value in medical diagnostics as biomarkers.
One example presented in this talk is the ABCG2 xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the gout patients, correlating with a genetic polymorphism, causing a Q141K variant protein. In addition, based on RBC expression levels, we found stop and frameshift mutations, as well as a relatively frequent, novel ABCG2 mutation (ABCG2-M71V). This variant, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules, with a potential in allele-specific gout treatment.
Another example is the plasma membrane calcium pump (PMCA4b) of the RBC membrane, which showed heterogeneous RBC expression levels in healthy volunteers. By genetic analysis we found a correlation of the low PMCA4b expression levels with a minor haplotype in the erythroid cell specific regulatory region of this protein. This minor haplotype, now characterized in detail, seems to be correlated with sensitivity to malaria infection.
These results suggest that personalized medicine should consider the genetic analysis linked to RBC membrane protein expression, and these studies may provide a new tool to uncover clinically important variants of membrane proteins.


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Decoding Signaling Circuits: Surveillance of Kinase Conformations & GPCR Interactions

Scaffolding proteins act as organizing centres for molecular switches such as kinases and GTPases to relay membrane receptor-sensed input signals. First, I will discuss the implementation of a phospho-proteomics approach to identify unanticipated physical connections between kinase and G protein-coupled receptor (GPCR) signalling. We identified that the GPCR Gpr161 is both, a protein kinase A (PKA) substrate and a high-affinity scaffolding protein for PKA holoenzymes. This function of the orphan GPCR is relevant for recruiting cAMP-sensing PKA complexes to the primary cilium where the Gpr161:PKA signalosome antagonizes Hedgehog signalling. Second, I will present a strategy to systematically track kinase conformations and interactions which are controlled by GTPases, cancer mutations, and clinical kinase inhibitors. The full-length RAF kinase is the basis for an extendable cell-based reporter platform which enables non-invasive recordings of (i) open (active) and closed (inactive) RAF conformations and (ii) transformations of binary GTPase:kinase interactions following lead molecule exposure. The in vivo recording of auto-inhibited enzyme conformations and interactions opens new opportunities for engineering biosensors for ‘undruggable’, novel, or neglected drug targets.


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Structural basis for the transport of antibacterial peptides across bacterial membranes

Bacteria under nutrient starvation produce antibacterial peptides that target closely related species for survival. These peptides can be either lytic or non-lytic. Lytic peptides disrupt the membrane that results in nutrient loss whereas non-lytic peptides hijack membrane bound receptors for internalisation. The lasso peptide MccJ25 is an interesting class of non-lytic peptide that can be used as novel antimicrobial. MccJ25 is also toxic to the producing bacteria, which utilise the ABC transporter McjD to transport it out of the cell and provide them with resistance. Once it has been secreted it hijacks the outer membrane siderophore receptor FhuA and inner membrane transporter SbmA for internalisation inside the target cell. Inside the cell it targets the RNA polymerase. My lab is interested to elucidate the structure and function of these transporters. We have determined the crystal structure of the ABC transporter McjD in distinct conformations. The structures are in novel conformations, apo inward-occluded and nucleotide-bound outward occluded that have allowed us to refine the mechanism of antibacterial peptide ABC transporters. Our functional data in proteoliposomes show that MccJ25 is specific to McjD and not to other antibacterial peptides or drugs. We are also investigating which portion of the peptide is important for recognition by NMR and non-denaturing mass spectrometry. We have also determined the structure of the sidero-phore receptor FhuA in complex with MccJ25. MccJ25 mimics the binding mode of the natural siderophore substrate. The structure has allowed us to identify a key hydrogen bond that is essential to induce a transport event. In addition, these data provide new avenues for the design of novel antibacterials to fight bacterial multi drug resistance.


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Mechanism and Inhibition of ABC Transporters that Mediate Multidrug Resistances in Cancer

Cancers recurring after chemotherapeutic treatment often present resistances to most of the currently available anti-cancer drugs. Similar resistances also occur in the chronic treatment of diseases like HIV-AIDS, epilepsy, and other important disorders. Such in-sensitivities to therapies pose immense problems to the treatment of the affected pa-tients. One cause of such drug resistances is the overproduction of members of the ABC transporter membrane proteins, P-glycoprotein (P-gp, ABCB1), breast cancer re-sistance protein (BCRP, ABCG2), and/or the Multidrug Resistance-associated Protein 1 (MRP1, ABCC1). These ABC-transporters pump therapeutics out of cells, thereby lower-ing effective intracellular concentrations of the drugs to sub-therapeutic levels. In this study, we have investigated the mechanism of transport by P-glycoprotein using mo-lecular simulation methods and have identified a number of inhibitors of P-glycoprotein and BCRP using massively parallel computational methods. Initial validation of the in silico identified inhibitors in biochemical, biophysical, and cell culture assays as well as novel optimization of hits will be reported. This work is supported by the US NIH NIGMS [R15GM09477102], SMU University Re-search Council, the SMU Center for Drug Discovery, Design and Delivery, the Commu-nities Foundation of Texas, and private gifts from Suzy Ruff of Dallas, Texas.


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The role of Solute Carriers (SLCs) in drug transport and their genetic interactions: a systematic approach

Solute carriers (SLCs) are the largest family of transmembrane transporters, controlling the movement of a variety of diverse molecules across cellular membranes. Despite the importance of this process, most chemical compounds still lack an associated protein transporter that explains their entry and distribution in cells and tissues. In order to prioritize new SLC-drug relationships, we have investigated a publicly available large pharmacogenomic dataset involving 1001 molecularly annotated cancer cell lines and 265 compounds using linear regression models. In parallel, we have also screened a large panel of cytotoxic approved drugs in HAP1 cells in order to address the long-standing question of whether all drugs require transporters to enter cells and at the same time identify novel and therapeutically relevant SLC-drug interactions. Furthermore, we have undertaken a large genetic screen by systematically looking for synthetic lethality interactions between all non-essential SLCs expressed in the human HAP1 cell line (136 genes, approximately 250 individual cell lines) and any other SLC. Mapping of the genetic interactions between SLCs holds the promise to be a powerful method to derive hypotheses on the function and potential ligands of orphan transporters and understand the degree of redundancy present within the family.


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Use of PET to study inhibition of efflux transporters at the blood-brain barrier to improve brain delivery of drugs

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis patients. It belongs to the family of ATP Binding Cassette (ABC) transporters, and shares their conserved core architecture comprising two transmembrane domains, which in CFTR form an ion translocation pathway, and two conserved cytosolic nucleotide binding domains (NBDs) which catalyze a cycle of ATP binding and hydrolysis. CFTR is unique among ion channels because opening and closing (gating) of its transmembrane pore is coupled to this irreversible cycle and hence operates far from equilibrium. A growing number of atomic crystal structures of ABC transporter homologs, together with observations of CFTR conformational transitions at a single-molecule level in patch-clamp recordings, have begun to outline the dynamics of molecular motions responsible for this unique coupling between an enzymatic cycle and gating of an ion-channel pore.


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Membrane-Spanning DNA Nanopores: Bottom-up Structures for Single-Molecule Research, Nanotechnology, and Synthetic Biology

Replicating the biological function of membrane proteins with synthetic components is scientifically and technologically exciting. I describe the design and generation of membrane nanopores assem-bled from DNA. The DNA nanopores consist of a bundle of six hexagonally arranged duplexes which are interconnected by cross-overs. The negatively charged nanobarrels carry lipid anchors to facili-tate the pores’ insertion into the hydrophobic bilayers1-3. The pores facilitate the control transport of molecular cargo across the membrane; both voltage-gated4 and ligand-gated ion-selective channels could be built3. Pores can also be engineered to kill cells5. Membrane-spanning DNA pores will open up the design of entirely new molecular devices for applications within single-molecule research, biosensing, catalysis, drug delivery, and nanofluidics6.

(1) Burns, J.; Stulz, E.; Howorka, S. Nano Lett. 2013, 13 (6), 2351-2356. (2) Burns, J. R.; Göpfrich, K.; Wood, J. W.; Thacker, V. V.; Stulz, E.; Keyser, U. F.; Howorka, S. Angew. Chem. Int. Ed. 2013, 52 (46), 12069–12072. (3) Burns, J. R.; Seifert, A.; Fertig, N.; Howorka, S. Nat. Nanotechnol. 2016, 11 (2), 152-156. (4) Seifert, A.; Göpfrich, K.; Burns, J. R.; Fertig, N.; Keyser, U. F.; Howorka, S. ACS Nano 2015, 9 (2), 1117-1126. (5) Burns, J. R.; Al-Juffali, N.; Janes, S. M.; Howorka, S. Angew. Chem. Int. Ed. 2014, 53 (46), 12466-12470. (6) Howorka, S. Science 2016, 352 (6288), 890-891.


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Gating mechanism of CFTR, an ion channel member of the ABC transporter family

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis patients. It belongs to the family of ATP Binding Cassette (ABC) transporters, and shares their conserved core architecture comprising two transmembrane domains, which in CFTR form an ion translocation pathway, and two conserved cytosolic nucleotide binding domains (NBDs) which catalyze a cycle of ATP binding and hydrolysis. CFTR is unique among ion channels because opening and closing (gating) of its transmembrane pore is coupled to this irreversible cycle and hence operates far from equilibrium. A growing number of atomic crystal structures of ABC transporter homologs, together with observations of CFTR conformational transitions at a single-molecule level in patch-clamp recordings, have begun to outline the dynamics of molecular motions responsible for this unique coupling between an enzymatic cycle and gating of an ion-channel pore.


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ABC transporters and complex disease

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins with the capability of transporting a variety of diverse compounds through membranes, with their membrane traverse relying on ATP hydrolysis. The human genome contains 48 ABC genes with almost one-third harboring mutations that produce inborn errors of metabolism; such errors have provided insight into their physiological roles and contribution to human disease. It has been postulated that functional alteration of many ABC transporters modifies disease state or progression. My presentation will focus on the role of ABC transporters to neonatal respiratory distress syndrome and myeloid leukemia.


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Experimental Studies employing MDMA (= ecstasy) and LSD

MDMA and LSD are widely used recreational drugs but have recently also been used as adjuncts to psychotherapy. However, only limited clinical pharmacological data is available on the acute effects of these psychoactive substances. Findings from placebo-controlled experimental studies using MDMA and LSD in healthy subjects will be presented including pharmacokinetics, acute subjective effects, effects on emotion processing, and on the mechanism of action in humans. In particular, the role of serotonin in the mechanism of action of these drugs in humans will be discussed.


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Ligand-binding mechanisms and substrate delivery in ATP-binding cassette transporters at the single-molecule level

Type-I ABC importers play a pivotal role in the physiology of microorganisms, incl. amino acid supply for nutrition, pathogenicity and cell volume regulation. We focus on the homodimeric GlnPQ with two different substrate-binding domains (SBDs) per membrane domain, which offers a unique model system to decipher how conformational changes drive membrane transport (1). We demonstrate that the two SBDs have evolved to capture different amino acids by a previously undocumented type of induced-fit mechanism. We show that, following amino acid capture, the closed conformation of each SBD interacts autonomously with the TMDs, and SBD1 and SBD2 compete with each other for substrate delivery. Remarkably, SBD2 competes more strongly with SBD1 in the open-unliganded than in the closed-liganded state, which impacts the rate of transport at low amino acid concentrations. We find that the rate-determining step(s) depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release. Our findings have widespread implications for the workings of substrate-binding protein-dependent ABC importers, and recent progress from single-molecule FRET studies will be presented. 1. Gouridis G, Schuurman-Wolters GK, Plotz E, Husada F, Vietrov R, de Boer, M, Cordes T and Poolman (2015) Conformational dynamics in substrate-binding domains influence transport in the ABC importer GlnPQ. Nature Struct Molec Biol, 22:57-64


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Receptor mobility in GPCR sensing and regulation

For the understanding of the regulation mechanisms of G protein-coupled receptor (GPCR) signaling it appears important to relate receptor output to it¹s mobility. We have investigated the interrelation between GPCR mobility and GPCR signal transduction in two different systems. In Ewing sarcoma-derived cells, mobility was related to the processes that are involved in regulating the function of the chemokine receptor CXCR4. In the slime-mold Dictyostelium discoideum, mobility appeared to relate to the initial amplification steps required for gradient sensing by the cAMP receptor, cAR1. We used single-molecule epi-fluorescence microscopy to analyze in detail GPCR mobility and the subsequent behavior of the downstream G proteins. The comparison between the two systems investigated revealed, that receptor mobility closely followed the activation state of the GPCR. However, the relationship between mobility and function appears to depend on the particular system.


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Mechanisms of vitamin transport

Energy-coupling factor (ECF) transporters belong to the ATP-binding cassette (ABC)-transporter family and mediate the uptake of essential micronutrients in many prokaryotic species. Crystal structures of bacterial ECF transporters have recently been obtained and suggest that transport involves an unprecedented re-orientation (“toppling”) of a mem-brane protein in the lipid bilayer during catalysis. I will present new structural insights, discuss the uniqueness of the toppling mechanism of transport, and consider the more general implications of these findings for our understanding of membrane transporters.


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Putting the Brakes on Trafficking: Ack1 Controls Dopamine Transporter Membrane Stability and Rescues an ADHD Coding Variant

The presynaptic dopamine (DA) transporter (DAT) temporally and spatially constrains DA neurotransmission by rapidly recapturing released DA. Aberrant DAT function is implicated in attention-deficit hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target and the rewarding properties of addictive psychostimulants strictly require their binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or “endocytic brake”, controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is not defined, nor is it known whether this braking mechanism is unique to DAT or common to monoamine transporters. We have discovered that the cdc42-activated, non-receptor tyrosine kinase, Ack1, is a DAT-specific endocytic brake that stabilizes DAT, but not SERT, at the plasma membrane and is released in response to PKC activation. Intriguingly, constitutive Ack1 activation rescues the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT mutants that exhibit trafficking dysregulation.


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"Structure and mechanism of respiratory complex I, a giant molecular proton pump"

NADH-ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain of mitochondria and many bacteria. It couples electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It is a major contributor to the proton flux used for ATP generation in mitochondria, being one of the key enzymes essential for life as we know it. Mutations in complex I lead to the most common human genetic disorders. It is an L-shaped assembly formed by membrane and hydrophilic arms. Mitochondrial complex I consists of 44 subunits of about 1 MDa in total, whilst the prokaryotic enzyme is simpler and generally consists of 14 conserved “core” subunits. We use the bacterial enzyme as a “minimal” model to understand the mechanism of complex I. We have determined first atomic structures of complex I, starting with the hydrophilic domain, followed by the membrane domain and, finally, the recent structure of the entire Thermus thermophilus complex (536 kDa, 16 subunits, 9 Fe-S clusters, 64 TM helices). Structures suggest a unique mechanism of coupling between electron transfer in the hydrophilic domain and proton translocation in the membrane domain, via long-range (up to ~200 Å) conformational changes. It resembles a steam engine, with coupling elements (akin to coupling rods) linking parts of this molecular machine, including several antiporter-like subunits. I will discuss our current work, which is aimed at elucidating the molecular details of the coupling


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"Untargeted metabolomics uncovers N-lactoyl-amino acids and glutamate conjugates as substrates of ABCC5"

Our group studies ATP-binding cassette (ABC) efflux transporters using metabolomics-based approaches. ABC transporters are integral membrane proteins and use the energy provided by the hydrolysis of ATP to transport specific substrates across membranes. Despite the fact that ABC transporters have been extensively studied, their physiological function is incompletely understood. This is mainly due to a lack of knowledge about their in vivo substrate spectrum. ABCC5, for instance, is present at high levels in the blood-brain barrier, neurons and glia, but its preferred substrates are unknown. In my talk I will show how we used untargeted metabolomics to shed more light on the in vivo substrate spectrum of this ubiquitous efflux transporter. Using HEK293-ABCC5 cells we, for instance, found that N-lactoyl-amino acids are transported by ABCC5. N-lactoyl-amino acids are ubiquitous pseudo-dipeptides of lactic acid and amino acids and were not known to exist in mammals. Intriguingly, we found that N-lactoyl-amino acids are formed by CNDP2-mediated reverse proteolysis a process previously considered negligible in vivo. The function of N-lactoyl-amino acids, if any, is unknown. We also applied our metabolomics screens to tissue extracts of Abcc5-/- and wild-type mice and uncovered ABCC5 as a general carrier for compounds containing a glutamate-moiety. Glutamate conjugates are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. Interestingly, ABCC5 also transports exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid and N-methyl-D-aspartate (NMDA), and the therapeutic glutamate analog ZJ43. In conclusion, in my presentation I will demonstrate the power of metabolomics to identify new substrates of (orphan) transporters. The presented methodology is not limited to ABC transporters but can be applied to any transporter of interest.


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"In-vivo tissue characterization: a Nuclear Medicine approach"

Noncoding variants in the human genome facilitate adaptation in a changing environment, but the vast majority of noncoding variants remains unknown. With widely diverse effects, only a small fraction of variants is likely to affect clinically relevant phenotypes. This is a critical challenge to understanding the genetics of complex disorders and treatments. We have developed an integrated computational and experimental approach to characterize and prioritize causal variants, with a focus on variants having clinical relevance. Such variants affect key genes in network hubs, tend to be under evolutionary constraints, and participate in epistatic interactions. I will present examples of noncoding variants in drug transporters, enzymes, and receptors with clinical relevance.


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Glutamate transporter-associated anion channels: molecular mechanisms, physiology and pathophysiology

Excitatory amino acid transporters (EAATs) form a class of glial and neuronal glutamate transporters which remove glutamate from the synaptic cleft to terminate glutamatergic synaptic transmission and to prevent neuronal damage by excessive glutamate receptor activation. EAATs are not only secondary-active glutamate transporters, but also anion-selective channels. EAAT anion channels are perfectly anion-selective, prefer large and polyatomic over small anions, and exhibit unitary current amplitudes that are small but in the range of conventional anion channels. Whereas key processes underlying glutamate transport have been identified in recent years, molecular determinants of the EAAT anion conductance still need to be clarified. We have used a combination of atomistic Molecular Dynamics simulation, fluorescence spectroscopy and cellular electrophysiology to identify a novel anion-conducting conformation that accounts for all experimental findings on EAAT anion currents. To understand the physiological role of EAAT anion channels we are studying human diseases that are associated with mutations in genes encoding such transporters. We recently demonstrated that a point mutation identified in the SLC1A3 gene in patients with episodic ataxia type 6 results in gain-of-function EAAT1 anion channels and postulated that EAAT anion channels might regulate intracellular [Cl-] concentrations (Winter et al. (2012) Brain 135 3416-3425). To test this hypothesis, we are currently determining intracellular chloride concentrations in glial cells from WT and Slc1a3-/- mice as well as from animal models for episodic ataxia.


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"Membrane models to investigate lipid-dependent protein dynamics"

A notorious dilemma of biophysical methods is that the precision with which they can capture and analyse processes in biological systems is constantly increasing, but at the same time, the reproducibility and predictability of measurements taken in living systems worsens – due to the inherently stochastic nature of living systems on the molecular level. One obvious solution is to reconstitute biological processes of interest in simplified and better controllable cell-free environments, but whether the insights obtained there also apply under physiological conditions has to be constantly scrutinized. In the last decade, supported membranes and synthetic Giant Vesicles (GUVs) have become invaluable tools in studying the influence of the membrane environment, in particular of lipid-dependent domain formation, on the function of membrane proteins. In my talk, I will discuss the current state of the art in model membrane design for single-molecule based biophysics, and highlight the pros and cons of different systems of relevance for molecular membrane biology. I will give examples of reconstituted protein self-organization on membranes in cell free systems and its dependence on membrane composition and topology.


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"Mitochondrial uncoupling proteins: insights in transport mechanism, structure and subcellular distribution"

Abstract. ATP generation is fueled by an electrical potential across the inner mitochondrial membrane, which can be decreased by proton leak facilitated by uncoupling proteins (UCPs). Apart from UCP1 which supports non-shivering thermogenesis, the function, transport mode and even tissue distribution of the other members of uncoupling protein subfamily (UCP2-UCP5) remain highly controversial. UCPs are implicated in the pathophysiology of different diseases such as obesity, diabetes, ische-mia, cancer and neurodegenerative disorders. We use different systems, including recombinant proteins reconstituted in planar bilayers, stem cells and knockout mice (1-4), to distinguish be-tween two main hypotheses suggesting the involvement of UCPs in either the regulation of ROS production or in cell metabolism. In my talk I will focus on our recent results that address the ex-pression pattern, transport mechanism and regulation of UCP1, UCP2 and UCP4.
References:
1. Beck V, Jaburek M, Demina T, Rupprecht A, Porter RK, Jezek P, Pohl EE (2007) Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers. FASEB J 21:1137-1144.
2. Zhu R, Rupprecht A, Ebner A, Haselgrubler T, Gruber HJ, Hinterdorfer P, Pohl EE (2013) Mapping the nucleotide binding site of uncoupling protein 1 using atomic force microscopy. J Am Chem Soc 135:3640
3. Rupprecht A, Sittner D, Smorodchenko A, Hilse KE, Goyn J, Moldzio R, Seiler AE, Brauer AU, Pohl EE (2014) Uncoupling protein 2 and 4 expression pattern during stem cell differentiation provides new Insight into their putative function. PLoS ONE 9:e88474.
4. Yu WM, Liu X, Shen J, Jovanovic O, Pohl EE, Gerson SL, Finkel T, Broxmeyer HE, Qu CK (2013) Metabolic regulation by the mitochondrial phosphatase PTPMT1 is required for hematopoietic stem cell differentiation. Cell Stem Cell 12:62-74.


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"Influence of drug transporters on PET and SPECT tracer kinetics: implication for imaging data interpretation"

It is now admitted that transporters of the ATP-binding cassette (ABC) and Solute carrier (SLC) superfamilies are key determinants of the pharmacokinetics of many drugs. These transporters may also influence the kinetics of radioligands used for SPECT and PET imaging. Several complementary methods have been proposed to investigate the transport of radioligands by drug transporters. These studies have revealed the influence of carrier-mediated transport systems on the body distribution of radioligands in humans and animals and showed how it may impact PET data interpretation. Furthermore, imaging studies using radiolabeled substrates of drug transporters have considerably improved the knowledge on the overall influence of drug transporters in humans. These studies have shown the potential impact of genetic polymorphism and transporter-based drug-drug interaction on the tissue kinetics of their substrates. Imaging studies were also useful to reveal and understand the influence of the regulation of transporter expression observed in various pathophysiological states.


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"Search for Non-coding Genetic Variants Relevant in Disease and Therapy"

Noncoding variants in the human genome facilitate adaptation in a changing environment, but the vast majority of noncoding variants remains unknown. With widely diverse effects, only a small fraction of variants is likely to affect clinically relevant phenotypes. This is a critical challenge to understanding the genetics of complex disorders and treatments. We have developed an integrated computational and experimental approach to characterize and prioritize causal variants, with a focus on variants having clinical relevance. Such variants affect key genes in network hubs, tend to be under evolutionary constraints, and participate in epistatic interactions. I will present examples of noncoding variants in drug transporters, enzymes, and receptors with clinical relevance.


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"Modulation of the efflux transporters ABCB1 and ABCG2: An approach to over-come the blood brain barrier and cancer chemo-resistance"

P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are physiologi-cally relevant efflux pumps, strongly affecting absorption, distribution and excretion of various drugs. The chemotherapy of malignant brain tumors is considerably compromised by the activity of the efflux transporters P-gp and BCRP, located at the blood-brain barrier. Furthermore, over-expression of ABC transporters by tumor cells is one of the major mechanisms of anticancer drug resistance. Hence, at least in theory, modulation of ABCB1 and/or ABCG2 is an attractive strategy to improve the therapy of both, tumors in the CNS and malignancies in the periphery. Previously, in a proof-of-concept study, we demonstrated that co-administration of the ABCB1 modulator valspodar resulted in a considerable increase in paclitaxel levels in the brains of nude mice [1]. The treatment of nude mice, bearing orthotopically growing human glioblastoma xeno-grafts, led to a decrease in tumor volume by 90 % [1]. Increased peripheral toxicity due to ele-vated plasma levels of the cytostatic, characteristic of combination treatment with valspodar, was not observed with the 3rd generation ABCB1 modulators tariquidar or elacridar [2]. Nevertheless, preclinical in vivo studies suggested that the physicochemical properties of the latter are not opti-mal. Aiming at more selective modulators with improved physicochemical properties, we synthe-sized, in an initial approach, compounds chemically derived from tariquidar and performed in vitro studies to explore their potential value in cancer chemotherapy. Surprisingly, in addition to ABCB1 inhibitors, we identified potent and highly selective inhibitors of the ABCG2 transporter [3-6]. Whereas first compounds were prone to rapid enzymatic cleavage and showed very poor solubility, bioisosteric approaches led to next generations ABCG2 modulators with improved properties. This lecture will give an overview of our approach, based on tumor pharmacology and medicinal chem-istry concepts. Examples of ABCB1 and ABCG2 modulators will be presented in the context with in vitro and in vivo investigations.
1. Fellner, S., et al. J. Clin. Invest. 2002, 110, 1309-1318.
2. Hubensack, M., et al., J. Cancer Res. Clin. Oncol. 2008, 134, 597-607.
3. Kühnle, M., et al., J. Med. Chem. 2009, 52, 1190-1197.
4. Ochoa-Puentes, C., et al. Bioorg. Med. Chem. Lett. 2011, 21, 3654–3657.
5. Ochoa-Puentes, C., et al., ACS Med. Chem. Lett. 2013, 4, 393−396.
6. Bauer, S., et al., ChemMedChem 2013, 8, 1773-1778.


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"Blood-Brain Barrier P-glycoprotein: A New Target for Alzheimer's disease?"

One hallmark of Alzheimer’s disease is accumulation of neurotoxic amyloid-beta (Abeta) in the brain. The mechanistic basis for this pathology is unknown, but reports indicate that elevated Abeta brain levels are in part due to a failure in clearing Abeta from the brain. Abeta brain clearance is a two-step process: Abeta must first pass through the abluminal plasma membrane and then through the luminal membrane of the brain capil-lary endothelium. Since Abeta is a peptide, both steps must be facilitated. At the ablu-minal membrane, LRP appears to be responsible for the first step of Abeta uptake. Our data indicate that the second step in clearing Abeta from the brain is mediated by P-glycoprotein, suggesting that this transporter plays an important role in AD pathology. We show that P-glycoprotein transports Abeta from brain capillaries into the vascular space. We also demonstrate in a transgenic mouse model of Alzheimer’s disease (Tg2576; Abeta-overproducing mice) that P-glycoprotein expression and transport activity are sub-stantially reduced in brain capillaries, suggesting a link between high Abeta levels and reduced brain capillary P-glycoprotein in Alzheimer’s disease. Using this Alzheimer’s dis-ease mouse model we show that restoring P-glycoprotein expression and transport activ-ity in brain capillaries significantly reduces Abeta brain levels within one week. We also found that blocking P-glycoprotein reduction by inhibiting the proteasome reduces Abeta brain levels. Thus, restoring blood-brain barrier P-glycoprotein and preventing its protea-somal degradation both have the potential to increase Abeta clearance and reduce Abeta brain accumulation. This mechanism could potentially be used as a new therapeutic strat-egy in Alzheimer’s disease.


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"Understanding Movement and Mechanism in Molecular Machines"

Research in the Henry Wellcome Unit for biological EPR focuses on the architecture and func-tional dynamics of membrane proteins, many medically relevant with a special interest on transporter systems and their interaction with intra-cellular signalling pathways. There is in-creasing evidence that membrane proteins do not act alone, but that they are organized as nano-machineries which functions through the concerted action of its individual components with high precision and specificity observed in both time and space. We are seeking to unravel the principles underlying the architecture and dynamics of these protein nano-machineries as well as their function and regulation. Our experimental approach focuses on the use of Electron Paramagnetic Resonance (EPR) techniques in combination with molecular biological, biochemi-cal and other biophysical methods including theoretical approaches. Our expertise lies in the development and application of novel EPR techniques to address these key questions. In this presentation I will introduce the primary concepts of the technique pointing out both its advan-tages and limitations as well as focusing on very recent method developments which are clearly shifting the focus of this technique away from being considered purely a niche technique to-wards a more universal structural biological tool. I will use examples from our recent work on multidrug efflux pumps (P‐glycoprotein) and bacterial pathogens (PsaA in Streptococcus pneumonia) to demonstrate the power of this technique to provide key mechanistic insight into e.g. how to observe conformational change within P-gp at a molecular level and on the molecu-lar determinants of metal binding by PsaA and the implications for host-pathogen interactions. This research is funded by The Royal Society, the Wellcome Trust as well as being embedded within the current EU COST Action CM1306 “Understanding Movement and Mechnism in Mo-lecular Machines”.


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"Structure function of transmembrane proteins at the single molecule level"

The main research theme at the Bio-Nanotechnology Laboratory is the nanoscale spatio-temporal organization of biological systems and its impact on normal and aberrant biological functions.

 We use quantitative fluorescence microscopy and characterize with single molecule resolution a number of processes taking place within or at the interface of biological membranes including membrane deformation, rafts, interfacially activated enzymes, SNAREs, transporters, GPCRs. We aim at identifying unifying biophysical mechanisms that control on the nanometer scale the structure and function of proteins and membranes. Here I introduce briefly few examples of past work and then focus on our recent unpublished work characterizing conformational dynamics and oligomerization of the b2AR at the single molecule level.

Selected References:

1. Journal of the American Chemical Society, 2012. 134 (22): p. 9296–9302 Single enzyme studies reveal the existence of discrete functional states for monomeric enzymes and how they are “selected” by allosteric interactions. Hatzakis, Nikos; Wei, Li; Jorgensen, Sune; Kunding, Andreas; Bolinger, Pierre-Yves; Ehrlich, Nicky; Makarov, Ivan; Skjøt, Michael; Svendsen, Allan; Hedegård, Per; Stamou, Dimitrios

2. Nature Nanotechnology, 2011. 7 (1): p. 51–55 Mixing sub-attolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics. S. M. Christensen; P.Y. Bolinger; N.S. Hatzakis; M.W. Mortensen and D. Stamou

3. FEBS Letters, 2010. 584: p. 1848, Invited Review BAR Domains, Amphipathic Helices and Membrane-Anchored Proteins use the same mechanism to sense membrane curvature. K.L Madsen, V.K. Bhatia, U. Gether and D. Stamou

4. Nature Chemical Biology, 2009. 5 (11): p. 835 How Curved Membranes Recognize Amphipathic Helices and Protein Anchoring Motifs. N. S. Hatzakis*, V. K. Bhatia*, J. Larsen, K. L. Madsen, P. Y. Bolinger, A. H. Kunding, J. Castillo, U. Gether, P. Hedegård and D. Stamou.

5. EMBO Journal, 2009, 28 (21), p. 3303 Amphipathic motifs in BAR domains are essential for membrane curvature sensing. V. K. Bhatia, K. L. Madsen, P. Y. Bolinger, P. Hedegård, U. Gether, D. Stamou.

6. Proceedings of the National Academy of Sciences. 2009. 106 (30): p. 12341 Quantification of nano-scale intermembrane contact areas using fluorescence resonance energy transfer. P. M. Bendix, M. S. Pedersen and D. Stamou.


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"The mighty methylenedioxy - a key structural feature of designer drugs of abuse"

Amphetamine-related compounds are popular drugs of abuse. In particular, the ring-substituted compound 3,4-methylenedioxymethamphetamine (MDMA, or Ecstasy) displays unique pharmacological actions when compared to its parent compound methamphetamine. In this presentation, it will be shown that the presence of a methylenedioxy moiety has two important effects: 1) it governs the potency and selectivity of MDMA for interacting at monoamine transporter proteins in nervous tissue, and 2) it dramatically impacts the hepatic metabolism of MDMA after systemic administration. Recently, a host of novel synthetic cathinones (i.e., so-called “bath salts”) have appeared in the recreational drug marketplace, including compounds that possess the methylenedioxy ring substitution. The pharmacological profile for two of these newer drugs, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV), will be compared to MDMA.


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"Navigating the landscape of membrane protein interactions: applications for human health and disease"

A focus of my lab is to understand the function of the majority of “druggable” yeast and human integral membrane proteins involved in cell signaling and membrane transport at a systems level. Despite extensive research in the past decade, there is a lack of in-depth understanding of protein networks associated with these integral membrane proteins because of their unique biochemical features, enormous complexity and multiplicity. This is a major obstacle for designing improved and more targeted therapies, and importantly, understanding the biology of deregulation of these integral membrane proteins which leads to numerous human diseases. To address this challenge, we are applying an in vivo genetic system previously developed in my lab, called the membrane yeast two-hybrid (MYTH) assay, to identify and characterize protein interactors of all yeast ABC transporters and human receptor tyrosine kinases (RTKs), as well as selected cancer stem cell receptors (CSCRs) and G-protein coupled receptors (GPCRs). During my talk, I will discuss exciting new findings indicating that the newly identified interactors of these various proteins play novel roles in regulating their activity both in vivo and in vitro. I will also be reporting on the development of the Mammalian Membrane Two-Hybrid (MaMTH) technology and its applications in analyzing the dynamic protein interaction networks regulating cell signaling in humans. In summary, our systematic MYTH and MaMTH approaches offer an unbiased systems level view that facilitates the identification of novel drug targets, thereby promising significant contributions to therapeutic research.


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"Genetics of mood and anxiety disoders: the need for new approaches?"

The risk for mood and anxiety disorders is determined by a combination of genetic and environmental factors. Up to now, the vast majority of genetic studies for these disorders have been restricted to cases/control associations. From these first studies, I will present results implicating the branched-chain amino acid transporter SLC6A15 in the pathogenesis of depression. The second part of the presentation will focus on gene x environment interactions as major factors in the the genetic of mood and anxiety disorders, using the co-chaperone FKBP5 as an example for which we now have an indepth understanding of these interactions on the molecular level.


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"Defeating drug resistant cancer"

Progress in the prevention, early diagnosis, and treatment of cancer has led to a steady decline in cancer death rates. Most who succumb to cancer die because their disseminated cancer doesn’t respond to available chemotherapies. We have learned much about drug action, and efforts to elu-cidate the molecular basis for resistance have revealed a variety of mechanisms that either pre-vent a drug from reaching its target, deploy compensatory mechanisms promoting survival, or lull cancer cells into a dormant state. In case of targeted therapies, mechanisms of resistance may be limited to the specific drugs whose action is dependent on a given cancer-specific target. Combina-tion treatments may lose efficacy due to cellular mechanisms that induce resistance to multiple cytotoxic agents. Of these mechanisms, the one that is most commonly encountered in the labora-tory is the increased efflux of a broad class of hydrophobic cytotoxic drugs that is mediated by ATP-binding cassette (ABC) transporters such as P-glycoprotein (Pgp). Conceivably, by circumventing resistance mechanisms, the efficacy of first line drugs could be re-stored. Strategies to circumvent the reduced drug accumulation conferred by ABC (“ATP-binding cassette”) transporters have relied on attempts to develop drugs that bypass extrusion (often with a sacrifice in activity); or the exploration of clinical inhibitors that, although showing promise in vitro, have not translated to the clinic. Our proposed approach is to target the Achilles' heel of can-cer cells by exploiting the paradoxical hypersensitivity of otherwise multidrug resistant (MDR) cells. Collateral sensitivity (CS) of MDR cells has been described as a curious anomaly, brought about by a diverse array of compounds including local anesthetics, steroid hormones and non-ionic deter-gents. Using a systematic approach we show that the activity of most of the reported CS com-pounds is cell-line specific, and is not influenced by P-glycoprotein. In contrast, MDR-selective compounds that we identify in the National Cancer Institute's DTP drug repository retain a P-glycoprotein –dependent toxic activity across diverse cell lines. Significant progress in reducing death and suffering from cancer will only come after means have been found to reduce the impact of drug resistance on current treatment protocols. The discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR.


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"Molecular diversity of retrograde signaling at cortical synapses"

Synaptic neurotransmission relies on precisely timed neurotransmitter release from the presynapse, which is followed by effective signal transduction in the postsynapse. Feedback mechanisms involving the production and release of “retrograde messengers” from the postsynapse effectively contribute to scaling presynaptic activity. Here, I will review data to show that “retrograde signaling” is increasingly appreciated as a general rule at cortical synapses. Nevertheless, the molecular identity of retrograde messengers and their metabolism are synapse specific. I will focus on dendritic glutamate and endocannabinoid release to discern vesicular (glutamate) and non-vesicular (endocannabinoid) forms of retrograde signaling, and their distinct circuit contributions. I will highlight a non-canonical role for vesicular glutamate transporter 3 (VGLUT3) in dendritic glutamate release. Moreover, I will use vesicular neurotransmitter transporters (particularly the vesicular GABA transporter) as selective markers of nerve cells, and show that recent advances in this research arena led to the development of “targeting tools” for the selective identification (labeling) or disruption (lesioning) of molecularly uniform neuronal subnetworks, allowing a fresh look at neuronal circuit assembly and functions. Finally, I will discuss the versatility of “targeting tools” developed towards vesicular neurotransmitter transporters to dissect how certain neurons might contribute to the pathogenesis of neuropsychiatric disorders.


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"Controlling synaptic function by glial GABA transporters"

Several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. Importantly, astrocytes possess the complete set of membrane proteins to detect GABA, the major inhibitory neurotransmitter of the brain. In addition to ionotropic and metabotropic GABA receptors, astrocytes also express GABA transporters the role of which has long remained uncertain. In this lecture I will present evidence that activation of the glial glutamate transporters by their endogenous substrates triggers the reversal of the closely localized glial GABA transporter subtypes GAT-2 or GAT-3 and lead to an increase of the extracellular GABA level. GAT reversal is initiated by the elevation of the local intracellular Na+ concentration that subsequently turns back the driving force for the GABA transporters. In addition, we explored the potential physiological and pathophysiological role of the Glu/GABA exchange process in freshly isolated hippocampal slices and in the hippocampus in vivo. We demonstrated that the glutamate uptake-induced release of putrescine-derived GABA through astrocytic GATs has a direct impact on the excitability of pyramidal neurons in the hippocampus. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner providing a tuneable, in situ negative feedback. We prove that the Glu/GABA exchange mechanism is functioning in the hippocampus under physiological conditions in vivo. Importantly, blockade of the mechanism increases the duration of seizure-like events and frequency of glial Ca2+ spikes in the low-[Mg2+] in vitro model of epilepsy, demonstrating that the negative feedback control of astrocytes on neuronal excitability offers significant neuro- and glioprotection in pathophysiologically overactivated states.


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"Pharmacogenomics of transporter proteins: clinical implications"

Variation in drug disposition and response is a major concern associated with many drugs used in all medical disciplines. The clinical relevance of variability is most evident with drugs with a narrow therapeutic window. Pharmacogenomics aims to elucidate the ge-nomic determinants of drug efficacy and toxicity. Variants in genes that are relevant for ADME processes such as drug transporters have been identified as important confound¬ers affecting therapy and patient outcome. In addition non-genetic, gene regulatory as well as epigenetic factors contribute significantly to the expression and function of human membrane transporters. For instance hepatic drug metabolism and elimination requires drug uptake that is determined by transporters in the sinusoidal membrane of hepato-cytes. Organic Anion Transporting Polypeptides (OATP) and Organic Cation Transporters (OCTs) are expressed in human liver (OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3), but also in kidney (OCT2) and other tissues, mediat¬ing the uptake of endogenous compounds (e.g. bile acid by OATPs) and of several drugs (e.g. statins, anti-diabetic and anticancer drugs). Numerous clinical studies support the relevance of common/rare variants in the respective genes (SLCO1B1, SLC22A1, SLC22A2) altering either pharmacokinetics and/or drug response of substrates. The common SLCO1B1 variant c.521T>C is highlighted by its association to an increased risk for simvastatin-induced myopathy. SLC22A1 variants have been considered to contribute to the anti-hyperglyce¬mic effect of metformin. Re-cently in addition to genetic variation of transporter genes a more comprehensive ap-proach including several -omics approaches (e.g. genomics, epigenomics, transcriptomics, proteomics, metabonomics) has been considered for the identification of further putative targets for better prediction of drug response. For example, next generation sequencing and metabonomics are promising for redefining disease diagnosis and predicting therapy response. The system’s pharmacology approach will support the integration process of the systems-level understanding of drug response and therefore promotes also the drug dis-covery process for personalized medicine.


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"Dopamine D2/3 receptor availability and function, addiction, and reward-related personality traits: neurobehavioural correlates in experimental animals"

Addictive behavior is importantly mediated by mesolimbic dopamine (DA) signaling. One current hypothesis in the field is that compulsive drug use is due to drug-induced neuroadaptations within the mesolimbic DA system, which cause hypersensitivity to drug-associated cues and increased motivation for drugs (incentive sensitization theory). Here will be presented results from studies on the effect of chronic exposure to ∆9-tetrahydrocannabinol (THC), the major psychoactive constituent of cannabis, on selected aspects of DA signaling, and DA-related behaviours. These include the availability and function of the DA D2 and D3 receptors, their gene expression, and other neurochemical measures of DA and endocannabinoid signaling, as well as tests measuring basal and drug-induced locomotor activity, conditioned place preference and sensorimotor gating. Moreover, recent evidence obtained in rodents and indicating that innate differences in DA D2/3R function are associated with phenotypic divergence in novelty-seeking trait and vulnerability to addiction will be presented.


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"Optical control of cellular signals"

A major challenge in biology is to understand how cells sense and process signals from the environment. To understand cellular signaling we require technologies that generate well-controlled temporal and local stimulation of cellular signaling pathways. Our past work focused on ionotropic glutamate receptors (GluRs), which are the primary mediators of excitatory synaptic transmission in the mammalian central nervous system. In order to remote control neuronal signaling, we designed a novel GluR that is K+-selective and light-gated. This hyperpolarizing ion channel termed HyLighter is activated by millisecond light pulses and allows manipulating neuronal activity with unprecedented spatio-temporal resolution. In optogenetic experiments, HyLighter reversibly and stably inhibits action po-tential firing in neuronal cultures and behavior in zebrafish. Inspired by the surprising compatibility of a K+-selective pore with a GluR revealed in HyLighter, we discovered a new family of invertebrate glutamate receptors that combine a K+ selectivity filter with glutamate sensing and represent missing links in GluR evolution. The goal of our ongoing work is to remote control signaling cascades related to G-protein coupled receptors and receptor tyrosine kinases with light to understand how cells orchestrate these signals into physiological responses.


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"Role of nicotinic acetylcholine receptors in circuits underlying complex behaviors in brain"

Nicotinic acetylcholine receptors (nAChRs) are critical mediators of the effects of ACh in the brain. Although we know a lot about the role of nAChRs in circuits underlying reward and addiction, nAChRs are expressed throughout the brain and are important for many behavioral processes, including those related to affect and appetite. Importantly, there are differences in the nicotinic receptor subtypes, the role of receptor activation and inactivation and the brain areas responsible for distinct behavioral functions of nAChRs. For example, both activation and desensitization of high affinity alpha4/beta2* nAChRs and activation of presynaptic alpha7 nAChRs in the ventral tegmental area is necessary for nicotine reward. In contrast, inhibition of acetylcholine signaling through beta2* nAChRs appears to be antidepressant-like and activation of beta4* nAChRs in the arcuate nucleus of the hypothalamus can decrease food intake and body weight. Overall, the identification of these diverse central effects of nAChRs provides new avenues for understanding the role of these receptors in brain function, and may contribute to the development of novel medications for addiction, depression and obesity.


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"Dopamine Transporter Cell Surface Dynamics: Mechanistic insights from novel imaging tools"

Dopamine (DA) neurotransmission is critical for a variety of neurological functions, including movement, mood and cognition. Aberrant dopaminergic transmission is implicated in several neuropsychiatric disorders such as Parkinson’s disease, addiction, schizophrenia and attention-deficit hyperactivity disorder. Following evoked release, extracellular DA is rapidly cleared from the synapse by presynaptic reuptake mediated by the plasma membrane dopamine transporter (DAT). The addictive and therapeutic psychostimulants amphetamine, cocaine and methylphenidate (Ritalin) competitively inhibit DAT function and, thus, increase extracellular DA levels. DAT is not static at the plasma membrane, but is subject to rapid endocytic trafficking to and from the cell surface, which is acutely modulated by protein kinase C (PKC) activation and amphetamine exposure. Investigations examining the molecular underpinnings of DAT trafficking have yielded conflicting results, with both clathrin-dependent and –independent mechanisms implicated in DAT internalization. Work from our laboratory investigates the cellular and molecular mechanisms that govern basal and modulated DAT membrane trafficking. In our current work, we are using a novel approach that directly couples fluorophore to DAT in order to visualize DAT surface dynamics in real time. This approach is enabling us, for the first time, to directly examine DAT internalization and to determine critical features of DAT internalization mechanisms.


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"Physico-Chemical Biology & Applications in Biomedical Research"

Theoretical concepts as much as methodological developments determine and even define the framework in which scientific investigations are performed as well as the questions possibly being addressed. Physico-chemical biology combines chemical rigour with an enthusiasm for biology and offers valuable opportunities in biomedical research. For example, although it has already been acknowledged that an increasing number of proteins are lacking stably folded tertiary structures and that this intrinsic flexibility has significant impact on biological functionality, an appropriate scientific conceptualization is still missing. In the lecture I will show that the existing binary order-disorder conceptual framework can be overcome by our recently introduced meta-structure approach. By adapting concepts from theoretical physics a higher level of description is obtained in which intrinsically disordered proteins reveal many more of their intricate physico-chemical properties and their relevance for biological functions. The theoretical predictions are validated with a diverse set of experimental data obtained on several proteins implicated in cell cycle regulation, tumour growth and metastasis. Additionally, it will be demonstrated that the combination with information-rich NMR spectroscopy offers unique tools for rational lead discovery in hitherto unaddressed protein systems and significantly extend the realm of rational drug discovery programs.


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"Single molecule imaging of membrane protein interactions and dynamics in living cells"

Due to improved equipment and a constantly increasing variety of fluorescent proteins and organic dyes, single molecule techniques developed from a topic for specialists in physics to a tool for many biological applications. One method that our group is helping to establish is the counting of GFP bleaching steps from subunits of oligomeric proteins in order to determine their multimerization state and the rules of their assembly. As an extension of the subunit counting approach , we use two colors to investigate more complex interactions of several different subunit types with each other. Recently, we analyzed the stoichiometry of synaptic ion channels and receptors because they are immobile in the membrane of Xenopus oocytes and therefore can be imaged easily. However, the subunit counting approach is more difficult for proteins that display high mobility in the cell membrane, because they show stronger fluctuations in intensity and can disappear from the field of view. A particular challenge is the analysis of GPCR interactions because the stoichiometry is not fixed, but there exists a finely tuned equilibrium between monomers and dimers that is postulated to be activation dependent for several clinically highly relevant receptors. Specific and effective labeling with multiple color tags is a prerequisite for the quantitative determination of this interaction, and we show how to tackle the problem of expressing multiple proteins at very low but equal levels in mammalian expression systems.


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"Molecular modeling of the human serotonin transporter and the leucine transporter – ligand binding and transport dynamics"

The serotonergic system utilizes serotonin as a neurotransmitter and is involved in the regulation of mood, aggression, anxiety, sleep, appetite, and body temperature.1 A key player in this system is the human serotonin transporter (hSERT) which is also the principal target for antidepressants.2 In recent years the structure of an evolutionary distant bacterial homologue, the leucine trans-porter (LeuT),3 has advanced the structural understanding of hSERT and related proteins.4 How-ever, some functional findings for LeuT5 are contradicting the findings from mammalian neuro-transmitter transporters.6 This inspired us to undertake a modeling studies aiming at producing models for the binding of the endogenous substrate serotonin (5-HT)7 as well as other ligands8 to hSERT and at studying the dynamics of the proteins during transport.9 In the talk, I will present our results from docking studies of several ligands to hSERT and from MD simulations of hSERT and LeuT revealing some characteristics of the transport mechanism.


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"ABC transporters regulate stem and progenitor cell proliferation, leukocytosis, thrombocytosis and atherosclerosis"

Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high fat, high cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and HDL, respectively, and are targets of LXR transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common beta subunit of the IL-3/GM-CSF receptor. ApoE is highly expressed on hematopoietic stem and progenitor cells where it interacts with ABCA1/G1 to control stem cell proliferation, monocytosis and accumulation of monocytes in atherosclerotic lesions. ABCG4 is the closest relative of ABCG1 and also promotes cholesterol efflux to HDL. ABCG4 has a restricted distribution in the bone marrow megakaryocyte progenitor (MkP) population, where it acts to control the cell surface levels of the thrombopoeitin (TPO) receptor, megakaryocyte formation, platelet counts and atherogenesis. The findings suggest a common theme wherein ABC transporters mediating cellular cholesterol efflux act to suppress cell surface expression of growth factor receptors and thus match cellular proliferative responses to cholesterol availability. When cholesterol efflux pathways are suppressed, excessive cellular proliferation may contribute to atherosclerosis and neoplasia.


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"Optochemical Genetics"

Transmembrane receptors allow a cell to communicate with its environment in response to a variety of input signals. These can be changes in the concentration of ligands (e.g. hormones or neurotransmitters), temperature, pressure (e.g. via acoustic waves or touch), transmembrane potential, or light intensity. Many important receptors have now been characterized in atomic detail and our understanding of their functional properties has markedly increased in recent years. As a consequence, these sophisticated molecular machines can be reprogrammed to respond to unnatural input signals. Arguably, the most useful of these signals is light. I will show how ligand-gated ion channels, G-protein cou-pled receptors, as well as voltage-gated ion channels, can be manipulated with synthetic photoswitches to become light-sensitive. The resulting hybrid photoreceptors can be used to optically control neurons with very high precision. They have been used to dissect neu-ral networks and have already found applications in the restoration of vision and the con-trol of other sensations (such as pain). This combination of synthetic photoswitches and receptor proteins augments the field of Optogenetics and adds a new functional dimension to Chemical Genetics. As such, we propose to call it “Optochemical Genetics”.


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"Metal Assisted Synthetic Approaches to Small Molecules Modifying Cell Differen-tiation"

Regenerative Medicine is understood as one of the most promising therapeutic approaches to alleviate a multitude of diseases. While there is significant progress in the experimental development of this ap-proach related to gene therapeutic intervention often in combination with employing embryonic stem cells, the regulatory implications of such therapies are yet unclear and ethical aspects represent a ma-jor obstacle in the further development of such strategies. Hence, small molecules capable to affect cell differentiation towards particular tissues have received significant attention, as the prospect of employ-ing such agents in regenerative medicine seem highly attractive. Within the past years we have been developing several compounds capable to trigger differentiation of certain progenitor cells towards particular tissues otherwise difficult to regenerate. Case studies will be presented in the area of small molecule induced cardiomyogenesis and neurogenesis, as well as in differentiation acceleration towards skeletal muscle cells. Within a very recent project we have identi-fied lead compounds capable to efficiently induce cardiomyogenesis starting from embryonic or pro-genitor cells, ultimately leading to independently beating heart cells. Considering the fact, that cardiac infarctation represents one of the major death causes in the developed world, the prospect of tissue regeneration of damaged heart muscle tissue offers a highly innovative perspective of regaining heart function, as this organ in not capable to functional repair on its own. Synthetic approaches towards the individual target compounds exploited modular strategies based on metal assisted catalysis, in particular sequential coupling strategies (C-C, C-N, and C-H activation). Optimization of functional decorations of the particular heterocyclic scaffolds will be discussed in detail.


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"Dopamine Transporter Mutant Mice in Experimental Neuropharmacology"

The dopamine transporter tightly regulates the extracellular dynamics of dopamine by recapturing released neurotransmitter into the presynaptic terminals, and genetic deletion of this protein results in profound alterations in both the presynaptic homeostasis and the extracellular dynamics of dopamine. By using this model of severe dopaminergic dysregulation, significant progress has been made in defining the major target of psychotropic drugs, understanding the mechanisms of their action, unraveling novel signaling events relevant for dopaminergic transmission, and mapping neuronal pathways involved in dopamine-related behaviors. Furthermore, DAT mutant mice provided an opportunity to model in vivo conditions of extreme dopaminergic dysfunction that could be relevant for human disorders such as ADHD, schizophrenia, and Parkinson's disease and, thus, could serve as test systems for developing novel treatments for these and related disorders.


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"Pendrin, a transporter involved in hearing loss and Asthma bronchiale"

The sequencing of the human genome was a milestone in science and marked the beginning of the post-genomic era. Genetic make-up, proteomic interaction and epigenetic regulation of gene expression are important for normal cellular physiology and play key roles in both disease progression and responses to therapeutic intervention. The knowledge of the genomic make-up, however, does not by itself give insight into the function of single genes; nor does it give conclusive answers on the effect of single genes or gene products on regulatory circuits established in living cells. The challenge now and in the immediate future is to combine the genomic and epigenetic information with the functional information already known regarding gene products in their native environment. The research in our laboratory is focused on the genomics, epigenetics and functional-proteomics of pendrin. The SLC26A4 gene codes for the pendrin protein, which exchanges bicarbonate, iodide and other anions for chloride. Pendrin is mainly expressed in the thyroid gland, inner ear and kidney. The identification of the pendrin coding gene led to investigations regarding the origin of some types of hereditary deafness. 170 mutations located both in exons and introns of the pendrin gene have been linked to various human diseases. Individuals with disease-causing mutations can present distinct phenotypes, collectively named Pendred syndrome (PS, OMIM n° 274600), DFNB4 (OMIM n° 600792) or LVAS (OMIM n° 603545). PS is a rare disease causing 5-13% of childhood deafness. It is an autosomal recessive disorder characterized by sensorineural.


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"Integrins modulate the serotonin system through a three-fold interaction with the serotonin transporter"

Integrins are differentially expressed in the brain, where they modulate the assembly of synapses through the recruitment of scaffolding proteins that influence receptor localization and synaptic vesicle function. The integrin b3 gene (ITGB3) has been associated with autism, with a strong synergistic association with the serotonin transporter gene (SLC6A4, SERT). While SERTs have been extensively studied in the central nervous system, little is known on integrin b3 function in the brain. In the brain, integrin b3 binds to the integrin av subunit to form the vitronectin receptor (avb3). Here we utilize mouse models to determine the role of integrin avb3 on the modulation of the serotonergic system. Integrin b3 and SERT haploinsufficiency leads to several behaviors associated to altered serotonergic signaling. These behavioral modifications are correlated with reductions in av expression and integrin-linked signaling pathways due to the synergistic genetic interaction between these two genes. The genetic interaction also points to a functional interaction, where the activation of integrin avb3 signaling pathways are associated with increases in SERT reuptake activity. Increased adhesive properties in cells expressing avb3 also increase SERT uptake of serotonin in heterologous cells. Finally, some, but not all of the changes associated with avb3 activity may result from a direct interaction with the carboxyl-terminus of SERT. While we are still in the process of deciphering the multiple levels of interaction between integrin b3 and SERTs, we have discovered a complex mechanism by which cell adhesion may influence serotonin synaptic function and plasticity in vivo.


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"Reading The Labels On Your Medication Targets: A Walk-Through From Serotonin Transporter Cloning To Brain Disorders"

Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and neurodegenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for normal amine homoeostasis. I will present our efforts to understand (1) how cellular protein kinases and phosphatases regulate serotonin transporter expression, activity, trafficking and degradation, (2) how transporter phosphorylation regulates amine clearance, (3) how transporter phosphorylation is dysregulated in human disease and (4) our translational approach from in vitro, ex vivo to in vivo animal model to understand the role of transporter phosphorylation in aminergic neurotransmission and in human disease. We believe that understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases.


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"Ion coupling and dynamics of amino acid transporters"

Amino acids, which are important for cellular function and metabolism, are transported across cell membranes by specific transporter proteins. If the transport is uphill, against a transmem-brane concentration gradient of the amino acid, it is often coupled to the co- or counter-transport of a driving inorganic ion, which provides free energy by flowing down its own trans-membrane concentration gradient. In mammalian cells, active amino acid transport is often driven by the co-transport of Na+ ion(s). The transporters are thought to catalyze transport by simultaneously binding extracellular amino acid and Na+ ion(s) in their transmembrane do-main, followed by a structural rearrangement of the transporter-amino-acid-Na+ complex to allow dissociation of the substrates into the intracellular space. Therefore, transport is a multistep process that is composed of a number of sequential, individual reaction steps. Here, I describe ongoing efforts in our laboratory to dissect the mechanism of this multistep transport reaction, by applying rapid chemical kinetic techniques and measuring kinetic and thermody-namic parameters of amino acid interaction with the transport protein in two different model systems, the glutamate transporter EAAC1, and the neutral amino acid transporter ASCT2. Using structure-function analysis, we have also identified novel sites of interaction with the cation in these transporters.


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"Single Molecule Biology - Studying Movements and Meetings within the Plasma Membrane"

Current scientific research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity of structures with dimensions between 1 and 100nm. In the life sciences, the diversity of this Nanocosm attracts more and more researchers to the emerging field of Nanobiotechnology. In my lecture, I will show examples how to obtain insights into the organization of the cellular Nanocosm by single molecule experiments. Our primary goal is to understand the molecular organization of the plasma membrane and its impact on signaling processes. For this, we apply single molecule tracking to resolve the plasma membrane structure at sub-diffraction-limited length-scales by employing the high precision for localizing biomolecules of ~15nm. Brightness and single molecule colocalization analysis allows for studying stable or transient molecular associations in vivo. In particular, we developed a technique to detect molecular cluster formation directly in the live cell plasma membrane. With this methodology, individual aggregates can be selectively imaged, and the load of each cluster can be determined. We applied this technique to investigate the association of a fluorescent lipid analogues and lipid anchored proteins (1). Aggregates containing up to 4 probe lipids were observed to diffuse freely as stable platforms, shedding new light on the current debate concerning the existence of “lipid rafts”. Using the same technique we could further quantify the subunit stoichiometry of ion channels (2). Next, single molecule tools enable the direct observation of the random transitions a biomolecule experiences during its movements through the plasma membrane. For example, transient proximity due to interactions with other proteins can be directly monitored and quantified. I will present results on the interaction between antigen-loaded MHC and the T cell receptor directly in the interface region of a T cell with a mimicry of an antigen-presenting cell, using single molecule FRET (3). Finally, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions (4). This technology brings together our interest in immune signaling, and the capability for ultra-sensitive readout of large biochip surfaces. Cells transfected with a fluorescent fusion protein (“prey”) are grown on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of a membrane protein (“bait”); the fluorescence copatterning is used as readout for the baitprey interaction. We applied this technology for the study of the interaction between CD4 – the major coreceptor for T cell activation – and Lck, an important tyrosine kinase in early T cell signaling. In addition to the well-known zinc-clasp structure, we found strong contributions of Lck membrane anchorage to the binding of the two proteins.

1. M. Brameshuber et al., J Biol Chem 285, 41765 (2010).

2. J. Madl et al., J Biol Chem 285, 41135 (2010).

3. J. B. Huppa et al., Nature 463, 963 (2010).

4. M. Schwarzenbacher et al., Nat Methods 5, 1053 (2008)


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"Mitochondrial K+/H+ exchange in Health and Disease: From Ion and Volume Homeostasis to Seizures in the Wolf Hirschhorn Syndrome"

Mitochondrial K+/H+ exchange in Health and Disease: From Ion and Volume Homeostasis to Seizures in the Wolf Hirschhorn Syndrome The protein family Mdm38/LETM1 is essential for the mitochondrial electro-neutral K+/H+ exchange (KHE), driven by the inside-directed H+ gradient. To qualify the proteins as a component of the mitochondrial KHE and measure the K+ fluxes, we have developed a novel method involving H+ and K+ sensitive fluorescent dyes entrapped in submitochondrial particles (SMPs). Our data showed that the KHE activity was nearly abolished in mutant SMPs. LETM1 is a part of a protein complex whose components are presently under study. In the absence of LETM1, yeast mitochondria swell due to an overload of K+, the most abundant cation in the organelle, and are subjected to autophagic degradation (´mitophagy´). Up- or downregulation of the human LETM1 expression results in early cell death. Importantly, nigericin, an ionophore catalyzing KHE, fully reverted all LETM1 deletion phenotypes. Moreover, transcriptional profiling associated to compromised mitochondrial K+ homeostasis in yeast revealed a significant induction of intracellular Zn2+ transporters and changes in ergosterol biosyn-thetic pathways. Human Wolf-Hirschhorn Syndrome (WHS) is a complex disease resulting from the hemizygous termi-nal deletion on chromosome 4. A critical region as been defined (WHSCR1) which correlates with the hallmark of WHS including growth and mental retardation and facial dysmorphismus. LETM1 is 80 kb distal to this region and is always deleted in WHS patients with seizures while it is preserved in patients without seizures. These findings suggest that LETM1 is the candidate gene for seizures events associated with WHS. Using Drosophila as a model organism for understanding the molecular defects in Wolf-Hirschhorn Syndrome we demonstrated the function of LETM1 as a mitochondrial osmoregulator through its KHE activity. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues showed that depletion of LETM1 resulted in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of LETM1 resulted in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. These results will prompt us to study in more details presynaptic mitochondria in absence of LETM1.


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"Structural Basis for Protein Translocation in Molecular Machines"

Abstract - following soon.


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"Investigation of the consequences of nucleotide binding to a multidrug re-sistance ABC transporter with magnetic resonance techniques"

The multidrug ATP Binding Cassette (ABC) transporter LmrA is an integral membrane protein and a functional homologue of human P-glycoprotein involved in resistance against anti-cancer drugs in chemotherapy. LmrA forms a homodimer comprising the typical ABC transporter architecture of two nucleotide binding domains (NBDs) and two transmembrane do-mains (TMDs). It utilizes ATP binding or hydrolysis at its NBDs to drive the extrusion of toxic hydrophobic compounds via its TMDs. Combined, NMR and EPR present excellent tools to investigate all aspects associated with membrane transporter functionality, such as dynamics (e.g. side-chain or domain mobility) or structural (e.g. domain movements, substrate binding) effects. With a tri-fold magnetic resonance approach including solid-state NMR on the recon-stituted full-length protein, solution NMR on the isolated NBD as well as pulsed EPR on cysteine mutants of the full-length protein, a comprehensive picture on the dynamics of LmrA throughout its catalytic cycle could be developed. In order to investigate the ATPase activity of reconstituted LmrA, a 31P solid-state NMR based assay was developed that allows the simultaneous observation of all phosphor resonances in the ATP hydrolysis reaction. Because solid-state NMR is not limited by phase separation effects, this assay can be utilized to investigate simultaneous reactions in the aqueous as well as the lipid phase.


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"Patho-Genetics of Anxiety"

Twin studies propose a strong genetic contribution to the pathogenesis of anxiety disorders and panic disorder in particular with a heritability of about 50%. The present talk will give an overview of linkage and cytogenetic studies in panic disorder as well as association studies yielding support for several candidate genes contributing to the genetic risk for panic disorder such as the catechol-O-methyltransferase (COMT), the serotonin receptor 1A (5-HT1A), the serotonin transporter (5-HTT) and neuropeptide Y (NPY) genes. Additionally, evidence for a gene-environment interaction between 5-HTT gene variation and life events, respectively, will be reported. On a system level, fMRI activation in brain regions critical for emotional and learning processes has been proposed as a promising intermediate phenotype for genetic studies in psychiatric disorders. Thus, recent imaging genetic findings with respect to the influence of COMT, 5-HT1A and neuropeptide Y gene variants on neuronal activation correlates of emotional processing will be presented. Finally, pharmacogenetic studies in the field of anxiety suggest 5-HTT and NPY gene variants to drive inter-individual differences in treatment response to antidepressants in panic disorder as well as in anxious depression. These studies will be discussed with respect to their potential benefit in future efforts to develop an individu-ally tailored therapy for patients with anxiety disorders.


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"Molecular modelling: connecting protein structure to function using in silico methods"

Molecular modelling is gaining importance in studying protein function as its application spectrum in-creases. More importantly, improved predictive capability now allows for thorough experimental scru-tiny and validation of obtained results. An overview of methods (homology modelling and simulations) will be given and their application will be discussed. Analyses of the function of proteins by experimental techniques provide us with a large body of data that highlight e.g. the importance of amino acids, their accessibility and changes in distances. A full understanding of the function of proteins at the molecular level requires knowledge about relevant con-formations and structural changes. Although the number of protein crystal structures increases expo-nentially (presently above 50.000), only about 200 unique membrane protein structures have been sol-ved. Structures of bacterial homologs of the ABC and the NSS transporter subfamilies have become available, while the structures of the human counterparts remain elusive. Molecular modelling techniques can be used to predict the structure of these human transporters if a suitable template is available. The models created by homology modelling allow to project biochemical information onto the transporter structure. Hypotheses about the molecular details of protein function can be elaborated. Validated models can serve as a catalyst for the design of biochemical experi-ments. This is highlighted by the example of its application to the ABC transporter P-glycoprotein. Even if the structure of the human membrane transporter will become available, the crystal structure represents a single snapshot that still might not reveal the functionally relevant conformational changes. Molecular dynamics simulations are an in silico method that allows to explore the accessible phase space (accessible conformations) while fulfilling the laws of physics. Dynamics and involved conformational transitions of membrane inserted proteins can be studied at atomic resolution. Examples of applications to ABC transporters will be discussed.


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"Symmetry and the art of transport: a role for inverted-topology structural repeats in neurotransmitter transport"

Transport of solutes such as neurotransmitters across cell membranes is carried out by specific trans-port proteins that, for example, couple solute flux to the movement of ions along their concentration gradients. This has been proposed to occur via alternate exposure of a substrate binding-site to either side of the membrane, requiring at least two distinct conformations of the protein. X-ray crystallographic data for several secondary transporters from diverse families support this hypothesis and provide key insights into specific transport mechanisms. However, to date, most of these structures have been solved for only one state in the transport cycle. An intriguing feature of these structures is the presence of internal repeats with inverted topologies with respect to the membrane. We have carried out computational modelling using these structural repeats in order to model the opposite conformations of two families of neurotransmitter transporters. Experimental support for these models is provided by biochemical accessibility measurements and cross-linking studies by our collaborators, and by very recent X-ray structural data. The implications for these results will be discussed in terms of a potentially general role of inverted-topology repeats – and of symmetry – in the pseudo-symmetric alternating-access mechanism of secondary transport.


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"Role of canalicular phospholipid export pump ABCB4 for pathogenesis and treatment of cholestatic liver diseases"

Bile is primarily formed by canalicular excretion of bile acids and other biliary constituents including cholesterol, phospholipids, and bilirubin conjugates via specific ATP-binding cassette (ABC) transport-ers. The canalicular phospholipid flippase ABCB4 mediates biliary excretion of phosphatidylcholine which forms mixed micelles with bile acids and cholesterol to protect the bile duct epithelium from the detergent properties of bile acids. Mice with targeted disruption of the Abcb4 gene (Abcb4-/- mice) spontaneously develop sclerosing cholangitis with macroscopic and microscopic features of human primary sclerosing cholangitis. Bile duct injury in these mice is linked to defective biliary phospholipid secretion resulting in an increased concentration of free non-micellar bile acids which subsequently cause bile duct epithelial cell (cholan-giocyte) injury with development of sclerosing cholangitis, severe liver fibrosis and liver tumors (hepa-tocellular carcinomas). In analogy to the Abcb4-/- mouse model of sclerosing cholangitis, hereditary and acquired ABCB4 defects may play a role in the pathogenesis of a broad spectrum of hepatobiliary diseases in humans ranging from progressive familial intrahepatic cholestasis in neonates to intra-hepatic cholestasis of pregnancy, drug-induced cholestasis, intrahepatic cholelithiasis, non-anastomotic biliary strictures after liver transplantation, sclerosing cholangitis and biliary cirrhosis in adults. Therapeutic strategies for these disorders may target bile composition/toxicity via key nuclear receptors (e.g., FXR, PPAR) regulating biliary phospholipid and bile acid excretion. The therapeutically used bile acid ursodeoxycholic acid (UDCA) shows some of these properties, but has limited efficacy in the treatment of sclerosing cholangitis and other cholestatic disorders in humans. In contrast to UDCA, its side chain-shortened homologue norUDCA undergoes cholehepatic shunting between cholangiocytes and hepatocytes leading to a bicarbonate-rich hypercholeresis counteracting bile acid toxicity. Moreover, norUDCA has direct has anti-inflammatory, anti-fibrotic and anti-proliferative effects in hepatocytes, cholangiocytes and myofibroblasts. norUDCA is able to reverse the disease phenotype in the Abcb4-/- mouse model of sclerosing cholangitis. Upcoming clinical trials will have to demonstrate whether norUDCA or other side chain-modified bile acids are also clinically effec-tive in humans.


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"Neurogenetic mechanisms and distributed neural processing in schizophrenia"

Disturbed connectivity has been hypothesized to be central for the pathogenesis of schizo-phrenia since Wernicke. Neuroimaging has confirmed and specified this hypothesis by dem-onstrating aspects of decreased, but also increased connectivity, with a focus on prefrontal cortex, the extended limbic system, and the connections between the two. A dysregulation of prefrontal control, impacting on dopaminergic neurotransmission and salience attribution, may be a final common pathway to psychosis, leading to positive symptoms of stimulation of pre-frontal cortex, and abnormal connectivity between prefrontal cortex and hippocampus has been found to be a regionally specific abnormality in schizophrenia. New methods to quantify the entire connectome, derived from graph theory, have extended these regional findings by showing a generalized abnormality in the hierarchical wiring of neocortex and limbic system in schizophrenia. Since the disease is highly heritable, the study of the neural effects of genetic variants implicated in risk for schizophrenia offers a research strategy to dissect the neuroge-netic risk architecture of the disorder leading to, and potentially preceding, first episode psy-chosis. In this talk we review results from the study of risk genes related to dopaminergic stimulation (COMT, RGS4), canonical (DARPP-32) and non-canonical, beta-arrestin mediated signal transduction (AKT1), as well as results from the study of newly discovered variants ge-nome-wide significantly associated with risk for schizophrenia (ZNF804A, CACNA1C) identify-ing systems of disturbed connectivity mediating genetic risk for the disease.


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"Correcting the Trafficking Defect of Mutant CFTR"

The main cause of Cystic Fibrosis (CF), one of the most common life-shortening recessive genetic diseases, is the F508del mutation, a deletion of 3 nucleotides in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. CFTR (also ABCC7) is a cAMP-activated chloride (Cl-) channel expressed at the apical mem-brane of most surface epithelial cells, like the airways, the gastrointestinal tract, and in cells lining the ducts of several glands, like the sweat gland, the pancreas and the submucosal glands. Due to the absence/ impairment of functional CFTR in epithelia of CF patients very little or no CFTR-mediated Cl- transport is generally observed. The molecular and cellular defects associated with the most prevalent mutation (F508del), is at the level of CFTR intracellular localization, since the mutant protein fails to traffic to the plasma membrane, being mostly retained and degraded at the level of the endoplasmic reticu-lum (ER) [1,2,3]. The focus of this talk will be on the mechanisms of the ER quality control which are responsi-ble for the intrecellular retention of F508el-CFTR as well as on the pharmacological strategies to rescue this mutant by promising therapeutic small molecules which have started to emerge from the research to the clinical setting [4].
Grants: TargetScreen2 EU-FP6-2005-LH-7-037365; FCT-POCTI/MGI/47382/2002 (Portugal/ EU).
References:
1. Farinha CM & Amaral MD (2005) Most F508del-CFTR is targeted to degradation at an early folding check-point and independently of calnexin. Mol Cell Biol 25, 5242-5252.
2. Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by dif-ferent mechanisms. Proc Natl Acad Sci USA 103, 17891-17896.
3. Scott-Ward TS, Amaral MD (2009) Deletion of F508 in the first nucleotide binding domain of CFTR increases its affinity to bind the Hsc70 chaperone. FEBS J 276, 7097-7109.
4. Amaral MD, Kunzelmann K (2007) Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci 28: 334-341.


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"Regulation of D-glucose absorption in the kidney and small intestine"

According to text books luminal D-glucose is reabsorbed by sodium-coupled secondary active transporters (SGLT-family) located in the apical brush border membrane of epithe-lial cells in the kidney and small intestine. D-glucose is then released into the blood stream by facilitated diffusion via transporters of the GLUT-family located in the basal-lateral pole of the epithelial cell. However, recent results suggest that D-glucose absorp-tion is a much more dynamic process. There is intensive trafficking of the transporters involved between the plasma membrane and intracellular sites. Furthermore, there is short-term redistribution of transporters in epithelial cells in response to a changing nutritional state of the individual. The mecha-nisms of D-glucose transporter trafficking as well as their potential role as therapeutic targets for the treatment of obesity and type2 diabetes will be discussed in this presentation.


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"Ion channels as drug targets"

The present talk will be divided into 3 parts: 1) A brief introduction to the biotech company NeuroSearch specialized in performing research and development of compounds targeting membrane proteins. This introduction will include drugs that targets transporters. 2) A general introduction to ion channels and 3) A detailed description of a research project aiming at developing new principles for the treatment of cardiac arrhythmias. A more elaborated introduction to point 3) is as follows: The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three current components named IKr, IKs and IK1 with party overlapping functions. The ion channel α-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1 (KCNJ2). Loss of function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new a principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported 4 years ago and a limit number of this new compound class is now available. The present talk will illustrate how a research program aiming at developing a new principle for anti-arrhythmic treatment can be accomplished. Results will include data from in vitro to in vivo. The biophysical mode of action for hERG1 channel activators will be revealed by different electrophysiological experiments after heterologous expression in both Xenopus laevis oocytes and mammalian cells. Patch clamp experiments will further be included in characterization of hERG1 channel activators using native cardiomyocytes. Finally, the physiological and anti-arrhythmic properties of hERG1 channel activators will be demonstrated in isolated hearts and in different in vivo models.


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"Understanding Dopamine Transmission through Protein-Protein Interactions"

Our research group focuses on how neurons in the brain regulate dopamine homeostasis and the role of neurotransmitter transporters. Transmitter re-uptake through plasma membrane transporters is crucial in terminating synaptic transmission and ensuring that vesicular pools of transmitter are available for subsequent release. The dopamine transporter (DAT) is the main target site for major psychostimulants and for drugs used to treat mental illnesses. We have revealed the critical role of protein-protein interactions in the cell biology of DAT. These findings included the role of the PDZ synaptic protein PICK1 in targeting DAT to presynaptic terminals; the role of the adaptor protein Hic-5 in DAT trafficking; and the identification of a domain involved in DAT oligomerization. Recently, we have discovered a physical and functional link between DAT and synaptic vesicles through an interaction with the synaptic vesicle protein synaptogyrin-3. Our findings suggest that this novel interaction facilitates synaptic vesicle docking at the plasma membrane near DAT to provide efficient vesicle loading of extracellular dopamine during the reuptake process. These findings may represent a novel target for the actions of psychostimulants.


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"Amino acid transport across epithelia and blood brain barrier"

Small intestine and kidney proximal tubule epithelia are important cellular barriers where transcellular (re)absorption of amino acid takes place. The defect of the luminal sodium-dependent amino acid transporter B0AT1 (SLC6A19) causes Hartnup disorder. This transporter and other members of the SLC6 family require for their efficient surface expression in kidney and intestine association with collectrin (Tmem27) or ACE2, respectively. Since some mutations of B0AT1 differentially interact with collectrin and ACE2, a new mechanism leading the variable phenotype of Hartnup disorder is proposed. The closely related and previously orphan transporter XT2 (SC6A18) is shown to function as a higher affinity neutral amino acid transporter and to localize to the late kidney proximal tubule. We thus propose to rename it B0AT3. The basolateral amino acid transport machinery of these epithelia is less well understood and involves the interplay of exchangers and facilitated diffusion pathways such that it can fulfil, next to the efflux function, a role for specialized metabolic tasks and cellular housekeeping. A similarly complex task is that of the brain microvascular endothelial cells of the blood brain barrier across which an amino acid concentration gradient is also maintained.


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"Molecular aspects of coupled, uncoupled and leak currents in glutamate and dopamine transporters"

L-glutamic acid (glutamate) is a ubiquitous metabolite from the mitochondria and a neu-rotransmitter in neurons that express vesicular glutamate transporters (vGLUTs). Dopa-mine on the other hand is a neurotransmitter in those neurons that contains tyrosine hy-droxylase and vesicular monoamine transporters (vMATs). vGLUT and vMAT accumulate their substrate into synaptic vesicles, which upon fusion with the pre-synaptic membrane release the neurotransmitter into the perisynaptic space. The released neurotransmitter elicits a postsynaptic response through its pre- and post-synaptic receptors. Excitatory amino acid transporters (EAATs) and the dopamine transporter (DAT) remove the re-leased glutamate and dopamine, respectively, whereby they modulate the postsynaptic response. The plasmamembrane localized EAATs utilize the electrochemical energy from the sodium and potassium gradients created by Na+-K+-ATPase. In each stoichiometric, i.e. coupled, cycle the EAATs translocate one glutamate together with one proton and three sodium ions in exchange for one potassium ion. In the presence of glutamate EAATs also mediate a chloride flux, that does not influence the coupled cycle, and is therefore termed uncou-pled. We investigated and characterized the coupled and uncoupled fluxes as part of structure-function investigations aimed at understanding the molecular basis of the cation dependence of EAATs. In contrast to glutamate transporters, DAT utilizes only the electrochemical energy from the sodium gradient created by the Na+-K+-ATPase. DATs translocate one dopamine to-gether with two sodium and one chloride ion and results in the coupled, i.e. stoichiomet-ric, current. Human DAT is the best characterized of the DATs and mediates a leak cur-rent in absence of dopamine and a poorly characterized uncoupled chloride current in presence of dopamine. I will present some recent unpublished electrophysiological data on the functional differences, and similarities, between species homologues of DAT with special focus on the leak and uncoupled currents.


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"Targeting of the peroxisomal ABC-transporter ALDP"

Peroxisomes are ubiquitous, single membrane bound organelles, which exert a variety of metabolic reactions such as the degradation of hydrogen peroxide and of various fatty acids, or the biosynthesis of plasmalogens, docosahexaenoic acid and bile acids. Perox-isomal membrane proteins are assumed to be imported from the cytosol and the respon-sible targeting signals are usually recognized by the protein PEX19. The peroxisomal membrane protein ALDP belongs to the family of ABC-transporter proteins and mutations in this protein are associated with the neurodegenerative disease X-linked adrenoleu-kodystrophy (X-ALD). ALDP harbours a peroxisomal targeting signal and a binding site for PEX19 was identified. However, we found that mutations that destroy this interaction do not block the transport of ALDP to peroxisomes. Further investigations of the targeting process of ALDP revealed another relevant sequence and improve the understanding of the transport of peroxisomal membrane proteins.


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"Sert gene variation and risk for emotional disorders - contributions from rodent studies"

Identifying biological mechanisms through which genes lead to individual differences in emotional behavior is paramount to our understanding of how such differences confer risk for neuropsychiatric illness. The emergence of techniques such as in vivo imaging of brain function in humans and genetic engineering in rodents has provided important new in-sights into the impact of serotonin (5-HT), a key modulator of emotional behavior, on neural systems subserving anxiety and depression. A major finding has been the discov-ery of genetic variation in a crucial regulatory molecule within the 5-HT system, the 5HT transporter (5-HTT), and its influence on emotional traits. The study of the 5-HTT pro-vides a new foundation for understanding the neurobiological and genetic basis of emo-tional regulation and affective illness.


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"ABC transporters in Cancer drug resistance and ADME-tox"

Chemotherapy resistance is a recurrent problem, and the mechanism of resistance should be clarified in order to help further drug development. The active extrusion of several therapeutic agents by the MDR-ABC transporters should be considered as a potential cause of clinical resistance. The three major types of multidrug resistance (MDR) transporter proteins in humans include members of the ABCB, the ABCC, and the ABCG subfamily. These pumps recognize a wide range of drug substrates, mostly hydrophobic compounds, but also a variety of amphipathic anions and cations. MDR-ABC proteins play an important role in cancer drug resistance, but also in the absorption, distribution, metabolism and toxicity (ADME-tox) of several pharmaceutical agents. This concept is summarized as a "chemoimmunity" network, protecting our body against hydrophobic toxic agents.
This talk will discuss the role of MDR-ABC transporters in ADME-Tox and in drug resistance phenomena, focusing on the potential role of the ABCG2 transporter. This protein provides tissue protection against numerous toxic compounds and accounts for multidrug-resistant phenotype in various cancer cells.
Selective tyrosine kinase inhibitors (TKI) are key agents in modern cancer chemotherapy, and combination of TKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. However, ABCG2 displays a high affinity interaction with several clinically effective small molecule tyrosine kinase inhibitors, including Iressa (Gefitinib), an inhibitor of EGF-receptor dependent signaling, or Imatinib (Glivec) that inhibits the Bcr-Abl fusion protein, the molecular basis of chronic myeloid leukemia (CML). Recently we investigated the interaction of ABCG2 with several second generation Bcr-Abl inhibitors, already in clinical trials. Our data suggest that TKI interactions with ABC transporters may modulate drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interactions with TKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. These interactions should be carefully considered in clinical application. Also, a combined MDR-ABC transporter and TKI effect may bring an advantage in future drug development.


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"Monoamine transporters as the targets of psychostimulants"

Cocaine is a powerful psychostimulant and an addictive drug of abuse. There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies sup-port the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to ex-pectations, DAT knockout mice (DAT-KO), and SERT or NET knockout mice still display the rewarding property of cocaine. These studies indicate that none of these transporters are required for the cocaine effects and led to the re-evaluation of the DA hypothesis and the proposal of redundant reward pathways. However, the knockout mice have very sig-nificant adaptive changes during development to compensate for the complete absence of a critical protein, which might have altered how cocaine produces its effects in these mice. Therefore, we have engineered a functional but cocaine-insensitive mutant of DAT and generated a knock-in mouse line carrying this DAT mutant (DAT-CI mice). Normal doses of cocaine still block SERT and NET but have little effect on DAT in these mice which provide a unique tool to study the role of DAT in mediating cocaine effects. In DAT-CI mice, cocaine did not elevate extracellular DA in the nucleus accumbens (NAc), cocaine did not stimulate locomotor activity but suppressed it, and cocaine failed to pro-duce reward as measured by conditioned place preference and by drug self administra-tion. In contrast, amphetamine, another psychostimulant, was able to stimulate locomo-tor activity and produce reward, indicating that the reward system functioned well in these mice. In addition, re-introducing wild type DAT back into the brains of fully devel-oped DAT-CI mice restored the ability of cocaine to stimulate locomotor activity and to produce conditioned place preference. Our results indicate that cocaine blockade of DAT is required for the stimulating and rewarding effects of cocaine in mice with a functional DAT. Furthermore, we are now in the process of generating mouse lines carry a cocaine-resistant SERT or NET mutant to study the roles of these transporters in the complex co-caine effects.


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"The Binding Site for Dopamine and Cocaine on the Dopamine Transporter are Overlapping"

Cocaine is a widely abused substance with psychostimulant effects attributed to inhibition of the dopamine transporter. It has been debated whether or not the cocaine binding site overlaps with that of dopamine. If the binding sites were separate, it would be possible, at least in theory, to generate a cocaine antidote that did not influence the transport of dopamine. Here we present molecular docking models of the dopamine- and cocaine binding site showing an almost complete overlap. The models were validated experimentally using site-directed mutagenesis, Zn2+-site engineering, and chemical cross-linkers. These data argue against the possibility of creating such a cocaine antagonist. However, there might be hope for the cocaine addicts after all...


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"Regulation of the serotonin transporter by interacting proteins"

The neurotransmitter serotonin (5-hydroxytrytamine, 5-HT) exerts broad modulatory con-trol over many fundamental aspects of physiology and behavior. Alterations in whole blood 5-HT levels are associated with a variety of neuropsychiatric disorders, suggesting that conserved molecular mechanisms regulate 5-HT homeostasis in brain and blood. One such example is the serotonin transporter (SERT), responsible for the maintenance of 5-HT levels in both the brain and in platelets. Peripheral findings suggest that genetic varia-tion impacting both platelet and brain 5HT levels contributes to multiple brain disorders and may underlie comorbidity with peripheral symptoms. We recently characterized a number of regulatory partners that dictate SERT trafficking and function, including in-tegrins and focal adhesion proteins. Hic-5 is an adaptor protein responsible for the mo-lecular organization of signaling complexes downstream of integrin activation. Hic-5 bind-ing to the carboxy-terminal of SERT leads to inactivation and internalization of the trans-porter. The interaction between SERT and Hic-5 is regulated by intracellular signaling pathways and 5HT availability. Integrins are cell surface receptors that interact with ex-tracellular matrix proteins and mediate the signal transduction cascades necessary for cell motility and shape change. Activation and clustering of integrins activate SERT transport activity through previously characterized p38MAPK-dependent mechanisms. Importantly, integrin genetic variation associated with 5HT blood levels also influence SERT uptake ac-tivity. These are but two examples of SERT-interacting proteins that dynamically interact and regulate transport function that are conserved in platelets and in the brain. Our latest efforts aim to parallel changes in 5HT signaling in adult brain with altered 5HT blood ho-meostasis and map the molecular changes in SERT interactions in disease. These studies reveal a framework for understanding the comorbidity of depression and cardiovascular disease.


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