Current Projects

Project Title: Intracellular trafficking of Y-Receptors Status: Active
Investigators Annette Beck-Sickinger Leipzig
Vsevolod Gurevich Vanderbilt
Description    
The neuropeptide Y2, Y4, and Y1 receptors do not internalize upon agonist stimulation; however this is not the case for the Y5 receptor. This project focuses on identification of interacting protein sequences that are involved in the internal trafficking of these receptors and molecular interactions with arrestins involved in NPY receptor trafficking. So far in this collaborative project, we investigated the internalization and recycling of the human neuropeptide Y2 receptor. The first joint publication of the VU/LU collaboration appeared in the Journal of Biological Chemistry in 2010. This project achieved success so quickly because significant preliminary data were generated due to student exchanges in 2010. This work continues to identify elements of other members of the NPY receptor family that determine their binding to arrestins.
 
Project Title: Prolactin releasing peptide receptor (PrRPR) modeling Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Jens Meiler (Vanderbilt)
Description    
This project focuses on the PrRPR as a model receptor system, with interest in both thefunction and structure of the receptor The aim of the project is to identify the bindingpocket by using peptide analogues and receptor mutants. This will be supported by a3D model of the PrRP receptor based on known X-ray structures of GPCRs. Anoutcome of the project may be the identification of interesting approaches with respectto the onset of activity.
 
Project Title: High-throughput screen to identify modulators of the neuropeptide Y4 receptor Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Jens Meiler (Vanderbilt)
David Weaver (Vanderbilt)
Description    
As a continuation of the project on the structural structural modeling of the neuropeptide Y4 receptor, the goal of this project is to develop a small molecule library screening system for positive modulators of the receptor. The first such small molecules have been identified and are currently under evaluation at VU and LU.
 
Project Title: Structural modeling of neuropeptide Y4 receptor Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Jens Meiler (Vanderbilt)
Description    
The main goal of this project is to better understand the neuropeptide Y4 receptor. The chemical modulation of the Y4 receptor provides an interesting topic for modeling and experimental studies. The aim of the project is to identify the binding pocket by using peptide analogues and receptor mutants. This will be supported by a 3D model of the Y4 receptor based on known X-ray structures of GPCRs.
 
Project Title: Characterization of the molecular structure of the Y2 receptor Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Daniel Huster (Leipzig)
Jens Meiler (Vanderbilt)
Description    
The aim of this project is to obtain detailed structural information on the interactions of the neuropeptide Y2 receptor, including ligand binding, bioactive conformation, and protein-protein interactions, particularly with arrestins. The project aims to investigate the accessibility of defined Cys mutants to EPR and fluorescence labels and to obtain specific NMR, EPR, and fluorescence measurements to study the structural changes of the receptor upon activation. So far, several potentially useful residues have been identified, and proof-of-concept experiments are underway. The Beck-Sickinger lab has provided ligands, partly labeled with EPR-probes, or 13C aminoacids for NMR studies. Furthermore, Cys residues were mutated to Ala/Val/Ser residues according to the model made by Dr. Jens Meiler. Cellular assays will be contributed to the project by the Beck-Sickinger group to test the biological activity of mutated and recombinantly produced receptors. Receptor-arrestin interactions will be studied by Dr. Gurevich’s lab.
 
Project Title: Carbaborane-based COX inhibitors Status: Active
Investigators Evamarie Hey-Hawkins (Leipzig)
Lawrence Marnett (Vanderbilt)
Terry Lybrand (Vanderbilt)
Description    
This project is directed toward the investigation of non-steroidal anti-inflammatory drugs (NSAIDs) based on substitution of the carbaborane skeleton for aromatic groups in the scaffold of currently used NSAIDs. Carbaborane-substituted NSAID analogs are synthesized in the Hey-Hawkins laboratory and evaluated for inhibitory activity against cyclooxygenase-2 (COX-2), the molecular target for NSAID action. The molecular determinants of carbaborane-NSAID binding to COX-2 are evaluated using a combination of site-directed mutagenesis and X-ray crystallography in the lMarnett lab. The collaborators have planned several investigations such as activity studies and cocrystallization of the inhibitor/enzyme complex. Additionally, computational calculations and molecular modeling of COX-1 and COX-2 interactions will be explored with Dr. Terry Lybrand.
 
Project Title: Studies on the structure-function relationship of the TSH receptor Status: Active
Investigators Ralf Paschke (Leipzig)
Jens Meiler (Vanderbilt)
Description    
To-date, X-ray diffraction experiments have revealed the structure of the leucine-rich repeat (LRR) domain of the follicle-stimulating hormone receptor (FSHR) and the thyroid-stimulating hormone receptor (TSHR) and the 7TM domain of related receptors. Yet, no structural model of the hinge region is available. Despite its suggested high flexibility and its low conservation among the GPCRs, the hinge region plays an essential role in hormone binding, receptor activation, and signal transduction. The existence of constitutively activating mutations (CAMs) located within the hinge region suggests that a conformational change of the hinge region results in receptor activation. Therefore the elucidation of the hinge region’s structure is a mandatory and important step in understanding receptor function. During the collaboration, a protocol will be developed in which the hinge region will be modeled de novo using the Rosetta Software Suite as extension of the LRR domain in the presence of the bound ligand, TSH. In addition, parameters will be adjusted to allow structural conformations induced by physicochemical features. The identified interactions will be verified or falsified using site-directed mutagenesis of the receptor and the hormone at the laboratory facilities in Leipzig. The resulting experimental data and additional data from Dr. Müller and Dr. Jäschke will be used for further refinement of the models. An iterative process between model refinement and experimental validation will result in a structural model for the hinge region together with the LRR domain and the bound hormone. This model represents the basis for a complete receptor model, including the transmembrane domain, which is an important tool in the development of agonists, antagonists, and inverse agonists to treat several disease associated with the TSHR of the thyroid gland.
 
Project Title: Conformational flexibility of 5′-nucleotidase Status: Active
Investigators Norbert Sträter (Leipzig)
Jens Meiler (Vanderbilt)
Hassane Mchaourab (Vanderbilt)
Description    
Dr. Norbert Sträter of LU and Drs. Mchaourab and Meiler of VU aim to study the domain orientation of E. coli 5′-nucleotidase in solution. The apo-enzyme, as well as the enzyme in the presence of substrates, products, and inhibitors will be studied.
 
Project Title: Solution and solid-state NMR studies of the amyloid precursor protein Status: Active
Investigators Daniel Huster (Leipzig)
Charles Sanders (Vanderbilt)
Description    
Dr. Huster of LU and Dr. Sanders of VU propose to study APP via different NMR methods. The Sanders lab will carry out high resolution solution NMR studies to determine the structure of the 99 residue transmembrane N-terminal domain of the amyloid precursor protein (C99) in detergent micelles. Studies of lipid-protein interactions will also be carried out under the same conditions. The Sanders lab will provide the Huster lab with expression vectors, purification protocols, and unpublished data so that they can carry out solid state NMR studies of C99 under conditions of actual lipid bilayers. These studies will provide a test of whether the structural and lipid binding properties of C99 as mapped out in detergent micelle conditions by the Sanders lab also apply to the more native environment of bilayered lipid vesicles. Currently, one graduate student from the Sanders lab has been identified for a potential student exchange.
 
Project Title: Structure of membrane and receptor bound ghrelin Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Description    
The neuropeptide Y2, Y5, and Y1 receptors dramatically differ in their ability to bind arrestins: Y1 binds both non-visual arrestins equally, Y2 is more selective, whereas Y5 shows little interaction with WT arrestins. A post-doctoral fellow in Dr. Gurevich’s lab, Luis Gimenez, recently tested a set of arrestin-3 mutants with enhanced specificity for particular GPCRs on the Y2 receptor and found a unique pattern of changes dependent on the mutations. Several of the mutants showed a high preference for Y1 over Y2. The use of Y1/Y2 and Y2/Y5 chimeric receptors (generated in Dr. Beck-Sickinger’s lab by graduate student Stefanie Babilon) will allow us to elucidate key structural determinants in Y receptors responsible for robust arrestin binding and receptor specificity of the mutants. The immediate goal of this project is to develop arrestin-based tools that can selectively regulate the signaling by different Y receptor subtypes. The ultimate goal is to test whether these novel molecular tools can significantly affect feeding behavior in vivo.
 
Project Title: Structure of membrane and receptor bound ghrelin Status: Active
Investigators Daniel Huster (Leipzig)
Jens Meiler (Vanderbilt)
Annette Beck-Sickinger (Leipzig)
Description    
We have determined structural constraints of membrane bound ghrelin. From these NMR data, we will calculate the 3D structure of the membrane-associated molecule. Studies of ghrelin on the growth hormone secretagogue receptor will follow.
 
Project Title: Structure determination of membrane proteins from solid-state NMR sparse data sets Status: Active
Investigators Daniel Huster (Leipzig)
Jens Meiler (Vanderbilt)
Description    
We are working on an algorithm that allows the determination of the structures of membrane proteins from solid-state NMR data sets.
 
Project Title: Structure determination of NPY bound to the Y1 and Y2 receptor Status: Active
Investigators Daniel Huster (Leipzig)
Jens Meiler (Vanderbilt)
Annette Beck-Sickinger (Leipzig)
Charles Sanders (Vanderbilt)
Description    
This project focuses on the determination of the 3D structure of neuropeptide Y on its Y1 and Y2 receptors by solid-state and solution state NMR spectroscopy.
 
Project Title: Modeling of the Y4 receptor and docking of allosteric modulators Status: Active
Investigators Annette Beck-Sickinger (Leipzig)
Jens Meiler (Vanderbilt)
Description    
As a continuation of the project on the structural modeling of the neuropeptide Y4 receptor, the goal of this project is to develop a small molecule library screening system for positive modulators of the receptor. The first such molecules have been identified and are currently under evaluation at LU and VU.
 
Project Title: Integration of liquid beam and droplet desorption with ion mobility mass spectrometry Status: Proposed
Investigators Bernd Abel (Leipzig)
John McLean (Vanderbilt)
Description    
Preparations are being made to combine the expertise of the Abel group in liquid beam and droplet desorption mass spectrometry with structural separations in the McLean group utilizing ion mobility-mass spectrometry instrumentation. Dr. Ales Charvat (LU) and Dr. Jody C. May (VU) plan to initially integrate these novel ion sources with both commercial and prototype IM-MS instrumentation at Vanderbilt in late Spring 2011.
 
Project Title: Prolactin releasing peptide receptor: Peptide Bioassays Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Kevin Niswender (Vanderbilt)
Description    
This potential project involves development of new methods to assay PrRPR activity.
 
Project Title: Pegylation of peptides for GPCR crystallization studies Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Heidi Hamm (Vanderbilt)
Description    
This proposed collaboration involves the crystallization of GPCRs using pegylated peptides that mimic the G-protein.
 
Project Title: Internalization of the neuropeptide Y-receptor system to be used to shuttle toxins into the cell Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Borden Lacy (Vanderbilt)
Description    
This proposed project will investigate the internalization of the neuropeptide Y receptor system and its potential to shuttle toxins into the cell.
 
Project Title: Mapping key residues in Y2 receptor-arrestin interactions by NMR Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Daniel Huster (Leipzig)
Charles Sanders (Vanderbilt)
Description    
The goals of this project are to study arrestin-receptor interactions using NMR. A labeled (13C, 15N, or both) receptor generated in the Huster lab will be allowed to interact with unlabeled arrestins (purified in the Gurevich lab) to map receptor residues involved in the interaction. In addition, labeled arrestins will be generated in the Sanders lab and incubated with unlabeled receptor, to map receptor-binding elements in arrestins. In both cases, selective spin labeling will be used to assist in peak assignments. The labs of Drs. Sanders and Gurevich will use the experience they gained studying 13C and/or 15N-labeled visual arrestin-1 with different functional forms of the prototypical class A GPCR, rhodopsin (this study was recently accepted for publication by PNAS).
 
Project Title: Poloxin and poloxipan Status: Proposed
Investigators Thorsten Berg (Leipzig)
Jens Meiler (Vanderbilt)
Description    
This proposed project outlines a cooperation in the area of the inhibitors of the polobox-domain of Plk1.
 
Project Title: Boron and metallo-organic chemistry Status: Proposed
Investigators Evamarie Hey-Hawkins (Leipzig)
Piotr Kaszynski (Vanderbilt)
Timothy Hanusa (Vanderbilt)
Description    
This project will involve a change of ideas and techniques involving carboboranes
 
Project Title: Quantification of native membrane-associated protein-ligand interactions, labelfree and at physiological concentrations Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Description    
Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of chemical modulation targets, a general method for the direct, label-free measurement of membrane-bound protein-ligand binding within the native bilayer environment has not been reported. Current methods for these studies often require larger quantities of the proteins, demands them to be modified, truncated, or labeled, and often cannot be done in a complex matrix or with surface immobilization strategies. Taken together, existing interaction methodologies significantly limit the speed or quality of the assays. This project proposes to use back-scattering interferometry (BSI) to quantify ligand binding to membrane-associated proteins in freesolution, at physiological concentrations, and in native preparations. Specifically, binding will be studied in several model systems, including intact cell membranes constituted into micelles, bicelles, or nanodisks. Dr. Bornhop has pioneered the BSI technology and will handle all aspects of this technique in his laboratory. Dr. Huster is an expert in the experimental preparation of the target proteins, and will provide the selected systems.
 
Project Title: Molecular evolution and phylogenetic analysis Status: Proposed
Investigators Martin Schlegel (Leipzig)
Antonis Rokas (Vanderbilt)
Description    
A collaboration is planned between the Schlegel and Rokas groups in the area of molecular evolution and phylogenetic analysis.
 
Project Title: Catalytic enantioselective synthesis of unnatural and functional amino acids Status: Proposed
Investigators Annette Beck-Sickinger (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Description    
The Johnston and Schneider groups share a common interest in developing methods to catalyze important C-C bond forming processes with high enantiocontrol. Specifically, both groups have recently established Brønsted acid-catalyzed asymmetric protocols to prepare b-diamino esters and d-esters through nitro-Mannich and vinylogous Mannich reactions, respectively. Starting out from these investigations, it is their intention to collaborate in a project aimed at synthesizing unnatural and functional amino acids, which may form interesting peptide analogues. Specifically, they plan to extend the chemistry shown above to the enantio- and diastereoselective synthesis of various mono and diamino acids such as g- and -substituted ones using chiral Brønsted acid catalysis. Through careful adjustment of substrate structure and judicious choice of the respective Brønsted acid catalyst as well as reaction conditions, these valuable target compounds should be directly accessible with high stereocontrol.
 
Project Title: Adaptation of the yeast GPCR signaling system to arrestin function Status: Proposed
Investigators Torsten Schöneberg (Leipzig)
Vsevolod Gurevich (Vanderbilt)
Description    
The primary goal of this proposed project is to design receptor-specific non-visual arrestins and use a yeast system for testing the generated arrestin mutants with multiple GPCRs. Dr. Gurevich’s lab identified five key exposed residues on the receptor-binding surface of arrestins that determine their receptor specificity. Preliminary data were obtained by Dr. Luis Gimenez in the Gurevich lab using a BRETbased assay in cultured mammalian cells. The data show that several mutants in these positions in the first series show significant changes in relative binding to the β2-adrenergic and M2 muscarinic cholinergic receptors. Dr. Schöneberg is using GPCR activation-dependent yeast to characterize GPCR mutations. The idea is to use this system for screening of multiple arrestin mutant-GPCR combinations to significantly increase the throughput of the assay. This will allow the generation of arrestin mutants with specificity for several clinically relevant receptors. Dr. Schöneberg sent yeast expression constructs to Dr. Gurevich’s lab, where Dr. Gimenez will generate necessary yeast expression plasmids with different selection markers to co-express arrestins and GPCRs. The assay will be first tested in Dr. Schöneberg’s lab. If it works as expected, Dr. Gimenez will learn the necessary techniques in Dr. Schöneberg’s lab, and the collaborative work will be done in both labs.
 
Project Title: Ensemble docking of small molecule libraries into GPCR mutant libraries Status: Proposed
Investigators Torsten Schöneberg (Leipzig)
Jens Meiler (Vanderbilt)
Description    
Dr. Torsten Schöneberg of LU will work with Dr. Meiler on modeling GPCR mutants and validating the Rosetta results.
 
Project Title: Screening of orphan GPCRs Status: Proposed
Investigators Torsten Schöneberg (Leipzig)
Lawrence Marnett (Vanderbilt)
Description    
Dr. Torsten Schöneberg of LU proposes to work with the group of Dr. Larry Marnett of VU screening of orphan GPCRs for activation by arachidonoylglycerol.