Max-Kade Scholarship 2014: Short-Reports

/ October 28, 2014/ News, Students

2014 eight students on Bachelor- or Master level got the Max-Kade Scholarship to participate in ongoing research projects between Vanderbilt University and the University of Leipzig. Hereby there short research and travel reports:


Luise Spormann (Undergraduate Student at Leipzig University):
CYCLOOXYGENASE STRUCTURE, FUNCTION AND INHIBITION
(Laboratory of Prof. Dr. Lawrence Marnett / July-October 2014)

I worked in Dr. Marnett’s lab, which is part of the Department of Biochemistry at the Vanderbilt University School of Medicine, as an intern from July 30th to October 10th. To contribute to the research of the Marnett group, I first had to learn how to deal with cell cultures. I received tissue culture training where I learned about the basics of tissue culture and sterile technique. I then learned how to do cell viability assays and apoptosis assays to test the effect of certain compounds on tumor cell growth. Figure 1 shows the result of a cell viability assay. The compound WN-RX-184 acts as a very weak COX inhibitor but obviously has a great impact on the viability of the tumor cells. I learned how to collect cells for lysates and determine protein concentration with a BCA assay. I also gained experience running Western Blots.
My second task was to measure certain compounds in mouse plasma and tissues. It was necessary to test the compounds in vivo that gave good results in cell culture to see how they would act in a more complex system. We examined in which tissue the compound is present and its concentration. For this project, the animal tissues needed to be homogenized, and the extracts were measured with HPLC and mass spectrometry.
This internship was a great opportunity not only to improve my scientific skills but also to consolidate my English. I learned a lot about American everyday life and the American point of view on certain things in diverse conversations. Nashville offered many occasions to experience American culture with all of the museums, bars, and live music. In addition, I got the chance to learn about American history and current politics with a trip to Washington, DC. Of course, I also enjoyed the great nature around Nashville. The canoe trip and hiking trips were good chances to meet other Vanderbilt students.     


Ulrike Kinkel (Graduate Student at Leipzig University):
CONSTRUCTION OF ARRESTIN MUTANTS WITH ENHANCED SPECIFICTY FOR INDIVIDUAL NPY RECEPTOR SUBTYPES
(Laboratory of Prof. Dr. Vesevold Gurevich / July-October 2014)

During my work on this project I made good progress and was able to work confidently on my own. I learned a lot concerning self-organization and self-determined working. I am now able to use a wide range of methods that are used to introduce point mutations in DNA such as PCR, double digest restriction and ligation. Additionally, I am able to perform and evaluate the results of Bioluminescence Resonance Energy Transfer Assays on my own. During my work in Prof. Gurevich´s laboratory I created six new mutants of arrestin-3, each with a point mutation in the receptor-binding area. The mutations are now ready for testing. Furthermore, my results from testing the Y239T-Arr yield further insights into the mechanisms of arrestin recruitment to NPY-receptors.
I had the chance to experience a lot of American culture outside the lab. I went to Downtown Nashville several times. There, it is nearly impossible to escape the spirit of country music. On weekend trips, I also saw other parts of America. During my stay in Washington D.C., I was able to see worldwide famous monuments and indulge myself in the history of former presidents and the development of the city itself. Furthermore, I was able to make my first American football experience. During a game of Vanderbilt against South Carolina, I really enjoyed the spirit of the Americans who support their college football team and I even understood most of the rules.      


Caroline Schindler (Graduate Student at Leipzig University):
The mechanism of NPY2 mediated Gαi Activation
(Laboratory of Prof. Dr. Heidi Hamm / August-October 2014)

Heterotrimeric G-proteins serve as molecular switches that turn on intracellular signaling cascades in response to the activation of G-protein coupled receptors (GPCRs). In case of Neuropeptide Y Receptors physiological and pathological processes such as food intake, obesity, depression and cancer are regulated. In this study local dynamics and distances between Gαi-protein and NPY2 receptor (NPY2R) upon receptor activation were addressed by electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) techniques using a site directed spin-labeling approach.
During my internship at Vanderbilt University the focus was set on the expression and spin-labeling of Gαi mutants for further DEER measurements. In addition, I have started to establish an in vitro assay to determine the ability of membranous NPY2R for Gαi protein binding, which had already been set up for Rhodopsin. During my time in Heidi Hamm’s lab I also gained insight into the rhodopsin system. Although rhodopsin and NPY2R belong to the same class of GPCRs, rhodopsin shows two crucial differences: its covalent attached ligand retinal and its unique spectroscopic properties. The internship enabled me to be part of an international research collaboration for the first time.
At Vanderbilt University there is a kind of team spirit connecting the entire campus which is not present in Germany. This identification with Vanderbilt University finds also expression in the awesome atmosphere of sport events. I was pretty impressed when I was watching a university football game. Unfortunately, the Vanderbilt Commodores lost. Furthermore, I experienced Nashville as a living city of music – especially during the Live On The Green music festival. The Broadway is the heart of Nashvegas, where you can always find some good country or bluegrass music. And of course, I did not miss the traditional burger experience. To ensure that I get the best burger in Nashville, the entire lab members went to Pharmacy burger where I was eating an old-style American burger and drinking an amazing self-made ginger soda.      


Elizabeth King (Undergraduate Student at Vanderbilt University):
EXPRESSION, PURIFICATION AND REFOLDING OF THE GHS-R RECEPTOR FOR LIGAND STRUCTURE DETERMINATION
(Laboratory of Prof. Dr. Daniel Huster & Dr. Gerrit Vortmeier / May-July 2014)

My research at Leipzig University and Vanderbilt University both focus on the peptide ghrelin and its receptor in the brain, GHS-R1a (growth hormone seratogue receptor 1a), both of which are believed to play a key role in obesity. Ideally, ghrelin maintains metabolic homeostasis by binding to the hunger and satiety centers of the hypothalamus (the lateral and ventromedial hypothalamus), causing a healthy individual to stop eating when he has consumed a meal, when he “feels full.” However, ghrelin also acts on a separate food reward pathway, which is the focus of my research. In this pathway, ghrelin binds at the VTA (ventral tegmental area). This binding leads to DA (dopamine) overflow in the nucleus accumbens, creating a feeling of reward when food is consumed, which may lead to overconsumption, food addiction, and obesity.
The specific goal of my work this summer was to study the structure of GHS-R1a and its interactions with ghrelin using solid state NMR. I spent most of the summer with my graduate student mentor, Gerrit Vortmeier, learning how to isolate the receptor for NMR. My work this summer gave me a better understanding of GHS-R1a and ghrelin that for when I return to Vanderbilt University in the fall. I will continue to collaborate with Gerrit and use the NMR data when I move to the next step of generating models in the Meiler Lab.
The members of Dr. Huster’s lab welcomed me. Many of them helped me when I was stuck on a particular technique or method. The latter half of the summer, we took turns making lunch in the lab kitchen. We also had a post World Cup party at a bar near the lab, and Dr. Huster invited me to his home for dinner. I later spent time with his 12-year-old daughter on a trip to the zoo. It was especially nice to be around a family because I was away from mine for so long. My mentor, Gerrit Vortmeier, especially went out of his way to welcome me to Leipzig. He showed me different spots around the city and introduced me to his group of friends.
I would live in Leipzig long-term. The sense of community among the people of Leipzig cannot be matched, and it is reinforced by the constant stream of festivals, concerts, and markets around the town. The public transportation system is fantastic, and I took advantage of it to travel to the lakes south of town multiple times. I can’t write about all my Leipzig experiences here or it would take 10 pages, but I felt completely welcomed by the town and I would love to live there.
Outside of Leipzig, I traveled to Prague, Berlin, Dresden, Munich, Interlaken, and London. My favorite place was Prague—I am part Czech, and I loved seeing how the Czech people are gaining a national identity they didn’t always have before. I also enjoyed Berlin and Dresden, although I felt that I didn’t get enough time in either place. (I know Dresden and Leipzig are in competition as cities—Leipzig wins). London was also amazing; I went to visit a few friends and covered as much as I could in a few days. Still, Leipzig was my favorite city from the summer. It was a privilege to live there.        


Aditya Karhade (Undergraduate Student at Vanderbilt University):
INVESTIGATING THE BINDING SITE OF CHEMOKINE RECEPTOR-LIKE 1 (CMKLR1)
(Laboratory of Prof. Dr. Annette Beck-Sickinger & Tristan Zellmann / May-July 2014)

Chemerin is an immunomodulating factor secreted predominantly by adipose tissue and skin. As a chemoattractant for multiple cells of the adaptive immune system, it has been implied as a link between obesity, type II diabetes and systemic inflammation. Chemerin’s actions are mediated by the G-protein coupled receptor CMKLR1, expressed by dendritic cells, macrophages, cardiomyocytes, adipocytes and endothelial cells. Present understanding of chemerin-CMKLR1 binding is limited to existing knowledge of the binding modes of the related N-formyl peptide receptors (FPR), FPR1 and FPR2, and the Complement Anaphylotoxin C3a Receptor (C3aR) and the Complement Anaphylotoxin C5a Receptor (C5aR).
By translating the existing knowledge from previous studies, this project aimed to elucidate important receptor residues for ligand binding by using techniques such as site-directed mutagenesis and signal transduction assays.
The first challenge in determining CMKLR1’s structure was creating large quantities of the ligand, chemerin, in various endogenously expressed forms. Endogenously, chemerin is secreted as a 143 amino acid precursor, prochemerin, which undergoes 6 amino acid cleavage by serine proteases at the C-terminus, chemerinS157, which can be further cleaved to form chemerinF156. Each of these forms of chemerin has different relative binding affinity on the CMKLR1 receptor and can be studied by drawing parallels between the ligands of the FPR receptors. In this project, large quantities of these desired forms of chemerin were generated by recombinant expression in E. coli bacteria. Large quantities of ligand were generated by using plasmids to transfect bacteria, using antibiotics to allow only the successfully transfected bacteria to survive, lysing bacteria at the peak of the exponential growth phase to obtain the highest yield of protein inclusion bodies, washing and purifying the protein of interest, and using spectrophotometry and SDS PAGE gels to assess the success of the steps along the way as well as the final product.
After successfully creating large quantities of ligand, site directed mutagenesis of CMKLR1 was attempted to assess the relative binding affinities of the mutants. The purpose of this study was to identify the individual residues in CMKLR1 of highest important for binding. By finding these residues and comparing their characteristics to similar residues in related structures such as GPR1, FPR1, C5aR and C3aR, software could be used to generate candidate CMKLR1 structures.
Future work will continue to investigate the important residues for binding and will assess the binding affinities of the mutants. Creating a three dimensional model of CMKLR1’s structure will not only increase the present scientific understanding of this molecule but also create invaluable opportunities for drug design to modulate this receptor. Such understanding holds incredible promise for those suffering from conditions such as type II diabetes, obesity and systemic inflammation and can contribute to the newly evolving era of personalized medicine in which genetic studies can be coupled with this understanding to prevent suffering in the first place.      


Zhewen Zhang (Undergraduate Student at Vanderbilt University):
DETERMINING THE “PHOSPHO-BARCODE” OF THE D1 DOPAMINE RECEPTOR
(Laboratory of Prof. Martin von Bergen & Dr. Janina Tomm / May-July 2014)

My 10 weeks spent in Leipzig, Germany turned out to be one of the most enriching and enjoyable experiences I have had, both intellectually and culturally.
My work this summer at the Helmholtz Centre for Environmental Research (UFZ) in Leipzig was on determining the “phosphor-barcode” of the D1 dopamine receptor. The D1 dopamine receptor is the most abundant and one of the most important dopamine receptors in the central nervous system. Its main responsibilities include regulation of behavioral responses, neuronal growth and development, and regulation of other dopamine receptors. The primary mechanism by which the receptor is activated is well known, but there are secondary regulation mechanisms that are not as well studied. Therefore, my research looked into isolating the receptors and then using mass spectrometry to analyze the secondary regulation mechanisms. The work turned out to be extremely rewarding, as I learned a great deal about the D1 receptor and was also able to glean several new lab techniques. The person who made me feel so welcomed was my lab P.I. at UFZ, Dr. Janina Tomm. She was very accommodating and patient and I could not have asked for a more welcoming supervisor.
In addition to my work, this internship allowed me the opportunity to travel throughout Europe. While I had the pleasure of going to several cities, the highlights of my travels were Berlin, Paris, and Prague. All three cities, and all the other cities I visited, had their own unique culture and history, and I had the pleasure to learn about it all. Overall, this internship has been perhaps the most memorable adventure of my life, and I would definitely recommend anyone interested to apply for this internship.


Caitlyn D. Le (Undergraduate Student at Vanderbilt University):
Allosteric Modulation of the human Y4 Receptor
(Laboratory of Prof. Dr. Annette Beck-Sickinger & Mario Schubert / March-July 2014)

Allosteric modulation is one of these methods to target GPCRs that have come to the fore in the last ten years. Under this approach, small molecule ligands interact with the receptor in order to modulate the activation pattern and thus allow fine-tuning of the receptor function. Upon binding, the ligand causes a conformational change in the receptor that can affect the receptor’s affinity for its agonist or the activation of downstream interacting proteins as the G protein or arrestins. Therefore, investigation of allosteric modulation of GPCRs is a possibility for identifying new ligands of GPCRs as future therapeutics and examination of the allosteric mechanism could provide more information about GPCR activation. It is in this direction that current studies of the hY4 receptor are currently moving.
In an effort to study the binding mechanism of hY4, the interaction between a small molecule allosteric modulator and the Y4R was examined. Because the binding pocket on the receptor has not been identified, a molecular modeling approach was performed to identify the key residues that comprise a potential binding site, out of an ensemble of 10 different docking simulations.