Present CBI Fellows
(Mentor: James Petersson, Ph.D., CHEM)
My research focuses on incorporation of thioamides into peptides to inhibit aggregation and proteolysis. Specifically, I want to examine how thioamide incorporation will affect the aggregation and proteolysis of amylin, a therapeutically relevant peptide. A derivative of amylin, known as Pramlintide, is currently used as a peptide therapeutic in the treatment of Type II Diabetes. Pramlintide has several proline mutations that inhibit aggregation, but not proteolysis. By incorporating thioamides into the sequence of amylin, both proteolysis and aggregation may be inhibited. This method is widely applicable to other peptide and protein systems, and will also allow me to examine the underlying biophysics of amylin aggregation and proteolysis.
(Mentor: Ronen Mamorstein, Ph.D., CHEM)
The conserved N-terminal acetyltransferases (NATs) catalyze the co-translational N-terminal acetylation of the majority of the proteome. Many NATs require regulatory subunits for both robust activity and substrate selectivity. This project aims to use enzymological and structural techniques to probe the molecular roles of the small regulatory subunits, HYPK and Naa38p, in NatA and NatC activity, respectively. NatA and NatC are particularly interesting as they are the only NATs that require a small regulatory subunit for efficient enzymatic activity. Preliminary work in our lab has demonstrated that Naa38p dramatically enhances NatC activity. Furthermore, the Huntingtin (Htt) yeast two-hybrid protein K (HYPK) has important implications for Huntington’s disease: HYPK overexpression as well as NatA knockdown have both led to the decrease of Htt aggregation associated with disease pathogenesis. Therefore, we are also interested in investigating the link between HYPK/NatA activity and Huntington’s disease.
(Mentor: Rahul Kohli, M.D., Ph.D., CMB)
I am interested in studying the dynamics of LexA, a repressor-protease that regulates the DNA damage response pathway in bacteria and mediates acquired antibiotic resistance. Activation of the DNA damage (or SOS) response is driven by the auto-proteolysis of LexA, a reaction that appears to require a large conformational change within the protease domain to permit self-cleavage. Through the use of minimally-perturbing fluorescent unnatural amino acids, I aim to characterize the conformational dynamics of LexA in order to decipher the complete kinetic pathway of LexA activation. Following LexA auto-proteolysis in bacteria, depletion of LexA protein appears to drive a coordinated program of SOS gene expression. Through protein engineering strategies and cutting-edge fluorescent-labeling techniques, I plan to monitor how the dynamics of LexA turnover are coupled to SOS gene expression at high temporal and spatial resolution. These studies will illuminate how the protein dynamics of a single enzyme equip bacteria with a tightly regulated program of gene expression in response to a variety of stresses.
(Mentor: Benjamin Garcia, Ph.D., BMB)
Nucleosomes are the fundamental repeating unit of chromatin and are critically important in regulating transcription, chromatin structure, and other vital nuclear processes. Nucleosomes are extensively and dynamically post-translationally modified, and these PTMs are often responsible in mediating nucleosome function. I am interested in determining how specific PTMs alter nucleosome structure and dynamics, which could lend insights into how these modifications alter function. I am currently developing hydrogen-deuterium exchange coupled to top-down mass spectrometry (top-down HDX-MS) methodology, which differs from standard bottom-up HDX-MS methodology in that full coverage of the protein is guaranteed and the resolution of the data is often improved. I plan to create "designer" nucleosomes containing PTMs at defined locations using chemical ligation strategies, and employ the top-down HDX-MS methodology to determine how these PTMs alter nucleosome dynamics compared to unmodified nucleosomes.
(Mentor: James Shorter, Ph.D., CHEM)
The overall goal of my research is to understand the molecular basis of Hsp104-mediated protein remodeling. Hsp104 is a hexametric AAA+ protein disaggregase that is conserved among nonmetazoan eukaryotes and eubacteria. Hsp104 from yeast can rapidly disassemble disordered aggregates, preamyloid oligomers, amyloids, and prions, and has enabled yeast to exploit beneficial prions for adaptive purposes. However, humans and metazoa lack a direct Hsp104 homologue, and are therefore vulnerable to protein misfolding, which causes several fatal neurodegenerative diseases including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). I am using a combination of genetic, biochemical, and cell biology approaches to dissect the molecular determinants of Hsp104 substrate selectivity. I am also using molecular evolution techniques to evolve enhanced disaggregases to combat protein misfolding in neurodegenerative disease.
(Mentor: Elizabeth Rhoades, Ph.D., CHEM)
Tau is an intrinsically disordered protein (IDP) decorated with multiple post-translational modifications that interacts with a diverse set of cellular components. Most notably, tau is a microtubule-associated protein that promotes microtubule assembly and controls microtubule dynamics. A particular region of tau, the proline-rich region (PRR), has been proposed to regulate tau’s binding affinity to microtubules and microtubule assembly dynamics. Furthermore, specific phosphorylation patterns of PRR are proposed to have different affects of the native function of tau. However, with over 80 possible phosphorylation sites throughout tau, studying the impact of specific yet complex phosphorylation patterns on tau’s conformation and function remains relatively unexplored due to both the promiscuity of kinases and the difficulties in biophysically characterizing an IDP. Therefore, I plan to use chemical ligation strategies to install specific phosphorylation patterns within the PRR domain and study the impact on 1) the global conformation of tau 2) microtubule and tubulin binding affinities and 3) microtubule assembly dynamics. In conclusion, these studies aim to understand the unique representation of the structure-function relationship of tau from the viewpoint of statistical ensembles biased by post-translational modification.
Past CBI Fellows
(Mentor: David Christianson, Ph.D., CHEM)
Cryptophanes represent an exciting class of xenon-encapsulating molecules that can be exploited as probes for nuclear magnetic resonance imaging. Julie has been working towards the targeting of these xenon-encapsulated crytophanes to a biological target. As a model system, Julia chemically linked a xenon-encapsulated crytophane to an inhibitor of carbonic anhydrase II, a benezenesulfonamide, and determined high-resolution crystal structure of this cryptophane-derivatized benezenesulfonamide complexed with human carbonic anhydrase II. The structure of the complex reveals how an encapsulated xenon atom can be directed to a specific biological target. The crystal structure also confirms binding measurements indicating that the cryptophane cage does not strongly interact with the surface of the protein, which may enhance the sensitivity of 129Xe NMR spectroscopic measurements in solution. These studies have been a collaboration between the Christianson (Chemistry Graduate Group) and Dmochowski (Chemistry Graduate Group) laboratories and are reported in two peer-reviewed manuscripts and have implications for nuclear magnetic resonance imaging of human specimens for diagnostic purposes.
(Mentor: Shelley Berger, Ph.D., BMB)
Ed is employing a “top-down” mass spectrometric method to determine the full combinatorial complement of histone modifications in yeast as they undergo the developmental program of sporulation. The goal of the studies are to identify new histone modifications that only appear during sporulation, determine whether the modifications are transient or persistent and whether they are present in combinations on the same histone or different histones. These mass spectrometric experiments will be an invaluable complement to concurrent biochemical and genetic studies on-going in the Berger lab.
(Mentor: David M. Chenoweth, Ph.D., CHEM)
Designing and synthesizing molecules that are able to control protein-protein interactions (PPIs) is an important and fundamental problem. We are interested in developing a new class of molecules that are able to mimic alpha-helices. Our molecules are based on a set of bicyclo amino acid building blocks which can be combined to target a broad number of PPIs. The biochemical and structural information on p53/MDM2 allows us to use it as a model system. After determining inhibition constants of the helix mimics, structural studies will be done to co-crystallize the most potent with MDM2 to provide insight in designing new helix mimics. Once the new scaffold is tested, we plan to move on to higher risk targets such as the p70S6K kinase C-helix and HIV gp41.
(Mentor: Barry Cooperman, Ph.D., CHEM)
Diana worked on the flourogenic labeling of tRNA molecules in order to facilitate Fluorescence Resonance Energy Transfer (FRET) experiments to study the kinetics of protein translation. Diana successfully labeled several tRNA molecules with high yield and efficiency. She collaborated with the Goldman laboratory (BMB of UPenn SOM) to use Internal Reflection Microscopy (TIRFM) to visualize immobilized ribosomes and their interaction with several labeled factors. These studies yielded novel information underlying distinct tRNA-ribosome binding events. Unfortunately, Diana decided to leave the graduate program for personal reasons so these studies are being carried forward by other members of the Cooperman and Goldman laboratories.
(Mentor: James Shorter, Ph.D., BMB)
I am interested in studying enzyme mechanisms. In the Shorter lab I am studying the mechanism of Hsp104, a hexameric yeast disaggregase that can dissolve amorphous aggregates and amyloids. Specifically, I am asking how Hsp104 regulates intersubunit coordination and I am studying the effects of substrate stability on the level of hydrolytic coordination.
(Mentor: Ivan Dmochowski, Ph.D., CHEM)
Understanding the modes of action of general anesthetics through the use of AMA/AZA bioimaging. I am using AZA as a potential anesthetic to selectively photo cross-link to particular areas of the tadpole anatomy. Through fluorescent imaging, my goal is to uncover cells critical to AZA performance and to isolate out particular proteins that AZA binds to, providing further insight into how anesthetics function.
(Mentor: Ronen Marmorstein, Ph.D., The Wistar Institute)
Human Papillomavirus is the etilogical agent for cervical cancer that is mediated by two small viral oncoproteins, HPV-E6 and HPV-E7. Although several HPV vaccines have been developed, there is currently no therapeutic treatment for patients that already have cervical cancer. The oncogenic activity of HPV-E7 works, in part, by its ability to bind and inactivate the activity of the endogenous pRb tumor suppressor protein. Daniela has been interested in identifying and characterizing small molecule inhibitors of HPV-E7. To this end, Daniela has developed a high throughput ELISA-based screen for small molecule compounds that disrupt HPV-E7 binding to pRb, and is currently carrying out an 100,000 compound screen. In parallel, Daniela has screened 90,000 compounds in silico for HPV-E7 binding and has obtained some promising lead compounds that she is analyzing in vitro for disrupting HPV-E7-pRb binding. Daniela is also using biochemistry and crystallography to characterize the mode of HPV-E7 inhibition of the p300 histone acetyltransferase enzyme. The ultimate goal of Daniela’s studies is to develop lead HPV-E7 compounds that might be further developed into therapeutic agents to treat cervical cancer.
(Mentor: Madeleine Joullie’, Ph.D., CHEM)
My research in Dr. Joullié’s lab is focused on the development of a biomimetic semi-synthesis to access a number of mycotoxins derived from the indole alkaloid roquefortine C. Fungal metabolites have played a prominent role in the pharmaceutical industry since the discovery of penicillin in 1928, and the study of these compounds has been and continues to be integral to the advancement of medicine and the understanding of biological processes. This semi-synthesis will feature a combination of traditional organic chemistry as well as chemoenzymatic transformations to achieve highly selective and efficient routes to each compound. Following the completion of this synthesis, I hope to explore the biological activity of the newly synthesized mycotoxins as well as the biological activity of other metabolites generated during the fermentation of roquefortine C. Roquefortine C as well as a number of other secondary metabolites of Penicillium fungi have been reported to induce inflammatory and cytotoxic responses in animals, and it is our hope that we might also perform simple modifications on these mycotoxins to reduce their toxicity.
(Mentor: Jeffrey Winkler, Ph.D., CHEM)
Research in the Winkler Group is focused on the design of small molecules that inhibit protein-protein or small molecule-protein interactions. I am currently designing small molecules that inhibit the viral entry of a rare, deadly strain of Enterovirus 71 (EV71). EV71 is a causative agent of Hand, Foot and Mouth Disease (HFMD), which is a self-limiting disease that leads to lesion formation on the hands, feet and mouth of those infected. Although EV71 typically enters host cells through the SCARB2 surface receptor, the more virulent strain of the virus is able to infect host leukocytes through an interaction between PSGL-1, a leukocyte-specific surface receptor, and VP-1, a pentameric viral protein on the EV71 capsid. With our small molecules we aim to interrupt this interaction and render EV71 non-infective to leukocytes.
(Mentor: William DeGrado, Ph.D., BMB)
ABC transporters are nature's most diverse protein family due to the modular design of their subunits. Gabe studies the thermodynamic and kinetic contributions of each subunit to the transport mechanism. Understanding the energetics of transport would enable the development of inhibitors to block the transport cycle and prevent tumor drug resistance, which is predominantly caused by multi-drug ABC exporters. Additionally, Gabe works on designing new substrate specificities for these transporters in order to develop custom import and export mechanisms for cell-based synthetic pathways.
(Mentor: Ronen Marmorstein, Ph.D., The Wistar Institute)
Biochemical, chemical and enzymatic studies are being used to probe the mechanism of N-terminal protein acetylation by the ternary NAT5/ARD1/NATH complex.
(Mentor: Ronen Mamorstein, Ph.D.)
The goal of my project is to determine the molecular mechanism of ribosome-associated acetylation by N-terminal acetyltransferases (NATs). In humans, over 80% of all proteins are N-terminally acetylated. This acetylation has been implicated in a wide array of biological activities including protein stability and degradation, enzyme regulation, protein localization, and protein-protein interaction. There are six known NAT complexes in humans, all of which associate with the ribosome and acetylate N-termini in a co-translational process. Through the use of biochemical pull down assays, I will determine whether NAT association with the ribosome is exclusionary, and if not, how many NATs can bind simultaneously. To determine the molecular basis for the interaction, I will employ cryo-electron microscopy, x-ray crystallography, and mutagenesis. Isothermal titration calorimetry will be used to determine the thermodynamic parameters of NAT binding to the ribosome. Finally, radiometric assays will be used to assess the steady state kinetics of the NATs while complexed to the ribosome.
(Mentor: A. Joshua Wand, Ph.D., BMB)
Reverse micelle encapsulation of proteins allows for the study of relatively large proteins using NMR spectroscopy due to the decrease of solvent viscosity (and hence the decrease of the tumbling time). A consequence of this encapsulation is the retardation of water dynamics and hydrogen exchange within the reverse micelle. This allows for the residue-specific study of a protein with the surrounding water molecules in the hydration layer which will provide insight on one of the driving forces of a protein’s functionality: its interaction with water. This will also allow the study of the solvent slaving model which states that protein motions are classified into three types: those that are slaved to the hydration layer solvent, those slaved to the viscosity of the bulk solvent, and those independent of solvent. This model, though often cited, is not supported by substantial, site-specific evidence. I will use reverse micelle encapsulation technology in order to study the hydration dynamics of multiple protein systems (hen egg-white lysozyme – 14.4kD, and maltose binding protein – 42kD) in order to study the solvent slaving model.
(Mentor: Doron Greenbaum, Ph.D., BMB)
In my project I am investigating the function of a human family of calcium regulated cysteine proteases called calpains through the design of highly specific inhibitors. Calpains are of biomedical interest because they have been implicated in a variety of diseases including neurodegeneration, cancer, and parasite infection. Currently, we are designing and testing novel inhibitors of calpains based on the structure of the endogenous calpain inhibitor, calpastatin. Our inhibitors mimic a two-turn alpha-helix, which binds to a unique area near the active site of calpains. This helix allows our inhibitors to be specific for calpains relative to other cysteine proteases such as the lysosomal cathepsins that do not have this helical binding pocket. We have minimized the size of the peptide relative to other calpastatin-based inhibitors by developing a novel method for stabilizing the helix of the unbound inhibitor thereby decreasing the free energy needed for binding. We have found that one inhibitor, in which the end loop of the two turn helix is stabilized, inhibits calpain 1 with a Ki of 300 nM. We are now working to add electrophiles, such as an diketo-amide, to the N-terminus of the peptide to enhance potency through a covalent, reversible interaction with the active site cysteine. We are also performing structural studies, in collaboration with the Davies laboratory (Queen’s University) to solve the co-crystal structures of our stabilized helical peptides bound to the protease in order to better understand the molecular basis for increasing potency, selectivity and decreasing the overall size of the inhibitor. Initially, we are using these helical inhibitors to kill malaria parasites by preventing their exit from their host human red blood cells, a process dependent on the red blood cell calpain. We hope to expand the use of these calpain inhibitors to other biological applications such as cancer.
(Mentor: Eric Meggers, Ph.D., CHEM)
Sean has been preparing organometallic compounds as novel potent and selective enzyme inhibitors. Specifically, Seann has developed a solid phase synthesis methodology to prepare a library of stable dicationic ruthenium polypridyl complexes. The idea behind his studies is that the increased coordination sphere of the metal ion would facilitate the preparation of a library of chemical entities that expand the region of synthetically accessible chemical space for the preparation of novel small molecule protein inhibitors. Indeed, Sean has exploited this methodology to prepare a potent (IC50 value in the mid nanomolar range) and selective acetylcholinesterase (AChE) inhibitor. Sean’s studies have already resulted in four peer-reviewed publications and pave the way for not only developing even more potent and selective AChE inhibitors, but also for using this methodology to develop novel potent and selective inhibitors for other protein families.
(Mentor: Marisa Kozlowski, Ph.D., CHEM)
My research focuses on the synthesis of a group of axially chiral bisanthraquinone natural products, such as skyrin or bisoranjidiol. The reported biological activity and physical properties of some of these compounds affects a variety of public health issues including treatment of cancer (suppression of tumor cell growth), diabetes, hepatitis, depression, and use as an antioxidant. Still, a great deal of information is lacking for the activity of many of these bisanthraquinones, citing a need for more biological studies as well as efficient stereoselective synthesis. Specifically, the generation of the compounds will be achieved via a concerted synthesis that diverges from the same chiral bisnaphthoquinone intermediate and involves key reactions such as a copper catalyzed enatioselective oxidative biaryl coupling, oxidation/quinone formation, and tandem Diels-Alder/aromatization reactions with various vinyl ketene acetals.
(Mentor: Ivan Dmochowski, Ph.D., CHEM)
Julia has been working on the design of light responsive regulatory switches of various nucleic acid templated biological processes. Specifically, Julia has designed photoactivatable oligonucleotides whose function is blocked by a caging group until removal by irradiation. In one study, Julia designed “RNA bandages” for the photoregulation of protein synthesis in vitro and in a second study; Julia successfully designed a caged fluorescent DNA to photoregulate DNA polymerase I. Each of these studies resulted in peer-reviewed publications. Julia’s studies have implications for the temporal regulation of gene expression in living cells with possible therapeutic application.
Harry Schroeder, III
(Mentor: Yale Goldman, Ph.D., BMB)
Trey is employing single molecule studies to study switching of cargo between actin filaments (AF) and microtubles (MT), a process that is critical for appropriate endocytosis and secretion. In particular, by using an optical trap to position the cargo attached to a bead with a limited number of actively engaged motors near the actin-microtubule intersections, Trey has been able to examine cytoskeletal switching as a function of motor number. These studies reveal that the number of motors (or overall force generated) can be used to regulate switching behavior. Trey’s data also shows that rotation of the cargo is specifically seen at the intersections, which implies a torque component and suggests a mechanism for switching. Some of Trey’s work has been published in two peer-reviewed manuscripts.
(Mentor: Harry Ischiropoulous, Ph.D., PHARM, BMB)
I am interested in studying and characterizing the native state of alpha synuclein in human brain. While much research over the past decade has focused on investigating the process by which monomeric alpha synuclein aggregates and forms toxic protein deposits, recent evidence suggests that in its native state alpha synuclein is actually a folded tetramer. While a series of elegant experiments indicate that destabilization of this tetramer is the key first step in the aggregation of alpha synuclein, others propose that it is natively an unfolded monomer. Resolving this controversy is critical to developing effective therapeutic remedies to combat diseases caused by toxic aggregation of alpha synuclein, most notably Parkinson’s Disease.
(Mentor: Jeffrey Saven, Ph.D., CHEM)
Our group is currently focused on developing theoretical and computational tools to explore protein sequence space for a given range of three-dimensional backbone configurations and orientations. This allows us to hone in on sequences that are likely to adopt stable folds and structures of interest, such as protein scaffolds and protein crystals. The goal of my project is to design and characterize new protein structures with ligand-binding motifs that can also crystallize. By studying these de novo structures by means of computational and experimental techniques, we aim to generate novel biomaterials, as well as gain understanding of ligand-binding and self-assembling properties of macromolecules in biology.
(Mentor: James Petersson, Ph.D., CHEM)
My research entails developing new methods for studying protein conformational changes using fluorescence spectroscopy. If a chromophore were small enough to be incorporable at any position of a protein without changing its native conformation, the structural resolution of fluorescence experiments could be greatly improved. We have demonstrated that thioamides are capable of quenching a wide array of fluorophores such as tyrosine, tryptophan, and the unnatural amino acid para-cyanophenylalanine in a distance-dependent fashion. Currently, I am working towards combining unnatural amino acid mutagenesis with semi-synthesis techniques to construct full-length proteins bearing these minimally perturbing chromophore pairs. I am also interested in developing new methods for unnatural amino acid incorporation.
(Mentor: Jeffrey D. Winkler, Ph.D., CHEM)
My research involves the design and synthesis of novel steroid-based inhibitors of the Sonic Hedgehog (SHH) signaling pathway, which is important for cellular growth and differentiation during embryogenesis, and has recently been implicated as an important pathway to target in human cancer. The Winkler group has synthesized potent SHH inhibitors based on the naturally occurring inhibitor, cyclopamine, and I will continue to explore the design, synthesis and biological evaluation of estrone-derived cyclopamine analogs and their potential use as chemotherapeutics.