Fellows
Present CBI Fellows
Olivia Bullock
(Mentors: Tobias Baumgart & Elizabeth Rhoades, CHEM)
Supported 1/1/2024- present
During my graduate studies, I have endeavored to integrate the fields of chemistry and biology in the labs of my co-PIs, Dr. Tobias Baumgart and Elizabeth Rhoades. I am currently researching lamellipodin (LPD), a protein involved in the fast endophilin-mediated endocytosis (FEME) pathway. FEME is a clathrin independent endocytosis pathway conserved across many species and is involved in hormonal signaling, “fight or flight” hormone response, and can be utilized by Shiga and Cholera toxins to infect cells. Though this pathway has been broadly researched, its discovery is more recent compared to more established endocytosis pathways. Understanding LPD’s involvement in this pathway is crucial in developing a more thorough understanding of endocytosis and the biochemical processes involved. My research currently focuses on the 1-239 N-terminal region of LPD (NLPD), which is intrinsically disordered (lacks stable secondary structure) and currently has an unknown role in the FEME pathway. I am utilizing fluorescent imaging and confocal microscopy to observe NLPD’s binding and structural effects on artificial cellular membranes. I plan to expand my investigation to better understand NLPD’s and LPD’s interactions with other proteins in the FEME pathway. I will expand my project to include native chemical ligation (NCL), which is necessary to purify the full length protein from piecemeal domains able to be produced in bacterial cells. Finally, I am very interested in investigating the many post translational modifications in LPD, which again would require NCL to link individual domains containing the modifications. This would better elucidate the importance of these modifications in the function of the full length protein within the FEME pathway as well as the influence of LPD on related proteins
Current position: in Training
Andres Fernandez del Castillo
(Mentor: Mark Sellmyer, BMB)
Supported 6/1/2023- present
Parkinson's disease is a neurodegenerative disease characterized by pathological aggregates. One component of those aggregates is a-Synuclein, an intrinsically disordered protein that has the propensity to form fibrils. In collaboration with the Petersson lab, I aim to create small-molecule heterobifunctional ligrands which harness cellular degradation systems, such as the proteasome and autophagy, to selectively degrade a-Synuclein fibrils. I hope to use those ligrands to interrogate the interplay between a-Synuclein fibril formation and neuronal oxidative stress.
Current position: in Training
Matthew Gaynes
(Mentor: David W. Christianson, CHEM)
Supported 6/1/2022- present
Terpenes comprise a vastly chemodiverse class of natural products abundant with industrial, environmental, and pharmaceutical application. These compounds are biosynthesized by enzymes collectively known as terpene synthases, which can be utilized to access these valuable natural products with many technical advantages. Furthermore, robust definition of the mechanistic strategies involved with terpene biosynthesis can lead to engineering of designer enzymes. In the Christianson Laboratory we strive to continually build the basis of structural understanding related to terpene synthase function and mechanism. Terpene biosynthesis begins with production of linear, achiral, and highly flexible isoprenoid diphosphates by prenyltransferases. Subsequently, terpene synthases accept an isoprenoid diphosphate as a substrate in a cyclization reaction towards a myriad of diversly complex, often polycyclic products formed with regio- and stereo- chemical precision. My current research in the Christianson group focuses on the structural and mechanistic study of a monoterpene cyclase that produces a strained bicyclic compound called sabinene. Structural comparison between sabinene synthase and its sesquiterpene counterpart sesquisabinene synthase is expected to give fundamental insight related to substrate selectivity by terpene cyclases.
Current position: In Training
Christina Hurley
(Mentor: Rahul Kohli, BMB)
Supported 6/1/2022- present
Antibiotic resistance has depleted our antibiotic arsenal and poses a critical threat to human health. Innovative strategies are required to address this growing problem. One potential approach is to target the conserved DNA damage (or SOS) response, which allows bacteria to survive upon antibiotic challenge. Most classes of antibiotics are known to induce DNA damage directly or indirectly, leading to activation of the SOS response. My research focuses on Y-family translesion synthesis (TLS) DNA polymerases, which are induced during the SOS response when survival demands that bacteria replicate over damaged DNA. To facilitate such replication, these TLS polymerases feature open active sites and lack proofreading ability. Notably, this error-prone replication contributes not only to survival, but also to mutations that can promote acquired antibiotic resistance. However, TLS activity does not always give bacteria a clear advantage. For example, one of E. coli’s TLS polymerases, DinB, readily misincorporates noncanonical nucleotides (nNTPs) such as the damaged base, 8-oxo-deoxyguanine. This activity can induce lethality upon DinB overexpression. Thus, the expression of TLS polymerases by bacteria with an activated SOS response could be an Achilles’ heel that I intend to exploit in a novel antimicrobial strategy. I plan to use nNTPs as probes to understand tolerance of DinB for different nNTP modifications and assess whether nNTPs can compete with natural nucleotides in vitro. I will also determine the effects of nNTPs on the growth of E. coli. Ultimately, I hope to identify nNTP candidates that can synergize with existing antibiotics to kill E. coli.
Current position: In Training
Ryann Perez
(Mentor: E. james Petersson, CHEM)
Supported 6/1/2022- present
Alpha-synuclein (αS) is a protein that is found abundantly in the brain and is believed to play a role in the development of Parkinson's disease (PD). In healthy individuals, αS is thought to be involved in the formation of lipid vesicles at presynaptic termini. However, in individuals with Parkinson's disease, αS misfolds and forms clumps known as fibrils, which can lead to the death of neurons in the brain. In the Petersson Lab, one of our focuses is on developing therapeutics for PD by targeting the αS protein. The lab uses a combination of experimental and computational techniques to identify compounds that can inhibit the formation of αS fibrils or promote their disassembly. I am focused on creating experimental techniques adapted for this protein, such as high-throughput screening using fluorescence anisotropy, which may quickly identify compounds that bind to αS and affect its aggregation behavior. Within the computational space, I implement various machine learning algorithms and simulations to analyze large datasets and gain insights into the molecular mechanisms that underlie αS aggregation. By combining experimental and computational approaches, I hope to develop new therapeutics that can prevent or reverse the effects of αS aggregation in PD and further understand the underlying mechanisms which cause protein self-assembly.
Current position: In Training
Evan Yanagawa
(Mentor: E. James Petersson, CHEM)
Supported 1/1/2024- present
Neurodegenerative diseases such as Parkinson's disease (PD) are known to be associated with the aggregation of the neuronal protein, alpha-synuclein. Alpha-synuclein is an intrinsically disordered protein that can adopt a B-sheet rich, fibrillar structure in PD pathology. A central focus of the Petersson lab is analyzing how various post-translational modifications (PTMs) effect the formation of fibrillar aggregates of alpha-synuclein. My work in the Petersson lab focuses on studying how the N-terminus of alpha-synuclein, and associated PTMs, impacts fibril formation. Preliminary data has shown that the N-terminus, and acetylation of the N-terminus may play a major role in the formation of alpha-synuclein fibrillary aggregates, providing the basis for the two projects that I have worked on thus far. Through the incorporation of thioamide isosteres, I hope to better understand how each of the three main regions of alpha-synuclein (N-terminal, C-terminal, and NAC region) contribute to fibril formation. In collaboration with the Rhoades, Shalem, and Marmorstein lab I am working to develop a bisubstrate inhibitor of a native acetyltransferase of alpha-synuclein, NatB, to attenuate N-terminal acetylation and possibly perturb fibril formation.
Current position: in Training
Past CBI Fellows
Julie Aaron
julie.himmelberger@desales.edu
(Mentor: David Christianson, Ph.D., CHEM)
Supported 9/1/2006–8/31/2009
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.
Current position: Associate Professor and Chair, DeSales Univ.
Mina Ahmadi
(Mentor: Ivan Dmochowski, Ph.D., CHEM)
Supported 6/1/2021- 8/31/2023
In 2020, Dmochowski's lab published a paper on using a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) to quantify the intracellular concentration of ribose. Using this technique in parallel with LC-MS analysis, it was determined that the ribose concentration in HeLa cells is 5 mM, which is 3 orders of magnitude higher than a previous estimate. This unexpectedly high ribose concentration raises multiple questions regarding the biodistribution and biosynthesis of ribose, including whether a high concentration of ribose is a cancer biomarker. To answer these questions, my main research goal is to focus on mass spectrometry to take advantage of the high throughput screening of numerous cell sample sets as well as the possibility of looking at multiple metabolites within the same sets. I aim to develop an analytical method to quantify both ribose and its counter partner, ribose-5-phosphate, to compare endogenous levels of these metabolites in different mammalian cells, healthy or cancerous. Following these initial goals, I want to investigate whether and how these metabolites are interchanging with each other. To address these further questions, this project will be expanded by designing and synthesizing a ‘clickable’ ribose probe containing a photo-reactive chemical group, such as diazirine, which could be used in metabolic labeling to identify ribose-interacting protein targets.
Current position: In Training
Edward Ballister
(Mentor: Shelley Berger, Ph.D., BMB)
Supported 7/1/2009–8/31/2010
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.
Current position: Research Scientist, New York, NY
Taylor Barrett
(Mentor: James Petersson, Ph.D., CHEM)
Supported 9/1/2016– 8/31/2018
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.
Current position: In Training
Stephanie Barros
(Mentor: David M. Chenoweth, Ph.D., CHEM)
Supported 1/1/2012–12/31/2013
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.
Current position: Postdoctoral Fellow with Paramjit Arora (New York University)
Katelyn Bustin
(Mentor: Megan Matthews, Ph.D., Chemistry)
Supported 1/1/2019-12/31/20
Reverse-Polarity Activity Based Protein Profiling (RP-ABPP)identified a novel N-terminal glyoxylyl modification derived from a cysteine residue on Secernin3 (SCRN3), an uncharacterized human protein predicted to use the cysteine as a nucleophile for hydrolase activity. My project in the Matthews lab aims to determine the installation and processing, structure and function, and scope of this novel modification using biological and chemical principles while utilizing advanced chemical proteomics methods for multiple analyses. Molybdenum cofactor sulfurase (Mocos), known to have both pyridoxal-5’-phosphate (PLP) and molybdopterin catalytic domains, was identified to have specific interactions with SCRN3 and is being investigated as an installation machinery for the glyoxylyl modification. Crystallization of wild-type SCRN3, and variants is being explored, while reconstitution of the glyoxylyl modification via meta-Periodate oxidation is conveying spatial and possibly functional information for the glyoxylyl group. To explore the prevalence of this novel glyoxylyl modification, I am working with Clostridium Difficile (in collaboration with Michael Abt’s lab), which is an obligate anaerobe and human pathogen known to use metabolic reductases for amino acid anaerobic-specific Stickland fermentation. These reductases possess elusive carbonyl cofactors, some cysteine derived, that function in substrate binding and specificity. The literature suggests that glyoxylyl moieties are likely to present. The activity of these reductases is currently being characterized utilizing RP-ABPP in vivo via conjugation to isotopically differentiated biotin tags.
Current position: In Training
Diana Cabral
(Mentor: Barry Cooperman, Ph.D., CHEM)
Supported 9/1/2006–2/1/2008
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.
Current position: Regulatory Affairs Analyst at Pegasus Laboratories Inc.
Morgan DeSantis
(Mentor: James Shorter, Ph.D., BMB)
Supported 6/1/2010–5/31/2012
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.
Current position: Assistant Professor, University of Michigan
Andrea Detlefsen
(Mentor: Trevor Penning, Ph.D., BMB)
Supported 6/1/2020- 5/31/2022
Prostate cancer (PC) is the second most common cancer among men in the United States. It occurs most often in men over 50, and disproportionately affects some ethnic groups, suggesting genetic predispositions and possible pharmacogenetic factors. PC tumor growth is fueled by activation of androgen receptor (AR) signaling. Despite initial treatment efforts, patients often develop the lethal form of the disease termed castration resistant prostate cancer (CRPC). To combat CRPC, second generation therapies have been developed to inhibit the AR directly or obstruct upstream androgen biosynthesis, termed androgen receptor signaling inhibitors (ARSI). Unfortunately, drug resistance to these therapeutics often emerges. The mechanism(s) of resistance are still under debate and must be understood to develop therapies to fight CRPC.
My research will address these unknowns by exploring two mechanistically-linked hypotheses of ARSI drug resistance. First, I will investigate the role AKR1C3, a prominent androgen biosynthesis enzyme, in the conversion of weak androgen precursors to potent AR activating androgens. I will achieve this by measuring androgens from a panel of prostate cancer cell lines using a quantitative mass spectrometry-based approach. Second, I will determine if naturally occurring variants of AKR1C3 have biochemical differences that merit their consideration in precision therapy. I will compare kinetic parameters of AKR1C3 variants to WT using RP-HPLC and stopped-flow analyses and investigate in vitro and in cell protein stability through various approaches including circular dichroism. The expected outcome of this research would validate AKR1C3 as having a causal role in resistance development and would offers advances in precision treatment of CRPC through recognition of patients who would or wouldn’t benefit from AKR1C3 targeted therapies based on identification of possible loss-of-function AKR1C3 variants.
Current position: In Training
Sebastian Dilones
(Mentor: George Burslem, BMB)
Supported 6/1/2022- present
Post-translational modifications (PTMs) drastically increase protein diversity and contribute to protein function in variety of ways, one prominent one being the agonism of protein-protein interactions. Acetylation, the addition of acetyl groups to predominantly lysine residues, is one of three most reported PTMs which, along with phosphorylation and ubiquitination, make up around 90% of all reported PTMs in cells. Recent developments in the chemical biology field seek to selectively install PTMs by harnessing the potential of chemically induced proximity; proteolysis targeting chimeras (PROTACs) is one prominent example of the power of this technology. Using PROTACs as proof of concept, I will be designing and synthesizing a heterobifunctional molecule capable of recruiting a deacetylase with the hope of inducing deacetylation on a target protein.
Current position: In Training
Daniel Emerson
(Mentor: Ivan Dmochowski, Ph.D., CHEM)
Supported 6/1/2011-5/31/2012
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.
Current position: Research Assistant, University of Pennsylvania
Jean Etersque
(Mentor: Mark Sellmyer, M.D., Ph.D., BMB)
Supported 6/1/2020- 6/1/2022
My research focuses on the development of molecular imaging probes for applications in Positron Emission Tomography (PET). My research goal is to engineer a library of unique protein-tags derived from E. coli dihydrofolate reductase to accept complementary radiolabeled probes, which are analogs of the small molecule, Trimethoprim. This library of reporter tools can be used to monitor distinct cell populations in deep tissue in vivo.
Current position: In Training
Daniela Fera
(Mentor: Ronen Marmorstein, Ph.D., The Wistar Institute)
Supported 9/1/2006–8/31/2008
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.
Current position: Assistant Professor, Swarthmore College
Clare Gober
(Mentor: Madeleine Joullie’, Ph.D., CHEM)
Supported 1/1/2014–6/30/2016
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.
Current position: Medical Writer, Synchrony Group
Sara Goldstein
(Mentor: Jeffrey Winkler, Ph.D., CHEM)
Supported 6/1/2014-5/31/2016
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.
Current position: Postdoctoral Fellow with Anthony Barrett (Imperial College UK)
Gabriel Gonzalez
(Mentor: William DeGrado, Ph.D., BMB)
Supported 6/1/10–5/31/2011
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.
Current position: Software Engineer, Awake Security
Leah Gottlieb
(Mentor: Ronen Mamorstein, Ph.D., CHEM)
Supported 1/1/2015-12/31/2016
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.
Current position: Postdoctoral fellow with Ian Blair (PSOM)
Zachary Hostetler
(Mentor: Rahul Kohli, M.D., Ph.D., CMB)
Supported 1/1/2016-12/31/2017
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.
Current position: Medical Resident, Weill Cornell Medical School
Christopher Johnny
(Mentor: David Chenoweth, Ph.D., Chemistry)
Supported 1/1/2020- 12/31/2021
Recently, the Chenoweth laboratory has shown the emerging versatility of the diazaxanthylidene scaffold as a promising basis for fluorescent dye development. In 2013 the lab synthesized the reported structure of Xylopyridine A (a diazaxanthylidene natural product). In 2015, the lab discovered Xylopyridine A represented a new emissive and photoconvertible dye class, displaying low toxicity and good cell permeability. Furthermore, the lab then showed that Xylopyridine A could be used to selectively image lysosomes in HeLa cells. Most recently, in 2018 the lab then showed that a derivative of Xylopyridine A could be alkylated with an azide handle and subsequently used for the in vivo tracking of alpha synuclein fibril formation via copper mediated click conjugation to alpha synuclein monomers.
My research efforts currently focus on (1) synthesizing and evaluating the utility of the diazaxanthylidene dye alkylated with an IEDDA (Inverse Electron-Demand Diels-Alder) reactive handle for fast copper free conjugation to biomolecules, (2) performing experimental and theoretical studies of “push-pull” analogs towards discovery of ultra-bright, ultra-responsive spatio-temporily photoconvertible dyes, (3) and continuing the investigation of diazaxanthylidene molecules as small molecule alternatives to photoconvertible proteins like Kaede
Current position: In Training
Hee Jong Kim
heejong@pennmedicine.upenn.edu
(Mentor(s): Kenji Murakami & Ben Garcia, Biochemistry/Biophysics
Supported 6/1/2018- 6/1/2019
Investigation of HIR Complex's structural and functional aspects through crosslinking mass spectrometry and cryo EM.
Current position: In Training
Kelly Karch
(Mentor: Benjamin Garcia, Ph.D., BMB)
Supported 1/1/2015–12/31/17
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.
Current position: Postdoctoral fellow, Ohio State University
Yekaterina Kori
(Mentor: Benjamin Garcia, Ph.D., BMB)
Supported 6/1/2018- 6/1/2019
Gene expression is mediated by nucleosomes, which are protein-DNA complexes consisting of 147 base pairs of DNA wrapped around a histone octamer containing two copies of the canonical histones H2A, H2B, H3, and H4. Histone variants, such as H3.3, can replace canonical histones in nucleosomes to alter gene accessibility. Gene expression can also be modulated through post-translational modifications (PTMs) to histone N-terminal tails, which serve to alter chromatin compaction and recruit transcriptional activators or repressors. I am interested in understanding the role of methylation at lysine 27 on the variant histone H3.3 (H3.3K27me3) in pluripotency and early development. Previous literature has implicated H3.3 as being essential in early embryonic development. Additionally, H3.3 is enriched at promoters, suggesting an important regulatory role for H3.3 in modulating gene expression. My preliminary work has detected methylation on H3.3K27 during pluripotency of mouse embryonic stem cells (mESC), but the levels of methylation decrease upon mESC differentiation. Since H3.3 has been shown to be associated with active genes, it is surprising to observe this activating histone variant with the repressive K27me3 mark. To probe the role of H3.3K27me3 during this early developmental transition of pluripotency to differentiation, I aim to study the dynamics, localization, and potential regulatory mechanism of this histone variant modification using a variety of methods in mass spectrometry, genomics, and biochemistry.
Current position: In Training
Glen Liszczak
(Mentor: Ronen Marmorstein, Ph.D., The Wistar Institute)
Supported 6/1/2010–5/31/2012
Biochemical, chemical and enzymatic studies are being used to probe the mechanism of N-terminal protein acetylation by the ternary NAT5/ARD1/NATH complex.
Current position: Assistant Professor. University of Texas, Southwestern
Robert Magin
(Mentor: Ronen Mamorstein, Ph.D.)
Supported 6/1/2014-5/31/2016
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.
Current position: Postdoctoral Fellow with Sara Buhrlage (Harvard University/Dana-Farber Cancer Institute)
Zach March
(Mentor: James Shorter, Ph.D., CHEM)
Supported 7/1/2016–6/31/2016
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.
Current position: In Training
Bryan Marques
(Mentor: A. Joshua Wand, Ph.D., BMB)
Supported 6/1/2013-5/31/2015
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.
Current position: Research Scientist, Janssen Pharmaceuticals
Kristen McKibben
(Mentor: Elizabeth Rhoades, Ph.D., CHEM)
Supported 9/1/2016–8/31/2018
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.
Current position: Les Diablerets, Switzerland
Nataline Meinhardt
(Mentor: Doron Greenbaum, Ph.D., BMB)
Supported 6/1/2010–5/31/2012
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.
Current position: Account Executive, Clinical Thinking
Sean Mulcahy
(Mentor: Eric Meggers, Ph.D., CHEM)
Supported 9/1/2005–8/31/2007
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.
Current position: Associate Professor, Providence College
Cristian Ochoa
(Mentor: Marisa Kozlowski, Ph. D., CHEM, BMB)
Supported 9/1/2017- present
Honokiol and its constitutional isomer, magnolol, are biologically active organic molecules. These bisphenols inhibit the growth of Streptococcus mutans, the bacteria that causes dental cavities. The Kozlowski group has devised a selective phenol cross-coupling strategy that has been used to accomplish syntheses of honokiol amenable to product scale. We have probed the structure-activity relationships of honokiol by generating different bisphenolic analogs using our cross-coupling method. We plan on growing and testing these analogs against a variety of bacteria in a microaerophilic environment that mimics the conditions of a human mouth. This would give us a better understanding of the features necessary for anti-bactericidal and anti-biofilm activity.
Current position: In Training
Erin Podlesny
(Mentor: Marisa Kozlowski, Ph.D., CHEM)
Supported 9/1/2009–8/31/2010
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.
Current position: Assistant Professor, Stockton University
Nick Porter
(Mentor: David Christianson, Ph.D. CHEM)
Supported 9/1/2017-8/31/2018
Reversible lysine acetylation is a critical and conserved molecular strategy for the functional regulation of thousands of histone and non-histone proteins. This post-translational modification is reversed by histone deacetylases (HDACs), which we study in the lab of Prof. David W. Christianson. Since aberrant lysine acetylation results in dysregulation of cellular processes, developing a chemical understanding of the proteins that govern this regulatory process is crucial. My research focuses on using X-ray crystallography along with various other analytical techniques, such as isothermal titration calorimetry and mass spectrometry, to study the class I enzyme HDAC8 as well as the class IIb enzyme HDAC6, also known as tubulin deacetylase. Using these as our paradigm systems, we solve crystal structures that yield new insight regarding atomic-level features of enzyme mechanism, substrate recognition, and isozyme-selective inhibition.
Current position: In Training
Jennifer Ramirez
(Mentor: Elizabeth Rhoades, Ph.D., CHEM)
Supported 6/1/2020- 5/31/2021
Age-related neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) take an overwhelming toll on individuals and society and remain incurable. Traditionally, misfolding and aggregation of a single protein (tau in AD and αS in PD) was thought to be responsible for causing these different pathologies. However, there is increasing evidence that the pathologies of these two diseases overlap and the individual proteins primarily associated with each disease promote each other’s aggregation, adding to the complexity of these pathologies. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary structures under physiological conditions. IDPs challenge the structure-function paradigm as they perform important functions in a variety of cellular processes. Studying the structural ensembles of IDPs requires the development of new biophysical methods, as IDPs often stymie traditional structural techniques. Further adding to the complexity is that many IDPs have post-translational modifications (PTMs), which can alter structure/function and often represent the physiologically relevant forms of the proteins. In vitro expression systems are typically unable to include PTMs; chemical biology approaches to introduce PTMs offer an attractive means of introducing PTMs to allow for the study their effects on the conformational ensembles of IDPs and their interactions with binding partners.
My project would investigate the αS-induced aggregation of tau to provide insight into the overlap of AD and PD pathology mechanisms. Taking into consideration the role of disease relevant PTMs and mutations through a combination of chemical biology and biophysical methods. Complementing traditional aggregation and cellular studies with a combination of chemical and biophysical approaches such as NCL, single molecule Förster resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) will provide mechanistic insights to characterize conformations and binding of these proteins.
Current position: In Training
Nicole Raniszewski
(Mentor: George Burslem, BMB)
Supported 9/1/2021- 8/31/2023
The Burslem lab is broadly interested in lysine post-translational modifications (PTMs), including acetylation and ubiquitination. Ubiquitin is a 76-amino acid small protein that is added post-translationally to target protein lysines, including its own 7 lysine residues, thereby generating poly-ubiquitin. Polyubiquitin chains linked at each of these lysines have been discovered in cells, and these chains each have unique topologies as well as diverse roles in cellular processes. For example, K48-linked poly-ubiquitin is implicated in protein degradation, while K63-linked poly-ubiquitin is implicated in DNA damage response. Despite efforts to understand the “ubiquitin code”, there are many unanswered questions in this field, largely due to limitations in the generation and detection of specifically-linked poly-ubiquitin chains. Through a chemically-controlled enzymatic approach, I will be developing a new strategy for robust and facile generation of custom poly-ubiquitin chains. For this project, I will be developing both 1) solution phase and 2) solid phase synthetic approaches, resulting in novel methods for convenient, high purity, and high yielding generation of custom poly-ubiquitin chains.
Current position: In Training
Julia Richards
(Mentor: Ivan Dmochowski, Ph.D., CHEM)
Supported 9/1/2005–8/31/2008
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.
Current position: Scientist, Eurofins
Harry Schroeder, III
(Mentor: Yale Goldman, Ph.D., BMB)
Supported 9/1/2008–8/31/2010
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.
Current position: Physician, Radiology, Massachusetts General Hospital
Kollin Schultz
(Mentor: Ronen Marmorstein, Ph.D., BMB)
Supported 6/1/2020 - 5/31/2022
My project focuses on the cytosolic enzyme Fatty Acid Synthase (FASN), which is a large multifunctional enzyme with 6 catalytic domains and a flexibly tethered acyl carrier protein (ACP) that shuttles covalently linked intermediates between catalytic domains to facilitate palmitate synthesis. In normal physiological conditions, FASN expression is tightly regulated and is abundant only in lipogenic tissues. Overexpression of FASN has been found in multiple metabolic diseases, including cancer. The low expression in healthy tissue and upregulation in cancer has made FASN a promising therapeutic target, but there are no currently approved FDA drugs targeting FASN. Inhibition of FASN with small molecules or genetic knock outs has shown promising pre-clinical results in multiple cancer types. As an essential enzyme with therapeutic potential, many previous studies have worked towards understanding the molecular mechanism of palmitate synthesis and regulation of FASN. While these projects have well characterized the mechanism of each catalytic domain, they failed to resolve the molecular basis for intermediate shuttling by the ACP. I am working towards filling this gap in knowledge on FASN using single particle analysis cryo-electron microscopy (cryo-EM), hydrogen deuterium exchange mass spectrometry (HDX-MS), and complementary enzyme assays to elucidate the molecular details of ACP interaction with the catalytic domains during palmitate synthesis. Ultimately, I hope my work will provide novel insights into the complex mechanism of palmitate synthesis by FASN with implications for FASN-mediated therapy.
Current position: In Training
Juan Serrano
juan.serrano@pennmedicine.upenn.edu
(Mentor: Rahul Kohli, M.D., Ph.D., BMB )
Supported 6/1/2019- present
Modifications of cytosine within genomic DNA have been found to play a role in embryogenesis, gene regulation and a potential pathological role in cancer. My research focuses on developing chemical tools to study the activities of two cytosine modifying enzymes, TET and APOBEC3A.
TET enzymes are involved in the iterative oxidation of methylcytosine into substrates that can undergo active demethylation or potentially serve as independent epigenetic markers themselves. Using my past synthetic experience, I am aiming to develop cytosine analogues which can serve as either reporters of activity or probes to trap TET in its active state. For the latter aspect, the goal will be to isolate a TET-DNA complex formed in vivo, which will be used to perform proteomic analysis to potential TET regulatory partners or to directly localize its site of action in genomes.
APOBEC3A is a DNA cytidine deaminase and is implicated in antiviral defense within human cells and has been recently shown to play a role in mutagenesis in a variety of malignancies. By incorporating the potential mechanism-based inhibitor into a DNA double hairpin, a secondary structure with particular affinity for APOBEC3A, I aim to develop stable and specific oligonucleotide inhibitors. These nucleic-acid based inhibitors would allow one to not only probe the activity of APOBEC3A in a cellular context, but serve as a platform for development of adjuvant anticancer therapy.
Current position: In Training
Scott Ugras
(Mentor: Harry Ischiropoulous, Ph.D., PHARM, BMB)
Supported 6/1/2012–5/31/2014
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.
Current position: Associate at McKinsey & Company
José Villegas
(Mentor: Jeffrey Saven, Ph.D., CHEM)
Supported 6/1/2013-5/31/2015
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.
Current position: “bridge to faculty” position at the University of Illinois, Chicago in the Department of Pharmaceutical Sciences
Sarah Welsh
(Mentor: Alessandro Gardini, Ph.D., WISTAR)
Supported 9/1/2017- present
My research in the Gardini lab focuses on the novel metazoan protein complex, Integrator (INT). This multi-‐subunit complex was discovered to associate with RNA Polymerase II and has been found to have important roles in the biogenesis of enhancer RNAs. The lab has identified that INT is necessary for correct differentiation of hematopoietic stem cells, such that loss of single subunits of INT is sufficient to push differentiation towards different cell types. However, not much is known about the structure of the complex. My preliminary findings indicate that INT may exist as multiple sub-‐complexes with different functions throughout stem cell specification. Using a myeloid progenitor cell line as a model of hematopoiesis, I aim to determine the subunit composition and the contribution of the individual subunits to the functions of INT throughout differentiation.
Current position: In Training
Rebecca Wissner
Mentor: James Petersson, Ph.D., CHEM)
Supported 1/1/2011–12/31/2012
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.
Current position: Postdoctoral Fellow with Alanna Schepartz (Yale University)
Lyndsay Wood
(Mentor: Jeffrey D. Winkler, Ph.D., CHEM)
Supported 1/1/2012–12/31/2013
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.
Current position: Research Scientist, Dow Pharmaceuticals