We received 15 nominations and selected five students. The V-CBI program aims to maintain a 50/50 ratio of trainees between chemistry and biological departments. The V-CBI proudly introduces these new trainees to training grant this year:
The following are the trainees appointed to the V-CBI training grant in 2021:
Crissey Cameron (Chemistry, PI Lars Plate)
Understanding protein interaction networks can give insight into the inner workings of systems biology, linking seemingly unrelated pathways to one another and deepening our understanding of the cellular processes controlled by protein interactions. Many approaches have been used in the literature to tease out static interactions of important proteins, but most fail to account for the dynamics and transient nature of many protein complexes and fail to map interactions with time resolution. Because of this, only a snapshot of the protein interactions at one point in time are accessible, painting an incomplete picture of the interactome. Chemical genetic probe molecules designed specifically to rescue a protein of interest from degradation by the proteosome allow for controlled accumulation of proteins in the cell, therefore allowing time-resolved study of a protein of interest. My research has designed a system in which a protein of interest is linked to a mutant dihydrofolate reductase (dDHFR) domain that is marked for degradation by the proteosome. This dDHFR can be rescued from degradation and allowed to accumulate in the cell using the small molecule trimethoprim. A cysteine mutation has been made near the active site in position L28, which allows for nucleophilic attack and covalent linking to nearby electrophiles. A probe molecule made up of a trimethoprim moiety, a terminal alkyne Click-chemistry handle, and an electrophile allows for rescue of the protein of interest, bioorthogonal derivatization of the probe using a fluorophore or biotin for visualization or isolation, and covalent linking of the protein of interest and the probe. This system has been validated using yellow fluorescent protein (YFP) as a model, showing time-dependent accumulation of YFP and the ability to isolate YFP from cell lysates. Further validation of this system is currently being conducted to study the time-resolved interactome of the proteins KRas and coronavirus nonstructural proteins. Mutations in KRas are associated with colorectal cancer and nonstructural proteins of coronaviruses SARS1, SARS2, and MHV are involved in organelle remodeling or immune suppression, making their interactomes relevant for investigation. By combining quantitative proteomics using TMT-tags with this time-resolved system, the sequential protein interactions can be studied.
Melanie Padalino (Chemistry, PI Jeff Johnston)
Fluorescently-labeled proteins and small molecules have been extensively adopted by biologists as powerful spectroscopic tools in microscopy. The ability to visualize the subcellular location of proteins informs mechanism of action studies, while other techniques (e.g. FRET) confirm target engagement. However, the de novo chemical synthesis of many fluorescent peptides requires a minimum of 10 steps, placing them well outside the reach of most chemical biologists. Expanding access to more structurally and functionally diverse fluorescent tools could drive the discovery of new disease pathways, as well as enhance the rigor of current approaches. Steps required for the enantioselective synthesis of non-natural amino acid donors, protection/deprotection schemes, and Fmoc-friendly chemistry for adaptation to solid phase peptide synthesis (SPPS) each contribute to overall length. The development of new chemistries that address each of these pressure points will shorten length from commercial material to custom-designed product, while delivering renewable amounts for broad deployment. In one example, we will develop a concise synthesis of a derivatized tryptophan-containing dipeptide that is projected to shorten the length by 50%. The impact is further enhanced by the ability to prepare analogues using the same tools while benefiting from the streamlined route.
Payton Stone (Chemical and Biomolecular Engineering, PI John Wilson)
Cancer immunotherapy has displayed the potential to overcome the limited therapeutic efficacy of traditional cancer treatments such as surgery, radiation, and chemotherapy. Specifically, delivery of a retinoic acid-inducible gene I (RIG-I) agonist such as 5’ triphosphate double-stranded RNA (5’ppp-dsRNA) to the tumor site can elicit a downstream signaling cascade resulting in the enhanced production of pro-inflammatory cytokines and anti-viral interferons. This in turn allows for the reprogramming of the tumor microenvironment (TME) to a more tumoricidal phenotype capable of more effectively eradicating tumor cells. However, 5’ppp-dsRNA displays poor tumoral delivery in vivo due to nuclease degradation, rapid clearance from the body, and limited access to the cell cytosol. Therefore, our lab has designed pH-responsive polymeric nanoparticles capable of encapsulating 5’ppp-dsRNA for improved delivery to the tumor site. Our lab has previously reported the ability of polymer vesicles (polymersomes) fabricated from poly(ethylene glycol)-block-[2-diethylamino)ethyl methacrylate-co-butyl methacrylate] (PEG-bl-[DEAEMA-co-BMA]) copolymers to effectively improve cytosolic delivery of drug cargo. However, this strategy has not yet been explored for delivery of 5’ppp-dsRNA. The goal of this work is to optimize a facile and highly scalable flash nanoprecipitation (FNP) method for loading of RNA into endosomolytic polymersomes. To accomplish this, we will evaluate the effect of polymer block ratio and composition on polymer self-assembly and the loading of diverse hydrophilic drug cargo, including RNA.
Jenny Tran (Biochemistry, PI Breann Brown)
Hemoglobin is a tetrameric protein essential to the oxygenation of the body by transporting oxygen from the lungs to other tissues. To perform its functions, it relies on heme, an iron-containing porphyrin that is a cofactor for each hemoglobin subunit, allowing for oxygen binding. The enzymatic pathway starts in mitochondria with the condensation of succinyl coenzyme A (sCoA) and glycine to produce 5’-aminolevulinic acid (ALA), an important heme intermediate. This reaction is catalyzed by 5’-aminolevulinic acid synthase (ALAS) and is the rate-limiting step in heme biosynthesis. Vertebrates have two isoforms of ALAS, the erythroid-specific ALAS2 and the ubiquitous ALAS1. Heme biosynthesis is strictly regulated, and under- or over-production can cause human disease. Upregulation of human ALAS1 is seen in acute hepatic porphyrias (AHPs), which encompasses four inherited diseases relating to heme biosynthesis. Since ALAS1 is a housekeeping enzyme, its function is important for basic cellular tasks, therefore mutations in ALAS1 may affect other heme-mediated processes, such as cytochrome P450 or myoglobin production. The ubiquitous quality of ALAS1 makes it a prime therapeutic target, particularly for AHPs since treatment for acute attacks is centered around reducing hepatic ALAS1 activity. Under the direction of Dr. Breann Brown, my thesis research is focused on determining a structure for ALAS1, identifying potential protein-protein interactions, and studying the biomedical implications. Furthering research on the ALAS1 structure will provide the necessary insight on its function and may introduce new opportunities for drug design.
Sarah Zelle (Chemical and Physical Biology, PIs Hassane Mchaourab and Kevin Schey)
Organelles in ocular lens fiber cells are degraded after differentiation to maintain transparency and focus light onto the retina without scattering. Therefore, it is important to preserve the clarity and correct refractive index of the lens for the entire lifetime of individuals so that vision is not affected. However, as a result of the aging process, proteins accumulate post-translational modifications and shift towards nonnative states and form hydrophobic aggregates, potentially forming cataracts. Previous work in the Mchaourab lab has studied the effect of oxidative stress on cataract formation using zebrafish lens models. Nrf2, a transcription factor that is important in the oxidative stress response, was mutated in the lens so that the fish could not respond to stress properly. These fish were crossed with knock-out crystallin fish and phenotyped. In the lens, crystallin is a highly soluble and stable protein that acts as a molecular chaperone and binds destabilized protein. My research will focus on quantifying the protein in these mutant zebrafish lenses and I will be involved in the development and optimization of the targeted mass spectrometry assay. Ultimately, the goal of this proteomics project is to investigate how the lens maintains proteostasis and reveal mechanistic clues as to how cataracts can form. A separate goal of my thesis project is to optimize an assay to measure heat shock protein activity in zebrafish lens lysate using a client protein. Once optimized, this assay can be used to quantify the binding activity of mutant zebrafish lens lines and study cataract formation.
The major requirements of the program are Fundamentals of Chemical Biology(CPB 8320), register two semesters for Graduate Seminar in Chemical Biology(CPB 8310), one cross-disciplinary course (chemistry or one from a Basic Science department or CPB/IGP programs), Rigor and Reproducibility (PHARM 8328), and participation/membership in the Chemical Biology Association of Students (CBAS) program.