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2017 ChEM-H Undergraduate Research

The 2017 ChEM-H Undergraduate Scholars and their postdoc mentors. Front row: Karen Dai, Dr. Joe Knoedler, Dr. Kasper Karlsson, Dr. Paul Allegretti, Matthew Caffet. Back row: Alli Keys, Dr. Daniel Le, Anthony Agbay, Dr. Ben Schumann, Matthew Carter, Dr. Fred Poitevin, Abigail Zuckerman.

Research Project Descriptions

Protein crystallization of bump-hole engineered polypeptide N-acetylgalatosaminyltransferase

Undergraduate Scholar: Anthony Agbay, Bioengineering Major
Postdoc Mentor: Dr. Ben Schumann
Research Group: Prof. Carolyn Bertozzi, Department of Chemistry

Mucin-type O-glycosylation is a post-translational modification of proteins that is involved in various biological processes, from development to cellular signaling, and has been linked to different disease states, such as cancer. Mucin-type O-glycosylation is initiated by the addition of a N-acetylgalactosamine (GalNAc) sugar to an amino acid residue on the protein through the action of an enzyme called polypeptide GalNAc transferase (ppGalNAcT). There are more than twenty different isoenzymes that catalyze the transfer of GalNAc to overlapping, but distinct sets of protein substrates, making it difficult to study the exact function of each isoenzyme. One approach to studying this enzyme that is being explored in the Bertozzi Lab is to use a technique called bump-hole engineering to modify one isoform to accept a specific synthetic substrate that can be labeled and imaged. After expressing and purifying the engineered protein in milligram quantities, we are working on obtaining the crystal structure in collaboration with the ChEM-H Macromolecular Structure Knowledge Center through x-ray diffraction. With this structure, we will be able to evaluate the conservation of substrate specificities and investigate the molecular and structural characteristics of the bump-hole engineered enzyme to optimize future substrates.

Molecular dynamics simulations of antibiotic binding to the 30S ribosomal subunit and subsequent structural response

Undergraduate Scholar: Abigail Zuckerman, Biology Major
Postdoc Mentor: Dr. Fred Poitevin
Research Group: Prof. Michael Levitt, Department of Structural Biology

The ribosome is at the crux of the central dogma of molecular biology, and as a target for drugs, particularly antibiotics designed to disrupt translation in bacteria, it has far-reaching consequences for human health. Aminoglycoside antibiotics act by binding to the 30S subunit of the ribosome, and though effective in killing bacteria some can also predispose infants with mitochondrial ribosome point mutations to hearing loss. Despite growing structural and experimental data, perturbations to the ribosome, such as antibiotic binding, still resist interpretation at a molecular level. Molecular dynamics can help bridge the gap between experimental data and proposed molecular movements because it can achieve a resolution not achievable through experiments. We used atomistic scale molecular simulations to the 30S ribosome bound with aminoglycoside antibiotics to better understand the mechanism of action of the antibiotics, and apply these data to develop a protocol of coarse grain simulations of this system. This allowed us to characterize the coupling between local and global conformational response to perturbations such as point mutation or antibiotic binding through simulation. We established a set of computational tools that can be given an atomic resolution ribosome structure as input, and output its dynamic response to a chemical perturbation, such as a drug. This will function as a predictive tool that will help explain the molecular basis of drug action, and specifically help guide the design of non-ototoxic ahminoglycosides that still retain their antibiotic activity.

Designing small molecules that promote human beta cell replication as a treatment for diabetes

Undergraduate Scholar: Matthew Caffet, Chemical Engineering Major
Postdoc Mentor: Dr. Paul Allegretti
Research Group: Prof. Justin Annes, Department of Medicine - Endocrinology

Diabetes mellitus is a rapidly growing disease that affects over 400 million adults worldwide, and is implicated in over 3.7 million deaths a year. It is characterized by an inability of the body to manage blood-glucose levels due to decreased insulin production stemming from a loss of pancreatic beta cell mass. The focus of this project is to develop a curative treatment for diabetes by creating a small molecule that takes advantage of the unique chemical environment of the beta cell in order to selectively promote pancreatic beta cell replication. It has been shown that inhibition of the kinase DYRK1A can promote pancreatic beta cell replication. We are currently synthesizing potential inhibitors of DYRK1A, and will assess them with a DYRK1A-luciferase assay as a test for efficacy. Promising compounds will be used in animal studies where they will be evaluated for their viability as potential drugs for human patients.

An investigation into the genetic and transcriptomic markers of drug resistance in HER2 positive breast cancer

Undergraduate Scholar: Matthew Carter, Bioengineering Major
Postdoc Mentor: Dr. Kasper Karlsson
Research Group: Prof. Christina Curtis, Department of Medicine - Oncology, Genetics

It is currently understood that cancer is a disease characterized by uncontrolled cellular growth which presents itself in the form of tumors. Since cell growth is inherently self-replicating, mutations which alter the genetic and phenotypic makeup will be propagated to the next generation daughter cells. These mutations result in a large amount of intratumoral heterogeneity (ITH) which has a profound effect on the tumor’s overall phenotypic characteristics, especially in regards to drug resistance. As tumor growth continues, additional mutations may occur which can cause a particular subset of the tumor population to genetically diverge from the general tumor population. This divergent sub-population, often referred to as a sub-clonal population, then continues to grow and replicate its mutated genes. As more of these sub-clonal populations arise, each carrying unique genetic mutations, it becomes increasingly possible that a mutation will occur that provides advantages for the cancer cells and contributes to the tumor’s overall fitness. These mutations can include the deactivation of tumor suppressor genes, the activation of oncogenes that contribute to cell growth, or the alteration of certain genes providing drug resistance in a particular sub-population. Utilizing two orthogonal methods: expressed cellular barcodes for lineage tracing and CROP-seq CRISPR-screening, combined with 10X single cell RNA sequencing, we are identifying specific genes that confer drug resistance in HER2+ breast cancer, a particularly treatment resistant form of breast cancer. Identified genes are then targeted with either small molecule drugs or CRISPR based gene silencing to confirm their importance for drug resistance.

Determining the role of transcription factor binding affinity in yeast gene expression

Undergraduate Scholar: Alli Keys, Chemistry Major
Postdoc Mentor: Dr. Daniel Le
Research Group: Prof. Polly Fordyce, Department of Genetics, Bioengineering

Cells within any single organism can vary widely in behavior and function, for example, human heart cells behave very differently from human skin cells even though they both contain the same DNA. Those differences are largely due to the dynamic activity of key proteins called transcription factors that bind to DNA and regulate gene expression. Scientists have been able to determine key 6-12 base pair binding preferences for many transcription factors; however, these motifs fail to fully explain gene expression variation. Preliminary data has shown that up to five base pairs flanking the core binding regions impact transcription factor binding affinity and some transcription activities. Thus, we aimed to systematically probe how differences in flanking sequences impact transcription factor binding in cells and how those differences influence cellular phenotype. In order to do so, we developed a yeast plasmid containing a promoter region for our transcription factor to bind with randomized sequences on either side of the core binding region of the promoter. Under the control of this promoter is a fluorescent protein that is expressed when the transcription factor binds to the plasmid. Fluorescence-based sorting will be utilized to characterize and isolate cells exhibiting differential promoter activity. The genotype of these classified cell groups will be determined by high-throughput sequencing, allowing for the creation of a model that predicts transcription factor activity in cells based on sequence.

Characterizing sexually dimorphic gene expression in Esr1 neurons in the medial amygdala

Undergraduate Scholar: Karen Dai, Bioengineering Major
Postdoc Mentor: Dr. Joe Knoedler
Research Group: Prof. Nirao Shah, Department of Psychiatry and Behavioral Sciences, Neurobiology

It is known that the sex hormone estrogen, acting through its cognate receptor (Esr1), regulates the development and function of neural circuits behind many sex-typical behaviors, such as aggression and mating. Early in life, males release testosterone, which is converted to estrogen in the brain by aromatase-expressing neurons, where it masculinizes neural circuits. Females do not release estrogen until later in development, protecting them from this masculinizing effect. It is believed that this temporal difference in the presence of estrogen leads to the development of sexually dimorphic neural circuitry, which in turn underlies sexually differentiated innate behaviors in adulthood. However, the underlying molecular mechanisms of this process remain poorly understood. To study this, we conducted RNA-seq to identify sex differences in gene expression in the Esr1-expressing neuronal population in the medial amygdala, which regulates many sexually differentiated behaviors. We investigated sex differences in gene expression in this population using Translating Ribosome Affinity Purification (TRAP) followed by RNA-seq. To label Esr1-expressing neurons, we crossed mice expressing Cre-recombinase under control of the Esr1 locus to mice expressing Cre-dependent HA tagged ribosomal subunit, allowing purification of RNA specifically from these cells. We found several candidate genes that have significantly higher expression in males, including Trhr, App, Pcp4, and Snap25. Currently, we are in the process of conducting in situ hybridization on these genes to confirm the sex differences in spatial distribution and expression level. These initial findings suggest that estrogen receptor neurons show distinct gene expression profiles between males and females, including differences in genes involved in neurotransmission, cell signaling and neurological disease.