Phillip Bartels, PhD | Mentor: Yitzhak Tor
Developing Guanidinylated Neomycin-Enzyme Conjugates to Treat Lyososomal Storage Disorders Linked to Parkinson’s Disease
β-glucocerebrosidase (GBA) is a lysosomal enzyme that degrades glucosylceramide, a biomolecule believed to stabilize the toxic protein aggregates that lead to Parkinson's disease. Genetic variations that decrease β-glucoce (GBA) activity have been linked to an increased risk of Parkinson's disease. One strategy to restore lysosomal function is to deliver active recombinant GBA directly to neurons, but lysosomal targeting can present a challenge. Guanidinylated neomycin (GNeo), however, facilitates lysosomal localization when covalently appended to proteins. Our approach is thus to prepare GNeo-GBA conjugates, test their activity
in vitro and confirm lysosomal localization and activity in cultured neurons. Once optimized, the
in vivo effectiveness of GB/GNeo conjugates can be assessed using a mouse model system.
Julia Callender, PhD | Mentor: Christina Sigurdson
Manipulating PrP glycan structure to understand toxic signaling pathways driving prion-induced neurodegeneration
Prions cause a rapidly progressive neurodegenerative disease characterized by an exponential increase in prion aggregates as well as a spongiform encephalopathy, dystrophic neurites, and neuronal death. These processes depend on the neuronal expression of the cellular glycoprotein, prion protein, PrPC, however, the mechanism through which PrP signaling or aggregation contributes to neurodegeneration remains unclear. Previous work has shown that glycan modifications may impact PrP aggregation and neuronal toxicity. To investigate the role of glycans in prion-induced toxicity, we have engineered a new knockin mouse model introduces an additional glycan on the amino terminus of PrP. This mouse develops spongiform encephalopathy in the absence of PrP aggregates, thus uncoupling PrP aggregation from neurotoxicity. Here we will use this new mouse model to investigate the mechanism through which PrP glycans contribute to prion-mediated neurotoxicity and neurodegeneration.
Dillon Chen, MD | Mentor: Ajit Varki
Sun-Mi Choi, MD, PhD |
Mentor: Victor Nizet
Upper airway mucosal responses to MRSA in CRS
Chronic Rhinosinusitis (CRS) affects 10% of adults and Staphylococcus aureus (SA) colonization is increased in CRS. One hypothesis to delineate the role of SA in pathogenesis of CRS is that SA stimulates production of c-type lectin Reg3g which binds to peptidoglycan layer of Gram+ bacteria along with type 2 inflammatory cytokines (thymic stromal lymphopoietin [TSLP] and IL-33) that causes hyper production of mucins (Muc5AC and Muc5B) resulting in chronic inflammatory state involving aberrantly activated innate immune and T cells.
Michelle Ducasa, PhD |
Mentor: Philip Gordts
The role of heparan sulfate proteoglycans in non-shivering thermogenesis
The present worldwide obesity epidemic is associated with a sharp increase in its metabolic complications, including a dramatic surge in Type 2 Diabetes. Current pharmacological therapies focus on the regulation of energy intake by curbing appetite or reducing the craving for food, although in many cases these therapies have significant and unacceptable cognitive or psychiatric side effects. Furthermore, reducing energy intake can lead to repression of energy expenditure. Thus, increasing energy expenditure has emerged as another approach to treat obesity. Most of this energy expenditure takes place in mitochondria, where energy is generated through the electron transport chain, which creates a proton gradient across the inner membrane to drive the synthesis of ATP. This process can be uncoupled, resulting in the consumption of energy and generation of heat. Thermogenic uncoupling of mitochondrial oxidative phosphorylation is largely but not exclusively mediated by the thermogenic protein UCP1 in brown and beige fat. Activating and recruiting brown and beige fat is a major pharmacological focus for obesity and the associated metabolic disorders. Previous studies have shown that adipocyte glypican-4 (GPC4), a GPI-anchored Heparan sulfate proteoglycan (HSPG), serves as an insulin sensitizing factor and marker for insulin sensitivity and syndecan-1 (SDC1), a transmembrane HSPG, is essential for intradermal adipocyte differentiation. Their role in cytokine and growth factor presentation and their abundance in the extracellular environment place HSPGs in a unique position to modulate adipocyte phenotypes. BMP7 is one of the key regulator of cold-induced BAT thermogenesis and beige adipocyte induction, but also a HS-binding protein that requires HSPG at the cell surface to exert its biological activity. We showed previously that activation of BAT leads to a reduction in atherosclerosis development and that LPL fueling of brown adipose tissue upon cold activation involves an initial heparin-sensitive binding. Based on these observations and some of our preliminary data we will investigate using genetic targeting of HS biosynthetic enzymes in cell and animal models in the following two aspects: 1) We will test if heparan sulfate regulates the thermogenic activity and capacity of established BAT depots. 2) We will evaluate to what extent adipose heparan sulfate influences recruitment of beige adipose tissue and transdifferentiate a white adipocyte into a beige adipocyte. The overall goal is to establish that natural variation in heparan sulfate composition can affect the thermogenic potential of brown and beige adipose tissues.
So-Young Kim, PhD | Mentor: Mark Fuster
Glycosaminoglycan-based lung cancer immunotherapy
Lung cancer is the leading cause of cancer death in the U.S. and worldwide. Dendritic cells (DCs) recognize tumor antigen and instruct T cells to initiate antitumor immunity. In a tumor microenvironment, however, dendritic cells are suppressed from properly expressing mature phenotype to carry on this function. Heparan sulfate (HS) glycosaminoglycan, when altered on the DC surface, restores mature DC phenotype and inhibits immature DC trafficking to the draining lymph nodes demonstrating anti-tumor therapeutic potential, however, its effect on T cell associated lung cancer immunity has not been previously investigated. In my studies, I will uncover how DC specific HS alteration will influence the antitumor associated T cell function and downstream signaling pathways. Additionally, genetic and chemical strategies will be developed targeting DC HS.
Ryan Porell, PhD | Mentor: Kamil Godula
Design and Synthesis of Fluorogenic Proteoglycan Mimetic Polymers
Our goal is to use these fluorescent polymers bearing heparan sulfate (HS) chains to better understand how heparan sulfate impacts the pathophysiology of Alzheimer's disease. This HS-polymer will be modified with a lysosome-targeting molecule to hopefully clear the amyloid plaques upon binding to the HS chains.
Ruth Siew, MD | Mentor: Victor Nizet
Sialic acid interactions in cystic fibrosis
Cystic fibrosis is caused by a mutation in the CFTR gene that results in a multi-organ system disease with the primary cause of morbidity from severe, chronic respiratory infections. The opportunistic pathogen,
Pseudomonas aeruginosa, is an early colonizer of the cystic fibrosis lung and causes inflammation and structural lung damage. It produces sialidase as a pathogenic virulence mechanism where desialylation of the airway mucosal surface allows for the colonization of
P. aeruginosa and biofilm formation. There has been limited research in the area of bacterial sialidase as it contributes to the pathogenesis of infection, therefore I am interested in further exploring bacterial sialidase as it relates to the host immune system. CFTR function has also been linked to the sialic acid
N-glycolylneuraminic acid (Neu5Gc), which differs from the human-dominant form N-acetylneuraminic acid (Neu5Ac) by one oxygen atom due to lack of the enzyme CMP-N-acetylneuraminic acid
hydroxylase (CMAH). I seek to understand how this difference applies to lung epithelial cells with the CFTR mutation. As the Varki lab has had extensive experience with this glycosylation change from Neu5Gc to Neu5Ac, they have also been successful in recapitulating a cholera-like phenotype in a mouse model using a
Cmah null mouse. There are currently no mouse models available that have been able to simulate chronic lower respiratory tract infections such as those seen in humans. By crossing the Cmah mouse line with the most common mutation in cystic fibrosis (CFTR delF508), I hope to create a humanized mouse model of disease for cystic fibrosis.
Richard Sanchez, PhD | Mentor: Gulcin Pekkurnaz
O-GlcNAc dependent cytoskeletal influences on mitochondrial localization under varying cellular metabolic states
Intracellular O-GlcNAc is a single unelongated glycolytic molecule that is added on to proteins as a post-translational modification (PTM) on serine and threonine residues. The enzymes responsible for the addition and removal of O-GlcNAc are O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) respectively. In eukaryotes OGT is a major nutrient sensing enzyme that is expressed in every cell type but is most abundantly expressed in the brain and pancreas. OGT’s catalytic activity is regulated by intracellular concentrations of UDP-O-GlcNAc which is synthesized by varying metabolic substrates through the hexosamine biosynthesis pathway allowing its concentration to fluctuate in response to nutrient availability. Our lab has shown that OGT can regulate mitochondrial localization and metabolic efficiency in neurons. Expanding upon these results we have identified cytoskeletal regulating protein candidates that are susceptible to O-GlcNAcylation using mass spectrometry on isolated neuronal mitochondria. My focus will be on the role of these cytoskeletal proteins in the context of mitochondrial positioning and metabolic compartmentalization under the influence of O-GlcNAcylation. In addition, even though OGT is a single gene it codes for three splice isoforms all of which are capable of O-GlcNAcylating proteins. I will define which OGT isoforms are influencing metabolic efficiency. Overall, these studies will provide a more comprehensive understanding of the metabolic regulatory factors that influence cellular metabolic homeostasis, helping in elucidating human metabolic disorders that range from diabetes to neurodegeneration.
Ryan Weiss, PhD | Mentor: Jeff Esko
CRISPR-Cas9 Dissection of Heparan Sulfate
Heparan sulfate proteoglycans (HSPGs) are expressed on all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with many matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, less information exists regarding the mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to understand the genetic factors that control the formation of heparan sulfate in mammalian cells. A genome-wide CRISPR/Cas9-mediated screen will be developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library will be utilized to knock down gene expression across the entire genome in a human cancer cell lines. Subsequently, a high-throughput screen will be adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs or decrease binding of HS-dependent ligands to the cell surface. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biosynthesis, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases.