果冻影院

Mentor: Daisy Sahoo, PhD
Year Entered 果冻影院: 2019
Previous Education: BS, Biochemistry, University of Wisconsin-La Crosse, 2019

Cardiovascular disease (CVD) is the leading global cause of death and often results from atherosclerotic plaque buildup in the arteries. Plaques are formed by accumulation of macrophage foam cells as an inflammatory response to oxidized low-density lipoprotein (oxLDL) infiltration into damaged endothelial cells. Prevention of foam cell formation may be a potential strategy to reduce deaths caused by CVD and is thus an area of research that needs attention. Free fatty acid receptor 4 (FFAR4), also known as G-protein coupled receptor 120 (GPR120), is a long-chain unsaturated fatty acid receptor expressed in macrophages that plays an important role in anti-inflammatory signaling. Recently, FFAR4 was identified as a potential target for protecting against the development of atherosclerosis and CVD. Our lab has generated some exciting preliminary data demonstrating that incubation of murine peritoneal macrophages with the FFAR4 agonist, GW9508: (i) decreased cholesterol accumulation and expression of CD36, an oxLDL receptor; (ii) increased macrophage cholesterol efflux and expression of cholesterol transporters, ABCA1 and ABCG1; and (iii) reduced secretion of inflammatory cytokines. While these studies used synthetic agonist treatment to demonstrate a protective role of FFAR4, the effects of global FFAR4 deficiency on processes that generate macrophage foam cells and atherosclerotic plaque remain unknown. Combining our intriguing preliminary findings with published studies, we hypothesize that FFAR4 plays a cardioprotective role by preventing pathways that lead to atherosclerosis. We have designed experiments that rely on macrophages harvested from wild-type or FFAR4-deficient mice, to investigate FFAR4-driven mechanisms that protect against macrophage foam cell formation and promote atheroprotective signaling pathways.

1. Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A
At the University of Wisconsin-La Crosse, I worked with Dr. Weaver to understand the structural and functional effects of altering the nonpolar core of hemolysin A (HpmA), a secreted protein found in Proteus mirabilis that functions as a hemolysin and targets the destruction of red blood cells. HpmA is a member of the two-partner secretion pathway, which is used by gram-negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A-component (HpmA) is translocated, folded, and activated by its cognate B-component (HpmB). To study HpmA, a truncated version of the protein, termed HpmA265, was implemented. HpmA265 consists of three structural subdomains associated with folding and unfolding: C-terminal, nonpolar core, and polar core. Together, these three subdomains form a right-handed, three sided, non-globular, parallel β-helix that functions in red blood cell lysis upon secretion outside of the cell. My research project aimed to identify parts of the nonpolar core that are important for the structure and function of HpmA265. To do this, locations within the nonpolar core were selectively targeted and modified using a standard molecular biology technique. The structural effects of mutagenesis on HpmA265 were measured by circular dichroism (CD) and the functional effects were measured via hemolysis experiments. My findings revealed that mutations in the nonpolar core destabilized the protein and increased template assisted hemolytic activity. I presented this work twice at national conferences earning an honorable mention at the 2018 American Society for Biochemistry and Molecular Biology (ASBMB)’s Undergraduate Poster Competition in San Diego.
a. Stuttgen GM, Grilley DP, Weaver TM. Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A. FASEB J. 2018;32:792.13-792.13.
b. Stuttgen GM, Grilley DP, Weaver TM. Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A. FASEB J. 2019;33(S1):779.41-779.41.

2. Closed fumarase C active-site structures reveal SS Loop residue contribution in catalysis.
Also in Dr. Weaver’s lab, I worked on a project designed to investigate the residues important for catalytic activity of fumarase C (FumC), an enzyme that catalyzes the reversible conversion of fumarate to S-malate. Investigating a dynamic structural segment, termed the SS Loop, we were interested in understanding how placement of the SS Loop affected participation of key residues during the reaction. We used structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. Our high-resolution X-ray crystallographic results revealed three distinct FumC active-site conformations; disordered-open, ordered-open, and the newly discovered ordered-closed. Surprisingly, each SS Loop variant Michaelis constants were unaffected despite reductions in turnover number. Based upon our structural and functional analyses, we were able to propose structural and catalytic roles for each of the aforementioned residues. My contributions to this project included setting of crystal trays, performing X-ray crystallography both remotely and onsite at Argonne National Laboratory, running CD denaturation studies, running CD temperature melts, and performing all enzyme kinetic experiments. This project resulted in my first publication for my contributions in collecting all of the data as well as aiding in writing the manuscript.
a. Weaver T, Stuttgen G, Grosskopf J, Berger C, May J, Bhattacharyya B. Alterations within fumarase C reveal newly observed SS Loop conformations. FASEB J. 2019;33(S1):468.7-468.7
b. Stuttgen GM, Grosskopf JD, Berger CR, May JF, Bhattacharyya B, Weaver TM. Closed fumarase C active site structures reveal SS Loop residue contribution in catalysis. FEBS Lett 2019;594(2):337–357.

3. Role of FFAR4 in foam cell formation
Free fatty acids (FFAs) are implicated in the pathogenesis of metabolic diseases that include obesity, type 2 diabetes mellitus, and cardiovascular disease (CVD). FFAs serve as ligands for free fatty acid receptors (FFARs) that belong to the family of rhodopsin-like G protein-coupled receptors (GPCRs) and are expressed throughout the body to maintain energy homeostasis under changing nutritional conditions. FFAR4, also known as G protein-coupled receptor 120 (GPR120), is a long-chain fatty acid receptor highly expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. Furthermore, dysfunction of FFAR4 is associated with insulin resistance and obesity in both humans and mice, making FFAR4 an attractive therapeutic target for treating or preventing metabolic diseases. While much of the previous literature on FFAR4 has focused on its role in obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and CVD. Most notably, FFAR4 activation reduces monocyte-endothelial cell interaction, enhances cholesterol efflux from macrophages, and reduces lesion size in atherogenic mouse models. I recently published a review focused on the role of FFAR4 in metabolic diseases and highlighted an underappreciated role of FFAR4 in the development of atherosclerosis and CVD.
a. Stuttgen GM, Sahoo D. FFAR4: A New Player in Cardiometabolic Disease? Endocrinology 2021;162(8). doi:10.1210/endocr/bqab111.