Iron (Fe) is crucial for various biochemical processes in most human cell types, each of which is expected to employ specialized mechanisms to fulfill its distinct iron requirements. Further, it’s hypothesized that tumor cells remodel Fe regulatory pathways in cell type specific manner to meet their elevated iron demand. However, these pathways remain largely uncharacterized, primarily due to the lack of data acquisition pipelines capable of detecting low-abundance proteins. To understand how different tissues regulate iron metabolism in response to changes in environmental iron levels, we utilize data-independent acquisition mass spectrometry (DIA-MS) workflow. DIA-MS provides a robust, reproducible, high-throughput method for deep, unbiased profiling of Fe-regulated proteomes across cell lines. To further enhance this workflow, we are investigating the use of sample fractionation to design experiment-specific spectral libraries for deeper proteome coverage. Our preliminary results suggest that key components of Fe metabolism, such as HIF1A and STEAP3, are broadly conserved across cell lines, though notable differences exist. To further delineate these differences we are acquiring more robust data from an increased number of cell lines. The data was processed using DIANN and FragPipe-Analyst to identify differentially expressed core Fe-responsive proteins, uncovering shared and unique pathways across cell types. Our research aims to enable targeted therapies for organ-specific iron dysregulations and oncological pathologies

AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolic pathways within mammals. AMPK has functional roles in glucose, protein, and lipid biosynthesis, insulin signaling, and gene expression. As a result, AMPK has become a major target for potential novel treatments of metabolic diseases. The exact mechanisms behind how AMPK relays downstream signals to highly specified subcellular locations are poorly understood. This study is an extension of previous work to confirm the effects of oxidative stress and ROS on the activation of AMPK activity through the addition of superoxide scavenger Tiron. The effects of ROS on the activation of AMPK activity is further confirmed through the controls performed using sublocalized ExRai exposed to H2O2. Results of the ROS activation of AMPK activity are evaluated based on the usage of fluorescent microscopy of Mouse Embryonic Fibroblast (MEF) cells transfected with localized ExRai.

Natural enzymes have the ability to effectively catalyze many reactions, and understanding the source of their catalytic power is the key to synthetic protein design. A theory named electrostatic preorganization was proposed by Ariel Warshel, stating that within protein scaffolds, electrons are organized in a way that creates an electric field that stabilizes the transition state, and/or destabilizes the reactant state, surrounding where reactions occur. Here we examine the application of the theory on an enzyme called chorismate mutase, which catalyzes a pericyclic Claisen Rearrangement reaction that converts chorismates to prephenates. We took six systems of natural chorismate mutases, performed molecular dynamics simulations, and computed for the electric field within their active sites, surrounding the reacting atoms on the chorismates embedded. The field data were then put through tensor-based clustering, where a representative field emerged, and the corresponding structures were put through QM/MM for further information, such as barrier energy and coulombic interaction energy. Lastly, principal component analysis and partial linear square regression were employed to find correlation between the electric fields and the enzyme’s activity. Together, these methods allow us to relate the difference in surrounding fields to the catalytic efficiency of the different systems. Understanding the effect of electrostatic preorganization in chorismate mutases and other enzymes may provide more basis for future protein design.

Several human pathogens bind the apical domain of transferrin receptor 1 (TfR1) to invade cells. This makes it appealing to target the apical domain of TfR1 as a strategy to broadly neutralize entry of those pathogens into human cells. To mount an effective blockade, we set out to analyze binding sites on the apical domain and create stronger binders. We examined interactions between TfR1 and OKT9, an antibody that binds the apical domain, to assess contacts that are critical for binding. We first assessed conservation of apical domain residues and compared binding of OKT9 to apical domains derived from different mammals via enzyme-linked immunosorbent assay (ELISA). We found that OKT9 only retained affinity for human apical domain. Residues R208 and Y247 (R13/Y52 in apical domain) were found to be unique to human TfR1 and likely essential for binding. By outlining predicted binding residues in OKT9-TfR1 models, the protein prediction software, AlphaFold 3, indicated R208, but not Y247, as essential for binding. An ELISA comparing R13S and Y52H modified apical domains confirmed R208 was uniquely necessary for binding. However, dissimilar apical domains, modified to present R13, lacked significant binding via AlphaFold and ELISA. This indicates that further analysis of the binding interface is needed to design improved binders that deny entry of pathogens. We anticipate that the same strategy may be advantageous for TfR1-mediated drug delivery into cells, and provide a way to shuttle drugs across the blood-brain barrier.

Oleogels made from vegetable oils with polysaccharide emulsifiers show promise as fat replacers in traditional or cultivated meat products. Oleogels have preferable nutritional profiles when compared to intramuscular fat in whole-cut bovine, porcine, and poultry meat. However relative stability during degradation and digestion as well as physical and chemical properties of vegetable oil oleogels during degradation is unknown. The goal of the present study was to compare vegetable oil based oleogel stability to traditional animal fats. Oleogels were produced using olive oil and emulsified in chitosan to form 3D samples. Sample stability was then compared to animal fat through simulated gastric degradation, thermogravimetric analysis, and oil loss in centrifuge. Results show oleogels have promise as fat replacers to bovine adipose tissue, as well as high oil-binding capacity. The potential application of oleogels as fat replacers in future “hybrid” cultured meat products is discussed.