bluuebunny/crossref_metadata_2025_split huggingface dataset:
Designing a Stapled Peptide Inhibitor of CHP‐NHE1 Protein‐Protein Interactions
Authors: Bell Isaac , Gufstason Jeffrey , Provost Joseph 
Published: June 2021

The Sodium Hydrogen Exchanger (NHE1) is a membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. NHE1 plays a critical role interacting with, directing, and regulating proteins that drive cell motility. We have shown that NHE1 plays a pivotal role in pH homeostasis and migration of lung tumor cells. The Calcineurin homologous B protein isoforms 1 and 2 (CHP1/2) interact with NHE1 at the same 40aa domain (CHP binding domain (CBD)) both impacting NHE1 function. CHP1 is ubiquitously expressed and its interaction with NHE promotes basal activity. While CHP2 expression is limited to the gut in non‐diseased tissue, it is highly expressed in lung and other tumor cells. When CHP2 or NHE1 expression is diminished, tumor cell growth is nearly abrogated. This supports that CHP2‐NHE1 interactions have potential as an anticancer target. Models of both CHP proteins bound to NHE1 show each bind in the CBD of NHE but with subtle structural differences. To analyze the potential differences between CHP‐NHE binding, recombinant proteins were generated and used to determine the thermodynamics of CHP/NHE interaction through CD thermal melts. Recombinant CHP2 bound to NHE1 exhibited a lower free energy of binding (−1.08 kJ mol−1) compared to CHP1 (−0.48 kJ mol−1), a ΔΔG of 0.60 kJ mol−1. Supporting this are in‐vitro ligand binding assays where we show a 2.23 fold greater affinity of CHP2‐NHE than CHP1‐NHE1. In order to create an effective peptide inhibitor, Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) computations were carried out using know crystal structures of each CHP isoform bound to NHE1. From these computations CHP1 was determined to have a two‐state binding interaction, whereas CHP2 had a one‐state binding interaction with NHE1. Unique initial binding contacts of NHE1 were determined for each CHP isoform. Stapled peptides mimicking the CHP‐NHE1 binding region were synthesized via a sulfenylation reaction with tryptophan amino acids. Peptides were then screened using a competitive fluorescent plate binding assay in which equal amounts of tagged CHP1/2 in the presence of NHE1. These peptide inhibitors will allow for increased CHP1‐NHE1 activity in targeted (cancer) cells while simultaneously reducing CHP2‐NHE1 activity.Support or Funding InformationSupported in part by NSF Che 1664565This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitoylation‐Dependent Regulation of RhoGTPase Signaling and Cardiac Pathophysiology
Authors: Brody Matthew J , Vagnozzi Ronald J , Molkentin Jeffery D 
Published: June 2021

S‐palmitoylation is a reversible lipid modification that regulates protein trafficking, localization, and function by providing a lipid tag that serves as a molecular instruction for association with specific cellular membranes. S‐palmitoylation is catalyzed by the zinc finger Asp‐His‐His‐Cys (zDHHC) family of S‐acyl transferases, however the role of these enzymes in the regulation of signal transduction in cardiomyocytes and its effects on cardiac pathophysiology are not well understood. To identify zDHHC enzymes with important regulatory functions in the heart, we used AAV9 to screen for the effects of overexpression of different zDHHC enzymes in the heart. Overexpression of the Golgi‐localized enzyme zDHHC3 resulted in severe cardiomyopathy, an effect that was not observed with overexpression of plasma membrane‐localized zDHHC5, endoplasmic reticulum‐localized zDHHC6, or Golgi‐localized zDHHC13. These data suggest important signaling mechanisms regulated by zDHHC3‐mediated palmitoylation at the cardiomyocyte Golgi apparatus. We next generated mice with cardiomyocyte‐specific overexpression of zDHHC3 to further investigate signaling mediated by this enzyme in the heart. Overexpression of zDHHC3 resulted in cardiomyopathy and congestive heart failure that was preceded by an unprecedented induction of all Rho family small GTPases, including RhoA, Rac1, Cdc42, and RhoGDI. Importantly, transgenic mice overexpressing an enzymatically‐dead mutant of zDHHC3 did not induce RhoGTPase signaling or develop cardiac disease. Quantitative palmitoyl proteomics in zDHHC3 overexpressing cells identified Rac1 as a novel substrate of zDHHC3. Rac1 palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts, implicating zDHHC3‐mediated palmitoylation of Rac1 as an important signaling circuit in cardiomyocytes. Taken together, these data indicate that zDHHC3 functions as a novel effector of Rho family of small GTPases and suggest that inhibition of zDHHC3 acyl transferase activity may represent a novel therapeutic strategy to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.Support or Funding InformationThis work was supported by NHLBI K99 HL136695 (MJB) and funding from the NIH and Howard Hughes Medical Institute (JDM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating Mammalian Anabolic Three‐Carbon Metabolism by Liquid Chromatography‐High Resolution Mass Spectrometry
Authors: Doan Mary T , Jiang Helen , Snyder Nathaniel W 
Published: June 2021

The metabolism of propionate, a 3‐carbon short chain fatty acid to 2‐methyl‐2‐pentenoic acid (2M2PE), a 6‐carbon intermediate, occurs in humans via propionyl‐Coenzyme A. Anabolic metabolism of propionate in humans is unexplored and the enzymology, control, and further metabolism of this pathway remains unclear. To elucidate and quantify this pathway, we employed both targeted and untargeted liquid chromatography‐mass spectrometry in combination with stable isotope labeling. Kinetic and dose response studies in human hepatocellular carcinoma HepG2 cells indicated that 2‐methyl‐2‐pentenoic acid was generated in a propionate‐dependent manner and underwent further metabolism over time. Fibroblasts cells from apparently healthy controls, as well as propionic acidemia and methylmalonic acidemia patients demonstrated similar results confirming this pathway is not dependent on entrance of carbon into the Krebs cycle. In these experiments, surprisingly, simultaneous untargeted metabolomics did not detect any propionate derived metabolites larger than 2M2PE. Rotenone, a complex I inhibitor, increased the production of 2M2PE suggesting that this three‐carbon pathway likely functions opposing anaplerosis. However, co‐incubation with glutamine reduced 2M2PE‐CoA, suggesting other metabolic influences on this pathway. To extend this work to study human metabolism, we also found that 2M2PE formed in human myocardial tissue incubated with propionate.Support or Funding InformationNIH grants R03HD092630 and K22ES026235This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microwell‐based 3D co‐culture model for drug screening by shotgun metabolomic profiling
Authors: Lu Xiyuan , Lodi Alessia , Tiziani Stefano 
Published: June 2021

The three‐dimensional (3D) cell culture systems, which mimic the in vivo cell environments, are well recognized as tissue and organ substitutes. High‐throughput metabolomics is increasingly applied to investigate different individuals and time‐dependent responses to environmental stimuli. Rapid data acquisition and improved detection limits of direct‐infusion mass spectrometry (DIMS) are paving the way for high‐content metabolomics in preclinical screening, opening new opportunities in drug discovery and personalized medicine.Here, we investigated cell viability and induction of reactive oxygen species in stromal cells cultured on various 3D systems as well as standard monolayer culture (2D) to evaluate which system provides the most favorable growing conditions. The selected 3D system was then tested for use in 3D co‐culture of leukemia and stromal cells for DIMS‐based high‐throughput/high‐content metabolic drug screens. The nanobiomatrix‐polycaprolactone (NBM‐PCL) induced the highest cell viability and the lowest induction of ROS and was suitable for use in a 96‐ and 384‐ well plate format. The DIMS results show that very relevant metabolic modulations are associated with co‐cultured cells. Doxorubicin treatment in leukemia co‐cultured with stromal cells induces some unique metabolic responses that drastically different from those observed in leukemia cells alone thereby showing the value of the 3D co‐culture high‐content metabolic drug screen.In conclusion, we optimized a high sample throughput method for the use of 3D co‐culture with a DIMS‐based high‐content metabolic drug screen and drug development.Support or Funding InformationThis research is supported by the MD Anderson Cancer Center Leukemia SPORE P50 CA100632, R01 CA206210‐01, and CPRIT RP180309.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NaAtm1: Studying a Heavy‐Metal ABC Transporter System
Authors: Hicks Dennis , Yang Janet 
Published: June 2021

Adenosine Triphosphate Binding Cassette (ABC) Transporters are a superfamily of integral membrane proteins that have been found embedded in both the cellular membranes and internal organelles of all species yet analyzed. This extreme conservation throughout the tree of life is due to their vitally critical and broad task of transporting all manner of things into and out of the cell and related organelles. This broadness of responsibility has led to an abundant diversity of this family, which in turn, has allowed life to adapt to the many environments found on Earth. While the importance and variability of this family is abundantly clear, many mechanistic and regulatory questions remain. The diversity which has made this family so useful in the natural world has resulted in a huge challenge in the scientific world: elucidating the many mechanisms this family has developed to perform its many functions. The scientific community has so far been able to identify overarching features of all ABC transporters such as the Walker A and Walker B motifs; however, the mechanisms of individual transporters remain undiscovered. In our research, we focus on a specific ABC transporter called NaAtm1 (Novosphingobium aromaticivorans ABC transporter of mitochondria 1), which has been shown to mediate transition metal export in its host species.While some transition metals (e.g. iron and copper) are essential for certain cellular processes such as respiration and enzyme catalysis, other metals (e.g. cadmium and mercury) can be very toxic to life. Due to this extreme dichotomy, a cell's ability to distinguish between friend and foe is of the utmost importance, and therefore should be a highly regulated process. Our goal is to better understand the full mechanism of the NaAtm1 transporter, to determine why it exports some metals while leaving others alone, and to understand the regulatory process that controls it. Toward this goal, we are conducting site‐directed mutagenesis, Fluorescent spectroscopy, and quantitative analyses of the NaAtm1 transport system. Here we present our preliminary work on examining NaAtm1 transport activity of substrates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the functional role of an ancient mitochondrial fatty acid synthesis pathway
Authors: Dove Katja K , Kadirova Malika F , Nowinski Sara M , Ouyang Yeyun , Van Vranken Jon G , Rutter Jared 
Published: June 2021

Fatty acid synthesis plays a crucial role in the cellular energetic landscape. Cytoplasmic Fatty Acid Synthesis (FAS) generates fatty acid intermediates that are used to store energy, build cellular structures such as membranes, and act as precursors for the biosynthesis of hormones. In addition to the cytoplasmic fatty acid synthesis pathway, eukaryotic cells have maintained a second, highly conserved mitochondrial fatty acid synthesis pathway (mtFAS), reminiscent of prokaryotic fatty acid synthesis pathways. Here, we explore the functional role of this ancient mitochondrial fatty acid synthesis pathway. Historically, a major function of mtFAS was considered to be the production of lipoic acid which is an essential co‐factor for key mitochondrial, metabolic enzymes such as pyruvate dehydrogenase. However, recently our lab has shown that mtFAS also plays a critical role in mitochondrial biogenesis by coupling Acetyl‐CoA levels with iron‐sulfur biogenesis as well as electron transport chain complex activation by promoting late‐stage assembly (Van Vranken et al., 2016; Van Vranken et al., 2018). These findings expanded the functional roles of the so far underappreciated mtFAS pathway into its broader role in the regulation of mitochondrial biogenesis and metabolic function. The identification of human patients with mutations in mtFAS genes further highlights the importance of the mtFAS pathway and the need to understand all aspects of its functions. However, many facets, especially regarding its own regulation, are still unknown. Here we present putative players in the regulation of the mtFAS pathway and their impact on cellular function.Support or Funding InformationCardiovascular Training Grant, University of Utah, 5T32HL007576‐32, Howard Hughes Medical InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Regions Involved in the Interactions between CYP1A2 and CYP2B4
Authors: Saha Aratrika , Connick J. Patrick , Reed James R , Backes Wayne 
Published: June 2021

The cytochromes P450s (P450 or CYP) that are involved in drug metabolism are membrane‐bound heme‐containing proteins that reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. P450s interact with limiting levels of cytochrome P450 reductase (CPR) to oxidize endogenous and exogenous substrates, converting them to water‐soluble products. Apart from competing for CPR, P450s also have been shown to form both homomeric and heteromeric complexes. Using Bioluminescence Resonance Energy Transfer (BRET), our lab has shown that CYP1A2 and CYP2B4 form physical complexes that, when formed, alter their metabolic characteristics. Interestingly, CYP1A2 and CYP2B4 form high affinity complexes. CYP1A2 also is capable of forming homomeric complexes; however, CYP2B4 does not appear to form such complexes. The goal of this study is to identify the regions that participate in the formation of CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes, focusing on surface residues on both proteins. This will be accomplished by generating chimeric proteins and inserting a fragment of CYP2B4 into CYP1A2. The N terminus of the P450 sits in the ER membrane and we have divided the protein into 5 faces, namely the proximal face (region close to heme that binds to CPR), distal face (opposite to heme) and three lateral faces (DE, GE, and KL1). Using molecular cloning we have generated chimeric proteins. In these chimeric proteins, parts of the proximal, distal and lateral regions have been mutated. These chimeric proteins are predominantly CYP1A2 with fragments of CYP2B4 inserted in them and vice‐versa. These chimeras were expressed in HEK‐293T cell line followed by BRET to determine whether complexes are formed. Preliminary data shows that regions of the proximal and lateral faces that were tested did not appear to be involved in the complex formation. Other fragments in these regions remain to be examined. However, we inserted the region K59‐D75 of CYP2B4 into the corresponding region of CYP1A2 and tested for both CYP1A2•CYP1A2 and CYP1A2•CYP2B4 interactions. Surprisingly, substitution of the K59‐D75 fragment of CYP2B4 into CYP1A2 enhanced the BRET signal for formation of the CYP1A2‐CYP1A2 complex, suggesting that this lateral face participates in CYP1A2•CYP1A2 complex formation. Subsequent experiments will be performed to examine whether protein function is affected.Support or Funding InformationSupported by NIH GM123253 & ES013648This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic insight into oxidized ribonucleotide (8‐oxo‐GTP) insertion by a DNA polymerase
Authors: Smith Mallory 
Published: June 2021

During each cell division, more than 1 million ribonucleotides are misinserted into the genome. A close examination of the ribo‐ and deoxyribo‐nucleotide pools reveals the presence of damaged and potentially mutagenic nucleobases. In particular, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxo‐dG) is well‐known as a prevalent and mutagenic oxidized nucleobase that contributes to carcinogenesis and several neurological diseases. However, the mutagenic properties of its ribonucleotide equivalent (r8‐oxo‐GTP) remain poorly understood. Furthermore, the biological relevance of r8‐oxoGTP is underscored by the attenuated ability of nucleotide pool sanitizing enzyme MTH1 to catabolize r8‐oxo‐GTP. Using model DNA polymerases, DNA polymerase beta and Bst DNA Polymerase I, we comprehensively characterized r8‐oxoGTP insertion opposite cytosine (non‐mutagenic) and adenine (mutagenic) using both kinetic and structural techniques. Our results show that the 2′‐OH, unique to the ribo‐ form of 8‐oxo‐GTP, heavily diminishes the rate of insertion for r8‐oxo‐GTP opposite adenine and nearly eliminates its insertion opposite cytosine. These results provide novel insight into the mechanistic strategies employed by DNA polymerases to minimize the insertion of damaged ribonucleotides into the genome.Support or Funding InformationThis work was supported by the National Institutes of Health (grant number R01‐ES027558).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Crowding Modulates Actin Filament Mechanics and Structure
Authors: Castaneda Nicholas , Lee Myeongsang , Rivera‐Jacquez Hector J , Marracino Ryan R , Girardi Eric , Tang Jay X , Merlino Theresa R , Kang Hyeran 
Published: June 2021

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce the accessible volume limiting biomolecule interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament conformations, dynamics, and mechanical properties is unknown. In this study, we investigate the effects of solution crowding on the modulations of filament mechanics and structure both in vitro and in silico. Direct visualization of filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging, allowing for the quantification of filament thermal bending dynamics and mechanics. Biophysical analysis indicate that molecular crowding modulates thermal fluctuations, enhances filament's effective bending stiffness, and reduces average filament lengths. Utilizing the explicit molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and structural properties indicating overall compaction and stabilization that are directly coupled to filament mechanics. Taken together, our study suggests the interplay between excluded volume effects and non‐specific interactions raised from molecular crowding may modulate actin filament mechanics and structure.Support or Funding InformationThis study was supported by the UCF start‐up fund and In‐House grant for H.K. The authors would like to thank the National Science Foundation for support of this work through REU site EEC 1560007. We acknowledge the computational time from UCF stokes cluster.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Substrate Recognition by Bacterial Enoyl Reductases
Authors: Zhang Xujie , Rafi Salma , Tonge Peter J 
Published: June 2021

Tuberculosis (TB) kills over two million people every year. We are currently studying a series of known or putative enzyme drug targets from MTB that includes InhA, the NADH‐dependent enoyl‐ACP reductase from the MTB fatty acid biosynthesis (FASII) pathway.InhA is a validated target for drug discovery and we are investigating the interaction with the natural acyl carrier protein substrate (AcpM). Based on studies with the E. coli enoyl reductase homologue, we hypothesized that three basic residues close to the substrate binding loop in InhA, R195, R225, and K233, interact with acidic residues in the AcpM recognition helix. We also have evidence that an additional set of basic residues close to AcpM Helix II, R45, R49, R53, play a critical role in AcpM recognition by InhA. Our studies also have implications for the ability of acyl carrier protein to recognize and interact with a diverse array of intracellular protein targets.
Epac1, an exchange protein activated by cAMP, increases cell migration through syndecan clustering, PI3K activation and heparan sulphate production.
Authors: Baljinnyam Erdene , Iwatsubo Kousaku , Wang Xu , Ulucan Coskun , Lagunoff David , Ishikawa Yoshihiro 
Published: June 2021

Background: The role of epac1, an exchange protein activated by cAMP, in melanoma migration is largely unknown. Heparan sulfate (HS), a major ECM, and syndecan2, a HS‐binding protein on the cell surface, play important roles in regulating such migration. Syndecan2, when activated, translocates to lipid‐rich plasma microdomains, known as lipid rafts. We thus examined the role of epac1 in HS production and syndecan2 translocation in melanoma migration.Methods: SK‐MEL‐2, a human melanoma cell line, and migration assays with Boyden chambers were employed.Results: Activation of Epac1 by adenoviral Epac1 overexpression or 8pMeOPT, an Epac‐specific cAMP analog, significantly increased cell migration in a time‐ and dose‐dependent manner. Epac1 also increased HS production by 3.5‐fold, and the removal of HS abolished Epac1‐induced migration. Epac1 also increased expression of N‐deacetylase/N‐sulfotransferase‐1 (NDST‐1), a key synthetic enzyme for HS. Sucrose gradient fractionation showed that Epac1 markedly increased syndecan2 translocation to rafts. In addition, disruption of rafts with lipid depletion abolished Epac1‐induced migration.Summary: Epac1 increased migration in malignant melanoma through HS production and syndecan2 translocation to rafts.