bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding the Interacting Mechanism For the Type III Secretion System Through Molecular Dynamics Simulations
Authors: Yang Jing , Gao Meng , Huang Yongqi
Published: June 2021

The type III secretion system is found in a large number of Gram‐negative bacterial pathogens. They are responsible for the delivery of effector proteins into the cytosol of host cells. It has been found that delivery of effector proteins depends on association with specific chaperones in the bacterial cytosol. The structures of effector/chaperone complex reveal that the chaperone forms a homo/heterodimer which the effector binds to. Upon binding with the chaperone, the intrinsically disordered effector protein forms an extended ordered conformation. Although the overall structures of the chaperones are similar, the structures of the effector proteins and interactions between the effectors and chaperones are rather different. In this work, the effector/chaperone interactions from Pseudomonas aeruginosa, Salmonella typhimurium, and Yersinia pestis were studied to compare their coupled folding‐binding mechanisms through molecular dynamics simulations. Although the sequence similarity among the effectors is very low, the interacting pattern and mechanism are similar. Our results showed that the hairpin structures from both terminuses were critical to stabilize the encounter complex. On the contrary, helices located at the central segment interacted with the chaperone when the binding free energy barrier was crossed. Therefore, preformation of central alpha‐helices has little effect on the association rate. However, interactions between central helices and the chaperone were critical for the complex stability. Our results provided new insights into the binding affinity and kinetics for effector/chaperone interactions.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TIP60 regulation of ΔNp63α is associated with SCC proliferation
Authors: Stacy Andrew J. , Zhang Jin , Craig Michael P. , Kadakia Madhavi P.
Published: June 2021

The transcription factor ΔNp63α, a p53 family member, is found primarily in the basal stratum of the skin where it functions to promote epithelial morphogenesis, cell proliferation, and cell survival. ΔNp63α is up‐regulated in non‐melanoma skin cancers such as squamous cell carcinoma (SCC), implicating it as a proto‐oncogene. Using cell culture and in vitro techniques, we have identified the acetyltransferase TIP60 as an upstream regulator of ΔNp63α. We observed that overexpression of ΔNp63α with increasing concentrations of TIP60 resulted in a dose‐dependent increase in ΔNp63α protein levels. Also, co‐expression of ΔNp63α with a TIP60 catalytic deficient mutant diminished this increase, thereby suggesting that TIP60 acetyltransferase activity contributed to increased ΔNp63α protein levels. Further, knocking down TIP60 reduced endogenous both ΔNp63α protein and transcript levels. Inhibition of de novo protein synthesis led to increased ΔNp63α protein half‐life in the presence of TIP60. Finally, TIP60 decreased the ubiquitination and proteasomal degradation of ΔNp63α. Cumulatively, these results indicate that ΔNp63α expression is regulated by TIP60 at both the transcriptional and post‐translational levels. We further demonstrated that TIP60 and ΔNp63α can interact with each other in cells as well using recombinant protein and co‐localize within the nucleus of an SCC cell line. We confirmed acetylation of ΔNp63α by TIP60 using an in vitro acetylation assay with recombinant protein and identified acetylated lysines via mass spectrometry. Moreover, site‐directed mutagenesis demonstrates that these lysines are critical to the ability of TIP60 to regulate ΔNp63α protein levels. We also observed that knockdown of both TIP60 and ΔNp63α led to a reduction in SCC proliferation. Using flow cytometry analysis, we found that both TIP60 and ΔNp63α expression promoted G2/M progression. Our results therefore indicate that TIP60 mediates SCC growth in part due to regulation of ΔNp63α. The identification of TIP60 as a novel upstream regulator of ΔNp63α provides researchers with a novel therapeutic target to combat this disease.

Support or Funding Information

The work presented from this project was supported by grant R01 CA154715‐02 (MK) from the National Institute of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the phosphoprotein of human metapneumovirus and its role in formation of viral liquid organelles
Authors: Thompson Rachel , Wilburn Amanda , Cifuentes‐Munoz Nicolas , Creamer Trevor , Dutch Rebecca
Published: June 2021

Human metapneumovirus (HMPV) is a recently identified RNA virus that frequently causes lower respiratory tract infections in infants, the elderly, and the immunocompromised, yet it has no FDA approved antivirals or vaccines. Understanding how HMPV replicates could help identify targets for development of new therapeutics. A key aspect of the viral replication cycle of HMPV is the formation of membrane‐less, liquid‐like replication and transcription centers termed inclusion bodies (IBs). A major component of IBs is the tetrameric HMPV phosphoprotein (P), which contains N and C terminal domains that lack defined secondary structures, called intrinsically disordered regions (IDRs). IDRs are thought to be involved in formation of liquid organelles through a process called liquid‐liquid phase separation (LLPS). However, the role P plays in IB formation and function is not understood. Our overall hypothesis is that the IDRs and post‐translational modifications of the phosphoprotein allow it to facilitate LLPS for IB formation. Western blots of cell lysate show two distinct bands for HMPV P, both of which contain phosphorylated protein and are detectable with either C terminal or N terminal HA tags. However, a C terminal HA tag disrupts IBs when co‐transfected with N, suggesting interactions through the C terminal domain are critical for IB formation. Purified P can form liquid‐like droplets without the presence of N, indicating it plays an important role in LLPS. Future studies will identify which domains of P are critical for LLPS and how phosphorylation and other modifications of P contribute to IB formation.

Support or Funding Information

We gratefully acknowledge the NIH/NIAID grant R01 AI3200001873 for supporting our research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post Translational Modifications in MDH1 May Affect Lung Cancer Survival
Authors: Alexander Sati Amrita , Sohl Christal , Weeks Joi , Hoang An
Published: June 2021

Cancer cells alter their metabolism by increasing their consumption of glucose and altering pathways to support proliferation. Cytosolic MDH1 is amplified in squamous cell lung carcinomas which corresponds to a 50% reduced rate of progression free survival for patients. The role of malate dehydrogenase (MDH) in normal cells is to catalyze the conversion of oxaloacetate (OAA) and malate using NAD+/NADH. MDH drives the glycolysis cycle by providing NAD+, which we know is crucial in cancer due to the increased use of NAD+ for glucose production. Post translational modifications may affect MDH1 activity in lung cancer. However, the effects of post translational modifications on MDH1 and cancer phenotypes are unclear. Our goal is to understand the mechanism of MDH1 in lung cancer, by mimicking a tumor microenvironment and to understand the consequences of downstream mutations in MDH1, which is sensitive to oxidative stress. We will be using kinetic assays and a cystine C137S post translational modification on MDH1. We hypothesize that the irreversible oxidation of Cystine can impact the folding and stability of a protein, leading to alterations in physiological structure and function. Examining the effect of this post translational modification can show the downstream effect of preventing oxidation and we can observe the degradation and aggregation of proteins linked to cancer. Ultimately, we hope to demonstrate MDH1 to be a future therapeutic target for squamous cell lung cancer.

Support or Funding Information

1. “Research reported in this [publication/press release] was supported by the National Cancer Institute of the National Institutes of Health under award numbers:U54CA132384 & U54CA132379.2.”The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”This work was funded by the National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU) and SDSU startup funds (C.D.S). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Kinetics of Glucan Phosphatases
Authors: Henao Tiffany , Raththagala Madushi Upendrika
Published: June 2021

Plants comprise two active glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). These phosphatases belong to the dual phosphatase (DSP) family of the protein tyrosine phosphatase (PTP) superfamily. DSP is a structurally diverse group and carbohydrate is the physiological substrate for glucan phosphatase family of enzymes. The X‐ray crystal structures of SEX4, LSF2 and human glucan phosphatase laforin revealed the primary mechanisms how glucan phosphatases bind carbohydrates and integrate them into the catalytic site. In vitro, enzymatic assays revealed that SEX4 preferentially removes phosphates attached to C6 position of glucose in starch while LSF2 and laforin remove phosphate groups attached to C3 position. However, little is known about the kinetics and regulatory mechanisms of carbohydrate dephosphorylation by SEX4 and LSF2. Here we define the enzymology of SEX4 and LSF2 using the generic substrate para‐nitrophenyl phosphate and physiologically relevant substrates, amylopectin, and glycogen.

Support or Funding Information

Skidmore College Start‐up Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding oxygen tolerant [Fe‐Fe] hydrogenase
Authors: Silakov Alexey , Corrigan Patrick Sullivan
Published: June 2021

[Fe‐Fe] hydrogenases are enzymes that perform a reversible heterolytic splitting of hydrogen into two protons and two electrons. The major roadblock in the utilization of these very efficient catalysts in renewable energy devices is the irreversible damage by oxygen and reactive oxygen species. The exact mechanism of the oxygen‐triggered inhibition is not known. However, it has been shown that in the presence of oxygen the active 6Fe metallocenter (H‐cluster) is irreversibly degraded, perhaps via a generation of a reactive oxygen species that react with components of the H‐cluster. In this work, we illustrate that an [Fe‐Fe] hydrogenase from Clostridium beijerinckii shows staggering tolerance to oxygen in atmospheric concentrations. Using activity assays and Fourier Transform infrared (FTIR) spectrometry we illustrate that the H‐cluster forms a stable adduct with oxygen that can be readily reactivated in the presence of hydrogen. Using protein film voltametry (PFV), IR spectroelectromemistry and EPR spectroscopy we provide a detailed description of the oxygen‐reacted state of the active center. Guided by bioinformatics analysis of the [Fe‐Fe] hydrogense family, we performed amino acid substitutions and subsequent biophysical characterization of the variants. The results of this study allow us to delineate the role of protein surrounding of the H‐cluster and of the auxiliary [4Fe‐4S] clusters in facilitating oxygen tolerance and catalytic bias observed in our PFV experiments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a detailed model of Hexokinase using wREFERASS to implement the King‐Altman method
Authors: Christmas Kevin M
Published: June 2021

A full kinetic model for enzymatic facilitation of substrate to product should account and provide equations for the binding for all reactants, the multiple enzyme‐reactant states, the inherent ordered or random binding mechanisms for substrates, ions, or protons that modulate the kinetic activity. Kinetic models of this quality are uncommon because the simpler Michaelis‐Menten (MM) representations have been considered adequate. MM approximations are for steady state conditions; they are inaccurate during transients; they cannot accommodate circumstances in which the concentration of reactants are close to the affinity or dissociation constant of the enzyme. The ease in implementing MM has simplified the general understanding of enzymes, biology, and stunted the exploration for better quantitative methods of higher accuracy in enzymology. Here we report the of an enzymatic tool wREFERASS (Garcia‐Sevilla et al., 2010) to develop a complex kinetic model of skeletal muscle hexokinase (EC 2.7.1.1) using the King‐Altman notation: all terms consist of rate constants and reactant concentrations. The wREFERASS tool derives detailed mathematical expressions based on an input file that describes explicitly the kinetic pathways. The expressions provide unitary balance, conserves mass, and require defining and parameterizing the dissociation constants for each binding step. Rate constants and dissociation constants were parameterized using enzymatic data obtained from Lueck and Fromm (1973) on rat skeletal muscle hexokinase II. The kinetic model accounts for all reactants described from the input file and has physical units for all biological elements. The kinetic behavior far surpasses any MM representation due to the consideration of the multiple enzyme states. It accounts for time delay due to binding, shifts in conformational states and product release. After parameterizing each dissociation and rate constant it becomes clear that there is a preferred kinetic pathway for phosphorylation of glucose even though the binding mechanism is referred to as ‘random ordered’. Even with some parameter uncertainty by using detailed kinetic models we can develop targeted hypotheses on the less secure parameters. This approach is also attractive because every element of the pathway is explicit, results in little to no simplification of terms, and allows parameters to better reflect the biology. These models reintroduce time delays that exist in biology and metabolism and increases the accuracy of their predictions. Considerable work goes into developing a kinetic model that is not limited by MM assumptions. This model is archived at www.Physiome.org/Models/providing source code and a model that can be run over the web or downloaded.

Support or Funding Information

Supported by NIH grant: U01‐HL122199

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Thermodynamic and Kinetic Properties of the Thermophilic Oxoglutarate Carboxylase BC domain to Increase Activity for use in Biofuel Crops
Authors: Dillard Lucas , Rose Robert
Published: June 2021

For many biofuel crops, carbon fixation is limiting –making the production of these alternative fuels economically unviable. The first green revolution has led to significant advances in crop yields in the past half century. Crop yield is now mostly limited by plant biology. To overcome this limitation, a condensed reverse TCA (crTCA) cycle has been developed in the workhorse species camelina that rapidly fixes atmospheric carbon. The biotin‐dependent carboxylase Oxoglutarate Carboxylase (OGC) represents the slow step of the crTCA cycle engineered in camelina. OGC –a thermophile –is 500‐fold less active at mesophilic temperatures than at its optimal 70 °C. Engineering OGC to preserve high activity at mesophilic temperatures presents an opportunity to further improve the rate of carbon fixation in the crTCA pathway, and possibly increase yields in oil crops –reducing the cost of biofuels.

Biotin‐dependent carboxylases such as OGC are a ubiquitously found family of proteins that consist of three domains: The Biotin Carboxylase (BC) domain, Biotin Carboxyl Carrier Protein (BCCP), and the Carboxy Transferase (CT) domain. We choose to analyze the BC domain as previous studies suggest the rate limiting step of the enzyme to result from 1) conformational changes associated with lid closure in the domain or 2) deprotonation of a species within the active site. We report kinetic and thermodynamic ATPase activity measurements of the thermophilic OGC BC domain measured via discontinuous assay coupled with direct quantitation of ADP production via HPLC. ATP concentration dependent activity was observed at temperatures ranging from 48 °C to 76°C, with kcat at 76 °C being 93 min−1. A subsequent Eyring plot was generated to measure the free energy of the transition state of OGC BC. Along with this, kinetic deuterium isotope effect experiments are being performed to measure energetics of deprotonation in the active site. Lid motions are also currently being measured with FRET. These studies will aid in the identification of mutations that may increase the activity of the BC domain at mesophilic temperatures and facilitate the use of OGC to increase biofuel crop yields.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Celiac Disease Autoantibodies with a Rapid and Noninvasive Diagnostic Biosensor
Authors: Maldonado Marcos , Nguyen Anna , Bonham Andrew J
Published: June 2021

Celiac Disease (also known as gluten‐sensitive enteropathy and nontropical sprue) is estimated to affect between 0.5–1.0% of the general population of the United States (1.6 million to 3.3 million during 2017) with many of these cases being under‐diagnosed. Celiac Disease (CD) is the genetic hypersensitivity to ingested exogenous gluten. Subsequent exposure to gluten related proteins lead to the development of an autoimmune response, causing damage to the lining of the gastrointestinal tract. Due to increased inflammation, symptomatic individuals present with abdominal pain, diarrhea, nausea, and many other gastrointestinal complications. However, some individuals affected by CD are asymptomatic creating increased diagnostic error. An accurate diagnosis of CD remains challenging; current diagnostic procedures involve multi‐day serological screening, followed by invasive gastrointestinal biopsies of suspected lesions. Baring this in mind, there is a pressing need for a rapid, non‐invasive, and accurate detection method to diagnose individuals suffering from this disease. Our method is based on the known autoimmune interactions related to CD. Specifically, we observe the formation of a structured complex between a unique epitope—a chimera of tissue transglutaminase 2 and truncated gluten peptide—and the autoantibodies produced in response to the presence of gluten related proteins. Conducted within our research, we demonstrate a CD‐related diagnostic tool for early, accurate, and rapid disease identification of affected individuals. This tool is a electrochemical DNA‐based biosensor (E‐DNA) that relies on peptide epitopes acting as capture probes to detect levels of disease‐specific antibodies. This commonly used biosensor is utilized to detect small changes in electrical conductivity to determine whether or not a target molecule has been bound. In principle, this sensor design can be utilized for unprocessed finger‐lancet blood samples to provide a rapid digital readout, allowing for efficient and accurate diagnoses of CD affected patients.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and Application of a Mutational Sensor for Characterizing AID Activity
Authors: Woods Allison Taylor , Zelazowska Monika , Plummer Joshua , Mu Yunxiang , McBride Kevin
Published: June 2021

Activation‐induced cytidine deaminase (AID) is a mutator enzyme that introduces uracil:guanine mismatches in Immunoglobulin (Igh) genes during the antibody diversification processes of class switch recombination and somatic hypermutation in B lymphocytes. When this happens, one of three pathways is possible. Firstly, the DNA can be directly replicated with the mutation, causing a transition (C → T) mutation. Secondly, during genomic mutation, the repair enzyme Uracil N‐Glycosylase (UNG) can act to base excise the mutation in either an error free fashion, replacing the original cytidine in the mutated spot, or thirdly, an error prone fashion, in which a transversion mutation (C → G/A) will then be introduced. Certain “hotspots” in the DNA sequence increase mutational frequency: cytosine residues are strongly favored when they are part of an RGYW sequence, or its reverse complement, sequence WRCY (where W=A/T, R=A/G, and Y=C/T). However, AID can also produce off target damage to genes, including oncogenes, that result in lymphomas. Therefore, tight regulation of AID mutator activity is necessary for genomic stability. In this study, we designed a cellular mutation reporter assay to determine the role of AID in regulating mutational activity. Mutations introduced by AID were measured with the use of constructs containing a green fluorescent protein (GFP) with premature STOP codons which‐ upon mutation‐ will “turn on” GFP and can be screened by flow cytometry. The use of this construct allows for mutational screening to be done in any cell type in which AID is naturally occurring, such as lymphomas and leukemias, rather than having one cell type with the reporter gene built into the cell line (current method). Once the constructs were successfully created via PCR SOEing reactions, we verified their ability to serve as mutational sensors. To achieve this, cells were co‐transfected with constructs and AID into BOSC cells and analyzed for rates of nucleotide reversion via flow cytometry. AID is shown to reproducibly revert two constructs with premature stop codons in contextual hotspots from GFP− to GFP+, as seen by the increased presence of GFP when analyzed via flow cytometry. The data show that the GFP mutational sensor works and serves as a functional system for determining mutational analysis among BOSC cells via flow cytometry analysis.

Support or Funding Information

1. Allison Woods is an undergraduate scholar supported in part by the MARC Program at St. Mary's University and the Summer Program in Cancer Research at MD Anderson Cancer Center support by National Cancer Institute (R25CA181004).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Mycoplasma secreted protein P48 via Electrochemical DNA‐based Biosensor
Authors: James Anika , Bonham Andrew J
Published: June 2021

Mycoplasma bacteria are highly infectious agents of human disease and laboratory contamination. For example, Mycoplasma pneumonia infects almost two million people every year with contagious upper respiratory infections also known as atypical or ‘walking pneumonia’. In addition to human disease, Mycoplasma is a major source of contamination of laboratory human cell cultures. However, current methods of detection for Mycoplasma strains, such as molecular‐based assays, PCR and serological analysis, are time consuming, expensive, and less suitable for working under stringent conditions such as extreme temperature or pH. To provide a rapid and accurate alternative, we have focused on detecting the presence of Mycoplasma with a diagnostic electrochemical biosensor that detects lipoprotein P48, which is shed from the surface of several strains into the surrounding blood serum or growth media. This biosensor is based on a previously identified DNA aptamer that binds to the secreted P48 protein with high affinity. We have expressed P48 recombinantly in E. coli to serve as a positive control, and our results show rapid and sensitive binding of the target, as well as convenient electrochemical signaling from these binding events. This approach will allow us to potentially improve both prevention and diagnosis of Mycoplasma in cell culture platforms as well as patients who present a proposed infection.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biodegradation of Surgical Polypropylene Meshes
Authors: Fiedler Anna K
Published: June 2021

Surgical meshes of a variety of materials, such as polypropylene, are commonly used in many procedures to support tissues. From our previous studies on polypropylene meshes, samples of mesh removed from patients often demonstrated biodegradation proportional to the amount of time that had passed since implantation. To expand this field of study, we tested the mesh sample in E. coli media to observe if degradation would occur in a controlled, single‐organism environment. Mesh samples were immersed in either an E. coli media or a control solution; the biodegradation was measured at one‐, two‐ and three‐month intervals. At this point the samples were harvested from both E. coli media and the control media for analysis, and results were then compared. A scanning electron microscope was used to identify surface defects and capture images of the material deterioration. Additionally, infrared spectroscopy was used to qualitatively identify oxygen present in the samples, while energy dispersive X‐ray spectroscopy quantifiably determined oxygen levels and degradation. Results from this study demonstrate that bioerosion and increased oxygen content can be observed in the mesh structure due to E. coli activity. Future plans include the synthesis of a monomer with collagen as a functional group to improve interactions with living tissues while maintaining structural integrity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microbial diversity of Baeg‐nyong cave and characterization of the antibiotics extracted from Streptomyces exfoliatus
Authors: Park Shinae , Cho Yong‐Joon , Jung Da‐yea , Jo Kyung‐nam , Lee Jung‐Shin
Published: June 2021

Baeg‐nyong (BN) cave is a one of the natural cave and critical zone observatory (CZO) in the Republic of Korea, but the role of microbial species in BN cave is still not well‐known. To analyze the microbial diversity of the cave, we collected a moonmilk, which is a white deposit of limestone cave. Because moonmilk has different textures depending the moisture contents, we performed metagenomics for two moonmilks (Dry and wet moonmilk) by next generation sequencing (NGS). The results of metagenomics revealed that moonmilks have very different microbial community and dominant species to each other. Specifically, Chromatiales, Pseudomonas, and Steroidobacter groups are present in high percentages in dry moonmilk, and Xanthomonadales and Arthrobacter group are dominant bacteria in wet moonmilk. Specifically, Streptomyces species of Actinobacteria are only present in dry moonmilk, and surface analysis showed that only dry moonmilk contained round‐shaped calcite structures, which might be affected by Streptomyces. Therefore, we isolated nine Streptomyces spp. from dry moonmilk and characterized their morphological, cultural, physiological, and biochemical properties. Among the nine Streptomyces species, only one species, as known as Streptomyces exfoliatus, showed the antimicrobial activity against to some bacteria including E. coli and B. subtilis. In addition, we extracted the antimicrobial compound from S. exfoliatus and analyzed HPLC and various assays to establish the antibiotics of S. exfoliatus isolated from BN cave.

Support or Funding Information

This research is supported by NRF‐2015R1D1A1A02061743 and NRF‐2015R1A4A1041105.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitic Acid Stimulates Apoptosis and Extracellular Vesicle Release in Renal Proximal Tubule Epithelial Cells
Authors: Cobbs Alyssa A , Zhang Yuanyuan , Chen Xiaoming , George Jasmine , Huang Ming Bo , Bond Vincent , Zhao Xueying
Published: June 2021

Abnormal levels of lipids in the blood are associated with a myriad of chronic diseases. Lipotoxicity contributes to kidney deterioration through cellular dysfunction and apoptosis in renal epithelial cells. A predominant saturated fatty acid, palmitic acid, stimulates the release of extracellular vesicles (EVs) in hepatocytes and induces an inflammatory phenotype in macrophages. However, the role of EVs in the progression of chronic kidney disease remains vaguely explored. This study was aimed to investigate whether palmitic acid increases the release of EVs in renal tubular epithelial cells. Rat proximal tubular epithelial cell line, NRK‐52E, was incubated with 1% bovine serum albumin (BSA), BSA‐conjugated palmitic acid (PA), and BSA‐conjugated oleic acid (OA) for 24–48 hours. The EVs released into conditioned media were isolated by ultracentrifugation and quantified by nanoparticle‐tracking analysis (NTA). According to NTA, the size distribution of EVs was 30–50 nm with similar mode sizes in all experimental groups. Moreover, BSA‐induced EV release was significantly enhanced in the presence of PA, whereas EV release was not altered by the addition of OA. PA‐enhanced EV release was associated with an induction of cell apoptosis reflected by an increase in cleaved caspase‐3 protein levels and Annexin V positive cells analyzed by flow cytometry. Additionally, confocal microscopy confirmed the uptake of lipid‐induced EVs by recipient renal proximal tubular cells. Collectively, our results indicate that PA stimulates EV release from cultured proximal tubular epithelial cells. Thus, extended characterization of lipid‐induced EVs may constitute new signaling paradigms contributing to chronic kidney disease pathology.

Support or Funding Information

This work was supported by NIH/T32 (5T32HL103104‐07), NIH (SC1DK112151), NIH/NCRR/RCMI (8G12MD007602) and (8U54MD007588)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Role of Zinc (Zn2+) in Cellular Transitions Under Acidic Conditions
Authors: Hu Yuli , Case Maddie , Lickfelt Megan , Li Yang V.
Published: June 2021

The maintenance of ionic homeostasis is critical for cell metabolism, cell growth, and cell survival. It has been shown that pH and zinc dyshomeostases are linked to pathophysiological conditions such as cancerous mutations, urinary diseases, gastric infections, and ischemia. In particular, we observed that there were concentration increases of intracellular free zinc after acidic treatments. We further hypothesize that zinc may be a mediator for the acidic treatment‐induced cellular changes. A spindle shape or fibroblast‐like shape has been used as an indicator of decreased cell‐to‐cell adhesion, increased cellular motility, and an increased possibility of metastasis in cancer. Our preliminary experiments showed that the accumulation of spindle cells occurred after acidic treatments and that zinc chelation reduced the accumulation, indicating the role of Zn2+ in the accumulation of spindle cells. Spindle cell accumulation is a sign for cancer cell synchronizations at a particular phase in cell cycle, which provide windows for potential cancer therapeutics that target cell cycles. Our preliminary experiments showed that acidic treatments enhanced the expression of vimentin, which was attenuated by an application of zinc chelator, suggesting that zinc plays an important role in cellular transitions under acidic conditions. The significance of this research is to provide a novel mechanism that zinc may be a mediator in acidic treatment‐induced cellular changes. Acidic environment is seen in physiological condition such as gastric ulcer, and in abnormal conditions such as ischemia and cancer. It will be interesting to see how zinc is involved in these processes.

Support or Funding Information

Thanks to the Osteopathic Heritage Foundations, Graduate Assistantship Program at Ohio University Heritage College of Osteopathic Medicine for supporting graduate student Yuli Hu.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LY294002‐induced growth inhibition of cancerous cells is inked to downregulation of MEK‐ERK pathway and adhesion molecules
Authors: Kavege Llona , Baquier Jocelyn , Duff Angela , Hu Tang
Published: June 2021

Phosphatidylinositol‐4,5‐bisphosphate 3‐kinases (PI3Ks) regulate several important cellular and subcellular processes including cell survival, proliferation, and differentiation. PI3K‐Akt is one of most commonly activated signal pathways in human cancer. Thus, targets PI3K provides a great therapeutic opportunity in cancer treatment. LY294002 was the first synthetic small molecule inhibitor that can reversibly target PI3K family members and several other lipid kinases. However, its effect on cellular adhesion molecules (CAMs) and its link to MEK/ERK pathway are largely unknown. In this study, we present evidence that LY294002 significantly inhibited proliferation of human myeloid leukemia (TF‐1a and MV4‐11) and epithelia cancer (Hep‐G2 and MCF‐7) cells in suspension and adherent cell culture, respectively, measured by MTT and XTT assays. LY294002 also dramatically inhibited the colony‐forming cells derived from TF‐1a, MV4‐11, and Hep‐G2 cell lines, measured by semi‐solid agar culture. The adherent culture of Hep‐G2 and MCF7 showed substantial cell detachment from the culture substrate and formation of aggregates suspended in the culture medium, leading to disruption of the cell monolayer. Similar but smaller aggregates were also found in suspension culture of TF‐1a and MV4‐11 cells. As expected that LY294002 downregulated the phosphorylation of PI3Kp85 and Akt analyzed by Western blot. Unexpectedly, the expression of MEK and ERK, two components of Ras‐Raf‐MAPK pathway and adhesion molecule, integrin‐linked kinase (ILK) and focal adhesion molecule (FAK) were also significantly downregulated in MV4‐11 cells in response to LY294002. In contrast, GM‐CSF and PMA stimulated the expression of MEK and ERK in MV4‐11 and TF‐1a cells, respectively. Addition of LY294002 and PD‐98059 blocked GM‐CSF‐and PMA‐induced MEK and ERK. Lastly, ERK immunoprecipitants from GM‐CSF‐treated cells stimulates the phosphorylation of transcription factor Elk‐1, whereas the immunoprecipitants from LY294002‐treated cells downregulates the phosphorylation of Elk‐1. Briefly, our data suggest that LY294002 inhibited growth of human cancerous cells partially due to its disruption of the adhesion molecules, which is linked to downregulation of MEK‐ERK pathway.

Support or Funding Information

Barry University Faculty Stimulus Grant and NIH‐NIGMS RISE R25 GM059244‐17

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Is Less Actually More? Investigating the Synergistic Relationship between Chemotherapy Agents and NHE1 Inhibitors in Ovarian Cancer Cells
Authors: Larson Allie C , Wallert Mark A.
Published: June 2021

Chemotherapeutic regimens continue to be the conventional therapy utilized in the treatment of ovarian cancer. These treatment plans often include the use of cytostatic medications such as paclitaxel, rucaparib, olaparib, carboplatin, and cisplatin. The mechanisms of action for each of these oncogenic therapeutics differs substantially. Paclitaxel prevents the polymerization of microtubules in an attempt to hinder mitotic completion. Rucaparib and olaparib both inhibit PARP, an enzyme associated with repairing damaged DNA. Carboplatin and cisplatin also initiate apoptosis through the inhibition of DNA repair. However, they do so by crosslinking the purine bases on DNA. Although the use of chemotherapy agents has been instrumental in reducing cancer‐related deaths, the approbation of these drugs has largely been mitigated due to the adverse side effects they procure. Furthermore, the capacity of chemotherapy drugs to repeatedly eradicate cancer is often limited because of developed resistance. It has thus become of interest to investigate methodologies for reducing not only the required chemotherapy drug dosage, but also the threat of disease recurrence. Such a synergistic effect is hypothesized to occur through the coupling of chemotherapy agents with direct inhibitors of NHE1. NHE1, or the Na+‐H+ Exchanger Isoform 1, is a transport protein that is often hyperactivated in cancer cells and contributes, through maintenance of a pH inversion, to cancer cell proliferation. Inhibition of this transport protein results in intracellular acidification, an environmental requirement for the initiation of apoptosis. Applying this inhibition with an accompanying chemotherapeutic has thus interested researchers as a possible therapeutic approach towards mitigating neoplastic development. The potential of these drug‐inhibitor pairs has been evaluated through a reduction in proliferation rates and quantified via XTT assays. The larger the growth inhibition, the more powerful the combination therapy. Ultimately, the drug‐inhibitor pairs will be applied in a spheroid assay to better assess the potential of the therapy in a three‐dimensional environment.

Support or Funding Information

Lueken Family Cancer Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic regulation of the Birc5 promoter explains mechanism of action of YM‐155 in synovial sarcoma
Authors: Luelling Sarah , Mika Aleksander , Heyneman Alexandra , Parkinson Neil , Barrott Jared
Published: June 2021

YM‐155 is an anti‐cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis‐inducing therapy indirectly affects the protein levels of Survivin (Gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is another cancer with high protein expression of Survivin. We investigated whether YM‐155 would be a viable therapeutic option to treat synovial sarcoma. We, therefore, applied YM‐155 therapy to human synovial sarcoma cell lines and a genetically engineered mouse model of synovial sarcoma. We discovered that YM‐155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM‐155 by looking at the change of modifications of the histone tails that were near the Birc5 promoter. Using chomatin immunoprecipitation (ChIP) we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM‐155 treatment. H3K27me3 increased whereas the H3K27ac decreased, highlighting the decrease of the protein Survivin occurs through epigenetic silencing of the gene's promoter. The treatment of YM‐155 was accompanied by an increase in NFkB protein expression, which indicates an attempt of the cell to initiate a positive feedback due to the decrease in Survivin expression. This combination of molecular events eventually resulted in Caspace 3/7/8 upregulation and death of the sarcoma cells.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
XRN2 Depletion is Synthetic Lethal with PARP1 Inhibition
Authors: Pay Louise , Motea Edward E , Patidar Praveen , Morales Julio C , Singh Naveen M , Khandelwal Megha , Ogas Talysa , Zhou Yunyun , Fattah Farjana J , Xie Yang , Boothman David A
Published: June 2021

Persistent R‐loops (RNA‐DNA hybrids with a displaced single‐stranded DNA) create DNA damage and lead to genomic instability. The 5’‐3’‐exoribonuclease 2 (XRN2) degrades RNA to resolve R‐loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. XRN2 function in DSB repair is not fully understood. We hypothesize that XRN2 plays a critical role in multiple DNA repair pathways. Here, using tandem affinity purification‐mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70‐Ku80, DNA‐PKcs, PARP1, MCM2‐7, PCNA, RPA1), and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non‐homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2‐deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2‐deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. The XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 loss and PARP1 inhibition.

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This work was largely supported by the National Institutes of Health/National Cancer Institute (NCI/NIH) [R01 CA139217] to D.A.B, a minority supplement [R01 CA139217‐05S1] and the Cancer Biology Training Grant T32CA124334‐06 to E.A.M. (PI: Dr. Jerry Shay), Simmons Comprehensive Cancer Center, UT Southwestern. This work was also supported by grant 5P30CA142543, NCI/NIH to the UT Southwestern Proteomics Core. The UT Southwestern Proteomics Core was also supported, in part, by a grant from the Cancer Prevention and Research Institute of Texas [CPRIT, RP1206130] to Dr. Hamid Mirzaei. Funding for open access charges was from NCI/NIH CA139217 to D.A.B. Research reported in this study was also supported by the UT Southwestern CCSG grant 5P30CA142543 from the NCI/NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteasome Inhibition Increases Interleukin‐8 Expression in Triple Negative Breast Cancer Cells, Resulting in Their Increased Survival, Proliferation, and Migration
Authors: Uddin Mohammad , Zou Yue , Gaire Bijaya , Padmanabhan Sveta , Vancurova Ivana
Published: June 2021

Interleukin‐8 (IL‐8, CXCL8) is a pro‐inflammatory and pro‐angiogenic chemokine that stimulates cancer progression by inducing tumor cell proliferation, survival, and migration. IL‐8 expression is increased in many types of advanced cancers, including triple negative breast cancer (TNBC), and correlates with poor prognosis. Because no targeted therapies are currently available, and majority of TNBC patients initially responding to cytotoxic chemotherapy become drug‐resistant, development of novel therapeutic strategies is essential. Proteasome inhibition by Bortezomib (BZ; Velcade; PS‐341) and Carfilzomib (CZ), developed for its ability to inhibit transcription of NFκB‐dependent anti‐apoptotic genes, has been effective in treating hematological malignancies. By contrast, as single agents, proteasome inhibitors (PI) have failed to show a significant clinical activity in solid tumors, including TNBC, but the mechanisms are not fully understood. Here, we demonstrate that proteasome inhibition increases expression of IL‐8, resulting in increased survival, proliferation, and migration of TNBC cells. The induced IL‐8 expression is mediated by IkB kinase (IKK), and associated with an increased nuclear accumulation of p65 NFκB, and IKK‐dependent p65 recruitment to IL‐8 promoter. Inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ‐treated TNBC cells. These results are the first to show that proteasome inhibition increases IL‐8 signaling in TNBC cells, suggesting that targeting IKK activity might increase PI effectiveness in TNBC treatment.

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Sponsor: Dr. Ivana Vancurova (ASBMB member ID: 32743) Funding : NIH CA202775 grant (to Dr. Ivana Vancurova)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combinatorial use of Lovastatin and FTI‐277 to alter Ras membrane association in A375 melanoma cells
Authors: Vagher Bay Lea
Published: June 2021

In 2018, 9,000 of those people will die from melanoma (American Cancer Society, 2018). In melanoma like many cancers, a mutation in the Ras protein has been implicated in excessive cell proliferation. A farnesyl transferase inhibitor, FTI‐277, has shown the ability to lower Ras membrane association by disrupting Ras prenylation. Additionally, Lovastatin, a cholesterol lowering drug, is associated with decreased Ras membrane association. Unprenylated Ras cannot attached to the cell membrane or initiate signals in downstream effector proteins. Previous studies have shown that these two drugs cannot kill melanoma tumors individually, but the combinatorial effects have not been examined. We hypothesize that FTI‐277 will be a more potent inhibitor of Ras prenylation/activation than Lovastatin due to Lovastatin's high cytotoxicity. A metastatic melanoma cell line (A375) was treated and Ras prenylation and membrane association examined using immunofluorescence. Flow cytometry showed that the minimum concentration of 10 mM of each drug individually and in combination did not result in more than 30% cell death. Results showed that high concentrations of both drugs induced cell toxicity and at 10 mM Lovastatin was more cytotoxic than FTI‐277 (mean= 30.67%, 10.69% respectively, p=0.017). Cell image analysis showed that the average corrected total cell fluorescence for individual treatment with each drug was significantly lower than both the vehicle control and treatment with both drugs (p<0.05). Together this shows that treatment with these drugs is efficacious for melanoma due to their abilities to induce apoptosis and alter Ras membrane association.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.