bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of immune molecule expression in mouse adipose tissue and deep fat
Authors: Juresic Sanny , Ochrietor Judith D
Published: November 2023

Many recent studies indicate that adipose tissue plays a role in innate immunity and inflammation. This lab has been studying the expression of Basigin gene products and the innate immune receptor TLR4 with respect to neural immune functions. Basigin gene products are members of the immunoglobulin superfamily. In the neural retina, Basigin forms a transport metabolon with monocarboxylate transporter 1 (MCT1), which is vital for the function of photoreceptor neurons in mice. Basigin is also expressed on monocytes and epithelial cells throughout the body, but has been less studied in connective tissues like adipose. Recent studies by this laboratory and others suggest that Basigin interacts with the innate immune receptor TLR4, which has the ability to initiate expression of pro‐inflammatory cytokines. The purpose of the present study was to examine the expression of Basigin gene products and TLR4 in mouse adipose and deep visceral fat tissues in an effort to determine whether they participate in immune functions of those tissues. Detergent‐solublized protein lysates and RNA were isolated from mouse adipose and deep visceral fat and used in ELISA and quantitative reverse transcriptase polymerase chain reaction (q‐RT‐PCR), respectively specific for Basigin gene products and TLR4. It was determined that the ubiquitously expressed Basigin is expressed within adipose tissue and deep fat, but the photoreceptor‐specific Basigin‐2 was not. In addition, TLR4 was observed in adipose tissue, but not in the visceral deep fat tissue. The results of this study are consistent with the literature and suggest that Basigin may play a role in obesity‐induced inflammation.

Support or Funding Information

University of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
The Roles of GPD1 in Maintaining Blood Glucose Levels under Fasting Conditions
Authors: Sato Tomoki , Morita Akihito , Mori Nobuko , Miura Shinji
Published: November 2023

Glucose is used as one of the energy sources in many organs. Under feeding conditions, glucose can be obtained from dietary carbohydrate. On the other hand, when external supply of energy is interrupted, such as fasting, carbohydrate preserved in liver and glycogenic precursor derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acid derived from adipocyte and skeletal muscle, respectively, are utilized as glycogenic precursor. But, it hasn't become clear which glycogenic precursor is more important in gluconeogenesis. The recent study revealed that Metformin, therapeutics for treating type 2 diabetes, depressed gluconeogenesis from glycerol by inhibition of glycerol‐3‐phosphate dehydrogenase (GPD) 2. This result suggests that glycerol have a potent impact on gluconeogenesis. In this study, we focused attention on GPD1, which is the enzyme that related gluconeogenesis using glycerol, and examined the roles of GPD1 in gluconeogenesis and importance of glycerol as glycogenic precursor. Using GPD1 null mutant model BALB/cHeA mice (HeA mice), we measured the change of blood glucose levels under fasting conditions and gene expressions related to gluconeogenesis in liver. BALB/cBy mice (By mice) were used as a control. In HeA mice, blood glucose levels at 1 to 4 hour after fasting were significantly higher than the By mice. The hepatic gene expressions involved in gluconeogenesis and aminotransferase such as glucose‐6‐phosphatase and alanine amino transferase were also higher than the By mice. Moreover, we examined availability of the glycogenic amino acid by alanine tolerance test. At 30 min after alanine administration, blood glucose level was significantly higher in the HeA mice. In short, these data suggests that lacking of GPD1 maintained blood glucose levels under fasting state by enhancing gluconeogenesis from glycogenic amino acid and release of glucose into the blood stream.

Support or Funding Information

This study was supported by the Council for Science, Technology and Innovation (CSTI), Cross‐ministerial Strategic Innovation Promotion Program (SIP, No.14533567), and “Technologies for creating next‐generation agriculture, forestry and fisheries” (funding agency: Bio‐oriented Technology Research Advancement Institution, NARO), Grants‐in‐Aid for Scientific Research (KAKENHI, No. 15J10165, 15K00827) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo), and University of Shizuoka Grant for Scientific and Educational Research.
Metabolomics reveals the Glycerophosphodiester Phosphodiesterase GDPD5 to regulate an invasive phenotype in lung cancer cells and potentially constitute a novel drug target
Authors: Nordstrom Anders , Broom Oliver , Witek Barbara
Published: November 2023

Study Objective

Epithelial to Mesenchymal Transition (EMT) is a process by which epithelial cells lose their cell polarity and cell‐cell adhesion, and gain migratory and invasive properties required for invasion and metastasis. The discovery of new targets affecting this process opens up for novel cancer therapies.

Methods

We explored the metabolic profiles of A549 lung cancer cells undergoing EMT induced by TGF‐β in a time series using LC‐MS based metabolomics. Follow up on the gene and protein leads suggested by changes in the metabolic profiles included gene expression measurement with qPCR, gene knockdown (lentiviral siRNA), gene over expression (V5 vector), protein analysis using FACS and western blotting. Phenotypic evaluation was performed by transwell invasion assay and zymography. The publically available gene expression database Kaplan‐Meier Plotter was queried for survival analysis correlating with GDPD5 gene expression.

Results

Intracellular glycerophosphocholine (GPC) levels were increased 3‐fold within 24 hours after TGF‐β application. GPC is an intermediate in the phosphocholine (PC) metabolism and is the substrate for a family of phosphodiesterase's. Administration of TGF‐β together with the phosphodiesterase inhibitor dipyridamole increased the GPC levels but not N‐cadherin, a marker for EMT, suggesting that GPC itself is no driver of EMT. Genes in the Glycerophosphodiesterase family were downregulated when treated with TGF‐β, supporting accumulation of GPC. One member, Glycerophosphodiester phosphodiesterase 2, GDE2 (GDPD5) displayed a pattern of increased mRNA and protein expression upon TGF‐β administration. Knockdown of GDE2 using siRNA decreased the invasive phenotype and the amount of protease excreted into the media whereas stable overproduction of GDE2 increased the invasive phenotype and protease excretion. Interestingly the phosphodiesterase inhibitor dipyridamole, an approved drug for blood vessel dilation and inhibition of blood clot formation, also reduced protease excretion in GDE2 over producing cells. Finally, querying a public gene expression database gave that high expression of GDE2 is associated with decreased overall survival in lung, breast and gastric cancers.

Conclusions

These data suggests a central role of phosphodiesterase's and GDE2 in particular for the invasion and metastasis process and identifies a previously unrecognized EMT signaling mechanism which potentially could constitute a therapeutic target.
Signaling pathways activated during acute aldosterone stimulation in kidney distal convoluted tubule cells
Authors: Cheng Lei , Wu Qi , Peng Li , Pistikun Trairak , Fenton Robert
Published: November 2023

The kidney distal convoluted tubule (DCT) plays an important role in modulating body sodium balance and blood pressure. Long‐term effects of aldosterone to increase sodium reabsorption in the DCT by modulating the function of the NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC) are well described. However, potential effects of aldosterone to acutely modulate NCC or ENaC function in the DCT and the signaling cascades responsible remain largely uncharacterized. Here, we used stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics to map global changes in protein phosphorylation in mouse kidney convoluted tubule cells (mpkDCT) following acute (30 min) treatment with aldosterone (1μM). A total of 8136 unique proteins, containing 14838 different phosphopeptides were identified. Of these, 148 phosphopeptides were significantly up regulated, and 96 phosphopeptides were significantly downregulated in abundance following acute aldosterone treatment. Motif analysis indicated that basophilic motifs were preferential targets for upregulated sites upon aldosterone stimulation, whereas proline‐directed motifs were prominent for downregulated sites. Analysis of motif prediction indicated that the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) and CAMKK dependent pathways may be involved in aldosterone‐induced signaling response by the DCT. Acute application of aldosterone did not significantly alter cAMP levels in mpkDCT cells. Ongoing analysis and pharmacological inhibition studies will help clarify and verify these systems biology findings and determine novel new acute aldosterone‐mediated signaling networks important for blood pressure regulation.
Effect of Cinobufotalin on Three Different Ovarian Cancer Cell Lines
Authors: Afroze Syeda H , Tobin Richard , Peddaboina Chander , McCormick Timothy C , Newell‐Rogers Martha K , Zawieja David C , Kuehl Thomas J , Uddin Mohammad N
Published: November 2023

Objective

Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan su). CINO has been used as a cardiotonic, diuretic and hemostatic agent. Previously we have shown that CINO inhibits cytotrophoblast cell function. Recently others have shown that CINO inhibits function of a lung cancer cell line. In this study, we assessed the effect of CINO on three different ovarian cancer cell lines; SK‐OV‐3, CRL‐1978 and CRL‐11731 to assess whether the effect of CINO is cell specific.

Study Design

We evaluated the effect of CINO on three ovarian cancer cells by treating cultures with 0.1, 1, 5, and 10 μM concentrations of CINO. Cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA) was evaluated in cell lysates of CINO treated cells by western blot analysis. Cell cycle arrest and cell viability were determined by fluorescence‐activated cell sorting (FACS) analysis. We performed Annexin V staining by immunofluorescence to evaluate the pro‐apoptotic protein expression. In addition mitochondrial membrane potential was measured using MMP kit and by FACS analysis.

Results

Concentrations of CINO at 0.5 μM inhibit SK‐OV‐3, CRL‐1978, and CRL‐11731 ovarian cancer cells proliferation, migration and invasion (p < 0.05) without cell death and loss of cell viability, but cell viability differed among cell lines. Each cell line differed in response to CINO doses for PCNA expression (p < 0.05) as well as Annexin V pro‐apoptotic protein expression. CINO decreases mitochondrial membrane potential (p < 0.05) for SK‐OV‐3. However, in CRL‐1978 and CRL‐11731, mitochondrial membrane potential (p < 0.05) is increased in response to CINO treatment.

Conclusion

CINO is cell specific, as each cancer cell line responds differently. These data demonstrate that the mode of action of CINO is different on these three types of ovarian cancer cells.
Src Family Kinases as Proteasome Regulators and Therapeutic Targets for Treatment of Diffuse Large B‐cell Lymphoma
Authors: Cann Marissa , Priestman Melanie , Wang Qunzhao , Lawrence David
Published: November 2023

Diffuse large B‐cell lymphoma (DLBCL) patients can be categorized into two clinically relevant subtypes: activated B‐cell (ABC) and germinal center B‐cell (GCB). Since patients with ABC DLBCL tend to have a much worse prognosis than their GCB DLBCL counterparts, there is great interest in identifying effective drug targets for the treatment of this subtype. Chronic, overactive signaling through the B‐cell receptor and the anti‐apoptotic NF‐κB pathway are hallmarks of the ABC, but not the GCB DLBCL subtype. Interestingly, the Src family kinases (SFKs) are known to play important roles in both of these signaling pathways. Additionally, since the proteasome is also important for maintaining NF‐κB activity and is a target of interest for the treatment of ABC DLBCL, we hypothesized that the SFKs play a role in the regulation of the proteasome. In this study we explored this role and its potential as a therapeutic target for the treatment of DLBCL. We treated a panel of ABC and GCB DLBCL cell lines with the SFK inhibitor dasatinib and found that the ABC DLBCL cell lines are significantly more sensitive to dasatinib than the GCB DLBCL cell lines. To determine if this sensitivity to dasatinib was linked to regulation of the proteasome by SFKs, a set of novel peptide sensors were used to monitor the proteasome's chymotrypsin‐like, trypsin‐like, and caspase‐like activities. Using these sensors, we demonstrate that treatment of DLBCL cell lines with the SFK inhibitor dasatinib results in a marked decrease in chymotrypsin‐like proteasome activity for the ABC DLBCL cell lines, whereas nominal changes are observed in the GCB DLBCL cell lines. Biochemical analyses demonstrate that while there is minimal change in expression of the β5 proteasome subunit, which is responsible for the proteasome's chymotrypsin‐like activity, dasatinib treatment induces a decrease in expression of the proteasome 19S regulatory particle in isolated proteasomes. However, 19S expression remains unchanged in whole lysates, indicating that the SFKs might regulate the proteasome through a chaperone protein that helps assemble or stabilize proteasome complexes. Taken together, our data suggest that inhibition of the SFKs with dasatinib affects proteasome assembly in addition to B‐cell receptor signaling, thus making ABC DLBCL cell lines particularly sensitive to dasatinib treatment.

Support or Funding Information

This project was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Award Number TL1TR001110. The project is currently supported by the National Cancer Institute of the National Institutes of Health under Award Number F31CA196153. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
mTORC2 Regulation of the Hexosamine Biosynthetic Pathway (HBP) in Response to Nutrients
Authors: Vega‐Cotto Nicole Marie , Moloughney Joseph , Kim Peter , Wu Chang‐Chih , Lynch Thomas , Zhang Sisi , Adlam Matthew , Guntaka Sai , Chou Po‐Chien , Rabinowitz Joshua D. , Werlen Guy , Jacinto Estela
Published: November 2023

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that forms two distinct protein complexes, mTORC1 and mTORC2. mTORC1 has emerged as a central controller of cell metabolism. The role of mTORC2 in cell metabolism is less clear, but so far it has been shown to be involved in the regulation of actin cytoskeleton organization, cell growth and proliferation, and cell survival. The extracellular signals that regulate mTORC2 and its downstream targets remain to be elucidated. In a screen to identify mTORC2 targets or effectors, our lab identified glutamine:fructose‐6‐phosphate amidotransferase (GFAT1) as a physical interactor of mTOR. GFAT1 is the rate limiting enzyme of the hexosamine biosynthetic pathway (HBP). The HBP utilizes glucose metabolites and glutamine as substrates to produce UDP‐GlcNAc, which is used for protein glycosylation. We found that mTORC2 is involved in regulating GFAT1 expression and the HBP. Metabolomics analysis of mTORC2‐disrupted cells also revealed defects in metabolites of the HBP. Our preliminary findings reveal that mTORC2 could regulate GFAT1 in response to the levels of nutrient metabolites. Our studies provide insights on how mTORC2 regulates a biosynthetic pathway in response to nutrients.

Support or Funding Information

NCI
Experimental Demonstration of Localized Excess Protons at a Water‐Membrane Interface and the Effect of other Cations on Their Stability
Authors: Saeed Haitham A. , Lee James W.
Published: November 2023

The electrostatic localization of excess protons at a water‐membrane interface has now been clearly demonstrated experimentally, which corrects the Mitchellian proton delocalization view. To demonstrate the fundamental behavior of localized excess protons in a pure water‐membrane‐water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated by utilizing an “open‐circuit” water‐electrolysis system and their distributions were tested using a proton‐sensing aluminum membrane. The proton‐sensing film placed at the membrane‐water interface displayed localized proton activity while that placed into the bulk water phase showed no excess proton activity during the entire experiment. These observations clearly match with the prediction from the proton‐electrostatics localization hypothesis that excess protons do not stay in water bulk phase; they localize at the water‐membrane interface in a manner similar to the behavior of excess electrons in a conductor. Proton‐electrostatics localization hypothesis predicts that high concentration of other cations such as Mg++, K+, or Na+ could partially delocalize the electrostatically localized protons. Therefore, determination of the cation exchange equilibrium constant is another significant way to test this hypothesis. Since protons are the smallest cations and can exist as part of the water molecules, the predicted equilibrium constant for protons to electrostatically occupy the cation sites at the water‐membrane interface is likely to be much larger than one. Results indicated that the equilibrium constant for protons (H+) to electrostatically occupy the cation sites at the water‐membrane interface was about 107 as predicted. Therefore, electrostatically localized protons at the water‐surface interface are quite stable. These findings have significance not only in the science of bioenergetics but also in the fundamental understanding for the importance of water to life in serving as a proton conductor for energy transduction.

Support or Funding Information

This research was supported by Dr. Lee's start‐up research funds provided by the Department of Chemistry and Biochemistry, the College of Sciences, the Office of Research at Old Dominion University, and the ODU Research Foundation.
Activity and Kinetic Characterization of Four Human CYP2D6 Polymorphisms using the Substrates Bufuralol and Dextromethorphan
Authors: Glass Sarah , Furge Laura Lowe
Published: November 2023

Human cytochrome P450 enzymes (CYPs) are a heme‐containing enzyme superfamily that have a major role in the metabolism of drugs along with other endogenous and exogenous chemicals. Human CYP2D6 is responsible for approximately 12% of CYP‐mediated drug metabolism. There are over 100 different allelic variants of CYP2D6 with metabolic profiles ranging from poor to ultra‐rapid. Four CYP2D6 allelic variants, three with a small series of distal mutations (*34, *17‐2, *17‐3) and one possible ultra‐metabolizer (*53), were expressed and purified in E. coli to further characterize their interactions with the substrates bufuralol and dextromethorphan using HPLC with UV detection. Km and vmax values were measured for comparison between variants and the reference *1. Previous molecular dynamics simulations of these allelic variants with and without a ligand bound suggested that varied flexibility and active site accessibility altered interaction with substrates. The current metabolic and kinetic studies are compared to these molecular dynamics models to broaden the understanding of substrate interactions for CYP2D6 variants with local and distal mutations relative to the active site.

Support or Funding Information

NIH 1R15‐GM086767‐02
Investigating the Mechanism of Wnt‐Induced Chromosomal Instability
Authors: Stiers Aubrey , Willert Karl
Published: November 2023

Wnt signaling is essential for early embryonic development, the maintenance of stem cell pluripotency, determination of cell polarity, and cellular differentiation. Alterations in Wnt signaling activity have dramatic effects, resulting in developmental defects and contributing to the development of cancer. We recently found that perturbations in Wnt signaling lead to chromosomal instability (CIN), which results in aneuploidy, a hallmark of many cancer cells. However, we currently do not understand the mechanism by which Wnt promotes this CIN. We hypothesize that the protein p53 (gene name = TP53), often described as “the guardian of the genome”, acts downstream of Wnt to influence the fidelity of chromosome segregation. The goals of my research are to further study the effect of Wnt signaling on CIN and to explore the potential involvement of p53 in this process. To this end, I am utilizing DNA fluorescence in situ hybridization (FISH) as a means to detect and quantify the frequency of chromosome mis‐segregation across a population of cells. This method will allow me to detect changes in chromosome numbers in response to Wnt stimulation over time. In addition, I am creating a human embryonic stem cell line (H1/WA01) that will express fusion proteins composed of a fluorescent protein and either a histone (H2B) or tubulin. These transgene expressing H1 cells will be used to visualize mitosis and chromosome mis‐segregation. To address the involvement of p53, I will monitor both changes in gene expression of TP53 and changes in p53 protein abundance and modification, including phosphorylation and acetylation. Any observed changes in TP53 gene expression or p53 protein in response to Wnt signaling may contribute to CIN and hence to the occurrence of aneuploidy. These studies into the mechanism of Wnt‐induced CIN and the role of p53 will provide valuable insights into the development of cancer and may help identify a novel target of cancer therapeutics.

Support or Funding Information

This work is supported by a grant from the CIRM (RB1‐01406, to K.W.) and was made possible in part by the California Institute for Regenerative Medicine (CIRM) Major Facilities Grant (FA1‐00607) to the Sanford Consortium for Regenerative Medicine.
Identification of Cell Signaling Pathways involved in the repression of mouse ES Neuronal Differentiation by RAR Beta
Authors: Joseph Serenthia , Moses Diamond , Martinez‐Ceballos Eduardo
Published: November 2023

Embryonic Stem (ES) cells are pluripotent cells that can be induced to differentiate into cells of all three lineages: mesodermal, endodermal, and ectodermal. In culture, ES cells can be differentiated into mature neurons by treatment with Retinoic Acid (RA), a Vitamin A derivative. The effect of RA on cells is mediated mainly through the activation of the RA nuclear receptors of which there are three types: RAR alpha, RAR beta, and RAR gamma. As preliminary evidence from our laboratory indicates that active RAR beta acts as an inhibitor of neuronal differentiation and, since we also found that neuronal differentiation depends upon the RA‐induced activation of Hoxa1, a homeobox transcription factor, we then first sought to investigate whether RAR beta is an inhibitor of Hoxa1. We found that treatment of mouse ES cells with AC55649, an RAR beta agonist, prevented the pharmacological activation of Hoxa1 with RA. Next, we asked whether the pharmacological inhibition of RAR beta, together with RA treatment, would increase the efficiency of mouse ES cell differentiation into neurons. To test this hypothesis, mouse ES cells were treated with 1 micromolar RA plus/minus LE135, an RAR beta inhibitor, at 1 or 5 micromolar concentrations. Results from immunofluorescence analyses using a B‐Tubulin III antibody indicate that the highest efficiency of neuronal differentiation was obtained with a combination of RA and 5 micromolar LE135 for an 8 day treatment. Finally, we performed proteomic analyses on ES cells treated with RA vs RA plus AC55649 in order to identify the signaling pathways activated by the RAR beta agonist. Our proteomic analyses using antibody microarrays indicated that proteins such as p38, AKT, and Erk1 were upregulated in cells treated with RA plus the agonist as compared to cells treated wit RA alone. Thus, our results indicate that RAR beta functions as a repressor of neuronal differentiation by activating major cell signaling pathways, and its pharmacological inhibition may constitute a novel method to increase the efficiency of ES to neuronal differentiation in culture.
Extracellular Adenosine‐induced Rac1 Activation in Pulmonary Endothelium: Molecular Mechanisms and Barrier‐protective Role
Authors: Kasa Anita , Kim Kyung Mi , Shaw Mary , Batori Robert , Kumar Sanjiv , Black Stephen M , Fulon David J , Verin Alexander
Published: November 2023

Adenosine is an endogenous purine nucleoside. Cellular signaling by adenosine occurs through four known adenosine receptor subtypes (A1, A2a, A2b, A3). Extracellular adenosine concentrations from normal cells are approximately 300 nM; however, in response to cellular damage, these concentrations are quickly elevated (600–1,200 nM). Locally adenosine concentration can reach ~100 μM. In regard to stress or injury, the function of adenosine is primarily that of cytoprotection preventing tissue damage during instances of hypoxia, ischemia. Recent data implicate the involvement of adenosine in endothelial cell (EC) barrier enhancement/protection. However, the adenosine‐induced EC barrier‐protective signaling is largely unknown. Our group has previously shown that Gs‐coupled A2a adenosine receptors are primarily involved in adenosine‐induced human pulmonary artery EC (HPAEC) enhancement in vitro. Our hypothesis is that the Gs‐mediated stimulation of cAMP‐dependent Epac1‐mediated signaling cascades is involved in adenosine‐induced Rac1 activation and EC barrier preservation. Our results demonstrate that the infection of HPAEC with constitutively‐active (C/A) Rac1 (V12 Rac1) adenoviral construct significantly increases transendothelial electrical resistance (TER) reflecting EC barrier enhancement. Conversely, infection of HPAEC with D/N Rac 1 (N17 Rac1) decreases TER reflecting EC barrier compromise. Importantly, adenosine‐induced increase in TER is accompanied by Rac 1 activation while Rac 1 inhibition attenuates adenosine‐induced increase in TER. These data reveal the involvement of Rac 1 activity in adenosine‐induced barrier enhancement. Next we examined the role of cAMP‐activated Epac 1 and its putative downstream targets; Rac1 guanine nucleotide exchange factors (GEFs), Rap1, Tiam and Vav2. The depletion of Epac 1 attenuates adenosine‐induced increase in TER and Rac1 activation in human pulmonary EC. Furthermore, the depletion of Vav2 and Rap1a significantly attenuates adenosine‐induced TER increase and Rac 1 activation in HPAEC. In contrast, depletion of Rap1b modestly affects both adenosine‐induced Rac1 activity and increase in TER, whereas Tiam depletion has no effect. These data strongly suggest that Rac 1 activity is directly linked to EC barrier enhancement. Moreover, the barrier‐protective effect of adenosine involved Rac 1 activation. Further, adenosine induces Rac1 activation and EC barrier strengthening, at least in part, via cAMP‐dependent Epac1/Vav2/Rap1‐mediated signaling.
Transcriptional Changes in Aging Photoreceptor Cells of Drosophila melanogaster
Authors: Brennan Kaelan Joseph , Hall Hana , Weake Vikki Marie
Published: November 2023

As the human population ages, there is an increased risk for ocular diseases such as age‐related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Specifically, AMD is characterized by deterioration of the macula of the eye and is the leading cause of the loss of central vision in those individuals over 60. Twin studies have previously shown that non‐genetic, environmental factors contribute more than 50% of the overall risk for developing age‐related ocular diseases such as AMD. For this reason, epigenetic mechanisms have been thought to play a major role in the environmental‐based risks associated with AMD. The epigenetic factors that contribute to vision loss in the aging eye have yet to be identified, but are thought to play a pivotal role in maintaining proper ocular ability, as misregulation of these factors could lead to transcriptional changes in gene expression. We hypothesize that as photoreceptor cells age, there are changes in gene expression that contribute to the loss of vision seen later in life. Because age‐related changes in gene expression are cell autonomous, Drosophila melanogaster photoreceptor neurons were chosen as a model system due to the likelihood that these post‐mitotic cells are more sensitive to changes in gene expression. Photoreceptor cells are also likely to be at an increased vulnerability to the effects of aging because they must constantly express high levels of proteins involved in phototransduction in order to maintain their function throughout the adult lifespan. Using these Drosophila photoreceptors, we are conducting a study on the effects of aging on gene expression. Experiments to globally characterize the transcriptome of isolated nuclei from the R1‐R6 photoreceptor cells of male Drosophila were performed using RNA‐sequencing technology. We then used cluster analysis on this sequencing data to identify genes that exhibited statistically significant changes in gene expression at specific fly‐age time points. Using this cluster analysis data, we will prioritize genes from each cluster to generate subsets of genes to test through qRT‐PCR experiments on both dissected eye and isolated nuclear samples. This qRT‐PCR will allow us to validate and quantify the age‐related changes in gene expression observed by RNA‐sequencing in photoreceptor nuclei. From this, we can begin to ask questions about the role of epigenetic factors in regulating these changes in gene expression and how these factors are affected by aging.

Support or Funding Information

National Institutes of Health
Regulation of Protein Kinase R (PKR) activation during cell stress by TRBP phosphorylation
Authors: Chukwurah Evelyn Ekene , Patel Rekha C
Published: November 2023

A crucial component of cellular stress response is the attenuationof protein synthesis by phosphorylation of the α‐subunit of the eukaryotictranslation initiation factor, eIF2. This allows the cell to mount anappropriate response to the stressful conditions, while minimizing energyexpended on protein translation. However, a prolonged eIF2α phosphorylation dueto sustained stressful conditions results in the induction of apoptosis.

PKR is one of four well‐characterized serine‐threoninekinases responsible for mediating translation inhibition in response to viralinfection, endoplasmic reticulum (ER) stress, oxidative stress, and serumstarvation. During these conditions, PKR is activated via interaction either withdouble stranded RNA (dsRNA) generated during viral infections or with itscellular activator PACT (Protein Activator of PKR), resulting in itsautophosphorylation, activation of its catalytic function, and subsequent eIF2αphosphorylation.

PKR's kinase activity is inhibited by the double stranded RNA binding protein TRBP (TAR RNA Binding Protein) by a direct interaction, as well as by sequestration of PKR's dsRNA activators and PACT, there by preventing PKR autophosphorylation and downstream protein translation inhibition. Various studies have linked dysregulation of PKR activation with various neuro degenerative and neuromuscular disorders.

Recent findings indicate that TRBP also inhibits PKR inother cellular contexts (i.e. mitosis, obesity), but suggest that its abilityto inhibit PKR is dependent on various post‐translational modifications that significantly alter its binding affinity for PKR and ultimately affect PKRactivation in those contexts.

We investigated changes in both PKR‐TRBP interactions and TRBP‐mediated PKR inhibition during cell stress. We used various biochemical methods to study TRBP phosphorylation, its interaction with PKR, and effect on PKR's kinase activity. Various phospho‐mimetic and phospho‐defective mutants of TRBP were used to examine the role of phosphorylation in mediating PKRinhibition. Our results indicate a functional role of TRBP phosphorylation inregulating PKR activation in response to cellular stress, and elucidate animportant mechanism by which the deleterious sustained response to stress is down regulated.
EXAMINING APOPTOSIS IN A SEPSIS CELL CULTURE MODEL
Authors: Davis Bethany , Del Gaizo Moore Victoria
Published: November 2023

Sepsis, a systemic inflammatory response to infection of the blood by pathogenic organisms, impacts millions of people around the world every year. Pro‐inflammatory cytokines are released to fight the infection but many times lead to organ damage such as Acute Kidney Injury (AKI). AKI, a disorder characterized by the abrupt deterioration in kidney function, has a broad spectrum of consequences ranging from mild kidney injury to permanent loss in renal function and kidney failure. Common amongst critically ill patients, the development of AKI is believed to be due, at lease in part, to apoptosis. Apoptosis, or programmed cell death, is controlled by a multitude of cellular mechanisms. Cytokines can initiate the extrinsic pathway of apoptosis by binding to death receptors on the cell surface. While it is definitively known that apoptosis plays a large role in immune cell decline under septic conditions, its influence in AKI is not entirely understood. The objective of this study is to determine an efficient means of examining the role of cytokines in inducing apoptosis in renal cells in order to develop greater insight into the pathogenesis of sepsis‐associated AKI. Human immune THP1 cells were treated with a synthetic LPS to stimulate cytokine secretions. Cultured human embryonic kidney (HEK) cells were treated with either these naturally secreted or recombinant cytokines. Then flow cytometry of the HEK cells stained with Annexin V‐FITC determined the amount of apoptosis in the culture sample. Flow cytometry data demonstrates that recombinant cytokines allow for quicker analysis than naturally secreted cytokines. Apoptosis amongst HEK cells treated with recombinant cytokines for 48 hours was comparable to treatment with naturally secreted cytokines for a duration of 120 hours. Treatment with recombinant cytokines results in much quicker analysis because it excludes the time necessary for THP1 stimulation and cytokine recovery as well as significantly reduces the duration of treatment. Recombinant cytokines also have the benefit of easily picking precise concentrations of TNF‐alpha and other cytokines. With the greater understanding of the apoptotic processes associated with sepsis, the biochemical mechanism at the foundation of AKI can be further elucidated and the creation of more effective treatments for this disease may be possible.

Support or Funding Information

Elon University Undergraduate Research Program (BD), Elon University College Fellows Program (BD), FR&D (VM)
Phage Display and Structural Studies Reveal Plasticity in Substrate Specificity and Allosteric Regulation of Caspase‐3 from Zebrafish
Authors: Clark Clay , Tucker Matthew B , MacKenzie Sarah H. , Maciag Joseph J , Dirscherl Hayley B , Swartz Paul B , Yoder Jeffrey B , Hamilton Paul B
Published: November 2023

The regulation of caspase enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. Caspase‐3, an effector caspase, is the primary executioner in apoptosis, but at sub‐threshold levels required for apoptosis, the activity of caspase‐3 is used by cells for a variety of physiological reactions (referred to as adaptive responses), from remodeling the cytoplasm, neuron pruning, receptor endocytosis, macrophage function, to development of eye lens and inner ear. The roles of caspases in apoptosis are well‐known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3 (zCP3) from a cDNA library and examined substrate specificity of the recombinant protein compared to human caspase‐3 (hCP3) by utilizing novel M13 bacteriophage substrate specificity libraries that incorporated either random amino acids at P5‐P1’ or Asp fixed at P1. The results show that zebrafish caspase‐3 has similar preferences for P3‐P1’ amino acids, but the P4 position also accommodates hydrophobic amino acids. We determined the structure of zCP3 to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that amino acid substitutions at two sites may result in plasticity of the P4 amino acid by increasing flexibility of one active site loop. In addition, an allosteric site on the surface of the protein more closely resembles a similar site in caspase‐6, suggesting differential regulation when compared to the hCP3. The data suggest that zCP3 may exhibit a broader substrate portfolio, and while the known phosphorylation sites of hCP3 are conserved in zCP3, changes in an allosteric site on the protein surface, together with changes in substrate specificity, suggest overlap with the functions of caspase‐6 in zebrafish development.
Site Specific Oxidation of Calmodulin by Methionine Sulfoxide Reductase A in Drosophila
Authors: TARAFDAR SREYA , Rusan Nasser M. , Levine Rodney L.
Published: November 2023

Methionine sulfoxide reductase A stereospecifically catalyzes the reduction of S‐methionine sulfoxide to methionine and is important in defense against oxidative stress. Recently, we reported that methionine sulfoxide reductase A is also a stereospecific methionine oxidase, capable of oxidizing free and peptide‐bound methionine to S‐methionine sulfoxide. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of methionines in proteins could function as a redox‐based mechanism of cellular regulation. We also recently reported that methionine sulfoxide reductase A stereospecifically and selectively oxidizes Met77 in calcium‐bound calmodulin and can fully reduce it as well. Taking advantage of the powerful genetic tools of Drosophila, we seek to assess the physiological significance of methionine sulfoxide reductase A‐mediated reversible oxidation of calmodulin Met77. To accomplish this, we used site directed mutagenesis to generate transgenic animals carrying calmodulin mutated at Met77. Calmodulin Met77 was mutated to glutamine, alanine, leucine, or valine. Glutamine was included because it mimics methionine sulfoxide, thus creating a constitutively oxidized methionine sulfoxide state. We are currently analyzing the effect of these mutations on life span, fertility, sensation, and viability in a calmodulin null background.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, NHLBI.
Lysophospholipid Receptors, As Novel Danger Associated Molecular Pattern Receptors And Homeostasis Associated Molecular Pattern Receptors Modulate Inflammation – Novel Paradigm And Therapeutic Potential
Authors: Wang Xin , Li Ya‐Feng , Fu Hangfei , Johnson Candice , Shao Ying , Cueto Ramon , Wang Hong , Yang Xiao‐Feng
Published: November 2023

To determine whether lysophospholipids (LPLs) serve as danger‐associated molecular pattern molecules (DAMPs) in initiating inflammations, we analyzed the tissue expressions of all 26 LPL receptors in 23 human tissues and 21 mouse tissues. We also examined the expression changes of the 26 LPL receptors in five prototypic chronic sterile inflammations including rheumatoid arthritis (RA), metabolic inflammatory diseases such as coronary artery disease (CAD), metabolic syndrome, type 2 diabetes and type 1 diabetes. We made the following important findings: 1) Three LPLs including lysophosphatidylinositol (LPI), lysophosphatidylserine (LysoPS) and lysophosphatidylethanolamine (LPE) bind to two specific G protein‐coupled LPLs receptors (LPL‐GPCRs) while other four LPLs bind to more than five LPL‐GPCRs, respectively; 2) Lysophosphatidic acid (LPA), lysophingolipid sphingosine 1‐phosphate (S1P) and lysoyphosphatidylcholine (LPC) have ubiquitous physiological signaling functions among tissues whereas other LPLs, especially LPI, may have tissue‐specific functions; 3) The expressions of certain LPL‐GPCRs are significantly upregulated in human and mouse RA, CAD, and other metabolic inflammatory diseases; 4) Mechanistically, pro‐inflammatory cytokine signaling regulates the expressions of LPL receptors; 5) Toll‐like receptor (TLR), presumably bound by other DAMPs, regulates the expressions of LPL receptors; and 6) In cross‐talking, LPL receptor signaling regulates the expressions of TLRs; 7) We propose for the first time that, in parallel to the well‐accepted danger associated molecular pattern receptor model, novel homeostasis‐associated molecular pattern molecules (HAMPs) receptors (HAMPRs) are the receptors for binding to signals that are generated from endogenous metabolic processes and can initiate anti‐inflammatory/homeostatic signaling and promote inflammation resolution. From this point of view, we propose a new paradigm of that LPLs are either pro‐inflammatory DAMPs or anti‐inflammatory HAMPs for regulating the onset of inflammation, inhibition of inflammation, and resolution of inflammation. This new paradigm and new concept of HAMP receptors would significantly improve our understanding on the roles of LPLs and other endogenous metabolites in regulating the pathogenesis of inflammations and eventually lead to the future development of novel therapeutics for rheumatoid diseases, metabolic inflammatory diseases, cardiovascular diseases, and inflammatory cancers.

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NIH RO1 HL 108910‐01
FTY720 Induces Necroptosis by Regulating Ceramide Signaling at the Plasma Membrane
Authors: Ndeto Rose , Ogretmen Besim
Published: November 2023

Sphingolipids, important signaling molecules in cells, have recently been explored as cancer therapy targets. FTY720 (Fingolimod, Gilenya) is an FDA approved sphingosine analogue drug used for the treatment of multiple sclerosis (MS). FTY720 is phosphorylated by sphingosine kinase 2 (SK2), to generate P‐FTY720 to exert its immunosuppressive properties through binding to sphingosine 1‐phosphate receptors (S1PRs). FTY720 also exhibits anti‐cancer properties. Our previous studies indicated that one of the mechanism by which FTY720 induces cell death is through necroptosis. FTY720, similar to ceramide, directly binds to I2PP2A/SET (Inhibitor 2 of PP2A), consequently activating the tumor suppressor protein phosphatase 2A (PP2A). The activated PP2A then induces cell death by stimulating the activity of Receptor‐Interacting Protein Kinase‐1 (RIPK1), involved in necroptosis signaling. It is known that FTY720 can modulate sphingolipids metabolism in cells. However, little is known about the roles of FTY720 in ceramide signaling and regulation of necroptosis in lung cancer. We hereby seek to investigate the mechanisms of FTY720 in inducing necroptosis with regard to ceramide signaling. Preliminary cell viability data indicate that inhibitors of ceramide generation partially protect cells against FTY720‐induced cell death. Interestingly, using confocal microscopy, we show that FTY720 and its non‐phosphorylatable analogues lead to the formation of specific ceramide‐multi‐protein complexes at the plasma membrane involved in plasma membrane disruption for induction of necroptosis. Future mechanistic studies will help us understand the details of how these ceramide‐dependent lipid‐protein complexes are formed at the plasma membrane and how they are involved in the regulation of necroptosis in response to cellular stress evoked by FTY720 and other therapeutic agents, leading to tumor suppression.

Support or Funding Information

RO1CA088932, RO1CA173687, P30GM103339
The lipid transfer protein STARD3 modulates intracellular cholesterol localization
Authors: TOMASETTO Catherine Laure , WILHELM lea , DRIN Guillaume , ALPY Fabien
Published: November 2023

STARD3 [(StAR)‐related lipid transfer domain protein 3] belongs to the START domain protein family, a group of protein having lipid transfer properties. STARD3 (also known as MLN64 or CAB1), is overexpressed in ~25% of human breast cancer. On cancer tissue sections, we noted that STARD3 labelled large vesicles often at a perinuclear position. Consistently, we found that STARD3 is a resident protein from late‐endosomes (LE). Structurally, STARD3 is a multifunctional protein composed several conserved regions. The N‐terminal moiety called the MENTAL domain is anchored in the late endosome (LE) membrane and mediates oligomerization and cholesterol binding. At the C‐terminal end of this domain, STARD3 contains a FFAT‐like motif interacting with VAP proteins. The C‐terminal half of the protein bears a cytoplasmic START domain involved in cholesterol transfer. By interacting with the endoplasmic reticulum resident proteins VAPs, STARD3 creates membrane contact sites between endosomes and the endoplasmic reticulum, and remarkably remodels the subcellular architecture. We recently addressed how STARD3 expression modulates cholesterol localization within the cell. Using cholesterol‐specific fluorescent probes, we noted the presence of cholesterol‐enriched endosomes at the expense of other cellular compartments in STARD3 positive cells. Of interest, this STARD3‐dependent intracellular cholesterol redistribution relies both on the activity of the START domain and on the presence of membrane contact sites.

Support or Funding Information

The Institut National du Cancer ‐INCA (France) and The Ligue Nationale Française contre le Cancer