bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating host protein binding to SARS coronavirus untranslated region using immobilized RNA
Authors: Gonzalez Fabiola , Nag Anita 
Published: June 2021

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) encoded nonstructural protein 1 (Nsp1) is a key factor in dampening host gene expression, also known as host shut‐off. Nsp1 binds to the 40S ribosome, stalls ribosome assembly, and results cleavage of cellular messenger RNA (mRNA). Surprisingly, SCoV mRNA is resistant to cleavage under the same condition, allowing us to investigate host protein binding by SCoV mRNA that confers resistant to Nsp1‐initiated cleavage. The untranslated region of SCoV mRNA is about 100 nucleotides long and contain a RNA stem‐loop necessary for resisting endonucleolytic cleavage. We used T7 polymerase driven transcription and T4 RNA ligase to synthesize biotinylated RNA containing untranslated region of SCoV mRNA. The RNA was then incubated with cellular proteins in the presence of Nsp1 followed by UV crosslinking. RNA‐protein complexes were isolated using streptavidin‐coated magnetic beads. Proteins were separated by gel electrophoresis followed by their identification using LC‐MS/MS. Identification of these proteins will allow us to decipher the mechanism of host shutoff by Nsp1.Support or Funding InformationSC INBREFurman AdvantageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glyceraldehyde‐3‐phosphate dehydrogenase differentiates the cytotoxic mechanisms of cancer‐active electrophiles in a yeast model
Authors: Harach Janel L , Lynch Connor J , Montovano Giancarlo , Olsen Stefan H , Foley Timothy D 
Published: June 2021

Diverse examples of thiol‐reactive electrophiles, ranging from trivalent arsenicals to 3‐bromopyruvate (3BP), have shown promise as anti‐cancer agents. Inhibition of the glycolytic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has been identified as a primary mechanism underlying the anti‐cancer activity of 3BP but the importance of targeting GAPDH for the cellular actions of arsenicals remains uncertain. We have studied the effects of 3BP and the representative trivalent arsenical phenylarsine oxide (PAO) on the growth, viability, metabolism, and GAPDH activity of the yeast Saccharomyces cerevisiae, which reproduces metabolic features of Warburg‐positive tumor cells. Both 3BP and PAO abolished growth and, within hours, caused substantial cell death measured by propidium uptake and fluorescence. Neither compound increased ROS levels nor inhibited protein synthesis. Indeed, lower concentrations of PAO appeared to decrease cellular ROS. Critically, exposure of intact cells to 3BP produced a rapid and nearly complete inhibition of GAPDH activity. In contrast, PAO did not inhibit the cellular activity of GAPDH despite findings, also to be presented, that GAPDH was one of many proteins from yeast lysates able to bind immobilized PAO. These results suggest that changes in the activity of GAPDH, in situ, may differentiate the cytotoxic pathways of cancer‐active electrophiles. The mechanism(s) underlying the impairment of growth and viability of S. cerevisiae by arsenicals remains under investigation.Support or Funding InformationFunding was provided by the University of ScrantonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Calcineurin B Homologous Protein (CHP) Isoform Specific Function
Authors: Davis Shane , Provost Joseph 
Published: June 2021

Mammalian cells ubiquitously express the Sodium‐Hydrogen Exchanger Isoform 1 (NHE1) a membrane transporter responsible for intracellular pHi, motility and proliferation. Calcineurin B Homologous Protein (CHP) in part regulates activation and kinetics of the NHE1. CHP has two isoforms, CHP1 and CHP2, whose physiological function remains unclear but both have a unique role in nascent tumor survival. CHP binds NHE via the N and C terminal domains. There is a considerable tertiary structure between both the N terminal domains (amino acids 1–90) and the C terminal domain (aa 111/110–195/196) with a 72% and 74% conserved homology, an intervening loop domain is unique to each CUR. This signature domain CHP unique region (CUR: CHP1 R91 to S110 and CHP2 R91 to S111) when bound to NHE has a specific and distinct structure indicating its potential role in effecting CHP‐NHE1 related functions. The purpose of this study is to investigate how the these CHP unique regions contribute to CHP isoform cell function, specifically cell proliferation and location. Using site directed mutagenesis, we have generated epitope tagged (DDK and MYC) wild type versions of both isoforms of CHP and a variant with the respective CUR domain deleted (CHP1 ΔCUR1 and CHP2ΔCUR2). To distinguish the importance of the CUR domains we have swapped CUR domains between each CHP (CHP2(CUR1) and CHP1(CUR2). These mutations will be expressed in lung fibroblasts expressing NHE1 (CCL39) and PS120 cells (null NHE1 expressing CCL39 derivatives) to determine the impact of each CUR domain on cellular location in the absence and presence of NHE1 and on cell proliferation. We hypothesize that the CUR domain is critical for CHP isoform specific function and NHE1 interactions and present the role of both CHP isoforms on NHE1 related behavior.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Vesicle Formation at the Golgi Complex by the Arf GEFs Gea1 and Gea2
Authors: Muccini Arnold , Brownfield Bryce , Gustafson Margaret , Fromme J. Christopher 
Published: June 2021

In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs) Gea1, Gea2, and Sec7. Sec7 activates Arf1 at the trans‐Golgi network, and mechanisms regulating the activity and recruitment of Sec7 to the Golgi have been well characterized. For example, Sec7 is recruited to the TGN by four small GTPases including its substrate Arf1. However, Gea1 and Gea2, which activate Arf1 at earlier Golgi compartments, do not share the same regulatory mechanisms as Sec7. While initially thought to be redundant paralogs, we have found that Gea1 and Gea2 localize to separate regions of the Golgi despite sharing common regulatory elements. As such, the purpose and mechanism for their differential localization remains unclear. I aim to understand the functional relevance for their distinct localization and will present preliminary evidence suggesting that Gea recruitment is coordinated by Golgi SNARE complexes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel p53 barrier destruction mechanism through MMP‐7 mediating nucleolar matrix assembly & intra‐nucleolar cleaving p53 during cancer stem cell transformation
Authors: Yu wei‐hsuan , Wu Erxi , Li Yongqing , Yu Shuan Su C. , Huang Po Tsang 
Published: June 2021

The molecular mechanism underlying the striking enlarged nucleolus instead of disperse small nucleolus in cancer stem cells remains elusive. Our novel findings that MUC‐1 C‐ter participates in nucleolus matrix assembly has further prompted the idea that the cell surface receptor MUC‐1, via MMP‐7 proteolytic cleavage of the MUC‐1 SEA domain releases cryptic bioactive fragments that orchestrate nucleolar activities. Furthermore, MUC‐1 C‐ter induces a unique porous bulky ball‐shaped, cage‐like nucleolus that functions as a nucleus molecular “garage”for potent transcription factors, such as γ‐catenin and p53. Specifically, this cage segregates the transcription factors from its target promoter. In addition, the intra nucleolus degradation of p53 by MMP‐7 to generate a 35 kDa p53 fragment suggests that the nucleolus could also facilitate the novel subnucleus compartment for proteolytic processing tumor suppressor protein, p53 in cancer stem like cell, which implicate the novel molecular mechanism for destructing the p53 barrier and inducing cancer stem cell transformation. Salinomycin, an anti‐cancer stem cell agent, can disrupt the nucleolus inducing translocation of p53 and sensitize cancer stem cell to chemotherapy drug. These data provide important new insights into the dysregulations of cancer stem cells as well as highlight targets for cancer therapeutics.Support or Funding InformationTaiwan Ministry of Science and technology 94‐2320‐B‐002 ‐120This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CaMKIIa regulates let‐7 microRNAs in opioid tolerance
Authors: He Ying , Wang Zaijie Jim 
Published: June 2021

We have previously reported that let‐7 miRNAs modulate opioid tolerance, through complex mechanisms that involve the μ opioid receptor and other signaling molecules. Chronic opioid treatment up‐regulated brain let‐7 miRNA expression, which was functionally correlated with the development of opioid tolerance. The aim of this study was to understand the mechanisms by which opioids regulate let‐7 miRNA. We determined the let‐7 transcript and found that the expression of primary let‐7 (pri‐let‐7) remained unchanged in human neuroblastoma SH‐SY5Y cells that were treated with morphine (1 mM, for 48 h). In agreement with the in vitro findings, chronic morphine treatment induced opioid tolerance, but did not alter the level of pri‐let‐7 in mouse brain. These data suggested that the robust elevation of let‐7 occurred at the post‐transcriptional level. In the presence of KN93, inhibitor of Ca2+ /calmodulin‐dependent protein kinase II (CaMKII), chronic morphine treatment failed to generate let‐7 up‐regulation in SH‐SY5Y cells. In CaMKIIaT286A point mutation mice lacking functional CaMKIIa, opioid tolerance was absent in −/− mice, but tolerance was full developed in littermate wildtype +/+ mice. Interestingly, let‐7 miRNA levels were much lower in CaMKIIaT286A mutant mice than those in wild‐type mice. Moreover, morphine failed to regulate let‐7 miRNA in CaMKIIaT286A mutant (−/−) mice. Taken together, these data suggested that CaMKIIa is required for the biogenesis and possibly regulation of let‐7 miRNA in opioid tolerance.Support or Funding InformationR01 DA041809This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Doxorubicin Mitochondrial Dysfunction and Cytotoxicity in Human Proximal Tubular Epithelial Cells is Attenuated by Resveratrol (RES)
Authors: Valentovic Monica A , Brown Kathleen C , Dial Mason , McGuffey Rachel 
Published: June 2021

Doxorubicin (DOX, Adriamycin), is a cancer chemotherapy agent used in the treatment regimen for breast, Hodgkin and Non‐Hodgkin lymphomas, small cell lung cancer and acute lymphoid leukemia. Unfortunately, doxorubicin clinical usage is associated with permanent cardiomyopathy and renal impairment. The cellular mechanisms for doxorubicin mediated renal cytotoxicity have yet to be understood. Resveratrol (RES) is a phytoalexin found in many fruits including grapes, blueberries, cranberries as well as chocolate and peanuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies in cancer patients as well as animal models. This study tested the hypothesis that RES protection for DOX renal cytotoxicity, in human noncancerous renal proximal tubular epithelial cells (HK‐2), is mediated by preserving mitochondrial function and attenuating oxidative stress. HK‐2 cells were plated and grown in cell culture equilibrated for 48 h. Cells were next pre‐incubated for 1 h with 0 (DMSO) or 5 or 7.5 uM RES followed by a 24 h co‐incubation with 0–5 uM DOX. All results were obtained from 3 independent experiments. Western analysis probed for protein carbonylation was used as an indicator of oxidative stress. Mitochondrial function was assessed using a Seahorse analyzer system. Prior to examining mitochondrial function, cell number and substrate concentrations were optimized based on the manufacturer's recommendations. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Western blot was conducted on whole cell lysate for SIRT1 and PGC1alpha. Viability was assessed using MTT leakage and tyrpan blue exclusion cell counts. RES did not alter cell viability as indicated by comparable MTT values between DMSO and RES groups (p>0.05). DOX was cytotoxic to HK‐2 cells within 24 h. Pretreatment with RES provided protection from DOX to HK‐2 cells. Mitochondrial respiration assessed as oxygen consumption rate (OCR) was diminished by DOX. DOX impaired mitochondria function within 24 h and RES provided protection. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial function. Further studies are needed to probe changes in specific mitochondrial proteins mediated by DOX.Support or Funding InformationSupported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence. M.D. and R.M. supported by WV NASA Undergraduate Research Fellowship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physical Interactions between Heme Oxygenase 1 and the Cytochromes P450 in the Endoplasmic Reticulum Membrane
Authors: Connick John Patrick , Reed James R , Backes Wayne L 
Published: June 2021

Heme oxygenase 1 (HO‐1) and the cytochromes P450 (P450s) are endoplasmic reticulum (ER)‐bound enzymes that rely on NADPH‐cytochrome P450 reductase (POR) to provide the electrons that fuel their catalytic cycle. Previous work has shown that under normal conditions POR levels are limiting and P450s must compete for POR binding. Our lab and others have also shown that, in addition to simple competition, certain P450s can bind POR as multimeric P450•P450 complexes that are associated with altered P450 activity. Because HO‐1 also relies on physical interaction with POR in the ER, we hypothesized that it too would compete with the P450s for POR binding. Additionally, the prevalence of P450•P450 interactions led us to consider the possibility that HO‐1 might form analogous homomeric (HO‐1•HO‐1) or heteromeric (HO‐1•P450) complexes that could affect POR binding and enzyme activity. To gain insight into how these enzymes are physically organized, we used bioluminescence resonance energy transfer (BRET). We implemented BRET by expressing a pair of tagged proteins, one with a green fluorescent protein (GFP) tag and one with a Renilla luciferase tag, in HEK293T cells. Energy transfer only occurs if the tags are in close proximity, allowing us to detect protein complexes by observing changes in the tag's emission spectrum. We are also able to assess the relative stability of protein complexes by observing how co‐expression of a third, untagged protein affects the BRET signal of the tagged pair. Using this technique, we have determined that HO‐1 forms homomeric complexes that are more stable when POR is limiting. We have also found that HO‐1 forms heteromeric complexes with several P450s including CYP1A1 and CYP2D6. The BRET signal for the HO‐1•CYP2D6 complex was robust in both the presence and absence of co‐transfected untagged POR, showing that the complex is stable when POR is present. Interestingly, the BRET signal for the HO‐1•CYP1A1 complex was low in the absence of POR but increased significantly when cells were co‐transfected with untagged POR. Doubling the amount of POR led to an attenuation of the BRET signal. Current and future work will use BRET to determine whether HO‐1 affects the physical interaction between POR and P450s and, conversely, whether the presence of a P450 can affect the HO‐1•POR complex.Support or Funding InformationNIH ES004344, NIH ES013648, NIH GM123253This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA
Authors: Riaz Maryam , Cushen Spencer C. , Phillips Nicole R. , Goulopoulou Styliani 
Published: June 2021

BackgroundVascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell‐free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell‐free mtDNA.HypothesisMitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death‐dependent mechanisms.Material and MethodsHuman umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80–90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM ‐ 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non‐apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real‐time PCR techniques.ResultsTreatment of HUVECs with rotenone increased early apoptosis (Fig. A) and late apoptosis/necrosis (Fig. B; 1‐way ANOVA followed by Sidak's post‐hoc test). Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone (Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.07; Sidak's post‐hoc test). Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p≥0.84).ConclusionsMitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.Support or Funding InformationResearch support: NIH (R25HL125447)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Egr‐1 contributes to the enhanced expression of Giα proteins and hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats
Authors: Polchtchikov Stephanie , Li Yuan , Anand‐Srivastava Madhu B. 
Published: June 2021

Early growth response, Egr‐1, is known to activate the expression of several genes implicated in the development of vascular dysfunction. Earlier studies have shown that angiotensin II (Ang II) enhances the expression of Egr‐1 and Giα proteins in vascular smooth muscle cells (VSMC). In addition, the role of enhanced levels of endogenous Ang II has been shown to contribute to the enhanced expression of Giα proteins in spontaneously hypertensive rats (SHR). The present study was therefore undertaken to examine if VSMC from SHR also exhibit enhanced expression of Egr‐1 proteins and explore its role and underlying mechanisms in the hyperproliferation of VSMC in SHR. The levels of Egr‐1 proteins in VSMC from SHR started increasing at 2 weeks as compared to their normotensive control, WKY (Wistar‐Kyoto rats) and at 12 weeks, the levels of Egr‐1 proteins were increased significantly by 80%. The enhanced levels of Egr‐1 proteins were restored to control levels by losartan (an AT1 receptor antagonist), BQ123 (an ETA receptor antagonist), BQ788 (an ETB receptor antagonist), as well as by antioxidants, such as N‐acetyl‐L‐cysteine (NAC) and diphenyleneiodonium (DPI) but not by PD123319 (an AT2 receptor antagonist). In addition, pharmacological inhibitors of platelet‐derived growth factor (PDGF), insulin‐like growth factor (IGF) and epidermal growth factor (EGF) receptors also inhibited the overexpression of Egr‐1 proteins in VSMC from SHR. Furthermore, the enhanced expression of Egr‐1 proteins was attenuated by PD098059, an inhibitor of mitogen‐activated protein kinase (MAPK), but not by wortmannin, an inhibitor of phosphatidylinositol‐3‐kinase (PI‐3‐K). VSMC from SHR exhibit hyperproliferation which was attenuated by knocking down Egr‐1 by siRNA. In addition, siRNA of Egr‐1 also inhibited the overexpression of Giα‐2 and Giα‐3 proteins, as well as cell cycle proteins, including cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4), Cdk2 and phosphorylated retinoblastoma protein (pRb). These results suggest that VSMC from SHR exhibit an overexpression of Egr‐1 proteins, which is attributed to the enhanced levels of endogenous Ang II and ET‐1, oxidative stress, growth factor receptors and MAPK and that the overexpression of Egr‐1, through the enhanced expression of Giα and cell cycle proteins, contributes to the hyperproliferation of VSMC from SHR.Support or Funding InformationGrant from CIHRThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.