bluuebunny/crossref_metadata_2025_split huggingface dataset:
Steroid Sulfatase activates the integrin signaling pathway in human cervical cancer cells
Authors: Ye Dong‐Jin , Kwon Yeo‐Jung , Baek Hyoung‐Seok , Chun Young‐Jin 
Published: June 2021

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a crucial function in the production of estrogens and androgens that induce the growth of hormone‐dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not elucidated clearly. To investigate the function of STS in cancer cell growth, we examined whether STS is able to enhance the integrin signaling pathway. We observed that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), the major metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. STS overexpression and DHEA treatment induced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Furthermore, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS enhances the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up‐regulation of integrin β1 expression and activation of FAK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blue light‐excited retinal delocalizes plasma membrane bound PIP2 and triggers stress responses challenging the cellular integrity
Authors: Ratnayake Kasun , Payton John L. , Karunarathne Ajith 
Published: June 2021

The photoreceptor chromophore, retinal, an aldehyde derivative of vitamin A, harvests light to generate visual perception. Upon photon absorption, the receptor bound 11‐cis‐retinylidene (11CR) isomerizes to all trans retinal (ATR), changing the electrostatic environment in the chromophore‐binding site, subsequently transforming the inactive receptor to a momentary active state. The subsequent release of ATR allows the receptor to be relaxed to the basal state, enables a new 11CR binding and continuous photon reception. Due to the defects in retinal clearance mechanisms, higher concentrations of 11CR and ATR can be found in the retina, and a fraction of excess retinal can enter the circulation, exposing the rest of the body to retinal as well, leading to pathophysiological effects. Nevertheless, visible light induced, photoreceptor independent signaling activities of retinals and mechanisms of retinal phototoxicity are less explored. Here, we show that, retinal, in the presence of blue light, irreversibly remove the plasma membrane (PM) bound phosphatidylinositol 4,5 bisphosphate (PIP2) in photoreceptor and non‐photoreceptor cells. Interestingly, addition of retinals alone without blue light exposure as well as exposing cells to only blue light without retinal, did not show any detectable PIP2 dislodging from PM. Additionally, even in the presence of retinal, the other wavelengths of lights failed to show any effect on PM bound PIP2. Moreover, compounds that are structurally similar to retinal such as retinol and retinoic acid did not show any effect on PIP2 in the presence of blue light. Data further show that, this PIP2 removal from the PM is not due to regular enzymatic hydrolysis of PIP2, but as a result of lipid peroxidation induced by the generation of singlet oxygen and reactive oxygen species. The resultant PIP2 dislodging is followed by cellular stress responses and cell death. Clinically used photodynamic therapeutic agents (photosensitizers), also showed a unique PIP2 dislodging effect from PM in the presence of blue light as well as in other wavelengths of light, similar to blue light excited retinal. This further confirms retinal's ability to harvest light to induce photosensitization that leads to lipid peroxidation which suggests its future potential in photodynamic therapeutics in cancer as well as identification of new molecular targets for phototoxicity. Overall, here we show that, retinal exhibit light sensitivity in non‐photoreceptor cells and trigger crucial signaling pathways altering the cellular fate.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IQGAP Proteins Do Not Regulate Mitogen‐Activated Protein Kinase (MAPK) Signaling through Direct Interaction with Ras
Authors: Morgan Chase J. , Li Zhigang , Hedman Andrew C. , Sacks David B. 
Published: June 2021

The Ras family of small GTPases modulates numerous essential processes, including development and tissue repair. Activating Ras mutations occur in approximately 30% of human malignancies and understanding the molecules that modulate Ras function is key to elucidating its role in oncogenesis. The IQGAP family of scaffold proteins regulates numerous signaling proteins, including members of the MAPK pathway and the Rho family GTPases RAC1 and CDC42. However, it is controversial whether IQGAP proteins interact with Ras family GTPases. While initial reports showed no binding between IQGAP1 and Ras proteins (EMBO J 1996; 15:2997), subsequent studies claim that IQGAP1 and IQGAP3 bind K‐Ras (Biochem Biophys Res Commun. 2014; 444:360) and H‐Ras (Nat Cell Biol 2008; 10:971), respectively. To resolve these discrepancies, we performed careful and thorough binding analyses. Here we demonstrate that neither IQGAP1 nor IQGAP3 interacts with the major Ras isoforms, namely H‐, K‐, and N‐Ras. No IQGAP1 or IQGAP3 was detected when flag‐tagged, constitutively active mutant (G12V) H‐, K‐, or N‐Ras proteins were immunoprecipated from HEK293 cells. Similarly, IQGAPs were not detected in pulldown experiments from HEK293 lysate with wild type or constitutively active (G12V) GST‐Ras proteins. By contrast, we immunoprecipitated Myc‐IQGAP3 from HEK293 cells and show for the first time that IQGAP3 binds B‐Raf, an established binding partner of IQGAP1 (Proc Natl Acad Sci U S A 2007; 104:10465). It is possible that IQGAP and Ras do not interact directly in cells, but rather are in a complex with effectors such as B‐Raf, which could explain the positive binding results at over‐expressed, nonphysiological protein concentrations. Knowledge of the binding partners of Ras is of considerable biological and medical importance, as the direct interactors of Ras provide potential therapeutic targets for Ras‐driven cancers.Support or Funding InformationThis work was supported by the National Institutes of Health Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Follicle Stimulating Hormone Stimulation of a Murine Monocyte Cell Line
Authors: Leamon Annie B. , Cohen Brian D. 
Published: June 2021

Osteoporosis, an illness resulting in weak and brittle bones, will soon cause over $25 billion dollars of economic damage to the US economy each year. For decades, menopausal osteoporosis was thought to be the result of estrogen loss due to depletion of ovarian follicles. Recent research, however, has shown that the hormone primarily responsible for estrogen synthesis, follicle stimulating hormone (FSH), can contribute to bone degeneration. What is unclear is the specific mechanism by which FSH has this effect. We hypothesized that FSH may contribute to monocyte differentiation to osteoclasts. To test this, we used the RAW 246.7 murine monocyte cell line which can differentiate into osteoclasts in the presence of RANKL. We were able to find evidence of FSH‐dependent signaling in cells FSH treated cells with or without RANKL. In particular, time dependent activation of the cyclic AMP Response Element Binding Protein (CREB) was observed. Interestingly, RANKL treatment changed the pattern of phosphorylation of CREB and the related transcription factor ATF1. Since CREB partially regulates osteoclast differentiation and function this could be one part of the mechanism by which FSH stimulates osteoclastogenesis and bone demineralization. With this in mind, future therapies targeting FSH action could be developed to prevent the onset of osteoporosis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis of the Strigolactone Signaling through Plant SCF E3 complex
Authors: Hinds Thomas Richard , Shabek Nitzan , Mao Haibin , Zheng Ning 
Published: June 2021

Strigolactones (SL) are plant hormones that play an essential role in the control of plant branching, and act as rhizospheric signals for communication with symbiotic fungi and parasitic plants. MORE AXILLARY GROWTH2 (MAX2/D3) and DWARF14 (D14), are crucial signalling components that regulate SL‐repressed plant branching. D14 is a α/β hydrolase that hydrolyses SL into an inactive intermediate. MAX2/D3 is an ubiquitin ligase that has a leucine‐rich (LRR) F‐box region, which interacts with D14 in a SL‐dependent fashion. Once formed the D14‐D3 complex can bind to the repressor DWARF53 (D53) for subsequent ubiquitination and proteasomal degradation. The D3‐D14‐D53 complex is proposed to be involved in SL signaling. However, the recruitment of D53 by the D3‐D14 complex and the role of SL perception and hydrolysis remains largely unclear. In this study, we determine the structures of rice MAX2/D3 and D14‐SL complexes. In addition we measure the hydrolytic activity of D14 with and with out its binding partners D3 and D53. We also measured the binding of these three components using ALPHA technology. Our structural and biochemical studies shed light on SL sensing mechanism that is largely regulated by MAX2/D3 ubiquitin ligase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PRL Phosphatases Promote Tumor Progression by Regulating the Level of Intracellular Magnesium
Authors: Hardy Serge , Kostantin Elie , Wang Shan Jin , Uetani Noriko , Tremblay Michel L. 
Published: June 2021

The Phosphatase of Regenerative Liver (PRL) enzymes (PRL‐1,‐2,‐3; gene name PTP4A1, PTP4A2, PTP4A3) are members of the protein tyrosine phosphatase family. These enzymes have been identified as key contributors to tumor progression and metastasis in several human cancers, yet the molecular basis of their pro‐oncogenic property is unclear. Using mass spectrometry studies, we identified the CNNM magnesium transporters as key binding partners of PRLs in an evolutionarily conserved complex that regulates the intracellular magnesium concentration. Structural analysis of this complex indicates that the second cystathionine β‐synthase (CBS) domain of CNNM underlines a binding loop unique to the CNNM family that only exists in organisms having PRL orthologs. Mutation of an evolutionarily conserved aspartic acid located in this CBS domain of CNNM3 was able to completely abolish the interaction with PRL‐2 resulting in reduced tumor growth. Also, either the abolishment of complex formation or PRL‐2 knockdown resulted in reduced magnesium transport. Since magnesium is a critical metabolic effector, we confirmed that mitochondrial respiration is altered in cells isolated from PRL2‐null animals and is associated with a lower ATP turnover. This is also consistent with the observed decreased body weight of PRL‐2−/− mice, suggesting that their cellular metabolism is inherently less efficient. As cellular magnesium transport is regulated by intrinsic mechanisms controlling various magnesium transporters and magnesium uptake in cells, we also aimed to examine the involvement of PRLs in the adaptive response related to changes in magnesium availability. Using various cell lines cultured under low magnesium conditions, we observed a decrease in intracellular magnesium levels correlating with a marked increase of PRL‐1 and ‐2 (PRL‐1/2) expression. Although the expression levels of the magnesium transporter CNNM3 remained unchanged after magnesium depletion, we found that the interaction between endogenous PRL‐1/2 and CNNM3 was also markedly increased, indicating a pivotal regulatory role for PRL‐1/2 in the complex to compensate for decreased magnesium availability. On the contrary, upon increasing intracellular magnesium levels by overexpressing the TRPM7 magnesium transporter, we observed a reduction of PRL‐1/2 expression. Consistent with this, using an in vivo reporter mouse model, PRL‐2 tissue expression was upregulated when dietary magnesium was limited. Overall, our findings suggest that PRL phosphatases regulate cellular magnesium levels, essential for cell survival/proliferation, which may explain much of the clinical relationship between PRLs and cancer.Support or Funding InformationThis research is supported by an operating grant from Canadian Institutes of Health Research (CIHR); CIHR MOP 142497.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the effects of novel positive allosteric modulators on the functionality of the α7 nicotinic acetylcholine receptor (nAChR)
Authors: Reilly‐Andujar Francis , Lasalde José A. , Colón José O. 
Published: June 2021

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in the development of various neurodegenerative disorders such as Alzheimer's, Parkinson's, Schizophrenia, and a series of inflammatory disorders. In recent years, there has been an increased interest in the pharmacological modulation of receptor function as a therapeutic target. However, these efforts have been limited due to poor temporal and tissue selectivity. Therefore, there is a growing need for the synthesis of new modulators that can address these problems. Positive allosteric modulators (PAMs) for the α7‐nAChR have grown in significance and are currently being developed by many pharmaceutical companies. The California Northstate University has provided us with three novel PAMs (PAM‐2, OCF‐3, CF‐3) in order to test their functionality on the α7‐nAChR. Previous tests on these compounds have shown indirect PAM activity through calcium influx tests using fluorescence microscopy. However, the direct effects of these drugs on the α7‐nAChR have not been tested. Taking advantage of the Xenopus oocyte expression system we are studying the direct effects of these drugs on the α7‐nAChR using Two‐Electrode Voltage Clamp (TEVC) electrophysiology. Our results show that pre‐application of 20 μM PAM‐2, OCF‐3, or CF‐3 on oocytes expressing the α7‐nAChR causes a significant increase in response amplitude compared to controls. Also, we observe a dose dependent increase in current amplitude for PAM‐2 and OCF‐3. These results are consistent with the idea of positive allosteric modulation. Reverse potentials were performed to verify the effects of PAM‐2 and OCF‐3 on calcium permeability of the α7‐nAChR. We have observed that for PAM‐2 the shift in Vrev (ΔVrev) is similar to that of our control. However, for OCF‐3 we observe a reduction in ΔVrev in a low vs high calcium solution. Further studies of these compounds will give us a better insight into their potential use as therapeutic targets of the α7‐nAChR.Support or Funding InformationThis project was funded by the MARC Program at the University of Puerto Rico, Rio Piedras NIGMS Grant #5T34GM007821‐38.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redox Modulation of NQO1
Authors: Siegel David , Dehn Donna , Quinn Kevin , Backos Donald , Di Francesco Andrea , Bernier Michel , Reisdorph Nichole , de Cabo Rafael , Ross David 
Published: June 2021

NQO1 is a FAD containing NAD(P)H‐dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C‐terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β‐lapachone which caused rapid oxidation of NAD(P)H, NQO1 could be readily pulled‐down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β‐lapachone demonstrating that under non‐denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)+/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co‐localize with acetyl α‐tubulin and the NAD+‐dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle, NQO1 may provide NAD+ for Sirt2‐mediated deacetylation of microtubules. Overall, the data suggest that NQO1 can act as a redox‐dependent switch where the protein responds to the NAD(P)+/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.Support or Funding InformationThis work was supported by NIH/NDDK grant 1R01DK109964‐01A.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Globally Monitoring Allosteric Coupling in the A2A Adenosine Receptor by NMR in Solution
Authors: Eddy Matthew , Jacobson Kenneth , Stevens Raymond , Wuthrich Kurt 
Published: June 2021

In human G protein‐coupled receptors (GPCRs), drug binding near the extracellular region drives conformational changes over 30 Å away at the intracellular surface, leading to complex formation with partner proteins that initiate signaling. GPCR crystal structures have revealed the need for deeper understanding of conformational dynamics to describe mechanisms of allosteric coupling between the extracellular and intracellular surfaces.Using nuclear magnetic resonance (NMR) in solution, we provide a structural basis for allosteric coupling in the human A2A adenosine receptor (A2AAR), a validated drug target for Parkinson's disease and cancer co‐immunotherapies. Production of A2AAR in Pichia pastoris allowed stable‐isotope labeling with 2H,15N, and 13C, yielding reproducible high‐quality NMR correlation spectra. Assignment of endogenous tryptophans and glycines provided numerous probes distributed over the receptor intracelluar surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The “toggle switch” Trp 2466.48 was especially sensitive to variable drug efficacy due to the changes in relative orientation of the nearby aromatic ring of Phe 2426.44 in the highly conserved Pro5.50‐Ile3.40‐Phe6.44 (P‐I‐F) activation motif, providing a highly responsive probe of changes in structure and dynamics upon agonist binding. The novel NMR probes also enabled us to observe ligand efficacy‐related local conformational polymorphisms near the intracellular surface.The distributed NMR probes were utilized to monitor the impact of a key mutation in the A2AAR allosteric center at Asp522.50. Amino acid replacement of Asp522.50 leads to immediate loss of intracellular signaling. In NMR studies, this mutation caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at Trp 2466.48 and the P‐I‐F motif, demonstrating an interplay between the allosteric center and Trp 2466.48, and providing novel insights into a signaling mechanism that may be applicable to a wide range of class A GPCRs.Support or Funding InformationAmerican Cancer Society (MTE)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Homogenization Methods for Extraction of Live Bacteria and Recombinant DNA in Soil
Authors: Proctor Caleb , Atwood James , Easparro Brandon , Morehouse Zachary 
Published: June 2021

In many soil types, ubiquitous bacteria are present very in low quantities due to the inherent challenges of microorganism survival in environmental conditions. While, in some cases, only present in low quantity, soil microorganisms are a critical component of our ecosystem. Soil microbiome studies aim to characterize the microbial repertoire and to correlate the microbial population with soil and ecological functions. While many studies involve culturing and isolation of bacteria harvested from the soil, some organisms are hard to propagate in the laboratory or require the sample to be processed directly to maintain analyte integrity. Given time involved and complexity of culture based enrichments, many modern studies involve direct cell lysis and DNA recovery from soil followed by PCR for target enrichment and detection. In order to infer absolute taxa abundance non‐conserved, targeted gene products are quantified by qPCR. While this approach is rapid and robust, qPCR is subject to inhibitory effects of soil based compounds such as humic acids and polysaccharides.In this study, we compare the recovery and qPCR detection of a modified bacterial standard from multiple types of spiked soil samples. The standard was composed of E. coli, Bacillus subtilis, and Pseudomonas fluorescens which were genetically modified with green florescent protein (GFP). The modified organism allowed the absolute subtraction of background signal originating from native organisms found in the soil samples. The soil samples were spiked with decreasing quantities of the standard. The samples were extracted through a combination of bead milling and silica spin column based DNA purification. The DNA was then quantified by qPCR against the GFP gene and the resulting data established limits of recovery for the combination of organisms. These limits were evaluated side by side with a common incubation lysis to evaluate the effects of mechanical lysis on bacterial cells in soil samples that vary in resistance to lysis. Along with the limits of recovery, cycle threshold values (ct values) were compared to evaluate the impact of PCR inhibitor release.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.