bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ribosome Recycling Factor in Translational Coupling as a Ribosome Releasing Factor
Authors: Inokuchi Yoshio , Quaglia Fabio , Kaji Hideko , Kaji Akira
Published: June 2021

The objective of this presentation is to study the in vivo actions of ribosome recycling factor (RRF) and compare them with those found in vitro. RRF is known to catalyze three separate reactions: release of tRNA and mRNA from the post‐termination complex (PoTC), and splitting of the ribosome of the PoTC. To study the role of RRF in translational coupling in vivo, we used E. coli harboring temperature sensitive (ts) RRF and assayed by following downstream reading of translationally coupled ORF. At the non‐permissive temperature, ribosomes remain on the termination codon of the junction sequence of coupled ORFs and translate downstream ORF without the Shine‐Dalgarno (SD) sequence. The readings were in all three frames due to thermal frameshift at the termination codon. Introduction of sequence complementary to the 3′‐terminal of 16S rRNA into the region surrounding the junction sequence increased downstream reading. When upstream ORF was short, translation of the downstream reading was abolished, suggesting that the ribosomes released by RRF are moving toward the SD sequence of the upstream ORF. The thermal frame shift at the stop codon was also stopped by the upstream SD sequence. Our data suggest that the ribosome‐bound mRNA may take a secondary structure around the junction sequence. For in vitro studies, we used mRNA that incorporated different radioactively labeled amino acids based on the frameshift at the junction sequence, UAAUG, of two translationally coupled ORFs. In the absence of RRF, the ribosome stayed on the mRNA and translated in frame with the termination codon UAA. In the presence of RRF, amino acid incorporation occurred in frame with the start codon AUG. This suggests that RRF releases the ribosome from UAA and the released ribosome binds to AUG and begins translation. With the use of tethered, unsplittable ribosomes (Ribo‐T) in the in vitro system, we showed that complete ribosomal splitting is not required for the action of RRF in translational coupling. Therefore, the main role of RRF in translational coupling appears to be the release of ribosomes from mRNA.

Support or Funding Information

Supported and funded by Creative Biomedical Research Institute (CBRI) in Philadelphia, Pennsylvania.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overcoming the Rapid Metabolism of the Promising Anticancer Natural Product Rooperol
Authors: Jemal Mauricio A , Schwartz Zachary T , Zhao Zhenze , Rodebaugh Mary , Christy Quinn M , Shelton Spencer , Du Liqin , Kerwin Sean M
Published: June 2021

Rooperol [1,5‐bis(3′,4′‐dihydroxyphenyl)pent‐1‐en‐4‐yne] is a naturally occurring bis‐catechol that was originally isolated from the African Potato (Hypoxis hemerocallidea). Rooperol shows promising anti‐cancer activity in vitro, but it is rapidly and completely metabolized to inactive phase‐II metabolites in vivo. To address this problem, analogues of rooperol have been designed in which the linker separating the catechol groups is varied or in which some or all of the catechol groups are replaced with more metabolically stable isosteres in order to explore the role of the bis‐catechol groups and the effect of catechol replacement on anticancer activity. Here we report the synthesis and cancer cell cytotoxicity of these rooperol analogs. Based upon the activity of these compounds in a cell growth inhibition assay with HeLa cells, we conclude that the bis‐catechol nature of rooperol is not an essential feature for its cytotoxicity and that an analog of rooperol that is predicted to be more resistant to metabolism yet which retains much of the anti‐cancer activity of the natural product can be identified.

Support or Funding Information

William and Ella Owens Medical Research Foundation and Texas State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Molecular Dynamics Simulations to Evaluate Ligand Binding of L, L‐diaminopimelate Aminotransferase, an Enzyme Involved in Protein and Peptidoglycan Biosynthesis
Authors: Adams Lily , Babbitt Gregory , Hudson André
Published: June 2021

A common protocol, Site Directed Mutagenesis (SDM) experiments facilitate the alterations to a gene sequence to study downstream effects on its product. In addition, inhibitor/antagonistic studies investigate the potential of common ligands to inhibit or inactivate enzymes. Both approaches have seen minor updates in recent years, but methods to streamline these processes are highly desired. For example, a rate‐limiting step in SDM lies in choosing key nucleotides that will subsequently affect enzymatic activity. To facilitate SDM studies and subsequent analyses to save time and resources, a comprehensive and comparative molecular dynamics (MD) software package – DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0 – has been applied. Here, we use a novel aminotransferase, L, L‐diaminopimelate aminotransferase (DapL) as a test to propose a method of augmenting SDM and related experiments using MD simulations. DapL is involved in peptidoglycan and protein metabolism. As such, the essentiality of the dapL gene in eubacteria is currently a key question yet to be answered. Information to supplement knowledge of this enzyme will aid in evaluating its potential as a narrow spectrum antibiotic target. We have used DROIDS 2.0 to investigate the impact of different point mutations and putative antagonistic ligands on the activity of the DapL enzyme, identifying those that most significantly impact its dynamic behavior. In addition, the DROIDS 2.0 protocol was taken further to predict mutations that lessen binding affinity to chosen inhibitors, which may have the potential to guide the rational design of inhibitory ligands. Comparative dynamics in DROIDS 2.0 can be visualized (Figure 1) in a variety of statistical plots, colored structures, and colored movies of protein movement, enhancing interpretation and communication to a broad audience.

Support or Funding Information

National Institutes of Health (NIH) award (R15GM120653)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Full‐Time Physioxic Cell Culture and Handling Improves MSC Proliferation Over Hypoxic Pre‐Conditioning in Vitro
Authors: Henn Alicia D. , Darou Shannon , Alm Kersti , Yerden Randy
Published: June 2021

Cells cultured and handled in traditional room air experience a dramatic shift in oxygen levels when administered in vivo. It has been reported that hypoxic pre‐conditioning of mesenchymal stem/stromal cells (MSC) in culture can change cell function, increasing cell survival in vivo after administration, changing cell migration and modulating cytokine and exosome secretion patterns. These treatments place cells in low oxygen atmospheres for lengths of time from 15 minutes to 36 hours, sometimes with a period of reoxygenation in room air conditions before administration, risking oxidative stress. Here, we compare hypoxic pre‐conditioning to full‐time physioxic conditions with the null hypothesis that in vitro culture conditions make no difference to MSC morphology and proliferation rates after the cells are introduced to physioxic conditions as if administered. MSC cultured and handled under completely controlled conditions in the Xvivo System were split into two groups: (1) cells cultured under full‐time physioxia (5%O2), and (2) cells cultured under supraphysioxic room air incubator conditions (18%O2) and then pre‐conditioned in low oxygen (1% O2) conditions for 24 hours before reoxygenation at supraphysioxic room air incubator oxygen (18% O2) for 24 hours. The cells were then exposed to venous blood oxygen levels (5% O2) as if injected in vivo. Using an immersion oxygen probe, we recorded vessel headspace and pericellular medium oxygen levels under each regimen. Using the PHI Holomonitor M4, we recorded holographic time‐lapse images of the cells for changes in cell morphology or proliferation rates. In looking at cell culture oxygen change kinetics, we found that sharp changes in atmospheric oxygen levels were followed by much slower oxygen changes in the vessel headspace, and pericellular oxygen. MSC were actually at low oxygen conditions for far less time than the pre‐conditioning period. Hypoxic pre‐conditioning after incubation at 18%O2 increased the numbers of cells that failed to divide at physioxic oxygen levels, disproving the null hypothesis. We concluded that full‐time physioxic conditions for cell handling as well as incubation may be more conducive to MSC proliferation than hypoxic pre‐conditioning.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of IGSF3 in Lung Epithelial Barrier Disruption Induced by Cigarette Smoke Exposure
Authors: Schweitzer Kelly , Petrache Irina
Published: June 2021

Rationale

Cigarette smoke (CS) exposure disrupts lung epithelial and endothelial cell barrier function which is linked to lung injury and inflammation. We identified a tetraspanin‐interacting protein, IGSF3, with roles in cell proliferation, adhesion, and migration via unclear mechanisms. We have shown that lung IGSF3 is decreased by cigarette smoke (CS) exposure and that loss of IGSF3 can disrupt sphingolipid metabolites. Of these, sphingosine‐1 phosphate (S1P) protects endothelial barrier function but may disrupt epithelial barrier via distinct receptors. We investigated if IGSF3 and S1P are required for maintaining the lung epithelial barrier in response to CS.

Methods

Human bronchial lung epithelial cells (Beas2b) were stably transfected with lentivirus control or IGSF3 shRNA (shIGSF3). Trans‐epithelial resistance (TER) was measured using electric cell‐substrate impedance sensing. CS extract was prepared using research cigarettes (3R4F, University of KY). Phosphatidylinositol lipid strips (Echelon) were used to study IGSF3‐lipid binding.

Results

Compared to control shRNA, knockdown of IGSF3 did not alter epithelial barrier function as measured by TER. Exposure to CS (7.5%), as expected, decreased epithelial cell monolayers TER in control cells by 35%. CS‐induced effects on TER were attenuated by treatment with N‐acetylcysteine or with (R)‐FTY720‐phosphonate (1R), a sphingosine‐1 phosphate receptor 1 (S1PR1) agonist, and were worsened by sphingosine kinase‐1 inhibition using SKI2. The CS‐induced decrease in TER was significantly worsened (by 55%, p<0.01 vs. control cells exposed to CS) in shIGSF3 ‐ transfected cells which responded in turn to the barrier‐enhancing effect of N‐acetylcysteine treatment (by 30%, p<0.05). A putative Pleckstrin‐homology (PH) domain was identified in the C‐terminal cytoplasmic domain of IGSF3 which predicted binding to PI(3,4)P2, which was then experimentally confirmed. Further, site directed mutagenesis within PH domain disrupted binding of IGSF3 with tetraspanin CD81.

Conclusions

IGSF3 is required for partial maintenance of the lung epithelial barrier function in response to CS injury. Loss of IGSF3 may worsen CS injury of the airways, contributing to inflammation, an effect that could be alleviated by antioxidants and S1P1‐mediated signaling. The mechanisms of IGSF3 barrier effects may include binding to and tethering PI(3,4)P2 and CD81 at the cell membrane via its PH domain.

Support or Funding Information

Funding: Wollowick Chair of Pulmonary Research (IP), Department of Medicine Research Microgrant (KSS) from National Jewish Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of calcium in RGS10 regulation of inflammatory signaling in microglia
Authors: Almutairi Faris , Alqinyah Mohammed , Wendimu Menbere , Vella Stephen , Moreno Silvia , Hooks Shelley B.
Published: June 2021

Regulator of G‐protein Signaling (RGS10) is a member of a family of proteins that canonically bind and terminate signaling downstream of G proteins. RGS10 is highly expressed in brain and immune cells, with specific enrichment in microglia. Microglial RGS10 serves an anti‐inflammatory and neuroprotective role by suppressing expression of pro‐inflammatory genes, such as tumor necrosis factor‐alpha (TNF‐α), and cyclooxygenase (COX‐2) in response to TLR4 receptor activation. We recently demonstrated that this anti‐inflammatory activity of RGS10 occurs through an undefined G protein‐independent mechanism. Further, RGS10 expression is suppressed by activation of TLR4 receptor, resulting in amplification of inflammatory gene expression. The molecular mechanisms directly linking TLR4 activation to RGS10 suppression are also undefined. In order to exploit the anti‐inflammatory effects of RGS10 in the development of therapeutics for the treatment of neuroinflammation and accompanying neurodegeneration, it is essential to define both the mechanism for RGS10 suppression of inflammatory gene expression, and the mechanism for RGS10 silencing. The current study implicates calcium signaling as playing a critical role in the regulation of both processes. We have identified STIM2 as a novel RGS10 interacting protein in BV2 microglia cells and in RAW macrophages. STIM2 is an ER‐resident calcium sensor that mediates store‐operated Ca2+ entry (SOCE) through Orai channels in the plasma membrane in response to ER calcium depletion. STIM2 activation and its coupling to Orai‐mediated calcium signaling is strongly implicated in regulation of inflammatory signaling in microglia. Therefore, we hypothesized that RGS10 suppression of TLR4‐stimulated inflammatory gene expression reflects regulation of STIM2‐Orai activity. Our results demonstrate that pharmacological inhibition of Orai channels completely blocks the amplification of TLR4‐stimulated COX‐2 and TNFα expression in response to loss of RGS10 expression. Further, RGS10 knockout microglia exhibit markedly enhanced SOCE in response to ER calcium depletion. These results suggest that RGS10 suppresses COX‐2 and TNF‐α expression downstream of TLR4 activation of Orai‐mediated SOCE. Further, we demonstrate that thapsigargin, which triggers transient rise in cytoplasmic calcium and ER calcium depletion, strongly enhanced LPS‐induced RGS10 silencing, suggesting a potential mechanism for feedback regulation of calcium signaling. These findings provide critical insight into the mechanism that RGS10 utilizes to regulate inflammatory signaling. Defining these mechanisms in microglia is essential to develop novel therapeutics that target RGS10 in treatment of neuroinflammatory diseases.

Support or Funding Information

National Institutes of Health

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Insights into Cytochromes P450 11B1 and 11B2 Interaction with Ligands and Redox Partner Proteins
Authors: Brixius‐Anderko Simone , Scott Emily E.
Published: June 2021

Human steroidogenic cytochrome P450 enzymes 11B1 (CYP11B1) and 11B2 (CYP11B2) generate cortisol and aldosterone, controlling stress/immune responses and blood pressure, respectively. Excessive cortisol production by CYP11B1 results in Cushing's disease, while aldosterone overproduction by CYP11B2 causes hypertension and cardiac disease. Treatment of each disease with drugs inhibiting the respective enzyme is impeded because CYP11B1 and CYP11B2 have 93% identical amino acid sequences and no structures of CYP11B1 are available to identify structural differences that could be exploited clinically. A two‐fold strategy is employed herein to support the development of more selective treatment options for Cushing's and hypertension. First, structural differences between human CYP11B1 and CYP11B2 were identified. The first X‐ray structure of human CYP11B1 was solved, doing so in complex with the non‐selective inhibitor fadrozole. Comparison with a previous CYP11B2 structure also binding fadrozole revealed large differences in inhibitor orientation in the respective active sites. The two CYP11B enzymes bind distinct enantiomers of fadrozole, with CYP11B1 binding (S)‐fadrozole and CYP11B2 binding (R)‐fadrozole. Knowledge of the distinct active site architectures will facilitate structure‐based drug design for the treatment of Cushing's and hypertension. A second strategy explores the concept of selective inhibition of CYP11B enzymes interactions with their redox partner protein adrenodoxin (Adx). Binding assays revealed that adrenodoxin binding on the CYP11B2 surface significantly increases CYP11B2 affinity for its substrate in the distant active site, with much less effect on CYP11B1. Any differences in the protein/protein interaction might be exploited to provide an orthogonal approach for selective inhibition of CYP11B enzymes. A combination of structural and functional studies is thus used to identify similarities and differences in CYP11B interactions with Adx and with ligands to facilitate the design of compounds useful in the treatment of Cushing's disease and hypertension.

Support or Funding Information

X‐ray data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL). The SSRL Structural Molecular Biology Program is supported by the US Department of Energy Office of Biological and Environmental Research and by the US National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program and the National Institute of General Medical Sciences. This research was funded by R37 GM 076343 and startup funds from the University of Michigan

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation
Authors: Gordon Joseph W. , da Silva Rosa Simone , Field Jared , Martens Matthew , Dolinsky Vernon W. , Aliani Michel
Published: June 2021

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that Nix is a lipotoxicity‐responsive gene that accumulates in response to diacylglycerols induced by high‐fat and sucrose (HFS) feeding and exacerbated by exposure to gestational diabetes (GDM) during fetal development. Genetic inhibition of stimulus‐activated autophagy results in impaired exercise tolerance and reduced glucose uptake, yet reducing excessive mitophagy improves insulin resistance following high‐fat feeding. To provide a better understanding of these observations, we undertook a gene expression array and shot‐gun metabolomics in soleus muscle from rodents on an HFS diet and/or exposed to GDM. Interestingly, we observed a modest reduction in several autophagy‐related genes including Beclin‐1, ATG3, ‐5, and ‐7. Moreover, we observed an alteration in the ratio of cardiolipin to phosphatidic acid and reduced mitofusin‐2 expression, suggesting aberrant mitochondrial dynamics underlies muscle insulin resistance. In a series of gain‐of‐function and loss‐of‐function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium‐dependent activation of DRP‐1, and mitophagy. In addition, Nix‐induced mitophagy leads to myotube insulin resistance through activation of mTOR‐S6K inhibition of IRS‐1. Finally, we demonstrate that Nix‐induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix‐induced mitophagy and muscle insulin resistance during an overfed state when overall autophagy‐related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome insulin resistance.

Support or Funding Information

Support by NSERC, DREAM‐CHRIM, and Research Manitoba.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipopolysaccharide at low concentration suppressed tight junction assembly in intestinal epithelial cells via AMPK suppression and MAPK pathway
Authors: Meesomboon Roojanaat , Wongkrasant Preedajit , Muanprasat Chatchai
Published: June 2021

Intestinal tight junction acts as a physical barrier and selective filter in intestine. Effect of low concentration of lipopolysaccharide (LPS) on AMPK activity, which is central to tight junction regulation, and its mechanism in intestinal epithelial cells (IEC) are not well characterized. This study aimed to investigate the effect of low‐concentration of LPS on AMPK suppression and intestinal tight junction permeability. To test whether low‐concentration of LPS inhibits AMPK phosphorylation, western blot analysis was performed. The result revealed that low dose LPS (1 ng/ml) suppresses AMPK activation in T84 cells. In order to investigate whether LPS suppressed intestinal tight junction assembly, calcium switch assays were performed. The result showed that LPS significantly suppressed tight junction assembly at 6 to 24 hours post‐treatment, indicating that low‐concentration of LPS inhibits tight junction assembly in IEC. Moreover, this effect was disappeared in the presence of U0126, a MAPK inhibitor. This data indicate that low‐concentration of LPS inhibits tight junction assembly via MAPK pathways. In conclusion, LPS induced AMPK suppression and tight junction permeability via MAPK pathway in IEC. This study shed light on the mechanism of intestinal diseases involving gram negative bacterial infection.

Support or Funding Information

This work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University. (BRG5980008)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optical tweezers with IRM, TIRF and Widefield: studying cytoskeletalprocesses with or without the need for fluorescence labeling
Authors: Simpson Trey , Raja Ali , Au Ernie , Tafoya Sara , Sitters Gerrit
Published: June 2021

Microtubules and actin filaments are dynamic cytoskeletal structures that interact with motor proteins and play a fundamental role in many essential biological processes including cell division, cell migration, and mechanosensing.

Single‐molecule analysis played a central role in revealing many aspects of these complex and dynamic interactions. During a typical dynamic single‐molecule experiment, cytoskeletal filaments are imaged for an extensive period of time using fluorescence methods. It can be highly desirable to study these individual protein filaments with high contrast and at high temporal resolution but without the need for fluorescence labeling, to make the assay set‐up less laborious, costly and – in some experiments – prevent inducing phototoxicity.

Here we present an experimental arrangement that includes optical tweezers in combination with Interference Reflection Microscopy (IRM) and Total Internal Reflection Fluorescence (TIRF) Microscopy. Interference Reflection Microscopy is a recently introduced imaging method that allows visualizing biological structures in 3D without the need of fluorescence labeling and with sensitivity exceeding DIC microscopy. Total Internal Reflection Fluorescence microscopy provides high resolution fluorescent imaging of specimen near the working surface with high signal to noise ratio resulting in improved single‐molecule surface assays. In addition, we demonstrate the faster 2D imaging capabilities obtained – compared to confocal and STED techniques – by introducing widefield microscopy methods to our system.

In this work, we will discuss the experimental design and show an overview of the latest results obtained using this single‐molecule approach.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comprehensive identification of micropeptides encoded by long noncoding RNAs in human tissues
Authors: Cao Huojun , Shao Fan , Li Mark , Sweat Mason , Qian Qingwen , Guo Yuwei , Amendt Brad , Yang Ling
Published: June 2021

Many poorly characterized long noncoding RNAs (lncRNA) are expressed in human pancreas. Recently, a few lncRNAs have been found encode very small open reading frame, so‐called micropeptides. By regulating larger protein complex, these micropeptides have been shown to have important roles in fundamental biological processes such as muscle performance, mTORC1 activation and cell movement. To determine if any micropeptide play critical role for pancreas functions, we have took a bioinformatics approach to comprehensively analyze protein coding potential for lncRNAs expressed in human pancreas. We found more than 30 lncRNAs that are likely encode micropeptides. We have verified one conserved micropeptide, which we named Beta cell Glucose Regulated Micropeptide 1(BGRM1). We found BGRM1 regulate calcium homoeostasis and insulin secretion in beta cell. We have also performed mass spec to identify its binding proteins. Our result shows that some of lncRNA expressed in human pancreas might actually be translated as micropeptides and they play important roles for pancreas functions. We have also used the same pipeline to analysis lncRNA expressed in other organs. These results provides foundations for future comprehensive analysis of micropeptides functions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long Noncoding RNA H19 Plays an Important Role in a4‐Regulated Intestinal Epithelial Barrier Function Through microRNA 675
Authors: Chung Hee K. , Lan Shaoyang , Wang Shelley R. , Kwon Min S. , Xiao Lan , Turner Douglas J. , Wang Jian‐Ying , Jaladanki Rao N.
Published: June 2021

The mammalian intestinal epithelial barrier protects the subepithelial tissue by preventing the intrusion of luminalnoxious substances and microbiota, and the barrier dysfunction occurs commonly in various pathologies. The protein phosphatase 2A‐associated protein α4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. We have recently reported that targeted α4 deletion in intestinal epithelial cells (IECs) disrupts the epithelium homeostasis and impairs the barrier function, but its exact mechanism remains largely unknown. The H19 long noncoding RNA regulates expression of different genes via its encoded microRNA 675 (miR‐675) and is involved in the regulation of gut epithelial barrier. The current study tested the hypothesis that α4 modulates intestinal epithelial barrier function through H19/miR‐675 axis.

Methods

Studies were conducted in IEC tissue‐specific α4 knockout (IE‐α4−/−)mice and differentiated IEC‐Cdx2L1 cells. Levels of H19 and miR‐675 were measured by quantitative real‐time RT‐PCR analysis, and various proteins were examined by Western immunoblotting analysis. Gut permeability was measured by FITC‐conjugated dextran in vivo and by transepithelial electrical resistance (TEER) and FITC‐dextran assays in vitro.

Results

Conditional deletion of α4 in IECs increased the levels of mucosal H19 and miR‐675 in the small intestine, which was associated with a significant increase in gut permeability in IE‐α4−/− mice relative to control littermates. The levels of several intercellular junction proteins, including ZO‐1 and E‐cadherin, also decreased in the α4‐deficient intestinal epithelium. α4 silencing in cultured IECs by transfection with specific siRNA (siα4) also increased cellular H19 and miR‐675 abundances and disrupted the epithelial barrier function as indicated by a decrease in TEER and an increase in paracellular flux of FITC‐dextran. Both α4‐deficient intestinal epithelium in IE‐α4−/− mice and α4‐silenced IECs exhibited the decreased levels of RNA‐binding protein HuR that prevented miR‐675 processing from H19. Ectopically expressed HuR in α4‐silenced cells not only abolished the increased miR‐675 but also restored the epithelial barrier function to near normal level.

Conclusions

These results indicate that increased H19 in the α4‐deficient intestinal mucosa plays an important role in the epithelial barrier dysfunction through miR‐675 and that the induction in H19/miR‐675 axis by α4 deletion is prevented by HuR overexpression.

Support or Funding Information

Department of Veterans Affairs Merit Award (RNJ, DJT, JYW) and NIH‐NIDDK (JYW)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NLRP3 Inflammasome as a Novel Target to Abrogate Nicotine‐Induced Podocyte Injury
Authors: Singh Gurinder Bir , Kshirasagar Naresh , Patibandla Sai , Hussain Tahir , Li Xiang , Li Pin‐Lan , Koka Saisudha , Boini Krishna M
Published: June 2021

Recent in vivo and in vitro studies have established the role of nicotine in chronic kidney diseases. However, the molecular mechanisms of how nicotine induces chronic kidney disease are still unclear. The present study tested whether nicotine induces NLRP3 inflammasome formation and activation and thereby contributes to podocyte injury and dysfunction. Confocal microscopic analysis showed that nicotine treatment increased the colocalization of NLRP3 with Asc in podocytes compared to control cells. Pretreatment with caspase‐1 inhibitor, WEHD abolished the nicotine‐induced colocalization of NLRP3 with Asc, suggesting the formation of Nlrp3 inflammasomes. Correspondingly, nicotine treatment significantly increased the caspase‐1 activity and IL‐1β production compared to control cells. The prior treatment with WEHD significantly attenuated the nicotine‐induced caspase‐1 activity and IL‐1β production. Immunofluorescence analysis showed that nicotine treatment significantly decreased the podocin and nephrin expression compared to control cells. However, prior treatment with WEHD attenuated the nicotine‐induced podocin and nephrin reduction. In addition, prior treatment with ROS inhibitor, NAC significantly attenuated the nicotine‐induced caspase‐1 activity, IL‐1β production and podocin and nephrin reduction in podocytes. Based on these results, it is concluded that nicotine‐induced the NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury and dysfunction (supported by NIH grant, DK104031).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Orbital shaking drives differential changes in OK proximal tubule cell metabolism and endocytosis
Authors: Ren Qidong , Gliozzi Megan L. , Rittenhouse Natalie L. , Edmunds Lia R. , Rbaibi Youssef , Poholek Amanda C. , Locker Joseph D. , Jurczak Michael J. , Baty Catherine J. , Weisz Ora A.
Published: June 2021

Kidney proximal tubule (PT) cells have high metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate (GFR) and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT metabolism is impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA Seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96h revealed significant time‐ and culture‐dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis and endocytic capacity in our culture system that highlight the complexity of O2‐dependent and mechanosensitive crosstalk to regulate PT cell function.

Support or Funding Information

Tsinghua MD Scholars Program

NIH R01 DK101484

NIH P30 DK079307

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epidermal Growth Factor‐Induced Phosphorylation Changes in Rat Inner Medullary Collecting Duct
Authors: Chou Chung‐Lin , Knepper Mark A
Published: June 2021

Epidermal growth factor (EGF) is a potent mitogenic agent promoting cell proliferation, differentiation and survival. The kidney thick ascending limb of Henle's loop and distal convoluted tubule were identified as major sites of EGF synthesis. Downstream from these sites, EGF has been shown to modulate the osmotic water permeability of the collecting duct. The present study sought to determine the EGF‐elicited phosphorylation changes in inner medullary collecting duct (IMCD). Rat IMCD suspensions were treated with 1 μM EGF or vehicle for 30 min. Samples from three replicates were analyzed by TMT isobaric labeling quantitative protein mass spectrometry after phosphopeptide enrichment. A total of 12260 phosphopeptides with unique phosphorylation sites were quantified in EGF‐treated IMCD, with 78 peptides increased and 100 peptides decreased in phosphorylation based on dual statistical criteria. The enriched motif of upregulated phosphopeptides is consistent with activation of proline‐directed kinases, with a unique motif of [S/T]‐P‐K. DAVID analysis of 178 peptides (in 142 proteins) that underwent significant phosphorylation change shows ErbB as the prominent KEGG signaling pathway, confirming that EGF activates ERK mitogen‐activated protein kinase (MAPK), Mapk1 at T183/Y185 and Mapk3 at T203/Y205, via the canonical Ras‐Raf‐MEK pathway. There are five proteins (Atf2, eIF4ebp1, Raf1, Slc9a1, SP1) with phosphorylation sites that are known ERK targets. Among cytosolic docking proteins that can direct the intracellular effects of EGF, several proteins that underwent phosphorylation changes are associated with the PI3k‐Akt‐Mtor pathway, which regulates translation initiation complex eIF4F formation and the translation elongation processes (Ago2, eIF2ak3, eIF2ak4, eIF4ebp1, Eef2, Eef2k, Fbxo6, Pdcd4). In addition, several proteins associated with the Plcγ calcium signaling pathway were significantly altered in phosphorylation. These include Plcg1, Plcb4, Itpr2, Camk2g, Ppp3cb (calcineurin B) and Nos1. EGF had no significant effect on known vasopressin‐regulated phosphorylation sites of the aquaporin‐2 water channel (AQP2) (S256, S261, S264, S269) or the vasopressin‐regulated urea channel (UT‐A1) (S84, S486, S499). However, several modulators of cytoskeleton rearrangement (Cfl1, Fnbp1, Macf1, Parva, Plec, Sipa1l1, Specc1l, Trip10) and trafficking proteins (Dnajc5, Epn3, Mvb12a, Stx6, Ston2, Sytl5, Trak2) underwent phosphorylation changes. EGF also induced phosphorylation changes in several RNA polymerase II‐associated nuclear proteins (Cdk12, Gtf2f1, Leo1, Iws1, Rbm5, Rpap3, SP1). This data can be useful in further investigation of the EGF's action in renal collecting duct cells.

This 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 Protein Phosphatase 1 prevents high potassium mediated downregulation of the thiazide‐sensitive sodium chloride cotransporter NCC
Authors: Kortenoeven Marleen L.A. , Wu Qi , Cheng Lei , Fenton Robert A.
Published: June 2021

The “aldosterone paradox” is a poorly understood physiological mechanism in which increases in the hormone aldosterone either serve to stabilize blood pressure by increasing sodium‐chloride (NaCl) reabsorption or to excrete potassium (K+). Altered NaCl transport via the NaCl cotransporter NCC in the renal distal convoluted tubule (DCT) has been suggested to play a central role in switching from one response to the other. Under low NaCl intake, NCC abundance and activity is high, whereas it is low during high K+ intake ‐ leading to increased delivery of NaCl to downstream segments and increased electrogenic K+ secretion. Despite its essential role, little is known about the molecular alterations in the DCT during the aldosterone paradox, and the proteins involved in the regulation of NCC during this response.

To define the proteome of the DCT and how it is modified by increased circulating aldosterone levels subsequent to long term low dietary NaCl or high K+ intake, transgenic mice with expression of enhanced green fluorescent protein (eGFP) under the control of the parvalbumin promoter were used. These mice have specific eGFP‐labeling of DCT cells, allowing high purity fluorescence‐activated cell sorting (FACS) of DCT cells. Mice were maintained on low NaCl or high K+ diets for 4 days, which resulted in increased aldosterone levels and a change in NCC abundance. Following FACS, the DCT proteome was determined by liquid chromatography‐coupled tandem mass spectrometry. 3078 unique proteins were identified in the DCT. 210 proteins were significantly altered in abundance following a low NaCl diet. 625 were significantly altered in abundance following a high K+ diet. Protein Phosphatase 1α (Pp1α) abundance was only increased by high K+ diet. In freshly isolated mouse tubules from the renal cortex, a higher K+ concentration in the medium for 1–4 days resulted in a significant decrease in NCC expression and increased expression of PP1α, mimicking the in vivo situation. The K+‐induced decrease of total NCC levels could be inhibited by treating the cells with the PP1‐blocker tautomycetin, suggesting that PP1 is involved in the K+‐induced downregulation of NCC. In summary, these results suggest that PP1α is involved in the decreased NCC abundance seen during long term high potassium intake.

Support or Funding Information

Danish Medical Research Council, Fondation LeDuq, Novo Nordisk Foundation, Aarhus University Research Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Apolipoprotein A‐IV Receptor: a Possibility of an Orphan GPCR?
Authors: Mahmood Ammar , Stein Lauren , Tso Patrick , Samson Willis , Yosten Gina
Published: June 2021

Apolipoprotein‐A4 (apoA‐IV) is a 46 kDa protein that participates in many physiological functions including glucose homeostasis, lipid metabolism, and satiation. Thus, apoA‐IV could serve as a potential target candidate for the treatment of obesity, diabetes, and cardiovascular diseases. A previous study showed that apoA‐IV binds to the plasma membrane of human hepatic cells, but the membrane receptor responsible for apoA‐IV binding remains unknown. In addition, apoA‐IV exerts its physiological roles through PI3K/Akt signaling pathway in rat hypothalamus and mouse adipocytes. We therefore hypothesize that apoA‐IV interacts with a G‐protein‐coupled receptor, more specifically an orphan GPCR. To test this hypothesis, we are employing our Deductive Reasoning Strategy to match apoA‐IV with its potential receptors. Human gastric carcinoma cells (KATO III) and human liver hepatocellular carcinoma cells (HEPG2) treated with apoA‐IV exhibited significant increases in c‐FOS mRNA expression as measured by qPCR. In addition, KATO III and HEPG2 cells expressed many of the overlapping orphan GPCRs. These results suggest that an orphan GPCR for apoA‐IV ligand may potentially exist. Testing other cell lines for their responsiveness to apoA‐IV treatment and expression for orphan GPCRs as well as employing siRNA‐mediated knockdown of orphan GPCRs that are expressed by all tested cell lines is being performed to identify a potentially specific orphan GPCR that interacts with apoA‐IV. Identification of apoA‐IV receptor would provide a novel platform for apoA‐IV‐based therapeutics.

Support or Funding Information

National Institute of Health

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Varying Cation Types Differentially Stabilizes Poly(dA:dT) DNA
Authors: Schultz Thomas , Grilley Dan
Published: June 2021

The two meters of DNA in your cells is packaged by eight histone proteins into a complex called a nucleosome, which allows it to fit into a space one millionth of a meter in diameter. Storage in nucleosomes makes DNA inaccessible to the cellular machinery that is responsible for reading the information contained within the DNA and expressing the genetic code. Nucleosomes form on only approximately 70% of the DNA, and are excluded from forming on poly(dA:dT) tracts (A‐tracts). In nucleosomes, AA base steps are preferentially found at positions of high bending towards the minor groove such that a pattern of AA base steps spaced 10 base pairs apart leads to a highly stable nucleosome. Conversely, many AA base steps linked together to form an A‐tract will exclude nucleosomes. This A‐tract nucleosome exclusion is biologically important as it allows A‐tracts to act as regulatory elements and provide access to DNA binding proteins such as transcription and replication factors. In the context of a nucleosome and in naked DNA, AA base steps are known to form a narrowed minor groove. This narrowed minor groove brings the negative charges of the phosphodiester backbone into closer proximity which is normally not favored due to like‐charges repulsing. Our research is focused on the energetics of forming the narrowed minor groove that occurs in A‐tracts with a specific focus on the stabilization caused by cations. Using absorbance and circular dichroism spectroscopy, we show that the structure and stability of A‐tract DNA is dependent on the size and hydrogen bonding capacity of the cations that are in solution. This cation dependence tracks with the size of the A‐tract DNA, supporting the idea that there is cooperativity between AA base steps. To test the biological importance of these findings, we performed in vitro nucleosome reconstitutions in which generic sequence DNA and A‐tract DNA compete for limited quantities of histone proteins. Using these competitive reconstitutions, we show that A‐tracts are excluded from nucleosomes in a manner that depends on the cation in solution. Our results show that A‐tract structure, unlike generic sequence DNA, is sensitive to the type of cation that is present inside of cells, and that this sensitivity is important for understanding the packaging of DNA into nucleosomes.

Support or Funding Information

This research was supported by an Undergraduate Research and Creativity grant to TS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Lysine Acetylation Regulates the Choice of the Okazaki Fragment Maturation Pathway
Authors: Balakrishnan Lata , Njeri Catherine , Ononye Onyekachi , Surendran Sneha , Howald Olivia , Kaur Jasmeet
Published: June 2021

Eukaryotic DNA replication machinery has evolved to accurately maintain the human genome through decades of life and across generational time. Mutations in the genome can initiate and promote human diseases, such as cancer and neurodegenerative disorders. During replication, lagging strand synthesis initiated by the error‐prone DNA polymerase alpha primes template DNA with a RNA/DNA initiator primer. This is further elongated by high‐fidelity DNA polymerase delta, creating short stretches of DNA, known as Okazaki fragments. Because millions of these fragments need to be created and accurately ligated per cell division, the risk of mutagenesis is higher in the lagging strand compared to the leading strand. Maturation of the Okazaki fragment necessitates the displacement of the initiator primer, to remove the RNA segment prior to ligation. Two redundant pathways have been proposed in this process, one, known as the short flap pathway, rapidly removes the RNA primer and allows ligation of the polymerase alpha synthesized DNA primer. The other pathway, known as the long flap pathway, promotes displacement of the entire initiator primer and the region is resynthesized by the high‐fidelity DNA polymerase delta. Many of the proteins involved in Okazaki fragment maturation pathway are post‐translationally modified by lysine acetylation. Biochemical studies analyzing enzymatic properties of in vitro acetylated Okazaki fragment proteins show alterations in protein‐DNA, protein‐protein interactions and enzyme function. Our preliminary analysis of in vitro acetylated recombinant Okazaki fragment processing (OFP) proteins show that lysine acetylation regulates enzyme activity in a manner consistent with promoting the long flap pathway. Lysine acetylation inhibits the function of the short flap pathway nuclease, flap endonuclease 1 (FEN1) and stimulates the functions of nearly all the proteins in the long flap pathway. Further, using reporter constructs, we showed that in mammalian cells, increasing cellular lysine acetylation correlated with use of the long flap pathway for repair processing. Our current model proposes that lysine acetylation of Okazaki fragment proteins regulates the choice of the long flap pathway, resulting in higher‐fidelity lagging strand synthesis.

Support or Funding Information

This work was supported by funding from National Institute of Health (GM098328) and IUPUI New Faculty Startup Award.

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