bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Interaction between Glucocorticoid Receptor and beta‐arrestin proteins
Authors: Petrillo Maria Grazia , Cidlowski John
Published: June 2021

Glucocorticoids (GCs), vital stress hormones released as the end products of the hypothalamic‐pituitary‐adrenal axis, are involved in controlling inflammation. The actions of glucocorticoids occur through the activation of the glucocorticoid receptor (GR) which, like other members of the steroid hormone receptor superfamily, functions as a ligand‐activated transcriptional regulator. Throughout its intracellular journey, liganded GR creates physical contacts with regulatory proteins, thus affecting its activity and function. The protein‐protein interactions of GR are decisive for the resolution of the glucocorticoid‐dependent physiological processes. We have recently discovered a new interaction between GR and beta‐arrestin‐1 (barr‐1). Beta‐arrestin‐1 plays a well‐established role in participating in agonist‐mediated desensitization of G‐protein‐coupled receptors (GPCRs). However, it has become increasingly appreciated as a scaffold protein, thus conferring novel signaling properties independent of GPCR activity. These functions include interfering with the ubiquitin‐proteasome machinery. Moreover, recent studies, from our lab, have demonstrated that glucocorticoids can promote the transcription of beta‐arrestin‐1, thereby contributing to modulating its function. These findings led us to investigate the possibility that barr‐1 and GR can either form a tightly regulated loop, or that barr‐1 may contribute to the activity of the GR. To investigate whether the association of barr‐1 with GR affects the activity of GR, RNA sequencing in control‐ and barr‐1 knock down‐ A549 cell line has been performed. The transcriptome analysis identified post‐translational modifications as one of the top‐ranked cellular functions significantly altered by the lack of barr‐1. Notably, lack of barr‐1 reshaped the GR gene signature, regulating a cohort of genes responsible for the activation of the ubiquitin‐proteasome machinery. Among these genes, PELI1 (Pellino E3 Ubiquitin Protein Ligase 1) resulted the most upregulated glucocorticoid‐responsive gene. Indeed, in vitro data demonstrated that when beta‐arrestin‐1 was knocked down, it accelerated GC‐induced GR degradation reducing its half‐life and promoting its ubiquitination via PELI1 activation. These observations strongly suggest that barr‐1 is required for regulating GR expression, by limiting proteasomal degradation of GR. Playing a pivotal role in homeostasis, GR is susceptible to numerous stimuli that modulate its activity, and discovering a novel protein partnership between GR and barr‐1 may help in developing new glucocorticoids, as well as combination therapies that sustain the beneficial effects of glucocorticoids limiting the risks.

Support or Funding Information

The research project is supported by the Intramural Research Program of the NIEHS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALLOSTERIC MODULATION OF THE PENTAMERIC LIGAND‐GATED ION CHANNEL ELIC BY FUNCTIONALLY ACTIVE NANOBODIES
Authors: Ulens Chris , Brams Marijke , De Peuter Hannelore , Spurny Radovan , Pardon Els , Bertrand Daniel , Steyaert Jan , Govaerts Cedric
Published: June 2021

Pentameric ligand‐gated ion channels (pLGICs) or Cys‐loop receptors are a class of ion channels involved in fast synaptic transmission in the central and peripheral nervous systems. Members of this family include the nicotinic acetylcholine receptors, serotonin type‐3 receptors, GABAA/C (gamma‐aminobutyric acid) receptors and glycine receptors. These receptors are the target for a wide variety of therapeutics, including benzodiazepines such as diazepam (Valium), which act as positive allosteric modulators (PAMs) of the GABAA receptor and are prescribed as a sedative, anxiolytic or anti‐epileptic. In this study, we employed the prokaryote ligand‐gated ion channel ELIC as a model to understand the structural mechanism of allosteric modulation. We raised nanobodies, which are antibody fragments derived from camelids and have the potential to trap conformationally transient states, and used them as crystallization chaperones for ELIC. Functional characterization of ELIC nanobodies using electrophysiological techniques demonstrated that these nanobodies are active as channel modulators. We demonstrate that nanobodies can either inhibit or potentiate agonist‐evoked channel responses, suggesting that nanobodies can act as negative (NAMs) or positive allosteric modulators (PAMs), respectively. The X‐ray crystal structure of ELIC in complex with a nanobody reveals that a nanobody binds at an allosteric binding site and near to a binding site previously known to mediate potentiation of ELIC by the benzodiazepine flurazepam. Given the structural conservation of allosteric binding sites between ELIC and human receptors our study reveals common mechanisms for allosteric modulation in this family of channels. Our expanded knowledge of the structural determinants of allosteric modulation opens opportunities for the development of new therapeutics targeting pLGICs in ion channel‐related disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adaptation to Supraphysiologic Room Air Oxygen Changes the Effects of Nitric Oxide on A549 Lung Cells In Vitro
Authors: Henn Alicia , Darou Shannon , Yerden Randy
Published: June 2021

Airway endothelial cells are exposed to some of the highest oxygen levels encountered in vivo (~10% O2), however, these levels are significantly lower than room air (21% O2). The culture and handling of airway cells in room air oxygen incubators can change the redox status of the cells, introducing artifact. Nitric oxide, which can affect HIF‐1a stabilization and reversibly bind cytochrome oxidase, has been shown to play multiple roles in the response of lung to hypoxic injury and inflammation. Here we explore whether pre‐adaptation of the commonly used human lung epithelial cell line A549 to room air oxygen levels changes cellular responsiveness to NO. The null hypothesis was that pre‐adaptation to different oxygen levels would have no effect on responses to NO. Using the Xvivo System to provide full‐time control of oxygen and CO2 levels during cell incubation as well as handling, we adapted A549 cells for growth at 18% supraphysiologic oxygen (standard room air incubator level with humidity and CO2), physiologically relevant oxygen (10%) or pathophysiologic hypoxia (2% O2). We assayed cellular responses to gaseous nitric oxide (NO) exposure in standard plate‐based cytotoxicity and migration assays and assayed local nitric oxide levels in the cell culture media. We found that adaptation to different oxygen levels had a concentration‐dependent effect on the responses of A549 cells to gaseous NO. We concluded that it is critical to consider the oxygen‐adapted state of the cell prior to assay to avoid oxygen artifact.

Support or Funding Information

All support was from BioSpherix, Ltd.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Moderate Voluntary Ethanol Consumption on the Rat and Human Gut Microbiome
Authors: Pinkowski Natalie J. , Kosnicki Kassi L. , Penprase Jerrold C. , Cintora Patricia , Torres Pedro J. , Harris Greg L. , Brasser Susan M. , Kelley Scott T.
Published: June 2021

Many alcohol‐induced health complications are directly attributable to the toxicity of alcohol or its metabolites, but another potential health impact of alcohol may be on the microbial communities of the human gut. Clear distinctions between healthy and diseased‐state gut microbiota have been observed in subjects with metabolic diseases and recent studies suggest that chronic alcoholism is linked to gut microbiome dysbiosis. Here, we investigated the effects of alcohol consumption on the gut microbiome in both rats and humans. The gut microbiota of rats voluntarily consuming a 20% ethanol solution, on alternate days, were compared to a non‐exposed control group to identify differential taxonomic and functional profiles. Gut microbial diversity profiles were determined using culture‐independent amplification, next‐generation sequencing and bioinformatic analysis of bacterial 16S ribosomal RNA gene sequence libraries. Our results showed that, compared to controls, ethanol‐consuming rats experienced a significant decline in the biodiversity of their gut microbiomes, a state generally associated with dysbiosis. We also observed significant shifts in the overall diversity of the gut microbial communities and a dramatic change in the relative abundance of particular microbes, such as the Lactobacilli. We also compared our results to human fecal microbiome data collected as part of the citizen science American Gut Project. In contrast to the rat data, human drinkers had significantly higher gut microbial biodiversity than non‐drinkers. However, we also observed that microbes that differed among the human subjects displayed similar trends in the rat model, including the Lactobacilli as well as bacteria implicated in metabolic disease.

Support or Funding Information

NIH AA023291

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of cell wall perturbation reagents and plant phenolic compounds on growth, colony morphology, and germination of the corn rot fungus Fusarium verticillioides
Authors: Pierson Paige N. , Hardtke Haley , Brown Daren W. , McQuade Kristi L.
Published: June 2021

Fusarium verticillioides is a filamentous fungus known to infect corn and produce toxins called fumonisins, resulting in crop losses around the globe. Our lab previously showed that an F. verticillioides strain unable to produce the disaccharide trehalose is not only less pathogenic to corn than the wild‐type fungus but also unable to synthesize fumonisins. We are exploring the origins of the observed reduction in pathogenicity of this trehalose‐deficient strain. To test whether cell wall integrity of this strain is compromised, we measured the effects of cell wall perturbation reagents on colony morphology, radial growth, and germination kinetics. Both Congo Red and Calcofluor‐white have a greater effect on the trehalose‐deficient strain than on wild type. Interestingly, Congo Red causes significant hyphal clumping in the trehalose‐deficient strain. A comparison of the effects of phenolic plant defense compounds (caffeic or vanillic acid) on growth of the two strains is also reported.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interdisciplinary undergraduate research program with a travel component at Mount Saint Mary's University Los Angeles to promote success of women in STEM
Authors: Nogaj Luiza A.
Published: June 2021

In response to the national need to produce skilled and globally aware STEM students, Mount Saint Mary's University, Los Angeles developed an interdisciplinary undergraduate research program with an international fieldwork experience in the summer. The goals of the program are: (1) to train undergraduates in interdisciplinary research methodologies (laboratory techniques, cross cultural survey research, and policy analysis); (2) to collect real fieldwork data in Peru; (3) to analyze and present the results in professional conferences and publications; and (4) to develop leadership skills and teamwork necessary for student success and retention in STEM fields.

Students enrolled in the program spend two semesters doing course work. In the first semester they learn about the molecular, environmental and nutritional causes of cancer from MSMU's STEM faculty and about the disparities in policy and healthcare access in the U.S. and Peru from political science faculty members. In the second semester, the students develop research skills in both STEM disciplines and the social sciences: laboratory research skills in biology and chemistry, and survey methodology in political science. The students develop an evidence gathering plan, design a survey, and seek an IRB approval in preparation for their global research experience over the summer.

During the summer, the students first interview women in urban and rural areas of Peru about factors contributing to their health, such as access to healthcare and nutritional habits.

During the culminating, third semester of the program, the students analyze their survey data, present their results at conferences and develop leadership skills. They also examine barriers to success for women in STEM. We expect this combination of regular course work with a summer research component, international field study, and leadership training to be an effective way to increase success and retention rates of undergraduate students in the STEM fields at MSMU.

This program is a model for future research topics thus creating a sustainable and meaningful undergraduate research training.

Support or Funding Information

MSMU LA Presidential Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Degradation of Metabolic Proteins during Starvation
Authors: Peterson Celeste , Maragos Nicholas , Papaj Agllai , Ngo Pauline , Mendes Amberly , Von Rosen Tatjana
Published: June 2021

When E. coli cells become starved for glucose, degradation of the stationary phase master regulator RpoS by the protease ClpXP ceases. However it unknown how other proteins in the cell are also affected by differential degradation by ClpXP. While many studies have examined how gene expression changes during starvation, fewer studies have identified changes in protein degradation in this condition. We used a ClpPtrap, which recognizes and unfolds ClpP substrates but does not release them to monitor substrate selection during starvation conditions. We then further analyzed candidates by Western blot analysis. Numerous differentially regulated substrates were involved in metabolism as well as protein synthesis and other processes such as replication. Many of these substrates had ClpX tags on their N or C termini. Thus changes in protein stability of metabolic and other proteins may be a more general way that cells alter their proteome during starvation.

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 Receptor Signaling is Regulated by Lipid Raft Residency
Authors: Wells Megan M. , Cohen Brian D.
Published: June 2021

Infertility affects 7.4 million women and their partners in the United States. One cause for infertility in women is human follicle stimulating hormone (hFSH) dysfunction. hFSH is involved in follicle maturation and estrogen production in females and spermatogenesis in males. Lack of normal hFSH function in either the ligand or receptor leads to infertility in women, however does not fully inhibit fertility in males. hFSH is secreted from the pituitary gland and binds to its g protein‐coupled receptor (hFSHR) located in the ovaries or the testis. Previous research in our lab has shown that hFSHR resides in cholesterol, sphingolipid, and caveolin enriched microdomains called lipid rafts. It has been previously shown that hFSHR interacts with caveolin although the specific mechanism of this interaction is unknown. One possibility is that the interaction is through a specific amino acid sequence in hFSHR consistent with caveolin interaction motifs identified in other proteins. The current study investigated how lipid rafts regulate hFSHR signaling. It was hypothesized that disruption of lipid rafts or interference with the caveolin interaction motif would alter hFSHR signaling. Lipid rafts in human granulosa cells (the hGrC1 cell line) were disrupted through cholesterol depletion with methyl‐β‐cyclodextrin (MβCD) and FSH signaling differences were observed by western blot. It was observed that MβCD treatment resulted in an increase of cAMP signaling compared to control cells. In parallel, mutations in the hFSHR caveolin interaction motif were made and expression plasmids were transiently transfected into HEK 293 cells. Western blot analysis revealed that some of the mutants showed increased basal signaling relative to the wild type receptor. Taken together, these results suggest that lipid rafts may be acting act as an inhibitory mechanism for hFSHR and that caveolin may play a role in receptor recruitment to the lipid raft. Researching hFSH signaling after lipid raft manipulation can provide a better understanding of the cellular mechanisms of hFSHR, which could lead to innovative ideas in infertility treatment for women.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification of Hepatocyte and Sinusoidal Endothelial Cells from Mouse Liver Perfusion
Authors: Cabral Fatima , Miller Colton M. , Kudrna Katrina M. , Hass Blake E. , Daubendiek Jocelyn G. , Keller Briana M. , Harris Edward N.
Published: June 2021

The liver is primarily composed of hepatocytes and the nonparenchymal cells which include stellate cells, Kupffer cells, sinusoidal endothelial cells and various other cell populations in very low numbers such as transient leukocytes. Cell isolation with high purity is required for many experiments related to the specific biology and biochemistry of these very different cell types. Here, we outline a relatively simple procedure to obtain a high yield of live hepatocytes and sinusoidal endothelial cells. Additionally, this procedure is relatively simple and does not require specialized equipment other than a refrigerated table top centrifuge and peristatic pump. The mouse portal vein is used as the catheterization site rather than the vena cava, limiting contamination of other cell types during the isolation process. Access to the vein is quick minimizing ischemia and reperfusion of the liver which reduces hepatic cell viability. Dead hepatocytes are also easily identified during centrifugation steps due to differences in cell density. This process allows for isolation of hepatocytes with 99% cell purity and isolation of sinusoidal endothelial cells with approximately 89.1% cell purity. This method will help to answer key questions in the hepatotology field such as determining specific cellular functions with in vitro assays or assessing individual cells after an in vivo treatment.

Support or Funding Information

Funding is provided in part by the NIH from grant R01HL130864.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The E3 Ubiquitin Ligase WWP2 Regulates Endosomal Trafficking of the Purinergic Receptor P2Y1
Authors: Kotsis Christopher R. , Singh Harjoyt , Dores Michael Robert
Published: June 2021

P2Y1 is a G protein‐coupled receptor (GPCR) for adenosine diphosphate (ADP) that signals to produce pro‐inflammatory effects in blood vessels. P2Y1 signaling contributes to vascular endothelial barrier disruption, leading to cardiovascular diseases such as atherosclerosis. When bound to ADP, P2Y1 internalizes and is either recycled for reuse at the cell surface or sorted to and degraded by lysosomes to attenuate signaling. P2Y1 lysosomal sorting is mediated by binding to the adaptor protein ALIX. Since ALIX activity is regulated by ubiquitination by the WW domain‐containing E3 ubiquitin protein ligase 2 (WWP2), we hypothesized that WWP2 is required for P2Y1 degradation. Here, we show that P2Y1 colocalizes with WWP2 at late endosomes upon ADP agonist stimulation. In addition, we demonstrate that depletion of endogenous WWP2 inhibits P2Y1 sorting into the lumen of late endosomes. We are currently examining how receptor signaling regulates WWP2 translocation to late endosomes and interaction with ALIX. Our evidence suggests that WWP2 is required at endosomes for P2Y1 degradation in response to agonist stimulation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ESCRT membrane scission revealed by optical tweezers.
Authors: Schöneberg Johannes , Yan Shannon , Bahrami Amir H. , Righini Maurizio , Lee Il‐Hyung , Pavlin Mark‐Remec , Carlson Lars‐Anders , Goldman Daniel , Hummer Gerhard , Bustamante Carlos , Hurley James
Published: June 2021

ESCRT proteins catalyze the function of membrane budding and scission from the inside of the cytosol to the outside. This process is fundamental in cell biology, comprising multivesicular endosome biogenesis, cytokinesis, viral budding (e.g. HIV, Ebola, Dengue) and other pathways. The mechanism underlying ESCRT‐III‐mediated membrane budding and scission remains elusive. We have encapsulated within giant unilamellar vesicles (GUVs) a minimal ESCRT module consisting of ESCRT‐III subunits and the AAA+ ATPase Vps4. Using optical tweezers, membrane nanotubes reflecting the correct topology of scission can be pulled from these GUVs. Upon photo‐uncaging of ATP, surprisingly large forces in the tens of piconewtons were recorded and tube scission could be observed. ESCRT subunit composition and concentration alter force generation and scission behavior. In combining confocal fluorescence microscopy and optical tweezers, the scission events can be observed in both force and fluorescence and studied in detail. For the first time, the biophysics of ESCRT membrane budding and scission are revealed.

Support or Funding Information

Marie Curie Fellowship to J.S.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Role of Annexin A6 in Cell Membrane Repair
Authors: Day Jenny , Sonnemann Kevin , Bement William
Published: June 2021

All cells are susceptible to stress or contraction‐induced membrane damage; any disruption to the plasma membrane, if left unrepaired, leads to cytoplasmic efflux, ion influx, and ultimately cell death. To maintain homeostasis, cells utilize a highly‐conserved, calcium‐dependent repair system that comprises multiple players that dispatch two fundamental operations: resealing of the plasma membrane and repair of the cortical cytoskeleton. Without an efficient repair system, human diseases like muscular dystrophy, diabetic myopathy, and acute lung damage result. To develop therapies to treat repair‐deficient pathologies, we are interested in identifying promoters of cell membrane repair. Due to their calcium‐dependent, lipid‐binding activity, the annexins have long attracted attention as potential participants in the repair response. Because the annexin isoform A6 has been genetically linked to muscular dystrophy, we predicted that annexin A6 plays an active role in the repair response and is a viable therapeutic target. To test whether annexin A6 promotes membrane repair, we investigated A6 function in a Xenopus laevis oocyte model of membrane repair. Specifically, we injected recombinant annexin A6 proteins into oocytes, generated a laser‐induced wound, and either measured protein efflux as a readout on repair or monitored A6 dynamic localization and behavior microscopically.

Consistent with a potential role in repair for A6, GFP A6 was intensely and rapidly recruited to wound sites. Its recruitment was significantly faster than the accumulation of f‐actin around wounds and annexin A6 concentrated interior to the bulk of f‐actin around wounds. Further, the biochemical assay demonstrated addition of exogenous annexin A6 promotes the wounding response. That is, annexin A6 addition resulted in significantly less leakage of cytoplasmic protein out of the cell during the repair process. To confirm the GFP‐A6 concentration used for imaging was comparable to the annexin A6 used in the biochemical assay, we tested the relative activity of the two constructs and found that the two indeed produced equivalent results. Next steps involve examining potential binding partners of annexin A6, such as alpha actinin and s100 proteins, as well as continuing to compare localization patterns to established wound repair proteins. Since the repair mechanism is highly conserved across kingdoms, we expect results from Xenopus oocytes to readily translate to our understanding of mammalian repair response.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nano‐scale size holes in ER sheets provide an alternative to tubules for highly‐curved membranes
Authors: Bahmanyar Shirin , Schroeder Lena , Barentine Andrew , Schweighofer Sarah , Baddeley David , Bewersdorf Joerg
Published: June 2021

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules.

Super‐resolution microscopy recently revealed densely packed, rapidly moving ER tubules, highlighting the importance of revisiting classical views of ER structure with high spatial resolution in living cells. Using live‐cell Stimulated Emission Depletion (STED) microscopy, we show highly dynamic, subdiffraction‐sized holes in ER sheets. Holes coexist with uniform sheet regions and are distinct from tubular ER structures. The curvature‐stabilizing reticulon protein Rtn4 localizes to these holes and the ER luminal tether Climp63 controls their diameter and mobility. Analytical modeling demonstrates that holes in ER sheets can serve as reservoirs for curvature‐stabilizing proteins to support ER tubule extension and retraction, thus providing an explanation for how the ER locally alters its morphology on fast time‐scales.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation the zinc‐mitophagy signaling in hypoxic cells
Authors: Lu Qiping , Li Yang
Published: June 2021

Mitochondria are the power plants of the cell, their proper function is crucial for cellular function and homeostasis. Mitophagy regulates mitochondrial content/quality by selectively removing damaged mitochondria. Zinc is one of the most essential trace elements in the body. The concentration of intracellular free zinc is strictly regulated maintained, known as zinc homeostasis. Zinc homeostasis is achieved by proteins and organelles which sequester zinc. The abnormal zinc concentration has been implicated in numerous clinical manifestations including ischemic stroke. Emerging evidence over the past decade has shown that that zinc affects mitochondria in response to ischemia. It is progressively clear that zinc‐mitochondrial interactions occur in and contribute to ischemic injury. Among the pathological effects of zinc accumulation on mitochondria, zinc induced release and accumulation of ROS draws special interest. ROS is a major cause of mitochondrial damage and initiates mitophagy. In addition, emerging evidence suggests that mitophagy plays critical roles in the pathophysiological process of cerebral ischemia. In the present study I analyzed the role of zinc and mitophagy in hypoxia‐induced mitophagy and investigated the signaling pathways between zinc and mitophagy under hypoxia condition.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial membrane potential and aging in the beer fermentation process
Authors: Hall David J.
Published: June 2021

The process of fermentation and the characteristic aromatic compounds elicited play a key role in the flavor of bread, beer and wine. However, the practice of fermentation, particularly as it applies to beer, is more art than science. The quality of the resulting products of fermentation rely on the physiological state of the yeast biomass, that is, its vitality. A central organelle to vitality that is often overlooked is the mitochondria. Mitochondria are amazingly dynamic in their morphology, distribution and membrane potential as they reflect the energy requirements of the cell. Two key measures of yeast vitality are mitochondrial membrane potential and aging. We hypothesized that there are differences in these two key measures of vitality based on the domestication of the yeast (lager, ale, Belgian), the number of times the yeast was used (generations) and the type of supplements used (Zn, Cu, lithocolate, diammonium phosphate). Our findings provide an explanation to several beer brewing observations and, coupled with bioinformatics of the mitochondrial genomes (lager, ale Belgian), provide an avenue to study the molecular basis of yeast vitality as it applies to beer fermentation.

Support or Funding Information

Lawrence University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulatory network analysis of endoplasmic reticulum–mitochondrial contacts
Authors: Cho Ginam , Cho Il‐taeg , Lim Youngshin , Golden Jeffrey
Published: June 2021

The contact of subcellular organelles serves as molecular signaling hubs to maintain cellular homeostasis. Endoplasmic Reticulum‐Mitochondria Contacts (ERMC) are one of these signaling hubs. They regulate diverse cellular activities such as Ca2+ or lipid exchange, and mitochondria dynamics including metabolism, apoptosis and autophagy. The significance of ERMCs has recently been highlighted its dysregulation is linked to metabolic disease and neurodegenerative diseases. We previously identified multiple physical components of ERMCs using engineered ascorbate peroxidase (APEX) to map the ERMCs proteome. The identified 405 proteins with a 2.0‐fold cut‐off ratio (log base 2) in culture (SILAC)‐LC/MS‐MS quantification are classified by gene set enrichment analysis (GSEA). The analysis shows protein biosynthesis‐related proteins are enriched along with several Rho/kinase proteins. To investigate ERMC regulatory network, we screened 1126 compounds and ORFs identified in the proteomic analysis with a Split‐Rluc8 complementation assay; a biosensor that measures ERMCs. We found several modulators of adrenergic signaling associated with increased ERMC. These results may be useful to elucidate the regulation of ERMCs, understand ERMC physiology and unravel the disease mechanism.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Mitochondrial Calpain 1 Leads to Degradation of PDH
Authors: Chen Qun , Thompson Jeremy , Hu Ying , Hollander John , Lesnefsky Edward
Published: June 2021

INTODUCTION

Calpain 1 (CPN1) is a ubiquitous calpain including one large subunit and a small regulatory subunit (calpain 4, CPN4). Genetic removal of CPN4 eliminates the CPN1 activity. CPN1 exists in both cytosol and mitochondria. Activation of CPN1 contributes to mitochondrial damage and cardiac injury during ischemia (ISC)‐reperfusion (REP). Pyruvate dehydrogenase (PDH) is degraded in mitochondria following ISC‐REP. Since mitochondrial CPN1 (mit‐CPN1) is found in the matrix, we propose that activation of mit‐CPN1 contributes to PDH degradation. Purified heart mitochondria were used to exclude potential cytosolic CPN1 contamination. CPN4 knockout mice were used to eliminate mit‐CPN1.

HYPOTHESIS

We hypothesize that activation of the mit‐CPN1 impairs oxidative phosphorylation (OXPHOS) through degradation of PDH in isolated mouse heart mitochondria.

METHODS

Purified cardiac mitochondria were isolated from wild‐type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mit‐CPN1. OXPHOS was determined in mitochondria with or without calcium treatment. Protein content of PDH E1 subunit was assessed by Western blotting.

RESULTS

In wild type mice, calcium treatment significantly decreased the rate of OXPHOS using pyruvate + malate as complex I substrates compared to non‐calcium‐treated control. Succinate oxidation was also slightly decreased in calcium treated mitochondria from wild type hearts (Table). In contrast, calcium treatment did not affect OXPHOS in mitochondria from CPN4 knockout mice in both substrates. PDH E1 subunit content was decreased in calcium‐treated mitochondria from wild type mice but not from CPN4 knockout mice. A representative image is shown in Figure 1.

CONCLUSION

Knockout of CPN4 prevents exogenous calcium‐mediated PDH degradation. Inhibition of CPN4 during calcium over load conditions including heart failure and endoplasmic reticulum stress may decrease cardiac injury by protecting mitochondria.

Support or Funding Information

A Grant‐in‐aid (15GRNT24480123) from the American Heart Association (QC), R21AG054975‐01 (QC), and Merit Review Award (2IO1BX001355‐01, EL)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the KDO Glycosyltransferase KpsS in the Biosynthesis of the Polysialyltransferase Acceptor for Escherichia coli K1
Authors: Lanz Nicholas , Thon Vireak , Vann Willie
Published: June 2021

Many pathogenic bacteria produce polysaccharide capsules to evade immune recognition and prevent lysis by the host immune response. Neisseria meningitidis groups B and C as well as Escherichia coli serogroups K1 and K92 are coated with a polysialic acid (PSA) capsule. The PSA chain is synthesized by polysialyltransferases that makes an α‐2,8 linkage for E. coli K1 and N. meningitidis serotype B, an α‐2,9 linkage for N. meningitidis serotype C, or an alternating α‐2,8, α‐2,9 linkage for E. coli K92. These transferases cannot initiate synthesis of PSA de novo, but rather, a more complex set of machinery is required to generate an appropriate acceptor. This acceptor has been proposed to be a phosphatidylglycerol lipid anchor with a short keto‐deoxyoctulosonate (KDO) linker to one or more sialic acid residues. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized KpsS, the first enzyme in the pathway for acceptor synthesis and a membrane associated beta KDO glycosyltransferase. Much of the machinery required to produced bacterial capsules consists of transmembrane or membrane‐associated enzymes. Isolation and characterization of these enzymes in an active state has proven challenging. Here, we have purified KpsS in a soluble and active form and have begun to investigate its function. We show that KpsS can transfer a KDO residue to a fluorescent labeled phosphatidylglycerol lipid substrate. The enzyme tolerates various lengths of lipid tails on the phosphatidylglycerol substrate, including fluorescent tags that facilitate the characterization of the substrate and product. We have also shown that the enzyme can transfer modified KDO residues. Furthermore, we have isolated the product of the KpsS reaction and analyzed its structure. That KpsS is the first KDO transferase in the pathway is confirmed by the further modification of this purified product by KpsC with additional KDO units.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genotype‐Phenotype Correlations for Protein O‐Linked Mannose NAcetylglucosaminyltransferase 1 in Congenital Muscular Dystrophy
Authors: Ho Jessica Ziling , Boyd Sally Riewe , Singh Danish , Halmo Stephanie Marie , Wells Lance
Published: June 2021

Congenital muscular dystrophy (CMD) is a heterogeneous family of inherited muscle disorders. A subtype of CMD known as dystroglycanopathy is classified by hypoglycosylation of alpha‐dystroglycan (α‐DG). α‐DG, an integral component of the dystrophin‐glycoprotein complex, is responsible for connecting the actin cytoskeleton to the extracellular matrix in muscle tissues. Hypoglycosylation of α‐DG arises from defects in the protein O‐mannosylation biosynthetic pathway. One enzyme involved in the O‐mannosylation pathway is protein O‐linked‐mannose β‐1,2‐N‐acetylglucosaminyltransferase (POMGNT1). On serine and threonine residues in α‐DG, POMGNT1 catalyzes addition of N‐acetyl‐D‐glucosamine (GlcNAc) to an O‐mannose structure in a β1,2‐linkage to create a core m1 glycan structure in the cis‐Golgi. Mutations in the gene encoding POMGNT1 have been observed in patients with various forms of dystroglycanopathy.

Our work examines the role of R311Q, P493R, T176P, P303L, C269Y, E223K, C490Y, G502A, R605P, I287S, E156K, D395N, and D556N mutations in POMGNT1 to define a genotype‐phenotype correlation. The POMGNT1 mutations present a range of clinical presentations, from muscle‐eye‐brain disease, an extremely severe condition characterized by muscle weakness, vision and brain abnormalities, and developmental disabilities, to limb‐girdle muscular dystrophy, a much milder phenotype that results in progressive muscle weakness and wasting but lacks neurological comorbidities. The effects of the selected mutations on enzyme characteristics are not well established in the literature. Therefore, we sought to identify mutation‐derived changes in enzyme kinetics and stability.

To do so, HEK293F cells were transfected with mutant plasmids generated by site‐directed mutagenesis. Preliminary data indicated that R311Q, D395N, R605P, and D556N POMGNT1 mutants maintained enzyme expression. Radiolabel transfer assays established that the R311Q, D395N, and R605P mutants were kinetically dead while the D556N mutant still exhibited transfer. Promega's UDP‐Glo™ assay was performed on the D556N mutant, which demonstrated reduced kinetic activity compared to wild‐type POMGNT1. A SYPRO Orange thermal shift assay revealed the aforementioned POMGNT1 mutants to be thermodynamically stable. We are currently experimenting with the rest of the mutants and are also investigating the ability of all mutants to rescue POMGNT1 knockout cell lines for IIH6 reactivity, laminin binding, and Lassa pseudovirus entry. Understanding genotype‐phenotype correlations in those glycosyltransferases will facilitate the design of more targeted treatments for individuals based on the mutation(s) they carry.

Support or Funding Information

This work was supported in part by NIGMS/NIH (R01GM111939, LW).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MMP‐12 knockout mouse: Is it a relevant animal model to study the role of MMP‐12 in post‐stroke brain damage?
Authors: Nalamolu Koteswara Rao , Chelluboina Bharath , Magruder Ian B. , Mohandas Adithya , Venkatesh Ishwarya , Klopfenstein Jeffrey D. , Pinson David M. , Wang David Z. , Veeravalli Krishna Kumar
Published: June 2021

We recently reported for the first time the detrimental role of matrix metalloproteinase (MMP)‐12 on post‐stroke brain damage. We hypothesize that the post‐stroke MMPs dysregulation is similar across species and the genetic deletion of MMP‐12 does not affect the post‐stroke expression of other MMPs. We tested our hypothesis by determining the pre‐ and post‐ischemic expression profile of MMPs in MMP‐12 knockout and respective wild type mice. Focal cerebral ischemia was induced in MMP‐12 knockout and wild type mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischemia, reperfusion was initiated by removing the monofilament. One‐day after reperfusion, ischemic brain tissues from various groups of mice were collected for real‐time PCR analysis and PCR analysis followed by agarose gel electrophoresis. Although the post‐stroke regulation of several MMPs was altered in wild type mice, huge upregulation of MMP‐12 (~763 fold over sham) was noticed in mice similar to rats as reported earlier by our group. Unlike in rats, MMP‐8 was prominently upregulated (~280 fold over sham) in ischemic mice brains. As expected, MMP‐12 expression is absent in the ischemic brains of MMP‐12 knockout mice. Except the expression of MMP‐9, the expression of several other MMPs such as MMP‐1a, −2, −8, −10, −15, −16, −17, −21 and −28 was altered in the ischemic brain of MMP‐12 knockout mice as compared to wild type mice under similar experimental conditions. In addition, the expression of various MMPs in MMP‐12 knockout mice under non‐ischemic conditions was altered as compared to the wild type mice that did not subject to ischemia. Based on our results, we conclude that the mice with genetic deletion of MMP‐12 are constitutive in nature that warrants the attention of their use in elucidating the role of MMP‐12 in post‐stroke brain damage.

Support or Funding Information

This work was supported in part by the OSF HealthCare Foundation and OSF Illinois Neurological Institute, Peoria, IL.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of microRNA‐9 in the pathogenesis of Parkinson's Disease
Authors: Goh Suh Yee , Yeo Xin Yi , Srinivasan Dinesh Kumar , Dheen S Thameem , Tay Samuel Sam Wah
Published: June 2021

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the aging population. Previously, our lab has shown that miR‐9 is upregulated in the substantia nigra pars compacta (SNc) in pre‐clinical models of PD [1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated mice]. Briggs et al (2015) has also reported this trend in the SNc of post‐mortem PD patients. miRNAs are post‐transcriptional regulators of gene expression that are involved in the maintenance of normal cellular functions. While miR‐9 is well‐studied as a regulator of neurogenesis during brain development, its upregulation observed in PD suggests that miR‐9 may have other functions that contributes to PD pathogenesis. In this study, we are interested to investigate the miR‐9‐mediated mechanisms involved in the development of PD. Using miRNA qPCR, we verified the increase in miR‐9 levels in in vitro PD models [MN9D and SH‐SY5Y neuronal cells treated with 1‐methyl‐4‐phenylpyridinium (MPP) iodide]. MPP treatment decreased neuronal cell viability by about 30% and the inhibition of miR‐9 significantly reversed this trend, suggesting that miR‐9 plays a role in neurodegeneration. In addition, using NanoSight technology, we discovered a significant increase in the concentration and size of exosomes released by neuronal cells treated with MPP iodide. Interestingly, these exosomes also contained higher miR‐9 levels as compared to exosomes released by neurons under control conditions. This suggests that miR‐9 may be transported to other cell types (such as microglia) to regulate other events in PD, for instance, neuroinflammation. These research findings implicate miR‐9 as a potential diagnostic biomarker as well as a potential therapeutic target for PD treatment.

Support or Funding Information

This work was supported by a research grant from the Singapore Ministry of Education Academic Research Fund Tier 1.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Comparison of Multiple Histotechnical and Morphological Methods for Measuring Hepatic Lipids in NASH Models
Authors: Opsahl Alan C. , Crowell Danielle M. , Zhan Yutian , Geoly Frank J. , O'Neil Shawn P.
Published: June 2021

Nonalcoholic steatohepatitis (NASH) is a progressive syndrome in which fat (usually in the form of triglycerides) accumulates in the liver (steatosis), accompanied by inflammation and fibrosis in non‐alcoholic patients. Lipid accumulation occurs in intracellular vesicles in hepatocytes, typically producing a macrovesicular histologic pattern, and can lead to ballooning degeneration. The injury to hepatocytes can induce inflammation with subsequent fibrosis, and may ultimately lead to cirrhosis and/or hepatocellular carcinoma. A number of rodent models of NASH have been developed and quantification of hepatic lipids in liver sections is a common study endpoint due to the significant role that lipid accumulation plays in NASH pathogenesis. Several histotechnical and morphological methods for measuring hepatic lipid content have been developed. In this study, we used digital image analysis to compare three histological methods for measuring relative lipid burden in liver sections: 1) Oil Red O (ORO) histochemical stain, 2) liver vacuole area by morphometry (digital pathology), and 3) adipophilin expression by immunohistochemistry (IHC). Thirty C57 BL6/J mice were fed a high fat diet for 16 weeks to induce steatosis, followed by 7 weeks of high fat diet feeding in conjunction with daily oral dosing with vehicle or low or high doses of a proprietary compound. After the 23 week study period, livers were collected at necropsy and fixed in formalin before routine processing to paraffin (for IHC or morphology), or cryopreservation and snap‐freezing in OCT compound for cryosectioning (for ORO staining). Serial sections from the left lateral lobe of the liver were prepared from paraffin blocks for hematoxylin and eosin staining (vacuole morphometry) or IHC (for adipophilin), while the ORO stain was performed on cryosections from OCT blocks. Image analysis was performed to measure the area percent of the liver section that was occupied by ORO stain, DAB chromogen (adipophilin IHC) or lipid vacuoles, using Visiopharm image analysis software, and a non‐parametric statistical analysis was performed on the results. There were no significant differences among vehicle, low dose or high dose groups measured using any of the three endpoints for lipid quantification (Kruskal‐Wallis ANOVA); however, the relative patterns of median area percent for the three endpoints were identical among assays (median area percent for low dose compound was highest, and median area percent for high dose compound was lowest), suggesting that the three assays performed similarly, and that any of these assays is adequate for histotechnical measurement of hepatic lipids.

Support or Funding Information

Pfizer

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.