bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationFunding 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 InformationMarie 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 InformationLawrence UniversityThis 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

INTODUCTIONCalpain 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.HYPOTHESISWe hypothesize that activation of the mit‐CPN1 impairs oxidative phosphorylation (OXPHOS) through degradation of PDH in isolated mouse heart mitochondria.METHODSPurified 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.RESULTSIn 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.CONCLUSIONKnockout 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 InformationA 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.