bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Relationship between Furin Cleavage and Cytotoxicity in a Recombinant Immunotoxin Based on Pseudomonas Exotoxin A
Authors: Reifer Danielle , Grossman Brian , Weldon John E. 
Published: June 2021

Recombinant immunotoxins (RITs) are anti‐cancer therapeutic proteins that combine an antibody and a protein toxin. RITs based on the bacterial toxin Pseudomonas exotoxin A (PE) are currently in clinical trials, with one (moxetumomab pasudotox) recently approved by the FDA for the treatment of Hairy Cell Leukemia. PE‐based RITs are internalized by receptor‐mediated endocytosis and undergo retrograde trafficking to the endoplasmic reticulum, from which they translocate to the cytosol and catalytically inhibit protein synthesis. Early on during their intracellular trafficking route, PE‐based RITs are hydrolyzed and activated by furin, a serine protease ubiquitously expressed in mammalian cells. The furin cleavage site in PE, however, has a very slow cleavage rate compared to other targets of furin. Because the furin cleavage site of PE is so poor, it has been proposed that altering the cleavage site to enhance the rate would increase the cytotoxicity of PE‐based RITs. Previous research has shown that furin site mutations can modestly enhance RIT activity, but no correlation exists between cleavage efficiency and cytotoxicity across the full site. Interestingly, the correlation becomes significant if only mutations in the N‐terminal region of the cleavage site are considered (Spearman's r = 0.986; p = 0.0056). To investigate this relationship further, three different forms of an anti‐transferrin receptor PE‐based recombinant immunotoxin have been expressed and isolated: one with mutations on the N‐terminal region of the cleavage site, one with mutations on the C‐terminal region, and one with mutations over the entire cleavage site. Each toxin will be refolded, purified by column chromatography, and evaluated in cytotoxicity assays against HEK293 cells to determine how they impact cytotoxicity. It is expected that the RIT with optimizing mutations across the entire furin cleavage site will exhibit the greatest increase in cytotoxicity, followed closely by the N‐terminal mutations alone. These data will provide a better understanding of the relationship between cleavage efficiency and cytotoxicity, permitting better design of PE‐based RITs to produce more effective cancer treatments. It will also help to elucidate an incompletely understood PE intoxication pathway.Support or Funding InformationThis project was supported by funds from the Linda Sweeting Summer Research Fellowship and the Fisher College of Science and Mathematics.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 RGS Homology and Pleckstrin Homology Domains in GPCR Kinase 2 Activation
Authors: Elkins Kaely Elizabeth , Cato M. Claire , Tesmer John J. G. , Sterne‐Marr Rachel 
Published: June 2021

G protein‐coupled receptors (GPCRs) are transmembrane proteins that act through multi‐step pathways to regulate metabolic and synaptic signaling, as well as many other processes. GPCR kinases (GRKs) regulate GPCR signaling via phosphorylation. The seven vertebrate GRKs map to three subfamilies: GRK1 (GRK1/7), GRK4 (GRK4/5/6), and GRK2 (GRK2/3). GRK2 is a modular protein composed of an AGC kinase domain (KD), a pleckstrin homology domain (PHD) that binds Gβγ subunits and phospholipids, and a regulator of G protein signaling homology domain (RHD). The RHD is the central domain and makes contacts with both PHD and KD. Although GRK2 is activated by Gβγ and membrane phospholipids, it is its interaction with an activated GPCR that most dramatically promotes kinase domain activation. We seek to understand how GPCRs activate GRK2. Previously we used site‐directed mutagenesis to test the hypothesis that the release of inter‐domain contacts might artificially activate GRK2. Our hypothesis was not supported: mutations at the RH/KD small lobe interface disrupted the structural integrity of GRK2, while a mutation at the RH/KD large lobe interface (E96A) did not impact rhodopsin phosphorylation (Lodowski et al., 2005). This contrasts recent in vitro kinetic studies and molecular dynamic simulations with GRK4 family members (Komolov et al., 2017; Yao et al., 2017) that suggest that disruption of the RHD/KD interface (“ionic lock”) leads to RHD separation from the kinase domain and allows the KD to acquire an active conformation. The PH domain, only found in the GRK2 subfamily of GRKs, is essential for localizing GRK2 to the site of an activated receptor. Comparison of crystal structures of monomeric GRK2 versus the GRK2–Gβγ complex suggests that Gβγ binding causes slight rotation of the RHD away from the KD (Lodowski et al., 2005). Single time‐point kinase assays showed that mutations at the RHD/PHD interface, particularly P638D, impaired GRK2 function. It is possible that the PHD in GRK2 subfamily plays a large role in receptor mediated KD activation. In this work, we use an intact cell phosphorylation assay as well as more detailed kinetic studies in combination with expanded mutagenesis to assess the role of the RHD and the RHD/PHD interface in GRK2 activation.Support or Funding InformationSiena College Center for Undergraduate Research and Creative ActivityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of yeast Pah1 phosphatidate phosphatase by casein kinase I
Authors: Hassaninasab Azam , Han Gil‐Soo , Carman George M 
Published: June 2021

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate) in the synthesis of triacylglycerol. The enzyme is subject to phosphorylation (e.g. cyclin‐dependent protein kinases Pho85‐Pho80 and Cdc28‐cyclin B, protein kinases A and C, and casein kinase II) and dephosphorylation (e.g. by the Nem1‐Spo7 phosphatase complex) for regulation of its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is also phosphorylated by casein kinase I. By mass spectrometry, phosphopeptide mapping, and phosphoamino acid analysis, we showed that the phosphorylation occurs on multiple serine and threonine residues. Using Pah1 as substrate, casein kinase I activity was dependent on the time of the reaction and the amount of protein kinase, as well as the concentrations of Pah1 and ATP. The prephosphorylation of Pah1 by casein kinase I reduced its subsequent phosphorylation by Pho85‐Pho80, and the prephosphorylation by Pho85‐Pho80 reduced its subsequent phosphorylation by casein kinase I. This was attributed to both protein kinases having phosphorylation sites on Pah1 in common. The prephosphorylation by casein kinase I stimulated subsequent phosphorylation by casein kinase II, but the prephosphorylation with casein kinase II did not affect subsequent phosphorylation by casein kinase I. This indicated that the sites phosphorylated by casein kinases I and II were not common. Prephosphorylation of Pah1 by casein kinase I had no effect on the phosphorylation by protein kinase A, and vice versa. Hierarchal phosphorylation experiments with protein kinase C, along with experiments to identify sites on Pah1 phosphorylated by casein kinase II are currently in progress.Support or Funding InformationGM050679This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism
Authors: Xian Hongxu , Yang Qiaoyun , Liou Yih‐Cherng 
Published: June 2021

Mitochondrial quality control is essential for cellular homeostasis and accumulating evidence show that mitochondria can be selectively targeted for autophagic degradation (mitophagy). Mitophagy allows for the removal of dysfunctional mitochondria, which is highly implicated in energetically demanding cells including muscle and nerve cells. However, despite the well characterization of PINK1/Parkin route of mitophagy, mechanisms concerning PINK1/Parkin‐independent mitophagy are still poorly understood. With imaging approaches including structure‐illumination microscopy (SR‐SIM), here we demonstrate that the autophagy protein Syntaxin 17 (STX17), initiates mitophagy upon the depletion of outer mitochondrial membrane protein Fis1. Using mass spectrometry analysis, we identify STX17 interacts with Fis1, which preferentially gatekeeps the dynamic shuffling of STX17 between ER and mitochondria. Loss of Fis1 results in the accumulation of STX17 on mitochondria and mitochondria associated membranes (MAM), exposing its N‐terminus to assemble and self‐oligomerize for mitophagy. Mitochondrial STX17 interacts with ATG14 and further recruits core autophagy proteins hierarchically to form mitophagosomes, followed by Rab7‐dependent mitophagosome‐lysosome fusion. In addition, our results reveal that Fis1 loss impairs mitochondrial metabolic function, and potentially sensitizes mitochondria to STX17‐mediated mitochondrial engulfment within autophagosomes, which is directly initiated through canonical autophagy machinery, closely linking non‐selective macroautophagy and mitochondria. Our findings uncover a novel PINK1/Parkin‐independent mitophagy mechanism, in which outer mitochondrial membrane protein Fis1 gates the elimination of mitochondria.Support or Funding InformationThis work is financially supported by grants Tier 2 MOE and NUS, Singapore to Y.‐C.L.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of figs fruits extracts on rotenone‐induced Parkinson's disease‐like cytotoxicity in SH‐SY5Y cells
Authors: Essa Musthafa Mohamed , Al‐Adawi Samir , Manivasagam Thamilarasan , Justin‐Thenmozhi Arokiasamy 
Published: June 2021

The current study was aimed to find out the effect of figs fruits extracts (FFE) on in vitro rotenone model of Parkinson's disease (PD) by evaluating redox status, mitochondrial membrane potential, cell viability, macromolecule oxidation, and apoptosis. Incubation of SH‐SY5Y cells with rotenone lead neuronal death, mitochondrial dysfunction and DNA fragmentation thorugh redox imbalance, condensation and apoptosis. Rotenone exposure also enhanced the expression of cytochrome C, caspase ‐3, ‐8, ‐9 and Bax (markers of apoptosis) and altered the anti‐apoptotic (Bcl‐2) markers. Reduction of the intracellular reactive oxygen species (ROS) levels ensued due to pretreatment of FFE along with improved antioxidant status, alleviation of rotenone induced mitochondrial dysfunction and apoptosis. Our current findings suggested that figs fruits extracts exhibit the marked neuro‐protective effects, which could be due to its antioxidant, mitochondrial protection and anti‐apoptotic properties. We thus conclude that FFE could be developed as a novel therapeutic molecule for the benefit of reducing the consequences of PD. However, further extensive preclinical and clinical studies are warranted.Support or Funding InformationThe funding support given by Sultan Qaboos University in the form of Internal grant (IG/AGR/FOOD/17/02) to Dr. Mohamed Essa is highly acknowledged.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 Basis of Anthracycline Drugs Transport by P‐Glycoprotein
Authors: Nguyen Phuong H. , Sigdel Krishna P. , Kings Gavin M. , Roberts Arthur G. 
Published: June 2021

P‐glycoprotein (Pgp) is an ATP‐dependent efflux transporter and plays a major role in anti‐cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti‐cancer drug efflux are not well understood. Even subtle differences in the anti‐cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, the focus of this study was on the closely‐related anthracycline drugs daunorubicin and doxorubicin. While only differing in the positions of their hydroxyl functional groups, daunorubicin has a 5‐fold higher transport rate than doxorubicin. They were both anticipated to activate Pgp‐mediated ATP hydrolysis, but instead they reduced Pgp‐mediated ATP hydrolysis below basal levels and to the same extent. This suggested that daunorubicin and doxorubicin mediated ATP hydrolysis of Pgp similarly. The KD values measured from from intrinsic tryptophan fluorescence of the transporter for both daunorubicin and doxorubicin are insignificantly different, implying that two drugs have similar affinity for the transporter. Interestingly, acrylamide quenching of Pgp tryptophan fluorescence revealed that daunorubicin had larger effects on the transporter conformation than doxorubicin. These changes are consistent with shifts in the punctate features of atomic force microscopy (AFM) images of Pgp in the absence and presence of the drugs. This information was used to develop a conformationally‐gated model for anthracycline transport by Pgp.Support or Funding InformationNIH 1RO1CA204846‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Electron Transport by Metformin in Cardiac Protection: Role of Complex I
Authors: Mohsin Ahmed A. , Maceyka Michael , Lesnefsky Edward Joseph 
Published: June 2021

RationaleModulation of mitochondrial complex I during reperfusion reduces cardiac injury following ischemia. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with reactive oxygen species (ROS) production. Metformin treatment may preserve the D form.ObjectiveWe hypothesize that maintenance of deactivation of complex I during early reperfusion will protect cardiomyocytes. The goal is to evaluate the potential of acute, high dose Metformin treatment to modulate complex I at the onset of reperfusion/reoxygenation and decrease subsequent cellular injury.MethodsH9c2 cells underwent 6 hr. simulated ischemia and 24 hr. reoxygenation with or without metformin treatment (1 mM) at the onset of reoxygenation. Cell death, ROS and thiol modification were studied. Additional experiments were performed with or without siRNA knockdown of AMPKa2 catalytic subunit.ResultsMetformin selectively inhibited complex I oxidative phosphorylation measured at reoxygenation (Metformin: 0.34±0.1 vs control: 0.59±0.05 nmolO2/min/106cells; ±SE; n=5–10; p<0.05) with decreased total cell death following simulated ischemia and reoxygenation (Metformin: 8.3±0.2 vs. control: 14.4±0.6%; n=3–6; p<0.05). Complex II driven oxidative phosphorylation was not reduced by metformin, supporting selectively to complex I. Apoptosis assessed by annexin‐V labeling and flow cytometry was also decreased by metformin treatment. The “burst” of ROS at reoxygenation was decreased and free thiol group content was preserved by metformin treatment at reoxygenation. Metformin decreased cell death in AMPK knockdown cells.ConclusionMetformin blockade of complex I at the onset of reoxygenation reduces cell injury. Metformin specifically inhibited complex I at reoxygenation with less oxidative injury, in line with protection against D→A reactivation of complex I. Metformin protection was AMPK independent, supporting a complex I mechanism of protection. Acute, high dose Metformin is a potentially safe translational complex I inhibitor to treat ischemia‐reperfusion injury.Support or Funding InformationThis work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs; National Institutes of Health NIH R21AG054975‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Importance of TFEB and TFE3 in Mediating Lysosomal and Mitochondrial Adaptations
Authors: Oliveira Ashley Nicole , Memme Jonathan Matthew , Tamura Yuki , Hoood David A 
Published: June 2021

Autophagy is the process through which damaged cellular components are degraded by the lysosome, and it is essential for the overall maintenance of the cell. Mitochondria are recycled through a selective form of autophagy, known as mitophagy, in which dysfunctional mitochondria are poly‐ubiquitinated, engulfed in an autophagosomal membrane and transported to the lysosome for degradation. Chronic contractile activity (CCA) alters mitophagy flux in muscle, and this coincides with the activation of lysosomal biogenesis, which precedes changes in mitochondrial content. Thus, it is proposed that the clearance of dysfunctional mitochondria through the lysosomes may play an important role in mediating mitochondrial adaptations to CCA. We sought to understand the transcriptional regulation of lysosomal biogenesis brought about by CCA, by investigating the transcriptional regulators TFEB and TFE3, and their relationship to mitochondrial content. TFEB and TFE3 were silenced using siRNA in C2C12 myotubes, and subjected to 3 days of stimulation (3hr/day) to induce CA. Following a single bout, TFEB and TFE3 were elevated in both the nuclear and cytosolic fractions, which coincided with increases in Cathepsin D, preceding the increases in VDAC seen following 3 days of stimulation. The loss of TFEB or TFE3 individually had no impact on lysosomal adaptations, since LAMP1 and Cathepsin D remained responsive to CCA, demonstrating redundancy in role of TFEB and TFE3 in mediating lysosomal biogenesis. However, in the absence of both transcription factors, CCA‐induced lysosomal adaptations were reduced, illustrating their collective importance in mediating lysosomal biogenesis. Interestingly, the absence of TFEB had no effect on mitochondrial adaptations, measured by COX I and IV protein content, however this adaptation to CCA was blunted in the absence of TFE3. These results suggest that while TFEB and TFE3 may exhibit compensatory roles in mediating lysosomal biogenesis, TFE3 alone appears to be important for the mitochondrial response to CCA. Since, lysosomal adaptations proceed in the absence of TFE3, these data suggest a more direct role of TFE3 in the regulation of mitochondria in response to chronic contractile activity.Support or Funding InformationANO is funded by an Ontario Graduate Scholarship (OGS). JMM is funded by Natural Science and Engineering Research (NSERC) scholarship. DAH is the holder of a Canada Research Chair in Cell Physiology with funding from NSERC and CIHR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional and Phenotypic Characterization of Vimentin Knockout Mouse during Aging
Authors: Hu Jennifer Yuan‐Shih , Gonzalez Francisco , Davis Jennifer , Cheng Yuan , Ciesielski Mark , Ridge Karen M. 
Published: June 2021

Vimentin is a type III intermediate filament (IF) that primarily found in mesenchymal cells and plays important roles in epithelial‐mesenchymal transition (EMT). IFs were originally thought to be a relatively static structure that mainly functioned as mechanical support. However, in the past decades, it has been shown that IF networks are highly dynamic structures and their roles have expanded from maintenance of cell shape and integrity to cellular migration, adhesion, and cancer metastasis. Vimentin knockout mouse was originally reported to have no overt phenotype, leading to the conclusion that vimentin IF represented an unnecessary cytoskeletal system. Interestingly, our group recently found that acute lung injury (ALI) is attenuated in mice lacking vimentin when challenged with either LPS, asbestos, or bleomycin. Moreover, these studies demonstrated vimentin to be a key player in the activation of the NACHT, LRR and PYD domains‐containing protein 3 (NLRP3) inflammasome, an intracellular protein complex that is activated in response to stimuli such as bacterial and viral pathogens, including influenza A virus. In this study, we thoroughly characterize young and aged vimentin knockout mice behaviorally using battery of neurocognitive tests and show that there are age‐dependent changes in neuromuscular, neurocognitive, and metabolic lung functions distinctively from the wild type mice. We also use RNA sequencing as an unbiased approach to study transcriptional changes and identify a significant number of differentially expressed genes between the two groups in various cell types and with aging. Additionally, histological analyses from multiple organs of these mice also suggest a crucial role of vimentin in maintaining cellular structures and functions during aging. In conclusion, our data suggests that vimentin intermediate filament plays dynamic roles in multiple organ systems and during aging.Support or Funding InformationNHLBI R01 HL128194This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative measurement of pH dynamics in living cells using the mCherry‐TYG red fluorescent protein as a lifetime sensor
Authors: Haynes Emily Patten , Tantama Mathew 
Published: June 2021

pH regulation plays a crucial role in mammalian and bacterial cell metabolism, protein function, apoptotic signaling, and a wide range of other physiological processes. Monitoring intracellular pH after environmental perturbations would further knowledge about cellular activities in both healthy and disease states in mammalian cells, as well as mechanism of infection and antibiotic resistance in bacterial cells. Non‐optical techniques such as x‐ray crystallography and NMR cannot resolve live‐cell dynamics, and many fluorescent dye or protein‐based sensors are susceptible to concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching, making lab‐to‐lab translation difficult without extensive calibration. Therefore, there remains a need for options to quantitatively measure pH dynamics in live specimens. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. The utilization of FRET pairs in fluorescence lifetime imaging microscopy (FLIM), has shown applicability of lifetime measurement for dynamic quantitation, however dynamic range can be limited. Here we present in vitro and live‐cell characterization of a red monomeric pH sensitive mCherry mutant mCherry‐TYG for robust measurement of live bacterial and mammalian pH in real‐time. We were successfully able to translate lifetime measurements with ease, showing pH calibration of both isolated protein and bacterial suspensions overlaying with a 2 ns dynamic range between pH 5.5 and 9.0. We further tested applicability in monitoring environmental perturbations by quantifying changes in pH as a function of energy source in e. coli. Additionally, we are currently exploring application for monitoring receptor trafficking, endocytosis and degradation in mammalian cells. Our results demonstrate the possibility of red shifted protein‐based lifetime sensors as a robust tool for pH calibration in live cells.Support or Funding InformationNIH/NINDS R21 NS092010, R21 NS106319NIH/NEI R21 EY026425This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.