bluuebunny/crossref_metadata_2025 huggingface dataset:
SPOP Regulates the Nuclear Pore Protein NupJ
Authors: Ong Joseph , Torres Jorge Z
Published: June 2021

Cell processes like growth and division are tightly regulated. One such mechanism of regulation is ubiquitination. Ubiquitination can change a protein's localization or activity, or it can mark the protein for degradation by the ubiquitin proteasome system. The final step of ubiquitination, the transfer of ubiquitin to the target protein, is mediated by E3 ubiquitin ligases and their substrate adaptors, proteins that allow E3 ligases to be selective in choosing their targets. Understanding the targets of E3 ligases and substrate adaptors, then, is crucial to understanding cell regulation and disease mechanisms linked to misregulation of protein levels and activity. SPOP is a Cul3 E3 ubiquitin ligase substrate adaptor whose targets, such as c‐Myc, PD‐L1, and ERG, are crucial for cell cycle progression and cancer proliferation. Through a mass spectrometry screen, we identified SPOP as a potential regulator of NupJ, a nuclear pore protein. Nuclear pore proteins play canonical roles in transport across the nuclear envelope and emerging roles in nuclear envelope morphology and cell division. SPOP and NupJ both co‐localize at the nuclear envelope via immunofluorescence microscopy, and co‐immunoprecipitation experiments demonstrate that SPOP and NupJ bind to each other in vitro and from cell lysates. Similar to overexpression of NupJ, siRNA against SPOP leads to an increase in the number of nuclear envelope defects. Moreoever, overexpressed NupJ leads to defects in cell division. Our results suggest that SPOP regulates NupJ activity and perhaps protein stability of NupJ.

Support or Funding Information

JYO is supported by Edwin W. Pauley Fellowship, NSF Graduate Research Fellowship DGE‐1650604, and Ruth L. Kirschstein National Research Service Award GM007185. This work was funded by NSF MCB1243645 (to JZT).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of structural factors controlling loop dynamics in acyl protein thioesterases
Authors: Johnson R. Jeremy , Hossain Asif
Published: June 2021

FTT258 is a close bacterial relative of human acyl protein thioesterases (APTs). Human APTs catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. For FTT258, catalysis was hypothesized to be regulated by the large‐scale movement of an essential dynamic loop, which transitioned the protein between active and inactive states. However, the causes for the movement of this loop are unknown. Herein, we investigated factors that trigger loop movement in APTs like FTT258. In particular, FTT258 was exposed to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.

Support or Funding Information

Funded by NSF MCB‐1812971

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annotation and Comparison of Drosophila takahashii Genes on the Dot Chromosome
Authors: Spencer Danielle Lynette , Allen LaDaja , Brown Cierra , Buckner Erin , Davis Tyonna , Gleason Khayvion , Jackson Joshua , Lampkin Letha , Williams Jovan , Williams Nikita , Winfrey Tyrese , Woodland Cedric , Woods Arreonna , Van Stry Melanie
Published: June 2021

The overall goal for this project is to understand how genes are expressed in a heterochromatic environment. Chromosome four (Dot chromosome; Muller F element) of Drosophila melanogaster is 1.3 Mb long with ~80 genes but is largely heterochromatic. In the Genomics Education Partnership (GEP) project, we are currently annotating a group of species that was diverged from D. melanogaster about 10 million years ago. Our project was to annotate genes on a 70,000 base pair region (contig40) of the D. takahashii Dot chromosome. In order to annotate the genes we used the BLASTX, gene predictors like Gene Scan, RNA‐sequence data, and the results from the Gene Model Checker. According to the gene predictor, Gene Scan, this contig contains five protein‐coding genes. It predicted the genes Slip1, Myo, CG11360, a gene of unknown function (GI25919), and another gene that appears to be a Mex3A‐Slip1 fusion. We annotated the genes Slip1, Myo, CG11360, and GI25919. We are currently annotating the gene Mex3A‐Slip1 fusion gene. The D. takahashii genes that are similar to D. melanogaster are Myo and Slip1. Myo has an identity of 73.1% and a similarity of 81.8%. Slip1 has an identity of 72.3% and a similarity of 82.2%. CG11360 is very different with an identity of 13.1% and a similarity of 26.8%. In contrast to the D. takahashii genes that are present in D. melanogaster, GI25919 is not present at all. It is predicted to be present in several other species including: D. mojavensis, Aedes albopictus, Stomoxys calcitrans, and Musca domestica. We compared GI25919 to D. mojavensis and it has an identity of 39% and a similarity of 55%. In conclusion, some D. takahashii Dot chromosome genes within this region are similar to D. melanogaster, but others are very divergent or missing in D. melanogaster. Our data will be combined with other gene annotations from students in the GEP for a comparative genomics study to identify conserved gene regulatory elements.

Support or Funding Information

NSF HRD#1623340NSF IUSE #1431407

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Structural Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin
Authors: Hall James , Copps Jeff , Barry Rachael , Zhang Ping , Blumenthal Donald K. , Taylor Susan
Published: June 2021

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform‐specific subcellular localization. The D/D domain interface of the R‐ subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in the regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calciuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79ct: RIIβ2‐C2 :CNt) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control. Support for this work was provided through NIH GM034921.

Support or Funding Information

James Hall is an IRACDA fellow and was supported by NIGMS/NIH award K12GM068524. This work was also supported by a grant from the National Institutes of Health GM034921 to S.S. Taylor. This Research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, which are DOE Office of Science User Facilities operated by Oak Ridge National Laboratory. This work utilized SasView software, originally developed by the DANSE project under NSF award DMR‐0520547.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic Analysis of Discoidal Nascent HDL Structure by IPET
Authors: Li Jennifer Jinping , Lei Dongsheng , Liu Jianfang , Zhang Meng , Ren Gang
Published: June 2021

Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. In the recent decades, several models of HDL are built and debated each other, but the exact structure of HDL can't be confirmed even now. In our previous work, we used cross‐linking chemistry and mass spectrometry to generate detailed models of apoA‐I in reconstituted particles as well as “real” HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt‐like arrangement. Thus, direct visible observation of the conformation of discoidal HDL with the help of electron microscopy is a possible way to solve the debate. Here we report a study of structure of HDL by using negative‐staining electron microscopy and cryo‐electron microscopy as well as electron tomography. The 3D structures of dynamical individual HDL molecules are reconstructed by the individual‐particle electron tomography reconstruction (IPET). The reconstructed density maps at ~ 2 nm resolution display the size heterogeneity and conformation variety of HDL particles, and reveal a fusion/release mechanism of discoidal HDL in lipid transfer. These results revealed that apoA‐I makes highly specific contacts with itself to form a molecular scaffold that stabilizes HDL and facilitates, through specific protein: protein interactions, the association of HDL partner proteins to define particle function. The notion is crucial for us to further understand how the major HDL protein, apolipoprotein (apo)A‐I are involved in cholesterol transfer and lipid metabolism and cardiovascular disease.

Support or Funding Information

This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (2R01 HL115153‐06, R01GM104427, and P01HL030086). Work conducted at the Molecular Foundry was supported by the Office of Basic Energy Sciences of the Office of Science at the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Reinforcing and Antinociceptive Effects of Agonists with Mixed NOP and MOP Receptor Agonist Action in Nonhuman Primates
Authors: Ding Huiping , Kiguchi Norikazu , Trapella Claudio , Czoty Paul W , Calo’ Girolamo , Husbands Stephen M , Zaveri Nurulain T , Ko Mei‐Chuan
Published: June 2021

Agonists with mixed nociceptin/orphanin FQ peptide (NOP) and mu opioid peptide (MOP) receptor agonist activities have been demonstrated as effective analgesics with fewer side effects. However, these newly developed compounds with different efficacies at the NOP and MOP receptors have not been compared in terms of their reinforcing and antinociceptive effects. The aim of this study was to determine and compare effects of AT‐121 and BU08028 (NOP/MOP partial agonists), AT‐175 (NOP full/MOP partial agonist), and cebranopadol (mixed NOP and opioid full agonist) in a series of behavioral assays established in rhesus monkeys (Macaca mutatta). Following systemic administration, AT‐121 (3–30 ug/kg), BU08028 (1–10 ug/kg), AT‐175 (1–10 ug/kg), and cebranopadol (1–5.6 ug/kg) all dose‐dependently produced antinociceptive effects against an acute noxious stimulus and attenuated capsaicin‐induced hypersensitivity. In the intravenous drug self‐administration assay, the relative reinforcing strengths (i.e., abuse potential) of these compounds were compared in monkeys under a progressive‐ratio schedule of reinforcement over a wide dose range. Unlike oxycodone and heroin producing robust reinforcing effects, AT‐121, BU08028, and AT‐175 did not significantly produce reinforcing effects. However, cebranopadol produced moderate reinforcing effects as compared to oxycodone and heroin. Taken together, these pharmacological studies in primates indicate that agonists with mixed NOP and MOP receptor agonist activities exert opioid‐like analgesic effects. Although the NOP receptor activation could attenuate the reinforcing effects mediated MOP partial agonist action, the NOP full agonist activity may not be able to completely attenuate the reinforcing effects mediated by MOP full agonist activity in primates.

Support or Funding Information

The US. NIH/NIDA grants (DA023281, DA027811, DA032568, and DA035359) and US. Department of Defense (W81XWH‐13‐2‐0045).

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 Pleiotropic GPCR Ligand Rotigotine Acts as a β‐arrestin Biased Ligand on Orphan Receptor GPR52
Authors: Filipeanu Catalin , Ogunlade Blessing , Ghita Ioana , Wood Michael W , Fang Yayin
Published: June 2021

Despite large progress made on understanding G protein‐coupled receptors (GPCRs) roles, there is still a large group in this family which has no identified endogenous ligand and thus are defined as ‘orphan’ GPCRs. One such orphan GPCR is GPR52, which has little homology to other members of this family, the highest identity being with another orphan GPCR, GPR21 (71%). GPR52 is a Gs‐coupled receptor which constitutively increases cellular cAMP levels and it has been proposed to play a role in locomotor activity through modulation of dopamine receptors. On the other hand, rotigotine, a non‐ergoline dopamine agonist, is a FDA approved drug for treatment of Parkinson's disease (PD) and restless leg syndrome. However, rotigotine binds to high affinity to other GPCRs, including α2‐adrenergic and HTR1A receptors, but the clinical relevance of these effects remains unknown. To find new ligands for GPCRs we examined the effects of multiple pleiotropic ligands, like rotigotine, against 45 orphan GPCRs. We found that rotigotine induced β‐arrestin mobilization by GPR52 (232 ± 28 %, n=8) but not by any other tested orphan GPR. Surprisingly, rotigotine did not change cAMP levels in GPR52‐transfected HEK293 cells (98.5 ± 23.38%, n=4). In contrast, FTBMT, a previously reported GPR52 agonist, strongly increased cAMP levels (290.85 ± 25.65%, n=4), but it failed to change β‐arrestin mobilization (86.88 ± 16%, n=4). Our data indicate that rotigotine and FTBMT act as biased ligands on GPR52 preferentially activating specific signaling pathways. This conclusion is supported by the effects on other signaling pathways: both rotigotine and FTBMT stimulated GPR52 mediated MAPK phosphorylation, but only FTBMT enhanced AKT phosphorylation. Although the overall sequence similarity between GPR52 and DRD2 dopamine receptor is less than 15%, the alignment of these two receptors in the transmembrane domains resembles 28% homology. Based on the crystal structure of risperidone binding to its receptor DRD2, the binding of rotigotine to DRD2 was modelled and the computational model was extended to rotigotine interaction with GPR52. Using this method, two key amino acids in the 3rd transmembrane domain of GPR52, I121 and K125, were identified as crucial sites for binding for both rotigotine and FTBMT. In conclusion, rotigotine and FTBMT are biased agonists of orphan GPR52, which can be used for further exploring its patho‐physiological functions.

Support or Funding Information

Supported by HUMAA Endowed Founder's Chair in Basic Science Award and Office of Naval Research (N00014‐17‐1‐2105 and N00014‐18‐1‐2145) to YF.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
No significant change of extracranial conduit vessel diameter during cerebral vasomotor reactivity test with moderately‐altered end‐tidal CO2
Authors: Liu Jie , Le Thuy Tien Cao , Knox Allan , Serrador Jorge M
Published: June 2021

Cerebral vasomotor reactivity (CVMR) is often assessed using transcranial Doppler (TCD) to measure the cerebral blood flow velocity (CBFV) in the middle cerebral artery (MCA). Recently the validity of this technique has been questioned because of the assumption that MCA diameter does not change. The goal of this work was to test if the extracranial cerebral arteries, i.e. internal carotid and vertebral arteries (ICA and VA), have a significant diameter change during moderate alteration of end‐tidal carbon dioxide (ETCO2). Fifteen healthy subjects (6 males; age: 32±10 yrs) were enrolled. The CBFV at the MCA was continuously recorded with TCD during an 8‐min CVMR test, i.e. 2‐min resting ventilation, 2‐min inspiration of 5% CO2, 21% O2, balance nitrogen, 2‐min recovery, and 2‐min mild hyperventilation. 8 min protocol was repeated on the same subjects, to allow measurement of ICA diameter during one run and VA during the other using high‐frequency ultrasound. CVMR is expressed as the percent change in MCA's CBFV per mmHg change in ETCO2, as quantified by the slope of this relationship. The time‐averaged diameter during each static stage was measured offline with echo‐tracking software. All results presented as Mean ± SD. The experimental protocol resulted in a moderated change of ETCO2 (mmHg) during 5% CO2 inspiration (+6.9±2.0) and mild hyperventilation (−14.8±5.8) relative to the baseline values (34.6±2.7). Both hypercapnic and hypocapnic stages showed no significant changes (relative to the baseline) in blood pressure, heart rate, diameters of ICA (5.23±0.83 and 5.21±0.71, vs. baseline 5.22 ±0.83 mm; P = 0.88 and 0.86) and VA (3.91±0.69 and 3.91±0.72, vs. baseline 3.87±0.65 mm; P = 0.40 and 0.55), except for a slight increase (+7±8 bpm, P < 0.001) of HR during hypocapnia. The individual variation of the diameter response showed no correlation with the concurrent blood pressure, heart rate, and CBFV responses. Based on these results, we concluded that the TCD appears to still be a reliable method to assess the CBF response during CVMR test with a moderate change of ETCO2, as no significant change of the conduit vessel diameter is likely to occur. However, future work is necessary to confirm that flow changes in the extracranial arteries are reflective of relative changes in TCD values.

Support or Funding Information

Supported by the Funding from Rutgers University and U.S. Department of Defense.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analgesics in the Pre‐Hospital Setting: Simulated Hemorrhagic Tolerance in Humans is not Impaired by Ketamine Administration
Authors: Huang Mu , Moralez Gilbert , Cramer Matthew N. , Hendrix Joseph M. , Hinojosa‐Laborde Carmen , Ryan Kathy L. , Crandall Craig G.
Published: June 2021

Hemorrhage is the leading cause of battlefield and civilian trauma deaths. Given that a hemorrhagic injury on the battlefield is virtually always associated with pain, it is paramount that the administered pain medication does not disrupt the physiological mechanisms that are beneficial towards the maintenance of blood pressure and vital organ blood flow during that hemorrhagic insult. Current guidelines from the US Army's Committee on Tactical Combat Casualty Care (CoTCCC) for the selection of pain medications administered to a hemorrhaging soldier are based upon limited scientific evidence, with the clear majority of supporting studies being conducted on anesthetized animals. Specifically, the influence of ketamine, one of three analgesics employed in the pre‐hospital setting by the US Army, on hemorrhagic tolerance in humans is entirely unknown. Thus, the purpose of this study is to test the hypothesis that ketamine will impair the capacity for a conscious human to tolerate a simulated hemorrhagic insult. Twelve subjects (4 females, 29±7 years old, 181±6 cm, 84±10 kg) participated in this double‐blinded, randomized, placebo‐controlled investigation. Tolerance to a simulated hemorrhage was performed using a progressive lower body negative pressure (LBNP) protocol to pre‐syncope following intravenous administration of Ketamine (20 mg—consistent with the US Army's CoTCCC guidelines) or placebo (saline). Tolerance was quantified as a cumulative stress index (CSI, mmHg•min). Tolerance to the LBNP challenge was not different between the ketamine and placebo trials (CSI: 746±368 mmHg•min and 795±316 mmHg•min respectively, P=0.61). These data, the first to be obtained in conscious humans, demonstrate that administration of ketamine does not compromise tolerance to a simulated hemorrhagic insult. These findings may be insightful in choosing the most suitable analgesic medication in the pre‐hospital setting during a hemorrhagic insult.

Support or Funding Information

Funding: Department of Defense – US Army, W81XWH1820012

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep
Authors: Reid Casey , Romero Monica , Dobyns Abigail , Hessinger David A , Puglisi Jose L , Wilson Christopher G , Zhang Lubo , Wilson Sean M
Published: June 2021

Gestational long term hypoxia (LTH) is a common prenatal stress caused by maternal anemia, high altitude living, smoking, and other disorders. Such gestational LTH can lead to a variety of cerebrovascular disorders in the neonate that compromise brain blood flow. Local and whole‐cell Ca2+ signals are important to the regulation of cerebrovascular tone. Rapid and localized Ca2+ transients, often referred to as sparks, activate large‐conductance K+ channels (BK), which dilate vessels. Whole‐cell Ca2+ oscillations, in comparison, are important to arterial contraction. Previous work shows that LTH and post‐natal maturity influence BK channel function in basilar arteries, arterial wall Ca2+ signals, cerebrovascular tone, as well as brain blood flow. We hypothesize that LTH‐dependent changes in spontaneous and depolarization mediated Ca2+ sparks and whole‐cell oscillations are important to BK channel activity, arterial wall Ca2+ signals, and vascular reactivity changes our group has previously observed. To begin evaluating the role of individual myocyte Ca2+ signals to the observed changes in cerebrovascular function, we isolated basilar arteries from adult and near term fetal (~141 gestational days) normoxic and hypoxic sheep that were raised at 3,800 m. for 110+ days. Cytosolic Ca2+ signals were examined in individual myocytes of intact arterial preparations loaded with fluo‐4 using laser scanning confocal microscopy techniques. Ca2+ spark activity as well as whole‐cell oscillations were enhanced by depolarizing myocytes with 30 mM K+. Maturation increased Ca2+ spark activity and depolarization‐dependent oscillations. LTH decreased oscillatory activity in fetal and adult animals but only spark frequency in adult myocytes. LTH decreased the area under the curve for Ca2+ oscillations independent of age as well as suppressed the ability of adult myocytes to respond to membrane depolarization. Depolarization, maturation, and LTH also influenced the spatial and temporal relationships of the Ca2+ oscillations. Overall, these observations illustrate that maturation and gestation at high‐altitude modify local and whole‐cell Ca2+ signaling, which likely contribute to adjustments in BK channel function, arterial wall Ca2+ signals, vasoreactivity, and cerebral blood flow that our group has examined in the past.

Support or Funding Information

This material is based upon work supported by NIH grants P01HD083132 (LZ). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI‐DBI 0923559 and the Loma Linda University School of Medicine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular CaseNet: Developing case studies using molecular representations for use in introductory chemistry, biology and biochemistry classes
Authors: Jakubowski Henry , Cortes Kimberly Linenberger , Lenahan Melanie , Marcey David , Marsteller Pat , Terrell Cassidy R. , Dutta Shuchusmita
Published: June 2021

Understanding “Structure and Function” relationships is foundational to learning biology, chemistry, and biochemistry. However, exploring and using biochemical data resources, especially the Protein Data Bank, to develop a deeper understanding of the relationship between structure and function are not common practices, especially in introductory courses. This project brings together biology and chemistry educators to develop case studies centered around molecular representations of biomolecules. We hope these studies, at the interface between both disciplines, will facilitate deeper understanding of structure/function in biology and chemistry and reduce conceptual barriers that hinder a common understanding. This presentation will focus on a hemoglobin case study that was developed and used in introductory chemistry and a one‐semester biochemistry course. Discussion will focus on implementation of using the same case at different levels of instruction, ideas for other content that could be used to bridge the disciplines of biology and chemistry using molecular representations and how you can get involved in the Molecular CaseNet.

Support or Funding Information

This work was supported by the National Science Foundation under award #1827011. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of the Pope Engagement Index to Measure the Cognitive Load of Biochemistry Students while Completing a 3D Physical Serine Protease Modeling Activity
Authors: Cortes Kimberly Linenberger , Terrell Cassidy , Calvert Jenifer , Randolph Adriane
Published: June 2021

Research has shown students have significant difficulty understanding enzyme‐substrate relationships. One way instructors have suggested to support students while learning this content is through the use of 3D physical models of the enzyme and substrate. However, little research has been conducted on how the models impact the learning process and the cognitive load of the students. This study aims to measure the cognitive load of biochemistry students while completing a modeling activity focused on understanding the relationship between the substrates and active site of serine proteases. During a simulated learning environment, biochemistry students completed an activity related to how enzymes and substrates interact based on electronics, stereochemistry and geometric complementary using the serine protease physical models. During the activity electroencephalography was used to capture student cognitive processing in order to assign cognitive load of the tasks in the activity using the Pope Engagement Index. This presentation will specifically focus on how spatial ability of the student impacted the level of engagement and in what region of the brain was engaged when completing various tasks. Additional discussion will focus on how changes to the models impacted the cognitive load of the participants when completing the activity. Suggestions will be provided for faculty to think about when developing modeling activities to decrease extraneous cognitive load for students.

Support or Funding Information

This project is supported by the National Science Foundation under award number IUSE 1711402/1711425 to University of Minnesota, Rochester and Kennesaw State University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fungicide‐induced melanin loss causes changes the transfer RNA modification pool in the radiation resistant fungi Cladosporium sphaerospermum
Authors: Kelley Melissa , Yoshida George , Myers Ryan , Alem Kibret , Chati Prathamesh , Jora Manasses , Addepalli Balasubrahmanyam , Limbach Patrick A
Published: June 2021

Fungi have been found in extreme environments all over the world. Of particular interest, Cladosporium sphaerospermum is a filamentous fungus found to grow around the nuclear reactor at Chernobyl. Thus far, little is known of the mechanistic details that allow these fungi to survive such harsh oxidative conditions. The current hypothesis is that the pigment melanin contributes to growth through enhancing energy production mechanisms. In this work, we aim to identify the effect melanin presence on post‐transcriptional modifications to transfer RNA (tRNA) in C. sphaerospermum. Post‐transcriptional modifications affect translation of response proteins and may be indicative of an underlying mechanism. Prior to this study, the tRNA genes of C. sphaerospermum were unknown. Through a bioinformatics approach, 195 tRNA genes have been identified throughout the genome. The identification of tRNA genes gives information on anticodon preference as well as composition of the tRNA pool. Furthermore, treatment with the fungicides pyroquilon and tricyclazole induce varying levels of melanin loss. Dose‐response curves were generated for optimal dosing. Both fungicides cause phenotype changes, growth rate differences, and tRNA modification profiles alterations. Next, the impact of melanin loss on C. sphaerospermum survival in the presence of ionizing radiation (IR) was determined through growth rates post‐IR. The effect of exposure to ionizing radiation on tRNA modifications has been qualitatively and quantitatively determined using a liquid chromatography coupled with mass spectrometry system.

Support or Funding Information

Funding support was provided by the Defense Threat Reduction Agency (DTRA) and the University of Cincinnati.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ultradeep Palmitoyl‐proteomic Analysis Uncovers Over 1,300 Novel Human Palmitoyl‐proteins
Authors: Yang Wei , Zhou Bo , Wang Yang , Yan Yiwu , Freeman Michael R
Published: June 2021

Protein palmitoylation (more accurately known as protein S‐acylation) is a reversible lipid modification that plays critical roles in protein localization, activity, stability, and complex formation. Aberrant protein palmitoylation is associated with many human diseases such as neurodegenerative disorders and cancer. The past decade has witnessed striking progress in studying the enzymes, substrates, and functions of protein palmitoylation. However, it remains unclear whether protein palmitoylation is a widespread post‐translational modification comparable to protein phosphorylation and ubiquitination. Previously, we developed a palmitoyl‐proteomics method termed Palmitoyl‐Protein Identification and Site Characterization (PalmPISC) and applied it to profile the palmitoyl‐proteomes in human cancer cells and platelets (Yang et al., Mol Cell Proteomics, 2010; Dowal et al., Blood, 2011). Nevertheless, similar to other acyl‐biotinyl exchange (ABE)‐based palmitoyl‐proteomics methods, one shortcoming of the PalmPISC method is that many non‐palmitoylated proteins are co‐isolated with palmitoyl‐proteins, resulting in a high background that masks the detection of low‐abundance palmitoyl‐proteins and interferes with accurate quantification of palmitoyl‐proteins across different conditions. In this study, we coupled stable isotope labeling by amino acid in culture (SILAC) with PalmPISC to quantify the extent of co‐isolated proteins and found that ~65% of identified proteins were co‐isolated contaminants. To address this issue, we developed a low‐background ABE enrichment method and applied it to comprehensively profile the palmitoyl‐proteome of a human prostate cancer LNCaP cell line. Using stringent cutoffs (false discovery rate < 1% and fold change >2), a total of 2,895 proteins, including 1,571 known and 1,324 novel human palmitoyl‐proteins, were found to be significantly enriched. Western blotting analysis confirmed the palmitoylation of six novel palmitoyl‐protein candidates that play important roles in prostate cancer. In summary, our low‐background ABE enrichment method enables proteome‐wide analysis of protein palmitoylation and the study demonstrates that protein palmitoylation is more widespread than we previously thought.

Support or Funding Information

This work was supported by the Department of Defense W81XWH‐15‐1‐0167 (to W.Y.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Falling Apart: the Self‐Catalyzed Process of Protein Splicing
Authors: Chavez Angel , Minteer Christopher J. , Jaramillo Mario , Mills Kenneth V.
Published: June 2021

Protein splicing is a self‐catalyzed process where an intervening intein is excised, with the concomitant ligation of the two flanking exteins to form a mature protein. The inteins we studied interrupt the DNA Pol II of Pyrococcus abyssi and Pyrococcus horikoshii. These extremophile inteins conditionally splice at high temperatures. The Hyperthermophilic and Thermophilic Hairpin (HTH) is only found in the structures of archaeal inteins. We hypothesize that the HTH increases stability by elongating the central beta‐sheet, and thus mutations that disrupt this hairpin would affect the stability toward unfolding and the reactivity at the active site. To further understand the protein splicing of the P. abyssi intein, an intein with an Arg40Glu mutation in the HTH was expressed, and we studied its activity. We also purified 15‐N labeled intein for NMR (HSQC) analysis of residue‐level changes induced by the mutation. We have also purified a variant of the P. horikoshii PolII intein in order to attempt to solve the structure of an unspliced precursor intein.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A structural investigation of NRZ mediated apoptosis regulation in zebrafish
Authors: Suraweera Chathura Dahamjith , Caria Sofia , Jarva Michael , Hinds Mark , Kvansakul Marc
Published: June 2021

Bcl‐2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host‐pathogen interactions and in disease. Dysregulation of Bcl‐2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intrafamily protein– protein interactions Bcl‐2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro‐survival Bcl‐2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here we examined the interactions of NRZ with pro‐apoptotic members of the Bcl‐2 family using a combination of isothermal calorimetry and mutational analysis of NRZ. We show that NRZ binds almost all zebrafish pro‐apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo ‐NRZ and a holo form bound to a peptide spanning the binding motif of the pro‐apoptotic zBad, a BH3‐only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl‐2 like fold observed for other cellular pro‐survival Bcl‐2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. NRZ engagement of Bad BH3 involves the canonical ionic interaction between NRZ R86 and Bad D104 and an additional ionic interaction between NRZ D79 and Bad R100, and substitution of either NRZ R86 or D79 to Ala reduces the binding to Bad BH3 tenfold or more. Our findings provide a detailed mechanistic understanding for NRZ mediated anti‐apoptotic activity in zebrafish by revealing binding to both Bad and Noxa, suggesting that NRZ is likely to occupy a unique mechanistic role in zebrafish apoptosis regulation by acting as a highly promiscuous pro‐apoptotic Bcl‐2 binder.

Support or Funding Information

This work was supported by the Australian Research Council (Fellowship FT130101349 to MK) and LaTrobe university (scholarship to CDS)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGE2 Augments Inflammasome Activation and M1 Polarization in Macrophages Infected with Salmonella Typhimurium and Yersinia enterocolitica
Authors: Sheppe Austin , Kummari Evangel , Walker Alyssa , Richards Angela , Hui Winnie , Lee Jung Hwa , Magnum Lauren , Borazjani Abdolsamad , Ross Matt , Edelmann Mariola
Published: June 2021

Prostaglandin E2 (PGE2) is an important immunomodulatory molecule that is part of a larger group of bioactive lipids known as eicosanoids. PGE2 has long been reported to have conflicting roles in the regulation of inflammation. For example, some effects are primarily anti‐inflammatory and other roles that mediate inflammation. During intracellular infection with Gram‐negative bacteria, host macrophages form complexes called inflammasomes. Inflammasomes drive activation and secretion of inflammatory cytokines such as IL‐1β that signal nearby cells in the process of inflammatory cell death called pyroptosis. PGE2 is reported to inhibit NLRP3 inflammasome activation and enhance NLRC4 inflammasome activation. Since both NLRP3 and NLRC4 inflammasomes mediate IL‐1β secretion, the PGE2's function in the inflammatory processes does not seem to be clear. Previous studies relied on LPS stimulation of macrophages in place of bacterial infection. In this study, we used an infection model of THP‐1 human macrophages with Salmonella enterica Typhimurium (St) and Yersinia enterocolitica (Ye) that suggests PGE2 plays a vital role in enhancing the transcription and secretion of IL‐1β upon inflammasome activation. Interestingly, distinct virulence strategies between St and Ye are reflected in the alteration of COX‐2 and resulting PGE2 biosynthesis by these pathogens. Ye virulence factors inhibit COX‐2 expression and thus decrease the amount of PGE2 secreted during infection. St, in contrast, activates COX‐2 and enhances PGE2 production. In our studies, PGE2 enhances bacterial load of St inside macrophages 48 hours post infection, but it decreases intracellular Ye. Finally, our results suggest that PGE2 primarily enhances the transcription of pro‐inflammatory cytokines while repressing anti‐inflammatory processes such as resource scavenging and matrix remodeling.

Support or Funding Information

This study was supported by a fund no. 2016‐31200‐06012 from the National Institute of Food and Agriculture (NIFA), https://doi.org/10.13039/100005825, National Institutes of Health (NIH) fund no. 5P20GM103646‐02, https://doi.org/10.13039/100000002, as well as MS Center for Food Safety and Post‐harvest Technology Grant Program (USDA‐ARS SCA 58‐6066‐7081) and NIH1R15ES015348‐02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of Algal Biofeedstocks in a Mixotrophic Batch Culture Supplemented with Exogenous Glycerol
Authors: Smythers Amanda L , Higginbotham Ethan , Holland Aaron , Stephenson Anthony , Kolling Derrick R.J.
Published: June 2021

Chlorella vulgaris is a fast growing, unicellular microalgae. With its limited nutritional requirements and ability to grow in adverse environments, it is a promising biodiesel feedstock that will not compete with agricultural resources.1 Published literature has shown C. vulgaris can produce up to 42% lipids per dry mass, the most desired of which are triacylglycerols (TAGs).2 TAGs consist of three fatty acids esterified to a glycerol backbone and are the most reduced, energy‐dense biomolecules. To generate drop‐in ready biodiesel, TAGs are transesterified to yield methyl‐ester alkyls and a glycerol byproduct.

To realistically produce fuel on an industrial scale, microalgal TAG production must be increased beyond current levels. Previous work published by the Kolling Lab increased lipid accumulation in C. vulgaris by using an exogenous carbon source to form a heteromixotrophic culture, in which the cells are energetically supplemented by both exogenous carbon as well as photosynthetic light reactions.3 This project seeks to further increase TAG production by supplying cells with exogenous glycerol, thus providing a use for the transesterification byproduct while reducing the cellular energy investment for TAG production.

While published studies have found 10 g/L glycerol to be optimal for lipid production in C. vulgaris, this was completed heterotrophically.4 While growing mixotrophically, we have increased lipid accumulation beyond previously published glycerol studies and have increased overall lipid productivity in glycerol concentrations of 31.5 and 63 g/L. We also monitored photosynthetic performance before and after glycerol addition and observed an increased reliance on cyclic electron transport (a mode of photosynthesis that does not result in the production of NADPH), indicating that the ATP need is greater than that of NADPH for TAG formation under photomixotrophic conditions. Finally, using isotopically labeled glycerol, we have used GC‐MS to determine the rate of glycerol uptake and that exogenously provided glycerol is directly used for the TAG backbone and not metabolized for the generation of fatty acids.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.

Amanda Smythers acknowledges a fellowship from the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant # NNX15AI01H.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Anoxia‐Tolerant Cell Line, AL4, Provides New Insight into Small Non‐Coding RNA Biology
Authors: Riggs Claire L , Podrabsky Jason E.
Published: June 2021

Small non‐coding RNAs (ncRNAs) can rapidly, reversibly, and specifically regulate gene expression. These characteristics make them compelling candidates for regulating gene expression in anoxia‐tolerant vertebrates, as survival of anoxia requires dramatic yet reversible changes in physiology. Recent small ncRNA sequencing in anoxia‐tolerant vertebrates revealed groups of candidate small ncRNAs for supporting anoxia tolerance. The expression of mitosRNAs, small ncRNAs derived from the mitochondria, was particularly striking in the most anoxia‐tolerant vertebrate, embryos of the annual killifish Austrofundulus limnaeus, and inspired further study. Cell culture is a particularly useful tool for detailed study of small ncRNA biology, due to the ease of subcellular imaging and chemical and genetic manipulation. Therefore, we aimed to establish and characterize an anoxia‐tolerant cell line from A. limnaeus to use as a tool to probe the biology of small ncRNAs of interest. The AL4 cell line was established from embryonic tissue of A. limnaeus using a tissue explant method. Proteomic phenotyping suggested a neuroepithelial identity. The cells can survive for over 40 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for green monkey kidney epithelial cells (Vero) and mouse neuroblastoma cells (N2A). Additionally, AL4 cells accumulate lactate during anoxia, which is consistent with whole embryos, as well as anoxia‐sensitive mammals. AL4 express many of the same small noncoding RNAs as the whole annual killifish embryos, including anoxia‐responsive mitosRNAs. Known mammalian stress‐responsive small ncRNAs also respond to anoxia in AL4. Therefore AL4 both reflects the biology of whole embryos and can be used in comparative studies with anoxia‐sensitive mammalian species. We used the cell line to investigate highly anoxia‐responsive mitosRNAs derived from mitochondrial tRNA cysteine. We found that these mitosRNAs are enriched in the mitochondria, particularly near the nucleus, and appear to be present in the cytoplasm as well. They also appear to be de novo transcribed in the mitochondria during anoxia. This body of work provides the first evidence for mitochondrial de novo transcription and cytoplasmic localization of mitosRNAs in response to stress. The establishment of the AL4 cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, including mitosRNAs, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.

Support or Funding Information

This work was supported by NSF GRFP DGE‐1057604 to Claire L. Riggs and NSF IOS‐1354549 to Jason E. Podrabsky.

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