bluuebunny/crossref_metadata_2025 huggingface dataset:
A free‐exchange mathematical model of EmrE
Authors: Hussey Grant , Thomas Nathan , Henzler‐Wildman Katherine
Published: June 2021

EmrE is a multidrug resistance transport protein from E. coli that couples the efflux of toxic, lipophilic cations to the influx of protons down their electrochemical gradient. Active transporters require alternating access between open‐in (binding pocket accessible to the cytoplasm) and open‐out (binding pocket accessible to the periplasm) conformations to move substrate across the membrane. Strict restrictions on alternating access or substrate binding lead to simplified models that achieve stoichiometric symport (coupling movement of both substrates in same direction) or “pure‐exchange” antiport (coupling movement of both substrates in opposite directions). However, recent data suggests that common strictly‐coupled models of transport may be oversimplified. For example, the EmrE point mutant, W63G, confers resistance to erythromycin, indicating H+‐coupled transport of that substrate, while also conferring susceptibility to polyamines, indicating uniport or H+‐coupled symport of those substrates. We recently developed a “free‐exchange” model of EmrE transport that can account for this behavior as well as NMR data showing that EmrE can simultaneously bind drug and proton and can alternate access when bound to both, one, or no substrates in violation of pure‐exchange models. Here, we develop a free‐exchange mathematical model to explore features that tip the balance between different H+‐coupled transport regimes. We find that under physiological conditions, transport is faster in the free‐exchange model and comparable substrate gradients can be produced. Using the available kinetic data for EmrE and our mathematical model, we show that EmrE can concentrate drug as experimentally observed in liposomal‐flux assays and also show how drug identity can switch directionality of transport, as observed for W63G‐EmrE.

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 Cell Penetrating Peptides to Deliver Cargo in Protists
Authors: Warner Max V. , Barnhill Zoie , LeCher Julia C. , Cooper Jennifer L.
Published: June 2021

Algae and plankton are important both ecologically as a food chain base and economically as nutraceutical “factories”. Despite their importance, technologies to transfect genes or proteins are limited or nonexistent in many protist species of interest, probably because cell walls present a barrier to macromolecular passage. We are using a novel cell penetrating peptide (CPP) system as a means to overcome this barrier. CPPs are a class of short peptides that can cross the cell membrane and sometimes the cell wall in a variety of species. Membrane translocation by CPPs involves endocytosis as well as other mechanisms. CPPs can be linked in a variety of ways to desired cargos to cause their entry into cells. Microscopic imaging of protist cells treated with CPP linked to fluorescently labeled cargo demonstrates that CPPs can cause the association of cargo with cells while fluorescently labeled cargo‐only treatment does not cause extensive cargo association with cells. Using fluorescence microscopy, we are currently optimizing the type of CPP and the CPP‐cargo uptake procedure to find the best conditions for cargo delivery to protists. We are also attempting proteolytic treatment to eliminate cargo stuck to the cell exterior and allow flow cytometric measurements of true cargo entry. Success of our method will provide a new means for rapid introduction of macromolecules into protist cells, allowing both investigation of their cellular function and potential manipulation of economically important products.

Support or Funding Information

Mentor Protégé Research Program, College of Science and Mathematics, Kennesaw State University; Research Opportunity Award, Office of Research, 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.
Plasmalogen Precursors Reverse Lipid Changes in a Barth Syndrome Cell Model
Authors: Bozelli Jose Carlos , Junior  , Epand Richard M
Published: June 2021

Barth syndrome is rare X chromosome‐linked genetic disorder that is caused by mutations in tafazzin, a phospholipid‐lysophospholipid transacylase responsible for the last step of cardiolipin (CL) biosynthesis in the mitochondria. The lipid alterations in Barth syndrome include loss in total CL content, an increase in the concentration of monolysocardiolipin (MLCL) and a greater heterogeneity of the acyl chains of CL. These changes are so characteristic of Barth syndrome that they have become the basis of diagnostic tests. Recently, one of us has demonstrated that the major change in the lipid composition of organs that have been depleted of tafazzin is the loss of plasmalogens. In several disease states where loss of plasmalogens has been reported, the condition could be reversed by the administration of plasmalogen precursors. Hence, we hypothesized that administration of plasmalogen precursors could reverse the phenotype of Barth syndrome. Here, we present the high‐resolution 31P‐NMR phospholipidomic study of the total lipid extract of a Barth syndrome cell model, i.e., lymphoblasts derived from Barth syndrome patients compared with controls in absence and presence of feeding plasmalogen precursors. Lymphoblasts derived from Barth patients in comparison to controls are ideal for studies of the mechanism as it is a single cell type that can be grown in culture and have all the characteristics of cells from patients with this Syndrome. In agreement with previous results we observed a marked decrease in plasmalogen in Barth lymphoblasts compared to controls together with other lipid alterations reported in those cells. Furthermore, as expected, feeding the cells with plasmalogen precursors restored plasmalogen levels. However, to our surprise the feeding also markedly increased the level of CL. Moreover, we also could show that the CL increase is not a consequence of a decrease in CL hydrolysis by a phospholipase (degradation) as evinced by the lack of MLCL accumulation in cells fed with plasmalogen precursors. In summary, we are able to reverse, at least in part, the lipid changes associated with Barth syndrome by feeding our cell model with plasmalogen precursors. We wish to extend these findings to determine how these changes in lipid composition affect the functioning of their mitochondria. This may present a simple and rapid process to bring these new strategies to the clinic since the precursors we are using are non‐toxic and have been administered to humans for other purposes.

Support or Funding Information

An award from the Barth Syndrome Foundation, Inc. and the Barth Syndrome Foundation of Canada.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic escape to glycolysis through SK channel activation inhibits ferroptosis and increases the life span of C. elegans in conditions of heat stress
Authors: Dolga Amalia , Krabbendam Inge , Honrath Birgit , Dilberger Benjamin , Schmidt Martina , Eckert Gunter , Culmsee Carsten
Published: June 2021

Metabolic flexibility is a characteristic of essentially all cells in order to cope with environmental alterations. One example of adaptation is represented by a mitochondrial switch from oxidative phosphorylation (OXPHOS) to glycolysis. Attenuation of mitochondrial respiration and cell metabolic shifts have been proposed as therapeutic strategies against oxidative stress and neurodegeneration. Activation of small‐conductance Ca2+‐activated K+(SK) channels contribute to neuroprotection in conditions of oxidative stress by attenuating mitochondrial calcium ([Ca2+]m), mitochondrial reactive oxygen species (ROS) generation and mitochondrial respiration in neuronal cells. To date, potential effects of SK channels have never been studied in relation to metabolic reprogramming nor the subsequent effects on survival in whole organisms in conditions of oxidative stress. Therefore, we aim to understand the mechanism by which SK channels confer neuroprotection during ferroptosis, a form of cell death associated with oxidative stress, and whether metabolic reprogramming can be part of neuroprotection.

Our data show that CyPPA, a positive modulator of SK channels, prevented cell death in conditions of ferroptosis and preserved mitochondrial function. High‐resolution respiratory revealed that opening of SK channels alone resulted in a mild decrease of mitochondrial complex I activity. These effects were accompanied by a slight increase in mitochondrial ROS production and depolarization of the mitochondrial membrane, suggesting preconditioning mechanisms to be also involved in the neuroprotection. Experiments where mitochondrial ROS were scavenged showed reduced capacity of CyPPA to mediate neuroprotection. Interestingly, extracellular flux analysis by Seahorse flux analysis technology revealed a CyPPA‐mediated initial increase in glycolysis followed by a slight reduction of OXPHOS activity. Induction of glycolysis was crucial for the neuroprotective effects of the SK channel opener, since substances that inhibit glycolysis were able to restore the cell sensitivity to ferroptosis even in the presence of CyPPA. Importantly, we found CyPPA to increase survival of C. elegans challenged with mitochondrial toxins in conditions of heat stress. These findings shed light on new molecular mechanisms mediated by SK channels that could explain the increased neuroprotective capacity in conditions of ferroptosis and neurodegeneration.

Support or Funding Information

A.M.D. is the recipient of a Rosalind Franklin Fellowship co‐funded by the European Union and the University of Groningen.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aloe‐emodin inhibits hypertension‐induced inter‐endothelial junction dysfunction through endothelial ROS‐TXNIP‐Nlrp3 inflammasome axle
Authors: Zhang Yi , Ye Lifeng , Wang Yunting , Zhang Yang , Li Pinlan , Chen Yang
Published: June 2021

Recent studies have revealed that Aloe‐emodin, a natural compound from the root and rhizome of Rheum palmatum L., exhibits significant pharmacological activities. However, the pharmacological relevance of the compound, particularly for cardiovascular disease, remains largely unknown. Here, we hypothesized that Aloe‐emodin could improve endothelial injury by inhibiting the activation of Nlrp3 inflammasome, and ultimately prevent cardiovascular disease. In vitro, our findings demonstrated that Aloe‐emodin inhibits AngII‐induced Nlrp3 inflammasome formation and activation in Microvascular Endothelial Cells (MECs), as shown by fluorescence confocal microscopy and Western blot. At the same time, the data shown that Aloe‐emodin exerts anti‐oxidation and ROS scavenged effects. Interestingly, thioredoxin‐interacting protein (TXNIP) protein and high mobility group box 1 (HMGB1) markedly decreased with Aloe‐emodin treatment. We also demonstrated that the decreased expression of the tight junction proteins ZO‐1/ZO‐2, which are related to endothelial permeability after stimulation by AngII in endothelial cells. Aloe‐emodin could recover the gap junction protein and decrease monolayer cell permeability in endothelial cells. Similarly, we used confocal microscopy to study the formation and activation of NOD‐like receptor family pyrin domain containing‐3 (Nlrp3) inflammasomes and expression of tight junction proteins in coronary arteries of AngII/DOCA‐treated mice. We found that Aloe‐emodin inhibited the formation and activation of Nlrp3 inflammasome, and also could restore the expression of the endothelial connective protein ZO1/2 and the release of HMGB1. Together with these changes, we revealed a new protection mechanism of Aloe‐emodin that inhibits Nlrp3 inflammasome activation and decreases subsequent caspase‐1 activation, triggering the release of HMGB1 by reducing ROS generation. Our findings indicate that Aloe‐emodin exhibits immense potential and specific therapeutic value in hypertension‐related cardiovascular disease and the development of innovative drugs.

Support or Funding Information

The National Key Research and Development Program of China (No. 2017YFC1700400); the project supported by National Natural Science Foundation of China (No. 81603587); Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2018B030306027); Science and Technology Development Plan of Guangdong Province (No. 2017A020211016)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of apolipoprotein A1‐ or Lecithin:cholesterol acyltransferase‐deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice
Authors: KYPREOS KYRIAKOS E , XEPAPADAKI EVA , SASSON SHLOMO , MAULUCCI GIUSEPPE , CONSTANTINOU CATERINA , KARAVIA ELENI A , ZVINTZOU EVANGELIA
Published: June 2021

High density lipoprotein (HDL) has attracted the attention of the biomedical community due to its well‐documented role in atheroprotection. HDL has been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral tissue insulin sensitivity. Indeed, data from numerous studies strongly indicate that β‐cells, as well as skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet‐induced obesity and type 2 diabetes we aimed at investigating how deficiency in Apoa1 or Lcat, two key proteins of peripheral HDL metabolic pathway, could affect these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western‐type diet results in increased sensitivity to body‐weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discreet effects on WAT metabolic activation and plasma glucose homeostasis. Apoa1‐deficiency results in reduced WAT mitochondrial non‐shivering thermogenesis. Lcat‐deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non‐shivering thermogenesis, rendering lcat−/− mice the most sensitive to weight gain out of the three strains tested, followed by apoa1−/− mice. Nevertheless, only apoa1−/− animals show disturbed plasma glucose homeostasis due to non‐responsive pancreatic β‐islets to glucose and insulin resistant skeletal muscles. Our analyses show that both apoa1−/− and lcat−/− mice fed high‐fat diet have no measurable Apoa1 levels in their plasma, suggesting that other HDL components may be responsible for the observed phenotypic differences among groups.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Allograft Inflammatory Factor 1‐Like (Aif1L) in Skeletal Muscle
Authors: Smith Anastasia , Waddell David
Published: June 2021

Skeletal muscle wasting, or atrophy, results from decreased protein synthesis and an associated increase in protein degradation. This shift towards a decrease in protein content of the cell can result from various physiological conditions, including immobilization, denervation, and aging. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has been found to be upregulated during almost all conditions of muscle wasting and is believed to play an important role in targeting proteins for degradation. However, preliminary data described in this study provides new evidence that MuRF1's role in the skeletal muscle atrophy cascade is likely more complex and it appears that MuRF1 is necessary for full transcriptional activation of a small subset of neurogenic atrophy‐induced genes, including allograft inflammatory factor 1‐like (Aif1L). Aif1L has been identified as a novel skeletal muscle gene that is upregulated in wild‐type mice, while MuRF1‐null mice show blunted upregulation in response to denervation. Aif1L is predicated to contain an EF‐hand domain, which can act as a calcium ion binding domain. Furthermore, Aif1L has also recently been implicated in regulating actomyosin contractility and filopodial extensions and may play a similar role in muscle cells. To confirm that Aif1L in expressed in muscle cells, qPCR was used to assess the expression levels in cultured muscle cells, while the cDNA of Aif1L was cloned from the C2C12 mouse myoblast cell line and fused with a green fluorescence protein (GFP) tag. C2C12 cells were then transfected with the Aif1L‐GFP expression plasmid in order to localize the Aif1L protein in proliferating myoblast cells. C2C12 cells transiently overexpressing Aif1L fused to GFP showed punctate fluorescence localized to the perinuclear region of the cytoplasm. The results presented here in combination with additional research into the transcriptional regulation and function of Aif1L will help contribute new insight into the molecular genetic mechanisms of skeletal muscle atrophy and may provide a more complete understanding of how MuRF1 may regulate the neurogenic atrophy cascade during muscle wasting.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BMP‐7 inhibits Pyroptosis in Vascular Smooth Muscle Cells in ATH
Authors: Garner Kaley , Singla Dinender
Published: June 2021

Atherosclerosis (ATH) is an inflammation‐mediated disease in which lipids, immune cells, and vascular cells contribute to the formation of plaque along vessel walls. Cell death by apoptosis and necrosis have been demonstrated in ATH; however, pyroptosis, an inflammation‐mediated cell death, has not yet been fully elucidated. We hypothesized that pyroptosis of vascular smooth muscle cells contributes to ATH progression. We further investigated whether administration of bone morphogenetic protein‐7 (BMP‐7) can inhibit these effects and demonstrate therapeutic potential. ApoE−/− mice (11±1 week old) were divided into three groups: Sham, PLCA, PLCA+BMP‐7 (200μg/kg; i.v). ATH was generated using our established partial ligation of the left carotid artery (PLCA) model and immunohistochemical staining and western blot analysis of arterial tissue was performed. ATH generated in PLCA mice demonstrated an upregulation in Toll‐like receptor‐4 (TLR‐4) and NLRP3 inflammasome components (NLRP3 and Caspase‐1), indicating the initiation and activation of pyroptosis in vascular smooth muscle (α‐SMA) cells. Further, inflammasome cleavage of pro‐IL‐1β and pro‐IL‐18 to mature forms released through cell membrane pores mediated by Caspase‐11 were investigated. IHC staining of arterial tissue demonstrated a significant increase in IL‐1β and IL‐18 expression following PLCA (p<0.05), which was significantly improved upon treatment with BMP‐7. Western blot analysis of arterial tissue supported these findings, indicating a significant upregulation in pyroptosis initiation via TLR‐4, inflammasome activation (Caspase‐1 and NLRP3) and release of pro‐inflammatory factors, IL‐1β and IL‐18 (p<0.05). Overall, this study indicates that pyroptosis occurs in the vascular smooth muscle cells in ATH and that BMP‐7 administration attenuates pyroptosis significantly.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Necroptosis Resulting from Activation of a RIP3‐dependent Pathway Contributes to Cardiomyocyte Death Induced by Isoproterenol
Authors: Wu Penglong , Wang Yibin , Liu Jinbao , Wang Xuejun
Published: June 2021

Background/Hypothesis

Excessive β‐adrenergic receptor activation causes maladaptive cardiac remodeling in humans with myocardial infarction. Cardiomyocyte apoptosis and necrosis are both considered as the main modes of cell death in β‐adrenergic receptor agonist isoproterenol (ISO)‐induced myocardial injury but the mechanism underlying necrosis remains undefined. Necroptosis is a newly discovered form of regulated cell death that exhibits morphological characteristics of necrosis but is regulated by the receptor interacting protein kinase (RIP) 3‐dependant pathway. Necroptosis has been suggested to play a role in the pathogenesis of common cardiovascular diseases. During necroptosis, the formation of necroptosomes consisting of proteins including RIP3 and RIP1 is critical. More recently, Ca2+/calmodulin‐dependent kinase II (CaMKII), which can be phosphorylated and thereby activated by β‐adrenergic receptor activation, was also reported to be involved in cardiomyocyte necroptosis by directly binding to and being phosphorylated by RIP3. However, whether ISO‐induced cardiomyocyte death involves necroptosis requires further investigation. Hence, we sought to test the hypothesis that RIP3‐depedent cardiomyocyte necroptosis contributes to ISO‐induced cardiac injury.

Methods and Results

Young adult mixed‐sex wild type (RIP3+/+) or RIP3 global knockout (RIP3−/−) C57BL/6 mice were subject to two consecutive subcutaneous injections of ISO (85 mg/kg body weight/injection, 24h interval) or vehicle, myocardial tissues were collected at 24h after the 2nd ISO injection. Evan's blue dye (EBD) was administered (100ug/g body weight, i.p.) at 18h before tissues collection for the EBD‐based detection of increased cardiomyocyte membrane permeability, a hallmark of necrosis. In RIP3+/+ mice, the ISO treatment significantly increased myocardial protein expression of RIP3 (both in the soluble and insoluble fractions), RIP1, RIP3 bund RIP1 (as detected by immunoprecipitation), and phosphorylated CaMKII, and resulted in increased EDB uptake by a large number of cardiomyocytes (p<0.01). By contrast, the elevation of RIP1 and the number of EBD‐positive cardiomyocytes, but not phosphorylated CaMKII, were significantly less in the RIP3−/− mice subject to the same ISO treatment (p<0.01).

Conclusion

Induction of myocardial injury by excessive β‐adrenergic receptor activation involves cardiomyocyte necroptosis via a RIP3‐dependent but CaMKII‐independent pathway.

Support or Funding Information

This work is supported in part by NIH grants HL072166, HL085629, and HL131667

This 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 Proximal Tubule Cell Endocytic Capacity by Nephrotic Levels of Albumin
Authors: Long Kimberly R , Rbaibi Youssef , Weisz Ora A
Published: June 2021

The glomerulus and proximal tubule (PT) of the kidney coordinate to prevent the excretion of albumin and other plasma proteins in the urine. Disruption of the glomerular filtration barrier in nephrotic disease overwhelms the endocytic capacity of the PT, leading to proteinuria. Nephrotic levels of albumin are cytotoxic to the PT and trigger cell signaling and adaptive responses. Recent studies have suggested that fatty acids carried by albumin cause mitochondrial oxidative damage that contribute to PT toxicity. However, fundamental questions about the PT response to albumin remain unanswered. Discordant results using different PT cell models, treatment conditions, and outcome measures have further complicated our understanding of how PT cells respond to albumin and its metabolic load in different disease conditions. We used a recently developed cell culture model that recapitulates key in vivo features of PT function to examine how PT cells adapt their endocytic capacity in response to nephrotic levels of albumin. We treated opossum kidney cells cultured under orbital shear stress with physiologic or nephrotic levels of apically‐added Fraction V bovine serum albumin (BSA), fatty acid‐free or palmitate‐loaded BSA, or human serum and monitored the cells for oxidative damage, megalin expression levels, and endocytic capacity. None of these parameters were altered by incubation with fatty acid‐free BSA, however, incubation with 20 mg/mL Fraction V BSA resulted in a rapid and reproducible decrease in endocytic capacity (within 3 h) followed by increased oxidative stress and decreased megalin expression measured after overnight treatment. Palmitate‐loaded BSA and human serum elicited very different profiles of cellular changes (relative to Fraction V BSA and each other) over a 3 – 48 h incubation period. These data suggest that PT cells initiate different adaptive signals in response to the composition of the metabolic load on endocytosed albumin. Our results have implications for the development of therapeutic strategies to mitigate disease‐dependent proteinuric tubular injury.

Support or Funding Information

NIH R01‐DK101484; NIH R01‐DK100357; NIH P30‐DK079307

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating Oligomeric State and Circadian Regulation of CTRP3
Authors: Trogen Greta , Shipley Madison M , peterson Jonathan
Published: June 2021

Adipose tissue secretes many important biologically active proteins called adipokines. A subset of adipokines, called C1q tumor necrosis factor (TNF) related proteins (CTRPs), play a key role in metabolism, inflammation, and cell signaling. C1q TNF Related Protein 3 (CTRP3) increases hepatic fatty acid oxidation, decreases inflammation, and aids in cardiovascular recovery following a myocardial infarction. However, the mechanisms behind CTRP3's protective effects on organ systems are unknown. This exploratory study aims to analyze the circulating oligomeric state of CTRP3 and the circadian regulation of CTRP3 to help understand the role of CTRP3 in preventing disease.

METHODS

The oligomeric state of CTRP3 was analyzed in serum separated by size exclusion filtration collected from non‐fasting high‐fat fed hyperglycemic mice as well as low‐fat fed normoglycemic mice. For analysis of the circadian regulation of CTRP3, serum samples were collected from mice at 4 different times throughout the day (early & late dark cycle and early & late light cycle). Circulating CTRP3 levels were then analyzed by immunoblot analysis.

RESULTS

In both high fat (HF) and low fat (LF) fed mice CTRP3 was found to circulate as oligomers above 300kDa as well as between 100 and 300kDa, with no detectable amount of CTRP3 observed below the 100 kDa cutoff. Interestingly, although there was no difference in the total amount of CTRP3 between the HF and LF fed mice, the proportion of CTRP3 above the 300 kDa cutoff was higher in the HF fed mice whereas the majority of CTRP3 in the LF fed mice was found between 100–300 kDa cutoffs. Additionally, we found that serum CTRP3 levels varied greatly throughout a 24‐hour time‐period within each mouse, but no consensus circadian pattern was observed.

CONCLUSION

In vitro mammalian produced recombinant CTRP3 protein exists as trimer, hexamer, and high molecule weight oligomer. This is the first study to indicate that CTRP3 circulates in different oligomeric states in vivo, and this is also the first study to observe a difference in the oligomeric state of CTRP3 related to metabolic state. Combined these findings indicate that oligomeric state of CTRP3 may be more metabolically relevant than total amount of circulating CTRP3. In addition, our finding of a high variability of CTRP3 within the same mouse at different times throughout the day indicates that is not regulated by circadian rhythms but is susceptible to variability due to some unknown regulatory factor. Combined, these findings have identified novel unknown aspects of CTRP3 which require further research to understand the role of CTRP3 in human health and disease.

Support or Funding Information

NIH‐R15DK105496

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Binding Interactions and Elucidating 3D Structure of Aptamer‐Based Biosensors
Authors: Armstrong Lindsay , Bonham Andrew J
Published: June 2021

Electrochemical DNA biosensors, whose signaling is based on signal change induced by the conformational dynamics of DNA aptamers upon target binding, have found success as sensitive and rapid methods to detect a wide variety of proteins, toxins, antibodies, and heavy metals. However, the precise mechanisms by which these surface‐bound DNA molecules change conformation upon target binding, thus changing the dynamics of an appended redox‐active tag and generating a measurable signal, is poorly understood. Here, we have used gel mobility shift assays and dimethyl sulfate footprinting fragment analysis to investigate 3D structural interactions of DNA aptamer biosensors in the presence and absence of binding targets. These have allowed us to better characterize the individual basepair interactions involved in binding targets, as well as offer clues to the dynamic three‐dimensional folded structures of these biosensors. These results will help inform the field of biosensors and aptamers on strategies for future optimization.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication
Authors: Dangerfield Tyler
Published: June 2021

The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity.

Support or Funding Information

The Welch Foundation (F‐1684)

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 LMAN1 (Ergic‐53) in the trafficking of GABAARs
Authors: Fu Yanlin
Published: June 2021

LMAN1, also called as ERGIC 53, is known as a cargo receptor that gathers mainly in the tubulovesicular cluster, also referred as ER‐to‐Golgi intermediate compartment (ERGIC), and recycles between ER and Golgi in a COPII and COPI dependent pathways (Hauri, Kappeler et al. 2000). Its C terminus contains a diphenylalanine motif that binds to COPII coat and facilitates the anterograde transport and a dilysine motif that binds to COPI coat and mediates the retrograde transport (Khoriaty, Vasievich et al. 2012). LMAN1 recognizes and binds to the high mannose structure on the glycoprotein through its luminal carbohydrate recognition domain (CRD) (Zheng, Liu et al. 2010). LMAN1 is the known specific cargo receptor for factor V and factor VIII, cathepsin C, cathepsin Z and α1‐antitrypsin (Hauri, Kappeler et al. 2000, Zheng, Liu et al. 2010).

Our lab's initial mass spec data shows that LMAN1 is one of the molecules that interact with GABAARs. We hypothesize that LMAN1 is a trafficking factor for GABAARS receptors. We have several new findings: firstly, endogenous LMAN1 interacts with at least α1 subunits in HEK293T cells overexpressed with WT GABAARs. This interaction is not dependent on the glycosylation status of α1 subunits. And the carbohydrate recognition domain (CRD) of LMAN1 is critical in the interaction between α1 subunits and LMAN1. Secondly, knocking‐down of LMAN1 in mouse hypothalamic GnRH neuronal (GT17) cells negatively affected the surface expression level of endogenous α1 and β3 subunits. We conclude that LMAN1 is also a trafficking molecule for multi‐subunits transmembrane GABAARs.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Chemical Compounds that Inhibit the Function of Initiation Factors I and III from Pseudomonas aeruginosa
Authors: Cantu Natalie M , Hu Yanmei , Bullard James M
Published: June 2021

Background

Pseudomonas aeruginosa is a bacterium that is known for its increasing rate of antibiotic resistance. As an essential metabolic process, protein synthesis is a target for the development of new antibiotics. Initiation of protein synthesis is stimulated by three initiation factors (IF), in which two of them (IF1 and IF3) act to facilitate formation of an active 30S initiation complex and are released from the 30S initiation complex as a result of the addition of the 50S ribosomal subunit to form the 70S ribosomal complex.

Methods and Results

We previously developed a protein synthesis system in the form of the aminoacylation/translation (A/T) assay which was used to screen for inhibitors of protein synthesis. This system allows the polyU mRNA dependent synthesis of poly‐Phe peptides and in the presence of low concentrations of IF1 and IF3 the activity is enhanced. Titration of either IF1 or IF3 into the A/T assay results in reduced activity. Treating IF1 and IF3 as inhibitors of activity of the A/T assay allows the IC50 for each to be determined (127 and 10.9 μM, respectively). The effect is additive and addition of both results in increased inhibition of protein synthesis. Holding IF3 at 10 μM, and titrating IF1 into the assay decreases the IC50 of IF1 to 6.6 μM. This assay can be used to detect inhibitory effects of the function of IF1 and IF3. If either IF1 or IF3 is inhibited the activity of the A/T assay is restored. Development of this assay into a screening platform to detect compounds with the ability to inhibit the function of IF1 or IF3 allowed the screening of a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds. A number of inhibitory compounds were confirmed and are currently being characterized for the ability to inhibit both the enzymatic activity (IC50) of the initiation factors and growth (MIC) of a panel of problematic pathogens in culture. They are also being analyzed to determine the potential toxicity to human cell cultures.

Conclusion

IF1 and IF3 at elevated concentrations block the activity of the P. aeruginosa protein synthesis assay. In this format they are being screened to identify chemical compounds that inhibit their function to interact with the 30S initiation complex and thus restore the ability of the A/T assay to carry out protein synthesis. This assay allows us to specifically identify inhibitors of the initiation phase of protein synthesis in P. aeruginosa and may lead to identification of potential broad spectrum antibacterials.

Support or Funding Information

National Institutes of Health (grant number: 1SC3GM098173)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification of Palmitoyl Protein Thioesterase and Acyl Protein Thioesterase for use in In Vitro Depalmitoylation Reactions
Authors: Esoe Dave‐Preston , Erickson Jeffery A , Hamann Michael J
Published: June 2021

Palmitoylation refers to the attachment of palmitate to protein cysteine residues and assists with protein localization to the plasma membrane. Like protein phosphorylation, palmitoylation is often a dynamic process, and it involves the attachment of the palmitate by palmitoyl transferases and removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Currently, the techniques to study palmitoylation are difficult to perform and involve the use of harsh chemical reagents like hydroxylamine (HAM) to depalmitate proteins, often leading to destruction of the proteins being studied. Recombinant, purified PPT/APTs could be used as an alternative reagent to depalmitate proteins and also to investigate their individual specificities in depalmitoylation reactions. To purify APTs and PPTs, the coding sequences of human variants were PCR amplified, cloned and expressed to produce either GST or MBP‐tagged fusion proteins. Expressed proteins have been analyzed using SDS‐PAGE and bands of expected molecular mass have been identified. Enzyme activity is currently being tested with the substrate 4‐methylumbelliferyl‐6‐thiopalmitoyl‐b‐glucoside (MU‐6S‐palm‐bGlc) in a fluorogenic enzyme assay, with the goal of eventually using the fusion proteins in depalmitolyation reactions involving palmitoylated protein substrates.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Degradation of Proteins Extracted from Teeth by Hydrogen Peroxide
Authors: Tadros Jonathan , Patel Feny , Keenan Kelly
Published: June 2021

The active ingredient in whitening strips and professionally done procedures is hydrogen peroxide (H2O2) which damages proteins. Teeth were either untreated or treated, either one or three rounds, with over‐the‐counter whitening strips in artificial saliva, and dialyzed against 10% EDTA pH 9.0 for two weeks. The dialysis fluid contained the non‐collagen protein from the enamel and dentin layers of the teeth. The amount of protein was measured using a Lowry assay and the individual proteins were separated using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) and stained with silver. The amount of protein extracted decreased with H2O2 treatments. The molecular weights (MW) for the proteins on the gel were correlated with the predicted MW for proteins known to be in the tooth and the impact of H2O2 was assessed. In order to demonstrate the effect of H2O2, pure proteins were pre incubated with either it or water for an hour and trypsin was added. Samples were collected, run on SDS PAGE and the impact on MW was assessed. The results show a loss of protein with a decrease in size when the proteins were treated with H2O2. These results suggest that if the active ingredient in whitening treatments can penetrate the enamel, it can damage the non‐collagen proteins in the teeth. This work was supported by Research and Professional Development Grant from Stockton 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.
Neural Stem Cell Fatty Acid Beta‐Oxidation and Autism Spectrum Disease
Authors: Bankaitis Vytas A. , Xie Zhigang
Published: June 2021

Inborn errors of metabolism occur with high incidence in human populations, and inborn deficiencies in fatty acid b‐oxidation (FAO) are clinically associated with neurological deficits such as speech weakness, motor delay, and autism. Using in vivo assays for analyzing neural stem cells (NSCs) in the developing mammalian forebrain, we find that neural stem cell (NSC)‐autonomous deficiencies in long‐chain FAO deplete neural stem cell (NSC) pools from mouse embryonic neocortex. Acute interference with activity of TMLHE (catalyzes the first step in carnitine biosynthesis), or with CPT1A (the rate‐limiting enzyme for carnitine‐dependent transport of long‐chain fatty acids into mitochondria), results in impaired NSC self‐renewal. Moreover, dorsal forebrain‐specific eviction of the Cpt1a gene resulted in reduced thickness of the murine neocortex at birth. Importantly, NSC deficits induced by TMLHE insufficiencies are rescued by exogenous carnitine in both cultured explants and by supplementation of the maternal diet. Taken together, the data argue for a direct role for FAO in controlling NSC self‐renewal vs differentiation in mammalian embryonic brain. The data further argue that deranged stem cell homeostasis is a significant mechanism for linking IEMs with human cognitive disorders such as autism. The implications of these findings for new strategies for management of autism risk will be discussed.

Support or Funding Information

This work was supported by the Robert A. Welch Foundation Award BE0017 and grant RO1GM112591 from the National Institute of Health

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiolipin Modulation in the Mammalian Heart
Authors: Bick Diane , Holmes Michael , Sparagna Genevieve
Published: June 2021

Cardiolipin (CL) is a phospholipid found in the inner mitochondrial membrane. It is essential for the optimal function of many of the protein components of the mitochondrial electron transport chain, metabolism and programmed cell death. CL has four fatty acyl chains. In the normal adult mammalian heart these fatty acyl chains are predominantly linoleate (18:2), making the dominant species tetralinoleoyl CL ((18:2)4CL). Research indicates that the composition of the CL acyl chains is altered in disease states associated with mitochondrial dysfunction like heart disease, ischemia and oxidative stress. The specificity of the CL side chain composition is not a result of specificity in CL biosynthesis. Specificity is determined by a remodeling process by which is either deacylation/reacylation or transacylated by enzymes using other phospholipids as acyl donors.

We hypothesized that observed changes in cardiac cardiolipin levels may be the result of altered expression of enzymes in involved in the synthesis and/or remodeling of cardiolipin. Quantitative RT‐PCR analysis of several of these genes indicates a significant change in expression as a result of changes in diet and induced heart failure, conditions where changes in CL composition are documented. Supported by NIH HL072770 to DB
Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase‐II
Authors: Miyamoto Shigeki , Murphy Anne N. , Brown Joan Heller
Published: June 2021

Akt activation supports survival of cardiomyocytes against cell death through opening of the mitochondrial permeability transition pore (PT‐pore). Mitochondrial depolarization induced by H202 is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect is observed even when cardiomyocytes treated with LIF are permeabilized and mitochondrial depolarization is elicited by elevating Ca2+. Cell fractionation studies demonstrate that LIF treatment increases both total and phosphorylated Akt in the mitochondrial fraction and that mitochondrial Akt associates with hexokinase‐II (HK‐II). HK‐II contains consensus sequences for phosphorylation by Akt and LIF treatment induces PI3K‐ and Akt‐dependent HK‐II phosphorylation. Addition of recombinant active Akt to adult mouse heart mitochondria directly stimulates phosphorylation of HK‐II and concomitantly inhibits the ability of Ca2+ to induce cytochrome‐c release; this effect of Akt is prevented when HK‐II is dissociated from mitochondria by incubation with glucose 6‐phosphate or HK‐II dissociating peptide. Finally LIF stabilizes HK‐II association with mitochondria and requires mitochondrial HK‐II to mediate cardiomyocyte protection. We conclude that Akt protects against oxidant or Ca2+‐stimulated PT‐pore opening through a direct effect at the mitochondria mediated via phosphorylation of mitochondrial HK‐II.

AHA Beginning Grant in Aide (Shigeki Miyamoto); NIH PPG 82521
Under the influence: Proof‐of‐concept chemical probe discovery in academia
Authors: Rosen Hugh
Published: June 2021

The Scripps Research Institute Molecular Screening Center is a Molecular Libraries Screening Center Network (MLSCN) facility that spans the Scripps campuses in La Jolla, California and Jupiter in Palm Beach County, Florida. Scripps has brought together an integrated combination of infrastructure, people and technologies that support the identification of proof‐of‐concept small molecules in the academic setting. By this we mean chemical probes of adequate potency, selectivity, physical properties and stability to show robust activities in cell‐based assays and in vivo, allowing pre‐competitive advancement to fields of breaking biology. Chemical probes developed for POC studies the sphingolipid receptors in physiology and pathology will be presented.
Emergence of reversible phosphorylation in the mitochondria
Authors: Rardin Matthew , Wiley Sandra E. , Dixon Jack E.
Published: June 2021

Reversible phosphorylation within mitochondria is a rapidly emerging topic in cell signaling. In order to determine which phosphatases are involved in this process we screened several protein tyrosine phosphatases (PTP) as potential candidates for mitochondrial localization. Of those examined, two of the atypical Dual‐Specificity Phosphatases (DSP) appeared to localize to mitochondria. DSP18 and DSP21 are catalytically active phosphatases. While DSP18 is widely expressed in rat tissues, DSP21 is selectively expressed in testes. We demonstrate that DSP18 and DSP21 are targeted to the mitochondria by unique internal localization signals. Electron microscopy and subfractionation of rat kidney mitochondria revealed endogenous DSP18 as being located within the mitochondria. Subsequent treatment of mitochondrial membranes established that DSP18 is located in the intermembrane space as a peripheral membrane protein of the inner membrane. In contrast, subfractionation of rat testes mitochondria revealed DSP21 is localized to the matrix as a peripheral membrane protein of the inner membrane. This work, which rigorously demonstrates the unique location of two highly similar DSPs on either side of the inner membrane, provides further evidence that reversible phosphorylation is an inherent aspect of mitochondrial function.