bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Dynamics Couple Substrate Recognition with Allosteric Domain Communication in Nonribosomal Peptide Synthetases
Authors: Frueh Dominique P , Mishra Subrata H , Kancherla Aswani K , Marincin Kenneth A , Nerli Santrupti , Sgourakis Nikolaos G , Dowling Daniel
Published: June 2021

Timely control of protein interactions is central to modulate biological activity such as gene activation, cell signaling or metabolism. Here, events such as post‐translational modifications and substrate or protein binding affect proteins so as to govern their interactions with partner proteins and repress or promote activity. In spite of progress in identifying protagonists of molecular communication and predicting the outcome of molecular events, it has proven more challenging to determine how molecular responses within proteins translate into responses between proteins, particularly when remote sites are involved within proteins. Such challenges are inherent to understanding microbial, multi‐domain enzymatic systems called nonribosomal peptide synthetases (NRPSs). NRPSs employ contiguous repeats of domains to covalently tether substrates onto carrier proteins (CP) and assemble them into complex products through intervening condensation or cyclization domains. The pharmaceutical properties of NRPS products (e.g. antibiotics) have spurred attempts at engineering NRPSs, but dynamics between domains have hampered progress, as it remains unclear whether and how the many catalytic steps required for synthesis relate with transient domain communication. Using NMR, we demonstrate that large scale structural dynamics within a 52 kDa cyclization domain sense substrates tethered to partner CPs to not only promote binding between both partner domains but also to remodel both the active site and a remote binding site for downstream partners, thus revealing a path for allosteric communication involving three partner domains. We provide a 3D structural description of the enzyme dynamics and demonstrate that mutations that impede catalysis injure the same dynamic allosteric network, suggesting that catalysis efficiency may be streamlined with productive domain communication and substrate recognition through dynamic responses. Our results reveal and describe at the molecular level a substrate driven allosteric response in NRPSs, and they exemplify how structural dynamics within proteins can couple substrate recognition with active site remodeling and promotion of both local and remote protein communication.

Support or Funding Information

NIH‐R01GM104257

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐activity relationships of fragment‐based inhibitors of Trichomonas vaginalis adenosine/guanosine preferring nucleoside ribohydrolase
Authors: Emilcar Melissa , Leonardo Angelica , Li Tian , Dulloo Zaafir , Kabir Nafeesathul Hanan , Gil Ari , VanAlstine‐Parris Melissa A. , Stockman Brian J.
Published: June 2021

Trichomoniasis is a sexually transmitted infection caused by the parasite, Trichomonas vaginalis. Infections are currently treated with 5‐nitroimidazole drugs, such as metronidazole and tinidazole. However, strains of the parasite with resistance to these drugs have emerged, indicating the need for new treatments with novel mechanisms. Since the parasite is unable to perform de novo synthesis of nucleobases, it must obtain them from its host using salvage pathway enzymes including adenosine/guanosine preferring nucleoside ribohydrolase (AGNH), an essential enzyme involved in the pyrimidine salvage pathway. We previously used fragment screening to identify ligand‐efficient fragment inhibitors of AGNH. Medicinal chemistry efforts were then focused on several fragment scaffolds including benzimidazoles and phenyl pyridines. IC50 values were determined using the same 1H NMR‐based activity assays as the fragment screens. The resulting structure‐activity relationships suggest that the fragment scaffolds interact primarily with the nucleobase regions of the active site rather than the ribose pocket. Collectively, the data define emerging structure‐activity relationships that suggest likely vectors and chemical modifications for improving inhibition potency while maintaining ligand efficiency. The data establishes a platform for ongoing medicinal chemistry development of compounds with nM potency that will provide the tools for in vitro target validation against both 5‐nitroimidazole‐sensitive and 5‐nitroimidazole‐resistant T. vaginalis strains.

Support or Funding Information

Research was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI128585 to BJS and MAVP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes 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.
Translation of 1H and 19F NMR‐based activity assays to in vitro characterization of nucleoside hydrolase activity in cell extracts and whole cells
Authors: Mahmood Maham , Canestrari Madison , Thuilot Samantha , Stockman Brian J.
Published: June 2021

Trichomoniasis, the most prevalent non‐viral sexually transmitted infection in the world, is caused by the parasitic protozoan Trichomonas vaginalis. Studies have indicated an association between T. vaginalis and a higher susceptibility to various other infections including chlamydia, HIV, and syphilis. The parasite has shown increasing resistance to the current treatment of 5‐nitroimidazole drugs such as metronidazole. T. vaginalis is incapable of de novo synthesis of purine and pyrimidine rings, so it must rely on salvage pathway enzymes such as adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH) to scavenge nucleobases. Both enzymes have been screened to identify fragment inhibitors with high ligand efficiencies to use as starting points for drug design. AGNH and UNH can be validated as antitrichomonal targets by demonstrating a correlation between enzyme inhibition and antitrichomonal activity. Escherichia coli cells with endogenous nucleoside ribohydrolase gene expression of rihA (ybeK), rihB (yieK) and rihC (yaaF) were used as a surrogate to develop protocols for observing in vitro enzyme activity. A 1H NMR‐based activity assay for AGNH using adenosine as the substrate and an 19F NMR‐based activity assay for UNH using 5‐fluorouridine as the substrate were previously developed for compound screening. These assays proved remarkably robust for observing nucleoside hydrolase activity in cell extracts and in whole cells. Signals for substrate and product are clearly distinguishable from background signals arising from the cell contents. Reactions have been shown to be cell‐dependent, indicating that both enzymes are intracellular and that substrate can rapidly enter the cells. Similar experiments are now in progress using T. vaginalis strains to validate the molecular mechanisms of inhibition.

Support or Funding Information

Research was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI128585 to BJS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes 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.
NITROGEN STARVATION‐INDUCED PHOSPHORYLATION OF GPA2 ACTS AS A MOLECULAR SWITCH IN PROMOTING SPORULATION
Authors: Huang Shan , Benben Alex , Green Robert , Keaveney Shannon , Wang Yuqi
Published: June 2021

G proteins are important molecular switches that facilitate the transmission of a variety of signals from outside to the inside of the cells. G proteins are highly conserved, which allows studying their mechanisms of regulation in model organisms such as the budding yeast Saccharomyces cerevisiae. Gpa2 is a yeast G alpha protein that functions in the nutrient signaling pathway. Through the usage of phos‐tag, we found that Gpa2 protein undergoes phosphorylation and its level of phosphorylation is markedly increased upon nitrogen starvation. Gpa2 interacts with glycogen synthase kinase (GSK), and disrupting GSK activity diminishes the level of Gpa2 phosphorylation. Functionally, phosphorylation enhances plasma membrane localization of Gpa2 and promotes sporulation in response to nutrient deprivation condition. Together, our study reveals a mechanism by which GSK‐and nutrient‐dependent phosphorylation of Gpa2 serves as a molecular switch that regulates sporulation in yeast.

Support or Funding Information

We thank Drs. Claudina Rodrigues‐Pousada, Henrik G. Dohlman, and Jeremy Thorner for generously providing strains, plasmids and antibodies.

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 Ellis van Creveld protein 1 binds and hydrolyzes GTP to a mixture of GMP and GDP and possesses the characteristics of large GTPases
Authors: ODUNUGA ODUTAYO O , NGUYEN GIANG T
Published: June 2021

Mutations in the genes that code for EVC1 and EVC2 proteins cause Ellis van Creveld (EvC) syndrome and its milder form, Weyers acrofacial dysostosis (WAD). EVC1 physically binds to EVC2 and together, they are tethered to the base of the primary cilium by the EFCAB7/IQCE protein complex. This multi‐protein assembly positively regulates the Sonic and Indian hedgehog (Hh) signaling pathways by promoting downstream processes after activation of the receptor, smoothened (smo), by the hedgehog proteins. Bioinformatic analysis revealed the presence of several structural motifs in EVC1 sequences including P‐loop, leucine zipper, transmembrane and nuclear localization signal. Using a fragment of EVC1 from mice and humans that contains P‐loop and leucine zipper regions, we present experimental evidence to show that EVC1 protein possesses a high intrinsic GTPase activity with a kcat of ~10 min−1. In addition, dimerization of EVC1 via its leucine zipper causes the protein to hydrolyze GTP to a mixture of GDP and GMP at unequal ratios. The protein binds GTP at milli to micro molar concentrations, indicating low substrate affinity. These kinetic parameters are similar to those displayed by large GTPases such as Dynamin. We propose that the GTP‐hydrolyzing activity of EVC1 protein is an important mechanistic step in the regulation of the hedgehog signaling pathways.

Support or Funding Information

Welch Foundation Grant # AN‐0008

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitoylation and Phosphorylation–Impact on NHE1 Transport
Authors: Bakker Clare , Hovde Moriah , Foster James , Wallert Mark , Provost Joseph
Published: June 2021

The sodium hydrogen exchanged isoform 1 (NHE1) has two key roles. First is to regulate intracellular pH (pHi) by exchanging an intracellular proton for an extracellular sodium. The extended intracellular carboxyl terminus binds several proteins that regulate NHE1 transport and act as a scaffold for proteins on the leading edge of a migrating cell. The regulation and interaction on the C‐terminus (amino acids 503–815) is complex and not fully understood. Several regulatory events are linked to multiple phosphorylation sites by kinases including AKT, ERK1/2, ROCK and several other protein kinases. Interacting proteins include isoforms 1 and 2 of calcineurin B homologous protein (CHP1 and CHP2) and 14‐3‐3. We have recently discovered that NHE1 is also reversibly lapidated. Palmitoylation is a unique post‐translational fatty acid modification (S‐acetylation) in that it is reversible and aids in binding specificity, folding, aggregation and clustering. The influence of both phosphorylation and palmitoylationsites upon NHE1 activity could influence both intracellular pH homeostasis and cellular behavior. To determine how both post translational modifications regulate NHE1 transport activity, we have determined the change in intracellular pH (pHi) after exposure to a known activator of NHE1 transport, lysophosphatidic acid (LPA), in the absence and presence of 2‐bromopalmitate (2BP) and specific inhibitors of protein kinases known to phosphorylate NHE1. Preincubation of cells with 0.1 mM 2BP inhibits NHE1, resulting in a significant reduction in LPA‐stimulated pHi. Inhibition of MEK, ERK1/2, AKT and ROCK all inhibit NHE1 transport by >50%. Assessing steady state and transport rate after acid load recovery of cells, with combinations of palmitoylation and protein kinase inhibitors, will help elucidate the relationship between the two post translational events in fibroblasts.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High exogenous NO inhibits proliferation and induces apoptosis by targeting PI3K/AKT/mTOR and MEK/ERK pathways in hepatocellular carcinoma cells
Authors: Liu Ling , Chen Jingjing , Cao Mengyao , Huang Zile , Li Ruifang , Wang Jiangang
Published: June 2021

PABA/NO (O2‐ {2,4‐dinitro‐5‐[4‐(Nmethylamino)benzoyloxy]phenyl} 1‐(N,N‐dimethylamino) diazen‐1‐ium‐1,2‐diolate) has N‐methyl‐p‐ aminobenzoic acid bound via its carboxyl oxygen as a 5‐substituent on the 2,4‐dinitrophenyl ring, a diazeniumdiolate‐based NO‐donor prodrug through releasing exogenous nitric oxide at high concentrations to induce apoptosis in many tumor cell lines. In the present study, the effects of PABA/NO on hepatocellular carcinoma proliferation and apoptosis induction were investigated both in vitro and in vivo experiments. PABA/NO dramatically inhibited the cell growth and significantly induced apoptosis in a concentration‐dependent manner, accompanied by down‐regulation of Bcl‐2, up‐regulation of Bax, and activation of cleaved‐caspase‐9 and cleaved‐caspase‐3. PABA/NO attenuated the PI3K/AKT/mTOR pathway through decreasing phosphorylation of PI3K p85 at Tyr458, AKT at Ser473, mTOR at Ser2448 and p70S6K at Thr389 in SMMC7721 cells. In addition, LY294002 as a specific PI3 kinase inhibitor can aggravate PABA/NO‐induced cell apoptosis. Furthermore, PABA/NO repressed the MEK/ERK pathway through decrease phosphorylation of MEK at Ser217/221, ERK at Thr202/Tyr204, and p90RSK at Ser380 in PABA/NO‐treated cells. ERK inhibitor U0126 could greatly intensify the apoptotic effects on PABA/NO‐treated cells compared to PABA/NO single treatment. Additionally, H22 tumor‐bearing mice experiments demonstrated that PABA/NO exerted good antitumor effects via reducing tumor volume, tumor weight and decreasing the expression of CD34. Furthermore, PABA/NO treatment strongly inhibited the phosphorylation of PI3K/AKT/mTOR and MEK/ERK signaling pathways in H22 hepatocellular carcinoma tissues. Collectively, these data clearly demonstrated PABA/NO ‐induced apoptosis through inhibition of PI3K/Akt/mTOR and MEK/ERK pathway in hepatocellular carcinoma cells.

Support or Funding Information

This work was financially supported by the National Natural Science Foundation of China (No.81502627) and the Young Backbone Teachers Assistance Scheme of Henan Province Colleges and Universities (No.2016GGJS‐065)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compartment‐specific neuron activity‐dependent redox dynamics
Authors: Tantama Mathew
Published: June 2021

Reactive oxygen species (ROS), redox active proteins, and molecules such as glutathione are central to the propagation of redox signals and oxidative stress. Propagation can occur between cells in the tissue environment, and it can also occur across organelles and cellular compartments within a single cell. However, simultaneous measurements of compartment‐specific redox dynamics at the single‐cell level have remained a challenge. We are therefore developing genetically‐encoded red fluorescent redox sensors that can be used together with currently available green fluorescent redox sensors in multiplexed live‐cell imaging experiments. Our protein engineering strategy employs Förster‐type resonance energy transfer (FRET) between the redox‐sensitive green fluorescent protein (roGFP) as the energy donor tethered to various red fluorescent proteins acceptors. This FRET‐based design generates a spectral relay which will ideally shift the green emission from roGFP to orange and red emission wavelengths. We have validated a set of second‐generation “rogRFP” sensors, and we have measured activity‐dependent redox changes in the cytosol and mitochondria of individual neurons that differ in compartment‐specific phenotypes. Our experiments with cultured primary mouse hippocampal neurons demonstrates that our sensors will be effective tools for studying compartment‐specific redox dynamics and signaling.

Support or Funding Information

NIH/NINDS R21 NS106319

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Bacterial Ribosome 30S and 50S Subunit Recognition and Modification by M. tuberculosis TlyA
Authors: Laughlin Zane Timothy , Conn Graeme
Published: June 2021

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.

Support or Funding Information

This work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Splicing of Perilipin 5
Authors: Jordan Spencer P , Tansey John T
Published: June 2021

Included among the most common diseases in the U.S., are metabolic diseases such as type II diabetes mellitus and nonalcoholic fatty liver disease. Understanding these diseases requires an understanding of neutral lipid storage and metabolism. Perilipins are a family of five conserved proteins that function in the regulation of neutral lipid metabolism. Of the perilipins, perilipin 5 is the most recently discovered and studied protein in the family. Perilipin 5 is a major regulator of lipolysis and is expressed in highly oxidative tissue such as heart, oxidative muscle, and fasting liver. It is primarily found localized to the surfaces of lipid storage droplets, but it can also be found in the cytosol. Alternative splicing has been observed in perilipins 1 and 3, resulting in several other proteins with various functions as these proteins lack domains which have the functions of the full‐length protein. We have identified a truncated form of perilipin 5 we have termed perilipin 5b using western blotting. Reverse transcriptase PCR indicate that this truncation results from a read through of one of several different introns, most likely the intron between introns 7 and 8. cDNA BLAST searches also provide evidence of this splice variant. Collectively, these data suggest that like other members of the family, perilipin 5 undergoes differential splicing resulting in shortened protein products with short, unique carboxy terminal tails.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
REDD1 upregulation via AP‐1 alleviates oxidative stress‐mediated liver injury
Authors: Ki Sung Hwan , Cho Sam Seok , Kim Jae Kwang , Cho Il Je
Published: June 2021

Regulated in development and DNA damage responses 1 (REDD1) is an inducible gene in response to various stresses, which functions as a negative regulator of the mammalian target of rapamycin protein kinase in complex 1. In the present study, we identified the role of REDD1 under the oxidative stress‐mediated hepatocyte injury and its regulatory mechanism. REDD1 protein was increased in H2O2 or tert‐butylhydroperoxide (t‐BHP)‐treated hepatocytes· H2O2 also elevated REDD1 mRNA levels. This event was inhibited by antioxidants such as diphenyleneiodonium chloride, N‐acetyl‐L‐cysteine, or butylated hydroxy anisole. Interestingly, we found that H2O2‐mediated REDD1 induction was transcriptionally regulated by activator protein‐1 (AP‐1), and that overexpression of c‐Jun increased REDD1 protein levels and REDD1 promoter‐driven luciferase activity. Deletion of the putative AP‐1 binding site in proximal region of the human REDD1 promoter significantly abolished REDD1 transactivation by c‐Jun. A NF‐E2‐related factor 2 activator, tert‐butylhydroquinone treatment also elevated REDD1 levels, but it was independent on NF‐E2‐related factor 2 activation. Furthermore, we observed that REDD1 overexpression attenuated H2O2 or t‐BHP‐derived reactive oxygen species formation as well as cytotoxicity. Conversely, siRNA against REDD1 aggravated t‐BHP‐induced reactive oxygen species generation and cell death. In addition, we showed that REDD1 was induced by in vitro or in vivo ischemia/reperfusion model. Our results demonstrate that REDD1 induction by oxidative stress is mainly transcriptionally regulated by AP‐1, and protects oxidative stress‐mediated hepatocyte injury. These findings suggest REDD1 as a novel molecule that reduced susceptibility to oxidant‐induced liver injury.

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 high‐throughput microfluidic microphysiological system (PREDICT‐96) to recapitulate hepatocyte function in dynamic flow conditions
Authors: Tan Kelly , Keegan Philip , Rogers Miles , Lu Mingjian , Gosset James , Charest Joseph , Bale Shyam Sundhar
Published: June 2021

Microphysiological systems (MPSs) are dynamic cell culture platforms that incorporate mechanical and chemical cues to recapitulate organ‐specific functions and architecture. Recent advances in microfabrication technologies and processes have enabled the development of enhanced in vitro models that capture human physiology more accurately while increasing throughput and reducing material‐based technical artifacts. A common candidate for MPS development is liver due to its crucial role in drug metabolism, inflammation and human disease. While several liver‐on‐chip models exist, few studies have combined long term culture with dynamic inputs, such as media flow, in a high‐throughput format. Herein, we have developed an oxygen permeable, high‐throughput (96‐well form factor), thermoplastic MPS (PREDICT‐96 array) integrated with an on‐board, ultra‐low volume, recirculating pumping system (PREDICT‐96 pump) to facilitate media flow in the system; collectively, these technologies comprise the PREDICT‐96 platform. To demonstrate usability of the PREDICT‐96 array, we introduce PHHs using standard handling procedures for multi‐well plates and maintain viable culture for up to 14 days. By incorporating media flow to mimic in vivo mass transport, we observe a robust increase in metabolic and secretory functions of PHHs when compared to static culture. The PHHs demonstrate reproducible baseline metabolic activity and secretion markers which underscores the utility of the system for drug screening applications. Furthermore, long‐term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and resolve cellular responses to chronic stimuli over time. The low‐volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi‐cellular co‐cultures and integration with high‐throughput analytical tools.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Platelet‐activating factor‐receptor dependent pathway mediates gemcitabine chemotherapy‐induced microvesicle particles release in pancreatic cancer cells
Authors: Thyagarajan Anita , Kadam Sayali M , Liu Langni , Kelly Lisa E , Rapp Christine M , Chen Yanfang , Sahu Ravi P
Published: June 2021

Studies including ours have shown that pro‐oxidative stressors such as chemotherapeutic agents generate oxidized lipids with platelet‐activating factor (PAF) agonistic activity. These PAF‐agonists bind with and activate the widely‐expressed G‐protein coupled, PAF‐receptor (PAF‐R), implicated in various pathophysiological diseases including cancer. Notably, recent reports from our group have demonstrated that these PAF‐agonists are transported extracellularly via microvesicle particles (MVP). While the role of PAF‐R in mediating chemotherapy effects has been documented, its significance in chemotherapy‐induced MVP release in pancreatic cancer model has not been studied. To that end, the current studies determined the functional significance of PAF‐R in clinically relevant gemcitabine chemotherapy‐induced MVP release in human pancreatic cancer cells. Our studies using PAF‐R‐expressing (PANC‐1), and deficient (Hs766T) cells demonstrate that gemcitabine induces MVP release in a PAF‐R‐dependent manner. Blocking of PAF‐R via PAF‐R antagonist or inhibition of MVP biogenesis via acid sphingomyelinase (aSMase) enzyme inhibitor, significantly attenuated gemcitabine‐induced MVP release from PANC‐1 cells, however, exerted no effects in Hs766T cells. Importantly, lipid extracts from MVP, isolated from gemcitabine‐treated PANC‐1 cells, contained appreciable amounts of PAF‐agonists. This was measured by a well‐defined and specific approach of accessing PAF‐agonists generation via interleukin‐8 (IL‐8) secretion from PAF‐R‐expressing human epidermoid KBP cells, and not from PAF‐R‐deficient KBM cells. These results indicate that gemcitabine‐generated PAF‐agonists are transported extracellularly via MVP. Mechanistically, pretreatment with extracellular signal‐regulated kinase (ERK1/2) or p38 inhibitors, significantly abrogated gemcitabine‐induced MVP release. These findings indicate the involvement of mitogen‐activated protein kinase (MAPK) pathway in PAF‐R‐dependent gemcitabine‐induced MVP release. In sum, these studies provide the significance of PAF‐R in gemcitabine‐induced MVP release as well as the rationale of evaluating PAF‐R targeting agents with gemcitabine or gemcitabine‐based chemotherapy approaches against pancreatic cancer.

Support or Funding Information

The financial support from the NIH K22 [ES023850] and Elsa U. Pardee Foundation [670645] grants are greatly acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nutritional Ketosis and Fasting: Metabolic Inferences for Mitochondrial Function in Human Health
Authors: Hernandez Melody M. , Einstein George P. , Tulp Orien L.
Published: June 2021

Cellular communication between healthy and sick cells during ketosis state is dynamic process. Converting our source of energy from glucose to fat is essential to morph sick cells into healthy cells. Changing our culture and environment from processed foods and toxins returning to our embryonic human; free of acquired diseases and disorders from genetic modification. The aim of this research is to free our society of the illnesses which become more and more prevalent with our daily contaminations. The research question is answered through an experiment which includes a questionnaire and lab results with case to case analysis. Achieving ketosis protects the healthy cells by regenerating our mitochondria with the appropriate fuel; which is responsible for the debilitation and apoptosis of the sick cells eliminating disease manifestations. Regenerating important signaling molecules helping regulate the expression of genes. The methodology of this research is achieving ketosis the dynamic metabolic state through different methods. One is through the fruit and vegetable diet and the other is through the ketogenic diet. Both achieve a level of ketosis in which allows the body to convert free fatty acids into ketone bodies to be used as the proper fuel for mitochondria to replenish the human body. The participants of this research are of all genders, backgrounds, and have individual health concerns and disorders with various goals for their outcome during the experiment. The results of the experiment showed overall improvement of health and wellness; some symptoms remedied while others ameliorated. Some challenges and modifications were made for children, pregnancy and severe illness.

Support or Funding Information

Support by Institutional Resources of USAT Montserrat, and the Einstein Medical Institute, NPB, FL

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 Effect of Chronic Contractile Activity and Retinoic Acid on Mitochondrial Turnover in C2C12 Myotubes
Authors: Erlich Avigail Tamar , Dovijarski Nemanja , Hood David A.
Published: June 2021

Mitochondrial turnover is necessary for the optimization and maintenance of a healthy pool of functional organelles, able to meet the metabolic needs of the muscle cell. Exercise is known to improve mitochondrial turnover by activating both mitochondrial biogenesis and mitochondrial‐specific autophagy, known as mitophagy. Moreover, research in adipocytes and liver tissue has shown the potential of the dietary supplement retinoic acid to activate mitochondrial biogenesis as well. Retinoids have also been shown to be effective in activating autophagy in cancer cells. However, the combined effect of retinoic acid isomers and exercise in facilitating mitochondrial turnover has not been described. The purpose of this project was to evaluate the effect of retinoic acid (RA) isomers combined with exercise on muscle mitochondrial turnover. Mouse C2C12 myoblasts were plated on 6‐well plates and allowed to differentiate into myotubes. Myotubes were then treated with either vehicle (DMSO), or the retinoic acid (RA) isomers 9‐cis RA or All‐trans RA (ATRA) in the absence or presence of electrical stimulation to elicit Chronic Contractile Activity (CCA; 4days, 3hrs/day). Sample collection took place 21h after the fourth day. Mitochondrial biogenesis was assessed by examining the protein content of PGC‐1α, as well as nuclear and mitochondrially‐encoded mitochondrial proteins. CCA led to a 3‐fold increase in COX subunit IV expression, as well as a 2‐fold increase in PGC‐1α. The mtDNA‐encoded COX subunit I was modestly changed by CCA, but increased 2‐fold in response to 9cis‐RA or ATRA treatment. These RA isomers also increased PGC‐1α by 3‐fold, but had no significant impact on COX IV expression. When RA isomers were combined with CCA, no additive or synergistic effects were observed. To evaluate mitochondrial degradation, we examined several lysosomal and mitophagy proteins. While autophagy proteins Beclin and p62 showed no changes with CCA or RA treatment, the LC3II to LC3I ratio was reduced 2‐fold with CCA, suggesting an enhanced clearance of autophagosomes. RA isomers had no effect on this ratio. The lysosomal fusion protein Lamp1 was increased 1.8‐fold by ATRA, but not further by CCA. Our data suggest that while RA isomers and CCA both promote mitochondrial biogenesis, RA appears to target mitochondrially‐encoded proteins, while CCA appears to have greater effects on the expression of target nuclear‐encoded proteins. CCA also may serve as a stimulus for the clearance of autophagosomes containing damaged cargo. The lack of synergy among these pathways suggests that they operate independently to stimulate mitochondrial turnover.

Support or Funding Information

Supported by NSERC, 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.
Month‐long AICAR Treatment Reverses Age‐related mTOR Pathway Hyperactivity
Authors: Wright Nathan A. , Wilcox Shalene H. , Thomson David M.
Published: June 2021

Sarcopenia is a condition defined by a loss of muscle mass with aging. It is associated with changes in many metabolic pathways. One of these is the mechanistic target of rapamycin (mTOR) pathway, which is chronically activated in aged skeletal muscle under basal conditions. This mTOR hyperactivity is thought to deactivate autophagy, leading to accumulation of defective cell machinery thereby potentially contributing to age‐related muscle dysfunction. AMP‐activated protein kinase (AMPK) acts as an inhibitor of the mTOR pathway. AMPK activation is generally suppressed in sarcopenic muscle, which may contribute to the age‐related increase in mTOR activity. The purpose of this study was to determine if chronic AICAR treatment could reverse mTOR hyperactivation in sarcopenic muscle. To do this, young adult (YA; 5 mo.) and old (O; 23 mo.) C57Bl/6 mice were injected with either AICAR (0.3 – 0.5 mg/g body weight) or saline for one month (31 days). Twenty‐four hours after the final AICAR injection, muscle was harvested and phosphorylation of mTOR‐related signaling components was measured. Sestrin2 protein, which promotes AMPK activity, was decreased in O vs. YA muscles, but was not affected by AICAR. Phosphorylation of both p70S6 kinase (S6k) and ribosomal protein S6 (rpS6) was significantly (p ≤ 0.05) elevated in saline‐treated O vs. YA muscles, similar to previous findings. S6k and rpS6 phosphorylation was unaffected by AICAR in YA muscles, but was decreased by AICAR in O muscles. Eukaryotic elongation factor 2 (eEF2) phosphorylation tended to be elevated in O vs. YA saline‐treated muscles, consistent with our previous results in rats. Since increased S6k activity would promote decreased eEF2 phosphorylation, any age‐related increase in eEF2 phosphorylation would be due to other factors besides S6k. In conclusion, chronic AICAR treatment in old mice reverses the age‐related change in resting mTOR pathway signaling that is observed in O skeletal muscle. This suggests that AMPK activation is a potential strategy for rescuing autophagy and other processes adversely affected by mTOR hyperactivation.

Support or Funding Information

This work was supported by an internal Gerontology Program Grant from Brigham Young 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.
Contribution of High Mobility Group Box 1 to Nicotine‐Induced Podocyte Injury
Authors: Boini Krishna M , Patibandla Sai , Singh Gurinder Bir , Puchchakayala Goverdhan , Koka Saisudha
Published: June 2021

High mobility group box 1 protein (HMGB1), a nuclear DNA binding protein is released under pathological conditions and locally act as one of the potent damage‐associated molecular patterns (DAMPs) to produce tissue injury and chronic inflammation. However, it remains unknown whether HMGB1 is implicated in nicotine‐induced podocyte injury. In the present study, we found that nicotine dose‐dependently increased the production of HMGB1 in cultured podocytes, and that HMGB1 binder and inhibitor, glycyrrhizin (Gly) completely blocked its release induced by nicotine. Immunofluorescence and western blot analysis showed that nicotine treatment significantly decreased the podocin expression compared to control cells. However, prior treatment with Gly attenuated the nicotine‐induced podocin reduction. Furthermore, western blot analysis showed that nicotine treatment significantly increased the TLR4 expression compared to control cells but not in Gly treated cells. In addition, prior treatment with TLR4 siRNA transfection significantly attenuated the nicotine‐induced podocin reduction in podocytes. Based on these results, it is concluded that HMGB1 is one of important mediators of nicotine‐induced podocyte injury. HMGB1 may be a therapeutic target for treatment or prevention of glomerulosclerosis associated with smoking.

Support or Funding Information

National Institutes of Health (DK104031)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fructooligosaccharides induce tight junction assembly in intestinal epithelial cells via AMPK activation
Authors: Ariyadamrongkwan Jutharat , Wongkrasant Preedajit , Muanprasat Chatchai
Published: June 2021

Several intestinal and metabolic diseases including inflammatory bowel diseases (IBD), malnutrition, necrotizing enterocolitis and diabetes mellitus (DM) involve impairment of intestinal tight junction assembly. AMP‐activating protein kinase (AMPK) is an energy sensor that enhances tight junction assembly. Fructooligosaccharides (FOS) are well known prebiotics protecting against intestinal and metabolic diseases with unclear mechanisms. This study aimed to investigates effect of FOS on AMPK and tight junction assembly in intestinal epithelial cells (IEC). Using western blot analyses, it was found that FOS with concentrations from 0.001 to 10 mg/ml induced AMPK activation on T84 cells. FOS at concentration of 0.1 mg/ml showed the greatest effect on AMPK activation and it was used in the subsequent experiments. Calcium‐switch assays and fluorescein isothiocyanate (FITC) leakage experiments were then performed. The results revealed that FOS significantly increased percentage of TER at 15 minute, 1 hour, 6 hour, 12 hour and 24 hour post‐treatment. Interestingly, STO‐609, a CaMKKβ inhibitor, suppressed the effect of FOS on increasing TER and reducing FITC leakage. These results indicate that FOS increased tight junction assembly via CaMKKβ‐AMPK activation.

Support or Funding Information

This work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University (BRG5980008)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Capacitance in Biochemical Networks: Sequential Reaction Delays
Authors: Bassingthwaighte James B
Published: June 2021

In an enzymatic reaction network there are four unavoidable, generally neglected, sources of delay: (1) diffusion of substrate S to the enzyme, (2) the rate k1 of binding S to the enzyme E, (3) the retention in the form of the ES complex as S is converted to product P, (4) “volume delay”, the transient delay for concentrations to reach the next steady state after each change in a flux, and (5)flux delay with reversible reactions. Michaelis‐Menten forward flux is the product of the maximal flux, Vmax, times the fractional occupancy of the active site, [S]/(Km +[S]), where Vmax= Etot • k2 , (Etot = enzyme concentration; k2 = rate of product release). This accounts for neither binding/unbinding nor the capacitance of the ES complex. We examine these delays for an unbranched unidirectional reaction sequence A→B→C→D→E, ignoring diffusion delay. The delays are nearly exponential single lags, each with time constant = exchanged mass/flux, e.g. t = ES/(k2 • ES) = 1/k2 . We consider four versions of this reaction set (available at www.physiome.org/Models): #423 forward first order reactions, #424 Michaelis‐Menten forward reaction, #425 M‐M coupled with a correcting time lag of 1/k2 , compensating virtually precisely for the lack of capacitance, and #426, the full enzyme binding and release kinetics. The programs include verification tests, and are open source, human readable differential equations in JSim's MML. Time constants for lags are: for binding substrate, 1/k1; for retention in ES complex 1/k2 ; for the “volume delay”, (Vol ml)/(Clearance in ml/sec) while going from one steady state to the next. A fifth model, #427, “flux reversal”, shows that with simultaneous forward and backward fluxes in an enzyme sequence such as A→B←→C←→D→E, there is increased capacitance or retention in the B←→D block of reactions that is dependent on the relative dissociation constants, Kd's, of the enzyme for binding substrate versus product. Such delays, and resultant flux limitations and increased susceptibility to system instabilities need to be accounted for in characterizing systems especially when branched or feedback‐controlled. Unidirectional M‐M models can be made temporally accurate by adding capacitance‐mimicking lags in series. In the full kinetic models the delays are fully accounted for by equations describing the kinetics directly, including the reversibility delays.

Support or Funding Information

(Supported by NIH/NHLBI U01‐HL122199)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory Effects of Novel Oridonin Analog CYD0617‐2 on Hepatic Stellate Cell Activation through NF‐κB and Stat3 Pathways
Authors: Wang Xiaofu , Cummins Claire , Gu Yanping , Vaishnavi Krishnamurthy , Zhou Jia , Radhakrishnan Ravi
Published: June 2021

Introduction

Inhibition of activated hepatic stellate cell (HSC) proliferation is recognized as a promising therapeutic strategy for liver fibrosis. Previously, we reported that natural compound oridonin exhibits potent anti‐hepatic fibrosis activity, and that NF‐κB and Stat3 signaling contribute to HSC activation. In this study, we determined the anti‐fibrotic effects of a novel oridonin analog CYD0617‐2 in activated HSCs and the underlying mechanism.

Methods

The activated human and rat HSC lines LX‐2 and HSC‐T6 were treated with CYD0617‐2. Cell viability was measured by alamarBlue assay. Apoptosis was determined by Cell death ELISA. Cellular proteins were analyzed with Western blot. Cytokines were determined with ELISA.

Results

CYD0617‐2 treatment significantly inhibited proliferation and induced apoptosis in both cell lines in a dose‐dependent manner approximately 10 fold more potent than the parent compound oridonin. CYD0617‐2 stimulated apoptotic proteins p21, p27, and cleaved‐PARP, while suppressing extracellular matrix proteins fibronectin and laminin. CYD0617‐2 blocked LPS‐induced NF‐κB p65 nuclear translocation and DNA binding activity, prevented LPS‐induced NF‐κB inhibitory protein IκBα phosphorylation and degradation, and suppressed LPS‐stimulated IKKα/β phosphorylation. LPS‐induced NF‐κB cytokines IL‐6, MCP‐1, and IL‐1β were attenuated by CYD0617‐2. The endogenous and LPS‐induced NF‐κB p65 S536 phosphorylation was inhibited by CYD0617‐2. Importantly, NF‐κB chemical inhibitor Bay‐11‐0782 was found to inhibit proliferation and promote apoptosis in both cell lines. Moreover, CYD0617‐2 attenuated Stat3 activation by suppressing pStat3/T705 phosphorylation and target gene c‐myc.

Conclusion

The potent anti‐hepatic fibrogenetic effect of CYD0617‐2 may be through suppression of IKK/IκBα/NF‐κB and Stat3 pathway.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BRCT domains contain an intrinsic post‐translational modification (PTM) recognition code that affects it stability
Authors: Davis Jamaine
Published: June 2021

BRCA1 C‐terminal (BRCT) domains are protein binding modules that relay signals throughout various cellular pathways. The human genome encodes 23 BRCT‐domain containing proteins that are commonly found in tandem and have been characterized as phosphoprotein binding domains with functions throughout the DNA damage response. Emerging evidence suggests these domains bind other post‐translationally modified (PTM) substrates although the details are not defined. Recently, the BRCT domains from BRCA1 were shown to bind an asymmetrically dimethylated arginine (ADMA) p300 substrate to regulate the transcription of the p21 in response to DNA damage. Based on this observation, we hypothesize that the methyl‐specific binding site for the ADMA interaction is distinct from the phospho‐substrate recognition interface. To test this hypothesis, we conducted peptide binding and thermal stability experiments to demonstrate the change in stability upon binding phospho‐versus methylated substrates. Our results show that phosphorylated substrates increase the stability of BRCA1's BRCT domains. Doubly phosphorylated substrates showed the greatest increase in stability while the methylated substrate demonstrated a decrease in stability. To define the BRCT‐ADMA binding interface, we set out to determine the three‐dimensional structure. High‐throughput co‐crystallization of the BRCA1 BRCT protein with a ADMA peptide derived from the p300 substrate identified one condition for crystal growth optimization. In silico modeling of the BRCA1 BRCT structure with the ADMA molecule has highlighted two potential binding sites for the ADMA substrate, which is not conserved throughout the BRCT domain family. Our results indicate that while BRCT domains share a conserved mode of binding phosphorylated substrates, which increases their stability, and their ability to binding methylated substrates may not be conserved. These finding will be critical in understanding how BRCT domains can accommodate binding various modified substrates and regulating signals throughout the DNA damage response.

Support or Funding Information

NCI K22 CA166544‐03

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