bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Human Ether‐a‐go‐go Related Gene (hERG) Potassium Channel by Nedd4 Family Interacting Proteins (Ndfips)
Authors: KANG YUDI , Guo Jun , Yang Tonghua , Li Wentao , Zhang Shetuan 
Published: June 2021

The cardiac electrical disorder long QT syndrome (LQTS) predisposes affected individuals to high risks of ventricular arrhythmias and sudden death. Dysfunction of the human ether‐a‐go‐go related gene (hERG)‐encoded rapidly activating delayed rectifier K+ channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by the anterograde trafficking of newly synthesized channels to and the retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin ligase Nedd4‐2 (neuronal precursor cell expressed, developmentally down‐regulated protein 4 type 2) targets the PY motif of hERG channels to initiate channel degradation. Although both premature and mature hERG channels contain the PY motif, Nedd4‐2 selectively mediates the degradation of mature hERG channels. Here, using whole‐cell patch clamp, Western blot and immunocytochemical analyses, we demonstrate that Nedd4‐2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Ndfip1 and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4‐2 to target mature hERG proteins for degradation during channel trafficking to the plasma membrane. Although Ndfip2 also recruits Nedd4‐2 to the Golgi apparatus to some extent, it mainly recruits Nedd4‐2 to the multivesicular bodies (MVBs), which impedes the degradation of internalized hERG proteins. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.(Supported by the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of Ontario)
Regulation of Lipid Droplet Formation by Group IVA Phospholipase A2
Authors: Balsinde Jesús , Guijas Carlos , Rodríguez Juan , Rubio Julio , Balboa María 
Published: June 2021

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased neutral lipid formation utilizing fatty acids coming from the de novo biosynthetic route. The latter predominates, accounting for 60‐70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2a, pointing out this enzyme as a key regulator of AA‐induced signaling. LD formation in AA‐treated monocytes can also be blocked by the combined inhibition of the mitogen‐activated protein kinase family member p38 and JNK, which correlates with inhibition of cPLA2a activation by phosphorylation. Since cPLA2a has no effect on neutral lipid formation there must be a step in the mechanism of LD biogenesis that is critically dependent on the enzyme, e.g. generation of a positive curvature at the organelle baseline.Supported by Grant SAF2013‐48201‐R from the Spanish Ministry of Economy and Competitivenes
Single Molecule Dynamics of Lipid Bilayer Associated PTEN
Authors: Gericke Arne , Harishchandra Rakesh , Bryant Anne , Ross Alonzo , Neumann Brittany 
Published: June 2021

ObjectiveStudy the dynamics of PTEN associated with laterally heterogeneous lipid bilayers using single molecule microscopy.BackgroundPhosphatase and tensin homologue deleted on chromosome 10 (PTEN), is one of the most frequent genes deleted/mutated in a wide variety of tumors. PTEN acts as an antagonist to PI 3‐kinase signaling, thereby affecting cellular processes such as cell proliferation and survival. PTEN activity is regulated by dynamic shuttling between the cytoplasm and the plasma membrane.MethodsWe use single‐molecule TIRF microscopy and stopped‐flow fluorescence measurements to observe PTEN molecules as they dynamically associate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN lipid binding is investigated using supported lipid bilayers of binary and ternary lipid mixtures of phosphatidylcholine (PC) with physiological relevant levels of phosphatidylserine (PS), phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) and/or phosphatidylinositol (PI).ResultsUsing single molecule trajectories of PTEN molecules we find significant differences in PTEN dynamic behavior when bound to different membrane environments. Interestingly, we determine the existence of three membrane bound states with different diffusion coefficients. To gain insight into the molecular mechanisms of PTEN membrane association, we compared the lipid binding of wt‐PTEN, PTEN‐(Δ1‐15aa) and the recently discovered, PTEN‐L with a 173 aa N‐terminal extension. We find profound differences in the dynamic behavior of these PTEN derivatives at the membrane. We also found that PI may replace PS as the second lipid component to which PTEN binds.
The PHO4 Transcription Factor Regulates Triacylglycerol Metabolism under Low Phosphate Conditions in Saccharomyces cerevisiae
Authors: Yadav Kamlesh , Singh Neelima , Rajasekharan Ram 
Published: June 2021

ObjectiveTo study the transcriptional regulation of lipid metabolism in Saccharomyces cerevisiae under low phosphate (Pi) conditions.Methodology[14C]Acetate labeling, confocal microscopy, gas chromatography, semi quantitative and quantitative PCR. Electromobility shift assay (EMSA) and β‐galactosidase assay. To validate the binding site‐directed mutagenesis was performed. Enzyme kinetics and characterization.ResultsThe expression of PHM8 is high under low phosphate conditions, which encodes a LPA phosphatase and nucleotidase. In an electrophoretic mobility shift assay, Pho4p was shown to bind specifically in PHM8 promoter. Analyses of PHM8 mRNA, LPA phosphatase activity and triacylglycerol levels under low phosphate conditions in phm8Δ showed that the plasmid harbouring mutated promoter along with the PHM8 gene had much lower PHM8 expression than the plasmid harbouring the native promoter with the gene. Enzyme kinetics with the differentially phosphorylated Phm8p using LPA and CMP as substrates showed that LPA is the preferred over CMP under low phosphate conditions. The results indicate that the transcriptional activator Pho4p binds to the PHM8 promoter under low phosphate conditions, activating PHM8 expression and leading to the formation of monoacylglycerol, which is successively acylated by Dga1p to form diacylglycerol and triacylglycerol.
imageConclusionThe phosphate‐mediated transcriptional activation of PHM8 by Pho4p affects triacylglycerol biosynthesis by controlling lysophosphatidic acid and MAG flux.
CD36 Deficiency Reduces Obesity‐Associated Inflammation and Oxidative Stress in Heart
Authors: Hajri Tahar , Gharib Mohamed , Tao Huan , Fungwe Thomas 
Published: June 2021

Mounting evidence shows that obesity and diabetes are associated with structural and functional changes in the heart. CD36 protein is highly expressed in heart and regulates lipid utilization in cardiac myocytes. In this study, we investigate the impact of CD36 expression in obesity‐associated inflammation in heart. Studies were conducted in control lean, obese leptin deficient (LepOb/Ob) and leptin deficient‐CD36 null (LepOb/Ob CD36‐/‐) mice. Histology and immunohistochemisty were conducted to examine heart structure and presence of macrophages. Uptake and oxidation glucose and fatty acids was tested in primary cultures of ventricular myocytes. Finally, the effects of fatty acid treatment on oxidative stress and activity of pro‐inflammatory kinases were examined in isolated cardiac myocytes. In LepOb/Ob, glucose uptake and oxidation in heart was lower than normal weight mice, while cardiac FA oxidation was strongly higher than normal. Silencing CD36 markedly improved insulin sensitivity and glucose uptake in heart, but marked reduced FA oxidation. Moreover, CD36 deficiency reduced macrophage content, oxidative stress markers and expression of pro‐inflammatory cytokines in heart. Chronic treatment with palmitate strongly induced oxidative stress and inflammatory markers in LepOb/Ob cardiac myocytes, but was clearly less effective in LepOb/Ob‐CD36‐/‐ cardiacmyocytes. These results show that CD36 deficiency induces a significant reduction of obesity‐associated oxidative stress and inflammation in heart, in parallel to a drop of lipid infiltration and improved insulin sensitivity
Oxidation of Salicylic Acid and Aspirin by Human Cytochromes P450
Authors: Sedgeman Carl , Bojic Mirza , Guengerich F. 
Published: June 2021

Aspirin (acetylsalicylic acid) is a commonly used analgesic. It is rapidly deacetylated both enzymatically and non‐enzymatically to salicylic acid. Salicylic acid can then be further metabolized by conjugation with glycine or glucuronides or by hydroxylation to form 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and 2,5‐dihydroxybenzoic acid (2,5‐DHBA). This oxidation has been suspected to occur from oxygen radicals via Fenton reactions as well as by cytochrome P450 enzymes. We analyzed the formation of 2,3‐DHBA and 2,5‐DHBA in the presence of human liver microsomes by HPLC with fluorescence detection. We were able to determine that microsomal oxidation was much faster for salicylic acid than aspirin. 2,3‐DHBA was found to be the major oxidative product formed from salicylic acid, whereas aspirin could detect only 2,5‐DHBA. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5‐hydroxylation of salicylic acid. Inhibitor studies were able to confirm that all six of these enzymes played a role in the formation of 2,3‐ and 2,5‐DHBA, as well as found that 2A6 and 2B6 played a role in the hydroxylation to 2,5‐DHBA. It was also shown in the inhibitor studies that P450 2E1 was the major enzyme contributing to the in both 3‐ and 5‐hydroxylations.This work was supported in part by National Institutes of Health grant R37 CA090426.
MD‐2 as the Target of a Novel Small Molecule, L6H21, in the Attenuation of LPS‐induced Inflammatory Response and Sepsis
Authors: Liang Guang , Wang Yi , Chen Gaozhi , Fang Qilu 
Published: June 2021

BackgroundMyeloid differentiation 2 (MD‐2) recognizes LPS, which is required for TLR4 activation, and represents an attractive therapeutic target for severe inflammatory disorders. We previously found that a chalcone derivative, L6H21, could inhibit LPS‐induced overexpression of TNF‐α and IL‐6 in macrophages.MethodsThe binding affinity of L6H21 to MD‐2 protein was analyzed using computer docking, surface plasmon resonance analysis, ELISA, fluorescence measurements, and flow cytometric analysis. The effects of L6H21 on MAPK and NF‐κB signaling were determined using EMSA, fluorescence staining, western blotting, and immunoprecipitation. The anti‐inflammatory effects of L6H21 were confirmed using ELISA and RT‐qPCR in vitro. Finally, the anti‐inflammatory effects of L6H21 in septic C57BL/6 mice were evaluated.ResultsCompound L6H21 was inserted into the hydrophobic region of MD‐2 pocket, forming hydrogen bonds with Arg90 and Tyr102 in the MD‐2 pocket. In vitro, L6H21 subsequently suppressed MAPK phosphorylation, NF‐κB activation, and cytokine expression in macrophages stimulated by LPS. In vivo, L6H21 pretreatment improved survival, protected lung injury, decreased serum and hepatic cytokine level in mice subjected to LPS. In addition, the MD‐2 gene knockout led to universal protection from LPS‐induced septic shock in mice.ConclusionsThis work identified a new chalcone derivative, L6H21, as a potential candidate in the treatment of sepsis; more importantly, the data confirmed that MD‐2 is an important therapeutic target against inflammatory disorders.
Targeted Inhibitors of P‐glycoprotein Overcome Multidrug Resistance in Human Cancer Cell Lines
Authors: Follit Courtney , Brewer Frances , Wise John , Vogel Pia 
Published: June 2021

Multidrug resistance (MDR), the intrinsic or acquired simultaneous resistance to structurally and chemically unrelated drugs, is arguably the largest barrier to the successful chemotherapeutic treatment of cancer. The best studied mechanism of MDR is due to the overexpression of ABC‐transporter member P‐glycoprotein (P‐gp). All ABC‐transporters utilize ATP hydrolysis to actively export substrates out of the cell, but P‐gp is exceptional in that its substrates are diverse enough to include the majority of clinically relevant chemotherapeutics. Researchers have searched for drugs to inhibit P‐gp for decades and largely failed because the majority interacted with the drug‐binding domain of P‐gp and were themselves transported. Using targeted molecular dynamic simulations and massively parallel drug docking studies, we identified novel compounds that inhibit P‐gp by specifically targeting the ATP binding domain of P‐gp. Biochemical and biophysical studies have confirmed ATP hydrolysis is inhibited by these novel compounds. In the studies reported here, we have evaluated these compounds for their ability to restore drug sensitivity to MDR human cancer cell lines that overexpress P‐gp. Effects on other ABC‐transporters capable of conferring the MDR phenotype are being evaluated and lead compounds are undergoing chemical optimization to develop co‐therapeutics capable of reversing MDR in human cancers.This work is supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, SMU Research Council, SMU Dean's Research Council, the SMU Center for Drug Discovery, Design and Delivery, and the Communities Foundation of Texas, Dallas.
Investigating the Use of Targeted Therapies for Triple‐Negative Breast Cancer
Authors: Bindeman Wendy , Redwood Abena , Lukose Benjamin , Piwnica‐Worms Helen 
Published: June 2021

Triple‐negative breast cancer (TNBC) accounts for 15‐20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and resistant to current chemotherapies. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We have evaluated the use of mammosphere cultures as an in vitro system for testing the sensitivity of TNBC tumor cells to chemotherapy and targeted therapy. We hypothesized that mammospheres would display higher levels of chemo‐resistance than adherent cells, due to their ability to enrich for tumor initiating cells (TICs), which have been shown to display chemo‐resistance. However, we reasoned that molecularly targeted therapies should maintain their effect in TICs, since the pathways that are targeted would remain vulnerable. To test these hypotheses, we evaluated the effect of cisplatin (DNA damaging agent, chemotherapy) and LY2606368 (inhibitor of Chk1, targeted therapy) on the viability of TNBC cells. Our data suggest that the TIC population of a tumor may retain its sensitivity to targeted therapy despite resistance to cytotoxic chemotherapy. Further studies are necessary to determine if targeted therapy can impair TIC self‐renewal capacity, to evaluate combination therapies, and to verify these results in vivo.Funding: Komen Foundation; Cancer Prevention & Research Institute of Texas
Pro‐tumorigenic Effects of Hydrogen Sulfide (H2S) on Normal Colonic Fibroblasts (NCF) and Colorectal (CRC) Cancer‐Associated Fibroblasts (CAF)
Authors: Chao Celia , Dikmen Aycan , Coletta Ciro , Pang Lan , Szabo Csaba , Hellmich Mark 
Published: June 2021

IntroductionBidirectional paracrine signaling between cancer cells and fibroblasts in the stroma facilitates tumor progression. We have shown that CRC cells upregulate cystathionine β synthase (CBS), resulting in H2S production, when compared to normal colonic epithelium, and that CRC‐derived H2S increases CRC growth, migration, and tumor angiogenesis. The aims of this study are to determine whether H2S can promote CAF proliferation and migration, and stimulate NCF to develop into a CAF‐like phenotype.MethodsHuman CAF from CRC and NCF were collected under an IRB approved protocol and established in culture. CAF were grown for up to 120h in the presence of vehicle or a slow‐release H2S donor, GYY4137. CAFs were allowed to migrate through transwell chambers toward conditioned media (CM) from HCT116 or normal colon mucosal cells (NCM356). Western blots for NCF and CAF were performed to determine CBS expression levels.ResultsH2S donor GYY4137 (30‐300μM) significantly increased proliferation of CAF, but not NCF. CAF migrate toward HCT116 CM, but not NCM356 CM. CAF migrate significantly less toward CM from HCT116 cells with shRNA‐mediated silencing of CBS or CBS inhibitor aminooxyacetic acid. NCF cells express less basal CBS protein compared to CAF cells. NCF cells can be induced to express CBS enzyme after exposure to HCT116 CM for 48h.ConclusionsH2S enhances CRC CAF cell proliferation and migration. Furthermore, when NCF cells are exposed to CRC CM, the NCF cells induce expression of CBS enzyme. The results support the hypothesis that the gasotransmitter H2S promotes pro‐tumorigenic activities in the CRC microenvironment.
Hypoxia‐induced carbonic anhydrase IX facilitates lactate transport in human breast cancer cells by non‐catalytic interaction
Authors: Becker Holger , Jamali Somayeh , Klier Michael , Barros Luis , McKenna Robert , Deitmer Joachim 
Published: June 2021

The most aggressive and invasive tumor cells, which often reside in hypoxic environments, rely on extensive glycolysis to meet their large demand for energy and biosynthetic precursors. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms that regulate lactate transport in MCF‐7 human breast cancer cells under normoxia and hypoxia. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Measurements of intracellular pH and lactate concentration with the FRET‐based lactate nanosensor laconic show that CAIX augments lactate flux via MCT1 by a non‐catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+‐shuttle His200, functions as a “proton‐collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of MCF‐7 cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.This work was supported by the Research Initiative Membrane Biology, the Stiftung Rheinland‐Pfalz für Innovation, the Graduiertenfördungskommission Rheinland‐Pfalz, and the DFG.
The APP Interactome: Identifying Targets in Alzheimer's Disease
Authors: Glucksman Marc , Philibert Keith , Marr Robert , Norstrom Eric 
Published: June 2021

Alzheimer's disease (AD) is a horrific neurodegenerative disorder affecting the elderly with progressive mental decline and profound effects on memory. Accumulating evidence indicates that the failure to clear the 40‐42 a.a. Aß peptides from the brain is a primary step in the pathology of sporadic AD. We utilized proteomic approaches involving isolation of physiologically relevant macromolecular complexes in vivo of proteins interacting with Amyloid Precursor Protein (APP) or the carboxy terminally located Amyloid IntraCellular Domain (AICD). After in vivo tandem affinity purification in cell culture models, downstream biophysical characterization and mass spectrometry identified individual interactors currently being validated. A new peptidase activity exhibited by metalloendopeptidase, EC 3.4.24.15 (EP24.15) was identified with the ability to degrade Aß at 3 sites and is different from identified activities of neprilysin 1/2 and insulysin. Transthyretin, a carrier protein transporting thyroid hormones was identified and interactions with Aß were quantified in the µM range with ramifications to inhibit Aß multimer formation. Validation has commenced with the target NEEP21, a neuronally expressed single‐pass transmembrane protein was expressed in lentiviral transduced cells and animals to examine the effects on Aß production. Taken together this data suggests that explicating interactions with APP and AICD can have effects on neurotoxic products and may aid in developing effective pharmacological approaches for people afflicted by AD.Support: RFUMS/DePaul Pilot program and NIH OD010662 (MJG) to the Midwest Proteome Center.
Role of Negative Charge on C2 Insert of Nonmuscle Myosin II‐C: Intermittent Structural Oscillation and Aggregation in Neuritogenesis
Authors: Saha Shekhar , Chattoraj Shyamtanu , Halder Debdatta , Goswami Swagata , Bhattacharyya Kankan , Jana Siddhartha 
Published: June 2021

Previous studies reported that inclusion of 41 aa (33 aa in human) insert in loop 2 of mouse nonmuscle myosin (NM) II‐C heavy chain makes the molecule to become independent of its regulatory light chain (RLC) phosphorylation in MgATPase activity and in vitro motility, and to localize in the neurites as puncta form. However, the molecular mechanism behind these unique properties among myosin II family is not known. Here, we show that deletion of the N‐terminal 12 aa, but not the C‐terminal 8 aa or the middle 21 aa of C2 insert, diminishes the fluorescence oscillation property of GFP tagged‐NM II‐C2 molecule in the neurites of neuro‐2a cells as determined by fluorescence lifetime imaging microscopy analysis. Also, we demonstrate that inclusion of 41 aa insert increases the population of extended monomers whereas deletion of N‐terminal 12 aa, middle 21 aa or C‐terminal 8 aa of 41 aa insert increases the folded autoinhibited NM II‐C2 monomers as revealed by fluorescence correlation spectroscopy (FCS) analysis in neuronal cells. Furthermore, FCS analysis reveals that NM II‐Δ12C2‐GFP significantly diffuses very slowly compared with NM II‐Δ8C2‐ or NM II‐Δ21C2‐GFP in the neurites, suggesting that radius (rH) of NM II‐Δ12 C2‐GFP is higher than that of wildtype or mutants Δ21 and Δ8C2‐GFP. These studies suggest that N‐terminal 12 aa (contributor of net charge of C2) plays a significant role in determining the oscillation and aggregation properties of the molecule.Research Support: Department of Science and Technology, India