bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential growth state‐dependent activation of p38MAPK by treatment of endothelial cells with docosahexaenoic acid (DHA)
Authors: Du Youjia , Taylor Carla G , Aukema Harold M , Zahradka Peter 
Published: November 2023

IntroductionEndothelial cells line the luminal surface of blood vessels and play an essential role in regulating vascular function. Endothelial cells quiescence is a primary property of the healthy endothelium, whereas cell proliferation is feature of vessel repair and progression of atherosclerosis. Docosahexaenoic acid (DHA), an omega‐3 fatty acid present in fish oil, is thought to exert an array of beneficial effects on vascular health by modulating endothelial function. However, a comparison of the effects of DHA on growing and confluent endothelial cells, which represent repairing and healthy states, respectively, has never been reported.ObjectiveTo investigate the effects of DHA on viability and proliferation of subconfluent (growing) and confluent (quiescent) human endothelial cells (EA.hy926) in relation to p38 mitogen‐activated protein kinase (MAPK) activation.MethodsEA.hy926 endothelial cells were seeded on Matrigel‐coated plates and grown up to 10 days. Cells were treated with DHA (1–150 μM) for 24 h when they were either subconfluent (4 d after seeding) or confluent (8 d after seeding). Viability and proliferation were assessed. Phosphorylation of p38MAPK and mitogen‐activated protein kinase kinase (MKK3/MKK6) was assessed after treating subconfluent and confluent cells with 125 μM DHA over 8 h.ResultsMaximum cell number and DNA synthesis rate were reached on day 8, and the cells subsequently became quiescent. DHA >125 μM reduced viability of both growing and confluent cells after 24 h, while 40 μM slowed proliferation of both subconfluent and confluent cells. In subconfluent cells, 125 μM DHA activated p38MAPK and MKK3/MKK6 (a known upstream kinase), producing peaks at both 10 min and 4 h. In contrast, phospho‐p38MAPK and phospho‐MKK3/MKK6 peaks were only observed at 10 min in confluent cells.ConclusionsOur study is the first to show a dose‐dependent effect of DHA on both proliferating and confluent endothelial cells with regards to viability and proliferation, as well as a growth state‐dependent activation of p38MAPK. This is highly relevant to how blood vessels respond to a dietary constituent such as DHA in various disease states. Since activation of stress‐stimulated protein p38MAPK, which is closely linked to pathological vascular remodeling, was observed in different growth conditions of cells, further studies to delineate the physiological effects of DHA on p38MAPK signal transduction in endothelial cells are warranted.
Tocopherols and tocotrienols prevent lipoxygenase‐driven phospholipid oxidation in ferroptosis
Authors: Mao Gaowei , Qu Feng , Angeli Jose Pedro , St Croix Claudette , Dar Haider , Tyurin Vladimir , Ritov Vladimir , Kapralov Alexandr , Amoscato Andrew , Anthonymuthu Tamil , Mohammadyani Dariush , Yang Qin , Stockwell Brent , Tyurina Yulia , Conrad Marcus , Bayır Hülya , Kagan Valerian 
Published: November 2023

Ferroptosis, a recently discovered regulated necrotic pathway, has been claimed to include a required stage of lipid peroxidation. However, neither the nature of oxidized lipid species nor their subcellular location and enzymatic generators have been identified. We established that inactivation or depletion of glutathione peroxidase 4 (Gpx4) induced ferroptosis in mouse embryonic fibroblasts (MEFs). Using mitochondria and endoplasmic reticulum (ER) targeted fluorogen‐activating proteins (Mito‐FAP and ER‐FAP), we demonstrated that generation of oxidized lipids predominantly occurred in the ER compartment. We further showed that oxidized arachidonoyl‐ and adrenoyl‐phosphatidylethanol‐amines (but not oxygenated free fatty acids) were the major players in the execution of ferroptosis. Formation of the oxidized phospholipids was predominantly catalyzed by 15‐lipoxygenases (LOX). Vitamin E homologues (tocopherols and tocotrienols) bind with the catalytic site of LOX and suppress ferroptosis, revealing a potential non‐canonical mechanism of action of vitamin E in protection against ferroptosis likely realized independently of radical scavenging. Taken together, these findings decipher molecular mechanisms of ferroptosis and provide new therapeutic targets.Support or Funding InformationUS National Institutes of Health (NS076511 to V.E.K., NS061817 to H.B., and ES020693 to Y.Y.T.), the Human Frontier Science Program (HFSP‐RGP0013/2014), and the Deutsche Forschungsgemeinschaft (CO 291/2‐3 and CO 291/5‐1) to M.C.
Differential effects of lipids on cellular activities of SH2 domain‐containing Proteins
Authors: Singaram Indira , Cho Wonhwa 
Published: November 2023

SH2 (Src Homology 2) domain is a prototypical protein interaction domain that specifically recognizes a phosphorylated tyrosine (pY) motif. Our recent study showed that a large majority of human SH2 domains bind lipids in the plasma membrane (PM) with high affinity using an alternate cationic patch (ACP) that is topologically distinct from the cationic pY‐binding pocket. However, some SH2 domains lacking ACP bind PM lipids, including phosphoinositides, in their pY‐binding pockets. To understand the differential effects of lipids on ACPs and pY pockets of SH2 domains on the cellular activities of their host proteins, we performed computational, biophysical and cellular activity and imaging analyses with various SH2 domain‐containing proteins and their lipid‐binding site mutants. Our results demonstrate highly variable mechanisms by which lipids cellular protein‐protein interactions and modulate signaling activities of these proteins in immune cells.Support or Funding InformationNIH GM68849
Targetting Cysteinyl Leukotriene 2 Receptor as a Therapeutic Target for Tumor Growth and Metastasis: A New Function for an Old Receptor
Authors: Teegala Lakshminarayan Reddy , Duah Ernest , Kondeti Vinay , Al‐Azzam Nosayba Zakariya , Adapala Ravi , Ghebreigziabher Samrawit , Thodeti Charles , Paruchuri Sailaja 
Published: November 2023

Excessive or insufficient angiogenesis can lead to number of diseases including atherosclerosis, diabetic retinopathy, age‐related macular degeneration and cancer. Although long‐standing inflammation secondary to chronic infection or irritation mediated by immune cells has been implicated in cancer progression, the contribution of endothelial cells (EC) in the generation of inflammatory mediators and their effects on angiogenesis is relatively unknown. One of the pro‐inflammatory mediators produced by EC are cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4), and they mediate their effects through two main receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast cancer and melanoma. Tumor microenvironment comprises numerous signaling molecules and pathways that influence angiogenic response leading to increased and aberrant vascularization. Further, angiogenesis is required for the supply of nutrients and oxygen for tumor growth and also serves as a route of tumor cells metastasis. Therefore, we investigated the role of CysLTR in angiogenesis, tumor progression and metastasis, using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO).We observed enhanced CysLT2R expression, and not CysLT1R in wild type (WT) tumors and tumor growth as well as angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in Lewis lung carcinoma (LLC) tumor model. Further, though few in number, tumor vessels in CysLT2RKO mice showed intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Furthermore, we found that activation of CysLT2R, but not CysLT1R increased EC contraction leading to junctional destabilization and permeability in vitro. Importantly, our results show that CysLT2R activates Rho kinase and inhibition of Rho kinase significantly attenuated CysLT2R‐induced EC contraction and permeability. Further, our preliminary results revealed that pericytes express CysLT2R and display calcium influx in response to LTD4. Taken together, our results suggest that CysLT2R promotes leaky blood vessel formation, enhancing tumor growth and metastasis to the lung. Blocking CysLT2R could offer novel CysLT2R‐targeted, therapeutic candidates for the treatment of cancer and other angiogenic disorders.Support or Funding InformationJames Foght Professorship Support, AHA GIA 15GRNT 25670004
Sac1 degrades its lipid substrate PI4P in the ER to maintain a steep electrochemical gradient on donor membranes
Authors: Hammond Gerry , Wills Rachel , Zewe James 
Published: November 2023

PI4P metabolism has been proposed to form the basis of an “inositide motive force”, whereby a steep electrochemical gradient of this lipid that is high in a target membrane and low in the endoplasmic reticulum (ER) drives counter transport of other lipids. This mechanism assumes that the ER‐resident Sac1 phosphatase degrades PI4P in the ER (i.e. acting in cis). However, several studies have proposed that the enzyme instead operates in trans at contact sites between the ER and other PI4P rich membranes. We therefore sought to establish whether Sac1 truly acts in cis or trans in living cells. Compared with bona fide plasma membrane (PM)‐ER contact site proteins and broadly distributed ER proteins, Sac1 exhibits a localization consistent with the latter. Strategies to test activity of the enzyme at the PM in living cells revealed far more robust activity when the enzyme met its substrate in cis than at contact sites in trans. Finally, we used chemical and genetic strategies to inactivate Sac1 in yeast and mammalian cells, revealing dramatic accumulation of PI4P in the ER. This result is consistent with a failure to degrade the lipid only after its delivery to the ER. Together, the data show that Sac1 acts in cis, establishing the plausibility of an inositide motive force, and implicating PI4P transfer proteins as essential to phosphoinositide and lipid homeostasis.Support or Funding Information1R35GM119412‐01: NIH(NIGMS) R35
Cholesterol Hydroperoxide Trafficking: Impairment of Macrophage Cholesterol Efflux with Implications for Atherogenesis under Oxidative Stress
Authors: Korytowski Witold , Wawak Katarzyna , Pabisz Pawel , Chadwick A C , Sahoo D , Girotti Albert W 
Published: November 2023

This study describes a type of “stealthy” delivery of cholesterol 7α/β hydroperoxides (7‐OOH) via a natural cholesterol trafficking pathway. In vascular macrophages this pathway normally delivers cholesterol to mitochondria to initiate export signaling when its level becomes excessive. Because 7‐OOHs are structurally similar to cholesterol, they can be recognized by steroidogenic acute regulatory (StAR) proteins and trafficked to mitochondria alongside cholesterol.Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides such as 7‐OOH, 7‐hydroxide (7‐OH), and 7‐ketone (7=O). Unlike 7=O and 7‐OH, 7‐OOH is redox‐active, giving rise to the others via potentially toxic free radical reactions. In the present study we tested the novel hypothesis that under oxidative stress conditions, StAR family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7‐OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. Stimulation of human monocyte‐derived THP‐1 macrophages with dibutyryl‐cAMP resulted in substantial upregulation of StarD1 and plasma membrane ABCA1 which exports cholesterol to acceptors such as ApoA1. siRNA‐induced StarD1 knockdown (kd) prior to stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1‐kd cells internalized 7‐OOH slower than non‐stimulated controls and underwent less 7‐OOH‐induced lipid peroxidation and membrane depolarization, as probed with C11‐BODIPY and JC‐1, respectively. Major functional consequences of 7‐OOH exposure were (i) loss of mitochondrial CYP27A1 activity, (ii) reduced 27‐hydroxycholesterol (27‐OH) output, and (iii) down‐regulation of ABCA1 and ABCG1. Consistently, 7‐OOH‐challenged macrophages exported less cholesterol to apoA‐I or HDL than did non‐challenged controls. StarD1‐mediated 7‐OOH transport was also found to be highly cytotoxic, whereas 7=O and 7‐OH were minimally toxic. These findings support our hypothesis that 7‐OOH found in oxidized low density lipoprotein gets delivered to macrophage mitochondria and promotes atherogenesis through impairment of cholesterol homeostasis.Support or Funding InformationThis work was supported by Polish National Science Center grant 2014/13/B/NZ3/00833 (WK) and by National Institutes of Health grants HL85677 (AWG) and HL58012 (DS). Support of American Heart Association Fellowship 14PRE185800221 (ACC) is also acknowledged
A Membrane Trafficking Screen To Identify Components Involved In Clathrin‐Independent Endocytosis
Authors: Dutta Dipannita , Wayt Jessica , Donaldson Julie G 
Published: November 2023

Endocytosis is an important event that cells utilize to internalize cell surface proteins and fluid into the cell. There are two main forms of endocytosis: clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE), amongst which CME is most studied. While CME is important for the internalization of many surface receptors, bulk trafficking of membrane and fluid occurs primarily in a clathrin‐independent manner. Our lab has focused on CIE associated with Arf6, although other CIE pathways exist for specific cargoes and in different cell types. While we have a fair understanding of the trafficking and sorting of the cargoes that utilize CIE, we lack understanding of the entry mechanism. We know that CIE occurs independently of clathrin, the clathrin adaptor protein (AP2), and dynamin. Among the types of CIE cargo proteins, we have focused on two ubiquitously expressed proteins, the major histocompatibility complex class I (MHCI) and CD59, a GPI‐anchored protein, with an objective to identify requirements for their entry. Therefore, we performed an siRNA screen using the Dharmacon™ Membrane Trafficking library, which targets 140 genes, to identify proteins essential for the internalization of CD59 and MHCI in Hela cells. Target genes were individually knocked down and cells were allowed to internalize CIE cargo proteins. Following internalization, the proteins were visualized by immunofluorescence and the amount of internalized protein was quantified by image analysis software. Depletion of each candidate gene was scored as either inhibiting or enhancing internalization based on the relative amount of internalization compared to that observed with the non‐target siRNA control cells. The knocked‐down genes that lead to inhibition of internalization might represent components involved in CIE entry and the knocked‐down genes that lead to “enhanced” internalization could be negative regulators of this process. The hits from the initial screen were followed up by other confirmatory tests with individual siRNA oligo sequences. Interestingly, different proteins appeared as hits for MHCI and CD59 suggesting the differential regulation of integral membrane proteins vs. GPI‐anchored proteins in CIE. Surprisingly, among them are distinct subunits of AP2 that inhibited MHCI internalization specifically, but not CD59. Knocking down individual alpha or beta subunits of AP2 inhibited MHCI internalization whereas knockdown of the mu subunit, which is typically knocked down to demonstrate a requirement for AP2/clathrin, had no such effect. This raises the question of whether components of AP2 can function in CIE entry independent of the involvement of CME. We hope to understand the function of these two AP2 subunits in CIE in future studies.Support or Funding InformationNational Institutes of Health
Palmitoylation Impact on the Sodium Hydrogen Exchanger Isoform 1 Function
Authors: Pritsch Emily , Holland Alexander , Kooiker Amanda J , Rastedt Danielle E , Vaughan Roxanne A , Foster James D , Wallert Mark Anthony , Provost Joseph John 
Published: November 2023

The sodium hydrogen exchanger isoform 1 (NHE1) regulates intracellular pH and directs protein motility as an anchor for proteins at the leading edge of migrating cells. NHE1 has three putative palmitoylation cysteine targets in its cytoplasmic tail. Palmitoylation is a reversible, post‐translational fatty acid modification (S‐acetylation) of cysteine residues altering cellular location and function of many proteins. 2‐bromopalmitate (2BP) is a non‐metabolizable palmitate analog that irreversibly inhibits the protein acyltransferases responsible for palmitoylation. Using a live‐dead assay we found that up to 50 μM 2BP had a minimal (8.5 −/+ 2.5 % SEM) effect on cell viability and was not significantly different than the control palmitate at all concentrations. A 25% reduction in NHE1 palmitoylation was observed after the addition of 15 μM 2BP over 18 hours of incubation. Incubation of 15 μM 2BP in lung fibroblasts expressing human NHE1 (PSN) abrogated cell proliferation in low serum (0.5% FBS) conditions and reduced proliferation four fold in the presence of 10% FBS. Interestingly cells incubated with 2BP show a diminished adhesive ability on quartz coverslips. LPA induced stress fiber formation was decreased in cells treated with 2BP compared to vehicle control. Transiently transfected RFP tagged NHE1 cellular location and migration was determined in the absence and presence 2BP. Finally, we show the impact of 2BP and its control, palmitate, on intracellular pH indicating a role of palmitoylation on NHE1 function. Together this work shows a novel and yet undiscovered regulation of an important protein involved in intracellular pH homeostasis and directed cell motility.Support or Funding InformationThis work was funded in part by the Beckman Scholars Program, National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)
Mutation of Follicle Ftimulating Hormone Receptor Putative Caveolin Binding Motif Results in Altered Signaling
Authors: Fleischer Justin H. , Cohen Brian D. 
Published: November 2023

The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein‐coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also within these domains, an intracellular protein, caveolin, is present. Our lab has shown that hFSHR also localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.To test our hypothesis, we have constructed CBM mutants and are creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type FSHR presents as a doublet on a western blot while the mutants only have a single band. We theorize that one of these bands may be a glycosylation variant however further investigation is required. The mutated receptors qualitatively show normal p44‐MAPK signaling in response to FSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in downstream PKA targets between the mutant and wild type hFSHR, suggesting that there might be a difference in the ability to activate downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.
Machinery Mediating Kiss‐and‐Run Mechanism of Cell Secretion
Authors: Jena Bhanu P. 
Published: November 2023

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950's, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of transient or so called ‘kiss‐and‐run’ mechanism that allows fractional discharge of intra‐vesicular contents during secretion. This proposed mechanism is mediated by a supramolecular cup‐shaped structure at the plasma membrane called ‘porosome’, which enable secretory vesicles to transiently fuse with the plasma membrane, expel a portion of its contents, and disengage. Porosomes range in size from 15 nm in neurons and astrocytes, to 180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex of >500 protein molecules. Porosome structure, its chemical composition, and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex to establish the fusion pore at the porosome base, and the molecular mechanism of secretory vesicle volume increase required for intravesicular content release during cell secretion, collectively provide a molecular understanding of cell secretion, resulting in a paradigm‐shift in our understanding of the process.Support or Funding InformationWork presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ)