bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Conserved Degron Containing an Amphipathic Helix Regulates the Cholesterol‐Mediated Turnover of Human Squalene Monooxygenase, a Rate‐Limiting Enzyme in Cholesterol Synthesis
Authors: Chua Ngee Kiat , Howe Vicky , Jatana Nidhi , Thukral Lipi , Brown Andrew J. 
Published: June 2021

Cholesterol biosynthesis is a key process in cellular cholesterol homeostasis and is known to be tightly regulated by several mechanisms. Of note, squalene monooxygenase (SM), the second rate‐limiting enzyme in cholesterol biosynthesis, is regulated by transcriptional and posttranslational mechanisms. SM is degraded by the ubiquitin proteasome system when cholesterol is in excess. The cholesterol‐mediated degradation of SM depends on the first 100 amino acids (designated SM N100), and this represents the shortest cholesterol‐regulated degron identified to date. Despite having an important function, how these 100 amino acids grant SM the ability to be degraded by cholesterol remains elusive. From our mutational analyses, we have identified a 12‐residue region (Gln‐62 to Leu‐73) which is necessary for this degron to be successfully degraded by excess cholesterol. Using molecular dynamics simulation and circular dichroism, we identified the 12‐residue region contains an amphipathic helix. Our analyses also revealed that mutating SM residues Phe‐35, Ser‐37, Leu‐65 and Ile‐69 into alanine based on the key residues of the well‐studied Doa10 yeast degron, Deg1, also blunted the cholesterol‐mediated degradation of SM. In this study, we propose a model whereby the amphipathic helix of SM N100 is associated with the endoplasmic reticulum membrane when cholesterol is not in excess, and that excess cholesterol triggers the dissociation of this helix, unraveling it and exposing a hydrophobic patch which promotes degradation. Our current study reveals a key feature of how this rate‐limiting enzyme can regulate cholesterol levels, by sensing endoplasmic reticulum membrane cholesterol levels via a newly identified amphipathic helix. More importantly, we identified conservation of a key degron feature, namely an amphipathic helix, from yeast to humans.Support or Funding InformationNational Health and Medical Research Council (1060515)Australian Research Council (DP170101178)DST‐INSPIRE Faculty Grant LSBM‐45Department of Biotechnology, Government of India, Research associateship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of PPARγ inhibits hepatic cholestasis with involvement of Nogo‐B expression
Authors: Zhang Shuang 
Published: June 2021

Hepatic cholestasis can develop to fibrosis, cirrhosis and liver failure. Currently, the therapeutic effect of ursodeoxycholic acid (UDCA) on primary biliary cirrhosis (PBC) is limited to early stages of the disease. Recently, obeticholic acid (OCA) has been approved by FDA for the treatment of PBC. However, the safety, efficacy and tolerance of OCA may need further investigation in clinics. Herein, we report the effect of peroxisome proliferator activated receptor γ (PPARγ), a ligand‐activated transcription factor, on hepatic cholestasis. Pre‐treatment of mice with a PPARγ ligand, rosiglitazone, substantially inhibited α‐naphthylisothiocyanate (ANIT)‐induced hepatic cholestasis in a PPARγ expression‐dependent manner. Rosiglitazone substantially reduced ANIT‐induced hepatic necrosis, restored muddy and dark colored bile and serum to normal. It also ameliorated serum biochemical parameters including ALT, AST, ALP, γ‐GT, total bilirubin and lipid profiles. In both mouse liver and HepG2 cells, activation of PPARγ induced expression of bile synthesis and secretion genes (CYP7A1, ABCB4, BSEP, ABCG5 and FXR) and Nogo‐B suggesting PPARγ enhances bile homeostasis and functions which might be involved with Nogo‐B expression. Surprisingly, we observed that deficiency of Nogo‐B expression in HepG2 cells greatly boosted expression of CYP7A1, ABCB4, ABCG5 and FXR while abolished PPARγ‐induced expression of these genes indicating the dual regulatory role of Nogo‐B in bile synthesis/secretion. In vivo, deficiency of Nogo‐B expression also increased expression of hepatic bile synthesis/secretion genes, and protected mice against ANIT‐induced hepatic cholestasis. Taken together, we identified that Nogo‐B is a target of PPARγ which might be involved in PPARγ‐inhibited cholestasis by its dual regulatory functions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression profiling of genes regulated by Sphingosine kinase 2 in a murine model of Pseudomonas aeruginosa mediated acute lung inflammation
Authors: Ebenezer David Lenin , Fu Panfeng , Krishnan Yashaswin H. , Jung Segun C. , Hu Hong , Arbieva Zarema , Madduri Ravi , Harijith Anantha K. , Natarajan Viswanathan 
Published: June 2021

BackgroundSphingosine‐ 1‐Phosphate (S1P) is an intracellular and extracellular signaling bioactive lipid mediator that signals by binding to five cell surface G‐protein‐coupled receptors S1P1–5. S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. S1P is a potent angiogenic factor and plays a key role in vascular development and maintenance of endothelial barrier integrity. However, the role of S1P in regulation of cellular functions depends on the pathology and type of S1P receptor involved. Here, we report that knock down of SphK2 (Sphk2−/−) in a murine model of Pseudomonas aeruginosa (PA) mediated lung infection conferred significant protection against lung inflammation and injury. To better understand the underlying molecular mechanism(s), genome‐wide gene mRNA expression profiling was performed with mouse lung tissue using Affymetrix MoGene 2.0 array.ResultsPathway enrichment analyses identified several signaling pathways that are likely to play a key role in PA‐induced lung inflammatory injury. These included pathways involved in adhesion/extracellular matrix (ECM) remodeling, immune responses to CCL 2 & 4 signaling, interleukin 3, 4, 6, 17 and 18 signaling and complement cascade regulation. Differential expression of genes in the lungs after PA infection were identified in Sphk2−/− mice and their wild type (WT) counterparts. PA infection up‐regulated expression of IL‐6, IL‐1β, TNF‐α and CCL‐2 genes in the lungs of WT mice compared to Sphk2−/− mice. Heat map analysis clearly showed a role for SphK2 in mediating up‐regulation of pro‐inflammatory genes after PA infection of mouse lung tissue.ConclusionUsing Sphk2 knockout mice and differential gene expression analysis, we show here that S1P/SphK2 signaling plays a key role in promoting PA lung inflammatory injury and may serve as a therapeutic target to combat bacterial lung inflammation.Support or Funding InformationNational Institutes of Health Grant P01 HL98050 to V.NatarajanThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CysLT2R is a novel therapeutic target for tumor angiogenesis, growth and metastasis
Authors: Teegala Lakshminarayan Reddy , Duah Ernest , Kondeti Vinay , Adapala Ravi , Thodeti Charles , Paruchuri Sailaja 
Published: June 2021

Angiogenesis plays a vital role in numerous diseases including cancer, atherosclerosis, diabetic retinopathy and age‐related macular degeneration. Although chronic inflammation mediated by immune cells has been known to play a role in neoplastic transformation, there exists limited information on how inflammatory mediators can cause endothelial dysfunction and their role in promoting angiogenesis. Cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4) are pro‐inflammatory mediators derived from arachidonic acid and signal through two well characterized receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast and melanoma. However, the receptors involved and the mechanism of action still remains elusive. We have recently shown that LTD4 can enhance endothelial cell (EC) contraction and permeability via CysLT2R in a Rho‐dependent pathway. Here, we present another seminal observation that LTD4 can enhance angiogenesis in vitro and in vivo via CysLT2R. Since enhanced angiogenesis is required for solid tumor growth, we investigated the role of CysLTR in tumor angiogenesis, tumor progression and metastasis using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO). We observed enhanced CysLT2R expression but not CysLT1R in wild type (WT) tumors, in line with the involvement of CysLT2R in endothelial dysfunction. Further, tumor growth and angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in subcutaneous Lewis lung carcinoma (LLC) tumor model. Furthermore, though few in number, tumor vessels in CysLT2RKO mice exhibited intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Finally, blocking CysLT2R using a specific CysLT2R antagonist BaycysLT2 recapitulated the results obtained with CysLT2RKO mice. Our results suggest that CysLT2R promotes tumor angiogenesis and growth as well as metastasis to the lung by disrupting vascular integrity and targeting CysLT2R could offer novel VEGF‐independent therapeutic strategy for the treatment of cancer and other angiogenic disorders.Support or Funding InformationJames Foght Professorship Support, AHA GIA 15GRNT 25670004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inactivation of dynamin 1 in Cln1−/− mouse brain contributes to declining synaptic pool size
Authors: Bagh Maria Bindu , Sadhukhan Tamal , Appu Abhilash P. , Casey Sydney , Zhang Zhongjian , Mukherjee Anil B. 
Published: June 2021

Lysosomal storage disorders (LSDs) are genetic disorders caused by impaired lysosomal function, which cause accumulation of undigested substrates in the lysosome causing pathogenesis. In the majority of LSDs, neurodegeneration is a devastating manifestation. Neuronal ceroid lipofuscinoses (NCLs), are a group of the most common neurodegenerative LSDs that mostly affect children. Mutations in >13 different genes (called CLNs) underlie various types of NCLs. The infantile NCL (or INCL), one of the most devastating neurodegenerative LSDs, is caused by mutations in the CLN1 gene encoding palmitoyl‐protein thioesterase‐1 (PPT1), a lysosomal depalmitoylating enzyme. Palmitoylation is the only reversible lipid modification, which regulates the function of many proteins, especially in the brain. Moreover, PPT1‐catalyzed depalmitoylation of S‐acylated proteins is essential for their degradation. Thus, PPT1‐deficiency causes lysosomal accumulation of lipid‐modified proteins (constituents of ceroid) leading to INCL. Previously, we reported that in Ppt1‐deficient Cln1−/− mice, which mimic INCL, synaptic vesicle (SV) pool‐size at the nerve terminals progressively decline with age. We found that this defect originates from impaired exocytosis and endocytosis of SV components at the presynaptic membrane impairing neurotransmission. Dynamin1 is a small neuron specific GTPase that plays critical roles in recycling and regenerating fresh SVs. Here we demonstrate that in Cln1−/− mice there is increased phosphorylation of dynamin‐1, which inactivates its GTPase activity required for regeneration of the SVs. Our results uncover an unexpected role of Ppt1 in maintaining dynamin1 activity and suggest inactivation of dynamin 1 in this mouse model of INCL contributes to impaired SV‐regeneration and progressive decline in SV‐pool size.Support or Funding InformationThis work was supported in full by the intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yvc1 acts as a Novel Suppressor of Vacuolar Membrane Fusion
Authors: Miner Gregory E. , Guo Annie , Sullivan Katherine D. , Fratti Rutilio A. 
Published: June 2021

Yeast vacuoles maintain homeostasis in part by balancing membrane fission and fusion; however the regulation of these events is not fully understood. Phosphoinositides (PIs), and the proteins they interact with, are crucial regulators of both fusion and fission. Our work has shown phosphoinositides are essential for membrane fusion, specifically phosphoinositide 3‐phosphate (PI3P), PI4P, and PI(4,5)P2. Comparatively the phosphoinositide PI(3,5)P2 is relatively scarce and uncharacterized in membrane fusion, however others have demonstrated its requirement in osmotic shock induced membrane fission. Creation of PI(3,5)P2 during osmotic shock is coupled with a release of Ca2+ from the vacuole into the cytosol. This Ca2+ release is thought to be caused by a direct interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Since Ca2+ release also plays a role in membrane fusion, we sought to determine whether Yvc1 or its activation by PI(3,5)P2 is involved in membrane fusion as well as fission. Utilizing isolated yeast vacuoles we found that the absence of Yvc1 caused a significant increase in membrane fusion. Our data suggest that the augmented fusion was due to elevated levels of PI3P on the vacuole leading to subsequent increased recruitment of fusion machinery. Specifically, we saw enrichment of the soluble SNARE Vam7, subunits of the HOPS tethering complex, as well as the Rab GTPase Ypt7 and its heterodimeric guanine exchange factor Mon1‐Ccz1. These proteins act as components of the tethering machinery and we found that their enrichment in yvc1D vacuoles led to enhanced trans‐SNARE pairing. The increase in SNARE complexes was not due to enhanced rates of priming, suggesting that the change was due to increased vacuole tethering linked to the increase in Mon1‐Ccz1, Ypt7 and HOPS. Taken together our data suggests Yvc1 acts as a novel suppressor of vacuole membrane fusion through its effects on phosphoinositide levels and recruitment of tethering factors.Support or Funding InformationNational Institutes of Health (R01‐GM101132) to RAFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gemcitabine and 5‐FU disrupt nuclear transport and subsequent localization of p21, p27 and p53 in cancer cells
Authors: Nickle Audrey , Resendes Karen K. 
Published: June 2021

Altered nuclear transport plays a role in the mislocalization of tumor suppressor proteins in certain cancers. In addition, the disruption of nuclear transport and the nuclear pore complex during apoptosis are important mechanisms of action in several different chemotherapies. We previously demonstrated that the chemotherapy 5‐Fluorouracil (5‐FU) alters nuclear transport by disrupting the Ran gradient and increasing nuclear pore permeability. This block in nuclear transport may be advantageous to overcome Crm1‐induced excessive nuclear export in some cancers or with certain cases of drug resistance. Other work has shown that an additional chemotherapy, gemcitabine, is more effective when in combination with Crm1 inhibition. Therefore, we were interested in determining if gemcitabine and 5‐FU work effectively alone and in combination to disrupt nuclear transport via mislocalization of the transport protein Ran. We utilized immunofluorescence to determine the localization of Ran, which is normally localized to the nucleus. We show here that while each of these drugs mislocalized Ran at both 6h and 24h time points, and that gemcitabine counteracts the effects of 5‐FU in combination treatments to induce no change in Ran localization. This may be due to gemcitabine inhibiting 5‐FU's calcium‐mediated mechanism for inducing nuclear pore permeability. Additionally, we were interested in determining how these drugs affected the localization of three cargo proteins regulated by Crm1‐mediated export: p53, p27 and p21. These proteins have important function in the nucleus to control cell cycle progression and induction of apoptosis. Therefore, inhibition of Crm1 export should lead to nuclear accumulation of these factors and a potential increase in their function as tumor suppressors. We show here that 5‐FU and gemcitabine have opposing effects on the nuclear localization of both p53 and p21, and that the drugs again have counteractive effects when in combination treatments. Both 5‐FU and gemcitabine decrease the nuclear localization and expression of p27, while the combination treatment shows an additive effect on both localization and expression. The effects of these drugs on protein localization may be related to an additional block in nuclear import, as well as an induction of nuclear pore permeability downstream of the induction of apoptosis. Further research will help to characterize the effects of 5‐FU and gemcitabine on nuclear transport and subsequent protein localization. This may help to determine how these drugs may be more effectively used in the treatment of specific cancers and in specific drug combinations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysophosphatidylcholine Analogues Alter Yeast Nuclear Envelope Architecture and Function
Authors: Ponce Maria Laura Sosa , Cobb Jennifer A. , Zaremberg Vanina 
Published: June 2021

The lysophosphatidylcholine analog edelfosine is an anticancer lipid drug that targets cellular membranes. Therefore, it is alleged that in contrast to other chemotherapeutic agents edelfosine does not impact DNA. Despite this claim, a genetic screen in Saccharomyces cerevisiae found that lack of silent information regulator (Sir) proteins, alleviated the toxic effect of edelfosine. Since Sir proteins are key architects and regulators of the genome, we decided to investigate if this unusual connection with edelfosine could be related to a potential effect of the lysophosphatidylcholine analog on the nuclear envelope (NE) structure and function. Indeed, we found that edelfosine alters NE morphology, leading to an abnormal elongation of the membrane. Since it is known that Sir proteins mediate tethering of subtelomeric chromatin to the NE, we investigated if this was affected by treatment of wild type cells with the drug. Results from chromatin immunoprecipitation of Sir4 showed a significant reduction in the association of the protein with telomeres when cells were treated with edelfosine. Current evidence also supports alterations of telomere clustering in the presence of the lysophosphatidylcholine analogue. Therefore, our results suggest that edelfosine alters NE morphology and function. We speculate that mislocalization of Sir proteins in the nucleus probably results in abnormal silencing of genes that may be implicated in pathways that confer resistance to this lysophosphatidylcholine analog.Support or Funding InformationThis work has been financially supported by the Natural Sciences and Engineering Research Council of Canada to V Zaremberg and JA Cobb.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel pharmacological modulator abrogates physiological mitochondrial fission though specifically inhibiting the Mff‐Drp1 protein‐protein interaction
Authors: Kornfeld Opher Shai , Qvit Nir , Shamloo Mehrdad , Mochly‐Rosen Daria 
Published: June 2021

Within a single mammalian cell, thousands of mitochondria form a dynamic network. The morphological state of this network is tightly linked to its function. Mitochondrial fission and fusion are at the core of mitochondrial dynamics. Cellular homeostasis depends on maintaining a balance between fission and fusion. An imbalance between these two processes is a hallmark of numerous pathologies. For example, excessive fission is found in cancers, cardiovascular disorders, and neurodegeneration. While mitochondrial fission poses as an attractive therapeutic target for these disorders, better tools are needed to differentiate physiological fission from excessive pathological fragmentation.The main regulator of fission is the cytosolic dynamin‐related protein 1 (Drp1), a large GTPase that uses mechanical force to constrict the mitochondrial membrane. Four identified adaptor proteins mediate Drp1 recruitment to the mitochondrial outer membrane, yet the function of Drp1 binding to each adaptor is unknown. We hypothesized that one adaptor, Mff, is primarily responsible for physiological fission. Therefore, a specific Mff‐Drp1 protein‐protein interaction inhibitor would differentiate physiological from pathological fission.Using a rational design approach, we identified a small peptide that specifically inhibits Drp1's interaction with Mff, but not with any of the other outer‐membrane adaptors: P259. Contrary to a Drp1/Fis1 inhibitor, treatment with P259 disrupted mitochondrial network morphology and function under basal conditions. Furthermore, inhibition of Mff‐specific Drp1 recruitment to the mitochondria halted mitochondrial motility, elucidating the mechanistic link between mitochondrial fission and transport. Mitochondrial motility is especially relevant in cell types with complex morphologies such as neurons. We therefore studied the effect of P259 in brains. In WT mouse brains, P259 limited mitochondrial recruitment of Drp1, resulting in a 30% decrease in ATP production and enlargement of striatal mitochondria by electron microscopy. Furthermore, treatment with P259 reduced the lifespan of R6/2 Huntington's Disease model mice and caused behavioral abnormalities in WT mice. Cumulatively, these results suggest that Mff orchestrates physiological recruitment of Drp1 to the mitochondrial membrane thereby maintaining mitochondrial network homeostasis through facilitating fission and motility. Results from this study suggest that Drp1's pathological or physiological roles are mediated through its specific interaction with adaptors on the mitochondrial outer membrane.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RESOLVING EXTENDED N‐GLYCANS BY NMR: NEW INSIGHTS INTO INFLUENZA HEMAGGLUTININ N‐GLYCAN INTERACTIONS
Authors: de Toro Ronda Beatriz Fernández , Peng Wenjie , Thompson Andrew , Domínguez Gema , Cañada F. Javier , Castells Javier Pérez , Barbero Jesús Jiménez , Paulson James C. , Canales Ángeles 
Published: June 2021

Seasonal flu (influenza virus infection) is one of the major threats to human health, causing of up to 500 thousands deaths worldwide annually.1,2 Hemagglutinin, a glycoprotein presented in the surface of the influenza virus, binds sialylated N‐glycans located on the surface of host epithelial cells.The structural elucidation and the conformational studies of N‐glycans remain a challenge due to the complexity, heterogeneity and flexibility of carbohydrates. Moreover, the isochronous chemical shifts presented for the same units located at different branches make impossible the distinction of their signals in the NMR spectra.Taking advantage of the lanthanide approach, an unambiguous assignment of each carbohydrate unit has been reached for bianntenary N‐glycans containing one and two LacNAc units, permitting their conformational elucidation and the recognition study of these N‐glycans by the HK/68 hemagglutinin strain.Support or Funding InformationThis research was financially supported by funding from Spanish government, MINECO, projects: CTQ2016‐76263‐P, CTQ2015‐64597‐C2‐1‐P and CTQ2015‐64597‐C2‐2‐P and FPI fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.