bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia potentiates the accessibility of cyclooxygenase‐2 gene promoter to transcription factors
Authors: Lai Yen‐Yu , Hsiao Kuei‐Yang , Tsai Shaw‐Jenq
Published: June 2021

Aberrant expression of cyclooxygenase‐2 (COX‐2) has been observed in many human diseases including cancer and endometriosis. Overproduction of COX‐2 derived eicosnoids, especially prostaglandin (PG) E2 not only enhances disease progression but also results in poor prognosis. However, mechanisms responsible for overexpression of COX‐2 in these tissues are largely unknown. Herein, we provide evidence to demonstrate that aberrant expression of COX‐2 in these tissues may be mediated by predisposition to hypoxia. Pre‐exposure of cells to hypoxic stress result in increased sensitivity of COX‐2 gene to IL‐1β stimulation even after 48 h of reoxygenation. Promoter activity assay revealed that induction of COX‐2 expression was mediated by upregulation of cox‐2 gene by hypoxia‐inducible factor‐1α (HIF‐1α). Similarly, treatment of cells with DFO, a chemical that will induce HIF‐1α accumulation, also increased promoter sensitivity of cox‐2 to IL‐1β. The increase in cox‐2 gene sensitivity by pre‐exposure to hypoxia is also evident when other cox‐2 stimulators such as EGF and PGE2 were used to treat the cells. Together, we have shown for the first time that overexpression of COX‐2 in endometriotic stromal cells is likely due to hypoxia‐mediated epigenomic modification of chromatin structure surrounding COX‐2 promoter.
PKC alpha‐Induced Derepression of the Human Luteinizing Hormone Receptor (LHR) through ERK‐Mediated Sp1 Phosphorylation
Authors: Liao Mingjuan , Zhang Ying , Dufau Maria L.
Published: June 2021

Transcription of the LHR is subject to repression/derepression through various modes and multiple effectors. The present studies revealed that PKCα/ERK signaling is essential for activation of the LHR gene induced by phorbol‐12‐myristate‐13‐acetate (PMA) in Hela cells. While this effect was attributable to PKCα activity, the ERK pathway was the downstream effector in LHR activation. PMA caused significant enhancement of Sp1 phosphorylation at serine residue(s), which was blocked by inhibition of PKCα or ERK. The interaction of activated phospho‐ERK with Sp1 and ERK's association with the LHR promoter points to Sp1 as a direct target of ERK. Following Sp1 phosphorylation the HDAC/mSin3A repressor complex dissociated from Sp1 sites, histone 3 was acetylated, and TFIIB and Pol II were recruited. In addition, overexpression of a constitutively active PKCα (PKCαCA) which strongly activated LHR transcription in MCF‐7 cells (devoid of PKCα), induced Sp1 phosphorylation at serine residue(s) and caused de‐recruitment of the HDAC1/mSin3A complex from the promoter. These effects were negated by co‐transfection of dominant negative PKCα. These studies have revealed a novel regulatory signaling mechanism of transcriptional control in which the LHR is derepressed through PKCα/ERK‐mediated Sp1 phosphorylation, causing the release of HDAC1/mSin3A complex from the promoter.
Regulation of Cholesterol Biosynthetic Genes by the EGR Family of Transcription Factors
Authors: Gokey Nolan Garrity , Svaren John
Published: June 2021

Recent work has shown that the Egr2 transcription factor plays a direct role in inducing transcription of cholesterol biosynthetic genes during myelination of peripheral nerves. Myelination requires a considerable amount of de novo cholesterol synthesis in Schwann cells, and mice lacking Egr2 show a decrease in cholesterol biosynthetic gene transcription despite little change in SREBP levels. To determine if Egr family members play a similar role in other tissues, we have investigated the role of Egr1 in regulation of cholesterol biosynthetic genes in liver. Egr1 is induced by insulin both in vitro and in vivo, and therefore could mediate the insulin‐dependent induction of hepatic cholesterol biosynthesis. Using chromatin immunoprecipitation assays, we have shown a >4‐fold insulin‐dependent increase in Egr1 binding to the Hmgcr and Cyp51 promoters in the H4IIE liver cell line. The analysis has been extended to detection of Egr1 binding to promoters in liver, which reveals a >5‐fold induction in Egr1 binding to the Hmgcr and Mod1 promoters in mouse liver in response to feeding as well as an increase in expression of these genes. Overall, we have identified Egr1 as a potential regulator of hepatic cholesterol biosynthesis. This work was supported by a grant from the American Heart Association, 0650102Z.
Enhanced expression of natriuretic peptide receptor‐A gene (Npr1) by Ets‐1 and all‐trans retinoic acid signaling
Authors: Kumar Prerna , Bolden Gevoni , Pandey Kailash N
Published: June 2021

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The present study was aimed at gaining insight into the function of Ets‐1 and retinoic acid signaling in the regulation of Npr1 gene transcription and expression. Npr1 promoter‐reporter deletion constructs were transiently transfected in mouse mesangial cells (MMCs) and transcriptional activity was measured by dual luciferase assay. Overexpression of Ets‐1 enhanced Npr1 promoter activity by almost 12‐fold, whereas mutagenesis of the two Ets‐1 binding sites decreased the promoter activity by 90%. Chromatin immunoprecipitation assays confirmed the in vivo binding of Ets‐1 to Npr1 promoter. All‐trans retinoic acid (ATRA) greatly enhanced Npr1 gene transcription using Ets‐1 binding sites in a dose‐dependent manner. Knockdown of Ets‐1 expression by siRNA significantly abrogated ATRA‐induced Npr1 gene transcription. ATRA also enhanced NPRA mRNA levels and ANP‐dependent intracellular accumulation of cGMP. In conclusion, Ets‐1 is an essential mediator of retinoic acid‐induced Npr1 gene transcription and expression and is likely to have important implications in the pathophysiology of hypertension and cardiovascular disorders. Supported by NIH grants HL 57531 and HL 62147.
Transferrin expression profile during bacteria infection in Aedes aegpyti mosquitoes
Authors: Velasquez Lissette S. , Zhou Guoli , Geiser Dawn L. , Winzerling Joy J.
Published: June 2021

Female mosquitoes require a blood meal containing iron for egg production; therefore discovering the role of iron and iron‐related proteins has significance in limiting mosquito reproduction. Transferrins are a family of iron‐binding proteins involved in iron uptake, immunity, cell growth, and blood and hormone responses during oogenesis. In hens, serum transferrin (Tf1) functions in iron transport, while ovotransferrin (Tf2) has an antimicrobial role in egg whites. The expression profile of Tf1 and Tf2 mRNA in the yellow fever mosquito, Aedes aegypti, was investigated at various life stages and during infection using real time RT‐PCR. In A. aegypti larval CCL‐125 cells treated with E. coli/B. subtilis and iron, Tf1 showed a time‐dependent, significant increase, while Tf2 expression decreased with bacteria treatment. Fat body (FB) tissues collected from blood‐fed females post bacteria injection contained more Tf1 and Tf2 transcripts than either midgut (MG) or ovary (OV). Infection caused a significant decrease in Tf1 and Tf2 levels in FB, a decrease in Tf1 and increase in Tf2 in MG, and an increase in Tf1 and decrease in Tf2 in OV at 72 hrs post blood meal. Further studies will measure Tf expression in eggs, larvae, pupae, males and sugar or blood‐fed females to reveal developmental, sex‐specific and reproductive roles. These findings may provide targets for mosquito vector and pathogen control strategies. This work was supported by funds from the National Institutes of Health (GM056812), the Agricultural Experiment Station, the College of Agriculture and Life Sciences and the BIO5 Institute for Collaborative Bioresearch at The University of Arizona.
RGS13 suppresses CREB‐dependent gene transcription
Authors: Xie Zhihui , Johnson Eric J , Druey Kirk M
Published: June 2021

cAMP response element binding protein (CREB) promotes transcription of genes involved in neuronal development, metabolism, and immunity. Norepinephrine (NE) secreted into lymphoid organs stimulates Gαs‐coupled β2‐adrenergic receptors on B lymphocytes, leading to CREB‐dependent transcription of the Oct 2 coactivator (OCA‐B). Regulator of G protein Signalling (RGS) proteins attenuate GPCR signaling by inhibiting Gαi and Gαq but not Gαs. We show that B‐cell‐enriched RGS13 is a direct transcriptional repressor of CREB. Phosphorylation of CREB induced RGS13 nuclear translocation, where it bound pCREB and inhibited CREB‐DNA contact. RGS13 abundance affected pCREB promoter occupancy and CREB target gene transcription in HEK293T cells. Two discontinuous sites on RGS13 (aa15‐33 and aa93‐117) were required for CREB binding and inhibition of CREB activity, which depended on Arg13, Glu15, and Ser104 within these two regions. RGS13 interacted with the kinase‐inducible domain (KID) of CREB, which required the α‐helical conformation of KID induced by CBP binding. Rgs13−/– B cells exhibited increased β2AR‐stimulated pCREB‐DNA binding and OCA‐B expression. Thus, RGS13 may impact humoral immune responses under sympathetic neural control and other physiological responses mediated by CREB.
Pyrophosphate (PPi) off‐rate from an E. coli RNA polymerase (RNAP) elongation complex in the absence of nucleotides
Authors: Johnson Ronald Sanders , Strausbauch Mark , Register J. Kristen
Published: June 2021

Nucleotide incorporation during transcription is accompanied by PPi formation. Because PPi at the active site blocks RNAP translocation, its release is a crucial step in transcription. Previously, we found that rapid PPi release after nucleotide incorporation only occurred if the next nucleotide required for incorporation was present. As an extension of these studies, we investigated PPi release from an elongation complex (EC) in the absence of nucleotides. The RNA primer in the EC contained a phosphorothioate internucleotide linkage at the 3′ end to block pyrophosphorolysis. The interaction of PPi with this EC was monitored by alterations in the intrinsic protein fluorescence in stopped‐flow kinetic studies. A plot of the observed first‐order rate constant against PPi concentration was hyperbolic. Data analysis indicated that the rate constant for PPi release (6.1±1.9 s−1) in the absence of NTPs was ∼100 fold less than that observed in the presence of the correct NTP for incorporation in previous studies. This supports a model for elongation in which the binding of the correct nucleotide (in either the entry or preinsertion site) triggers the rapid release of PPi followed by RNAP translocation.
Post‐transcriptional regulation of the U3 small nucleolar RNA
Authors: Nabavi Sadeq , Nazar Ross N.
Published: June 2021

A high copy shuttle vector was used to express a “tagged” U3 snoRNA gene in Schizosaccharomyces pombe to examine regulatory responses to a high gene dosage. RNA analyses utilizing RT‐PCR amplification and restriction fragment length polymorphism indicated that the “tagged” gene was both proportionally and highly expressed and that downstream processing and/or termination were critical to U3 snoRNA stability. In contrast, direct measurements of the total cellular U3 snoRNA showed essentially normal levels of mature RNA although measurements of precursor levels confirmed a highly expressed gene construct. Taken together the results indicated that the steady state amounts of mature U3 snoRNA were primarily regulated at the post‐transcriptional level. This regulatory mechanism prevents an over accumulation of the cellular U3 snoRNA and can efficiently degrade mutant RNA molecules. Together with past studies on other 3’ extended RNA precursors, the results support post‐transcriptional regulation as a quality control mechanism in which appropriate amounts of functional RNA are stabilized by protein interaction while excess or defective RNA is rapidly degraded. Precursor processing in vitro and mutational analyses were consistent with this model. Supported by the Natural Sciences and Engineering Research Council of Canada.
The role of a ubiquitin processing protease, UbpA, in the growth‐to‐development transition of Dictyostelium development
Authors: Semotiuk Andrew , Johnson Trudi , Sky David , Leanza Crystal , Lindsey David F
Published: June 2021

UbpA, a homolog of yeast Ubp14 and human IsoT, is required for multicellular development of Dictyostelium. When Dictyostelium cells starve, they initiate a development program whereby cells aggregate and form fruiting bodies. ubpA− cells are unable to make this growth‐to‐development transition. Starved ubpA− cells accumulate ubiquitin‐containing species which may include proteins that must be degraded for development to occur. To identify these proteins, we purified ubiquitin‐containing species by S5a‐affinity chromatography. MuDPIT mass spec analysis identified 134 proteins by more than one peptide and 26 proteins containing GlyGly‐modified lysine residues. These include components of phagosomes and PsmD8, a proteasome regulatory subunit repressed at the onset of development. ubpA− cells also mis‐regulate several genes involved in early development. Interestingly, UbpA is required for growth‐stage expression of lmcA and lmcB, genes which are involved in sensing nutrient levels and signaling cells to develop upon starvation. This suggests that UbpA‐dependent mechanisms function at a very early point in the growth‐to‐development transition. To elucidate LmcA/B‐dependent pathways and characterize their regulation by UbpA, we have identified interacting partners of LmcA/B. Our results suggest that LmcA and LmcB together form a complex and interact with a cystidine deaminase.
Targeting the Enoyl‐Reductase Enzyme (FabI): Modern Drug Discovery Effects to Combat Tularemia
Authors: LU HAO , amEnde Chris , Boyne Melissa , Luckner Sylvia , Slayden Richard , Kisker Caroline , Tonge Peter J.
Published: June 2021

Francisella tularensis is a highly virulent and contagious gram‐negativeintracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacteriumto survive for weeks in a cool, moist environment have raisedthe possibility that this organism can be exploited deliberatelyas a potential biological weapon. Fatty acid synthesis (FAS) is essential for bacterial viability and it has also been validatedas an attractive target for the discovery of novel antibacterials.

We have successfully cloned, overexpressed and purified the F. tularensis FAS enoyl reductase enzyme FabI (ftuFabI) and fully characterized the reaction mechanism through steady‐state kinetics. Based on the 2.5Å resolution crystal structure we firstly resolved and pre‐steady state kinetics, a two‐step mechanism coupled to ordering of the active site loop was proposed for inhibition of the enzyme by triclosan. We also identified a series of diphenyl ethers which are subnanomolar inhibitors of the enzyme and that inhibit bacterial growth with MIC90 values as low as 0.09 μg/ml. One of these promising compounds is active in a mouse model of tularemia infection.
Targeting the Dimerization of EGFR
Authors: Yang Rob , Pike Linda , Marshall Garland
Published: June 2021

It is estimated that one in eight American women will develop breast cancer in her lifetime and a significant number of these cases are due to misregulation of the epidermal growth factor receptor, EGFR. Current drugs based on antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) show promising yet highly variable clinical efficacy. Recent advancements in EGFR‐signaling suggest that these therapeutic limitations are due to underestimation of a complex system that relies on receptor dimerization. Here we report a class of small‐molecule inhibitors that target and disrupt the dimerization in living cells, which contrasts the traditional strategies of developing mAb‐ and TKI‐based inhibitors. Our approach is a top‐down protocol that combines in silico high‐throughput screen of a compound library and in vitro biochemical assays that experimentally test the putative candidates. Several compounds were identified as potent and specific inhibitor against the activation of EGFR in living cells. The discovery, and the on‐going characterization, of these inhibitors offer an alternative class of EGFR‐inhibitors and could serve as the building blocks of novel therapeutics against breast cancer.

NIH grant GM68460
Analysis of Genomic Instability in Colorectal Carcinoma
Authors: Flaherty Patrick , Davis Ronald W , Ji Hanlee
Published: June 2021

We are studying the genetic differences between colorectal carcinoma and normal somatic cells to understand the genomic instability events that lead to cancer progression in order to diagnose invasive carinomas in early stage two patients. We have compiled array comparative genomic hybridization data from 212 clinical samples of colorectal carcinoma and conducted a meta‐analysis of the data to search for regions of significant genomic loss or gain. Using robust logistic regression and cross‐validation procedures, we have isolated several regions that are associated with poor clinical outcome. This analysis has provided key genomic loci that we are using to interrogate allele‐specific gene conversion and copy number aberration events in a prospective clinical trial.
Functional analysis and positional cloning of futile cycle, a maternal‐effect gene involved in pronuclear congression following fertilization
Authors: Lindeman Robin , Pelegri Francisco
Published: June 2021

The recessive maternal‐effect mutation futile cycle (fue) was previously identified as part of a gynogenesis‐based screen for maternally‐supplied products acting during early developmental stages in zebrafish. Embryos from homozygous mutant fue females (fue embryos) fail to undergo pronuclear migration and fusion, never form mitotic spindles, and consequently fail to properly segregate chromosomes. Interestingly, in the majority of mutant embryos, cytokinesis proceeds normally during early cell divisions even though DNA is absent from most dividing cells. The goal of the current research is to further characterize the fue phenotype and determine the identity of the fue gene. The sperm‐derived monoaster and components of the pronuclear membranes are known to facilitate movement of the female pronucleus towards the male pronucleus. Here we show that fue embryos develop a normal monoaster, but show abnormal nuclear membrane dynamics and morphology shortly after fertilization. We also observed a partially penetrant defect in centrosomal alignment and furrowing in mutant embryos. We have been able to localize the fue gene to a region on linkage group 4 containing six predicted transcripts, all of which are currently being investigated as fue candidate genes. In conclusion, our data suggests that the fue gene may play a role in nuclear dynamics and organization in the early embryo. Support for this research comes from an NIH‐RO1 grant and an NSF graduate research fellowship.
Alpha and Gamma Isoforms of the Type I Phosphatidylinositol 4‐phosphate 5‐kinase Regulate Distinct Stages of the Ca2+ Response in Mast Cells
Authors: Vasudevan Lavanya , Jeromin Andreas , Volpicelli‐Daley Laura , De Camilli Pietro , Baird Barbara , Holowka David
Published: June 2021

Multivalent antigen crosslinking of IgE receptors in mast cells triggers the hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP2) to inositol 1,4,5‐trisphosphate (IP3). Ca2+ release from the endoplasmic reticulum (ER) via IP3 receptors and consequent store‐operated Ca2+ influx are critical for mast cell responses such as exocytotic release of inflammatory mediators. To investigate roles for isoforms of the type I phosphatidylinositol 4‐phosphate 5‐kinase family (PIP5kinase‐Iα,β,γ) in synthesizing PIP2 for Ca2+ mobilization, we overexpressed PIP5kinase‐Iα in RBL‐2H3 mast cells. Surprisingly, antigen stimulated Ca2+ influx was inhibited by wt PIP5kinase‐Iα, but stimulated release from ER stores was unaffected. Consistent with these results, stimulated Ca2+ influx was enhanced by expression of an inositol phosphatase, expected to reduce PIP2 levels at the plasma membrane. In contrast, antigen stimulated Ca2+ release from ER stores was substantially reduced in bone marrow derived mast cells from PIP5kinase‐Iγ KO mice. These results suggest that the kinase isoforms affect distinct phases of the Ca2+ response in mast cells: PIP5kinase‐Iγ positively regulates Ca2+ release from ER stores, whereas PIP5kinase‐Iα negatively regulates Ca2+ influx. The cellular localizations and mechanisms underlying these distinctive effects are currently being investigated. (Funded by NIHR01‐AI18603)
Immunosuppressive drug FTY720 sensitizes prostate cancer cells to radiotherapy by inhibition of sphingosine kinase‐1.
Authors: Pshezhetskiy Dmitry , Bohler Torsten , Doumerc Nicolas , Golzio Muriel , Brinkmann Volker , Waxman Jonathan , Malavaud Bernard , Cuvillier Olivier
Published: June 2021

A sphingolipid analogue FTY720 is a novel immunosuppressive drug – an agonist of sphingosine 1‐phosphate (S1P) receptors. Recently we identified S1P as a therapy target in prostate cancer. In the current study we have demonstrated that FTY720 can radiosensitize prostate cancer in cell and animal models.

FTY720 has induced apoptosis in several hormone‐ and radio‐resistant prostate cancer cell lines. FTY720 treatment induced a sustained inhibition of prosurvival sphingosine kinase‐1 (SK1) and a decrease in the intracellular S1P content. Enforced expression of SK1 in prostate cancer cells rendered them resistant to FTY720. In vitro sub‐lethal concentrations of FTY720 have dramatically sensitized PC‐3 cells to radiotherapy in p53‐independent manner.

Mice orthotopically engrafted with fluorescent PC‐3 cells were treated with FTY720 with or without ã‐Irradiation. FTY720 has demonstrated a synergy with ã‐irradiation significantly reducing tumor size, angiogenesis and formation of micrometastases as demonstrated by fluorescent in vivo imaging.

In conclusion, FTY720 can sensitize prostate cancer cells to radiotherapy both in vitro and in vivo, through inhibition of SK1 and modification of intracellular levels of lipid second messengers.
PLCε Selectively Transduces Thrombin Versus LPA Signals to Astrocyte Proliferation Through Rap1 and Rho
Authors: Citro Simona , Kelley Grant G. , Smrcka Alan , Brown Joan Heller
Published: June 2021

Phospholipase C epsilon (PLCε) is a recently discovered PLC that, in contrast to other PLCs, responds directly to inputs from Ras and Rho family small G‐proteins. Using primary astrocytes isolated from a PLCε‐deficient mouse strain we show that endogenous G‐protein coupled receptors for LPA, S1P and thrombin utilize predominantly PLCε for inositol phosphate (InsP) and DAG formation. LPA‐ and S1P‐mediated InsP production was sensitive to Pertussis toxin (65±8% and 74±9% inhibition respectively) whereas the thrombin response was reduced by C3 toxin (68±13% inhibition), suggesting that these ligands activate PLCε through different mechanisms, a Gi pathway for LPA and S1P and a Rho mediated pathway for thrombin. Thrombin stimulated [3H]thymidine incorporation mediated by was markedly reduced in KO astrocytes (66±2%) and further studies revealed that PLCε is required for thrombin‐induced sustained ERK phosphorylation. Activation of a Ras family protein, Rap1, was required for ERK phosphorylation and DNA synthesis and the activation of this small G‐protein was abolished in KO cells. Notably LPA‐induced ERK and mitogenic effects were not Rap1 or PLCε dependent. Thus PLCε is selectively utilized by thrombin vs. LPA to transduce mitogenic signals through mechanism distinct from its role in generation of PLC‐derived second messengers.

National Institute of Health Grant GM 36927 and GM 53536.
A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2‐Arachidonoylglycerol
Authors: Blankman Jacqueline Lorayne , Simon Gabriel M. , Cravatt Benjamin F.
Published: June 2021

The endocannabinoid system is the signaling network hijacked by the active component of marijuana, tetrahydrocannibinol (THC), which elicits its effects by activating the cannabinoid (CB) receptors. The endogenous ligands of the CB receptors (“endocannabinoids”) include the lipid transmitters anandamide and 2‐arachidonoyl glycerol (2‐AG). Endocannabinoids have been implicated a wide range of physiological processes including appetite, pain, anxiety and cognition. Endocannabinoid levels in the central nervous system are tightly controlled by enzymatic biosynthesis and degradation. Inactivation of anandamide by fatty acid amide hydrolase (FAAH) has been well characterized, but less is known about the degradation of 2‐AG, which can be hydrolyzed by multiple enzymes in vitro including FAAH and monoacylglyceride lipase (MAGL). We adopted a functional proteomic strategy including activity‐based protein profiling (ABPP) and pharmacological inhibition studies to comprehensively profile 2‐AG hydrolases in the mouse brain. Our results reveal that ~85% of 2‐AG hydrolase activity in mouse brain membranes is performed by MAGL, with the remaining ~15% being catalyzed by two previously uncharacterized enzymes ABHD6 and ABHD12. We determined that MGL, ABHD6, and ABHD12 occupy distinct subcellular localizations, suggesting that they may be responsible for regulating distinct pools of 2‐AG in the nervous system. This work was supported by the National Institutes of Health grants DA015197 and DA017259 (B.F.C), a Fletcher Jones Foundation Scholarship (J.L.B.), the Daniel Koshland Fellowship in Enzyme Biochemistry (G.M.S.), the Skaggs Institute for Chemical Biology, and the Helen L. Dorris Institute for the Study of Neurological and Psychiatric Disorders of Children and Adolescents.
The neonatal brain mitochondrial ribosomal protein subunit 6 gene expression is normal in the lethal Smit1 knockout and independent of the rescue of the phenotype with prenatal myo‐inositol treatment
Authors: Buccafusca Roberto , Venditti Charles , Kenyon Lawrence , Johanson Roy A , Golden Jeffrey A , Coady Michael J , Berry Gerard T
Published: June 2021

Myo‐inositol (Ins) is an essential component of phosphatidylinositol, polyphosphoinositides, and inositol polyphosphates including the second messenger, Ins‐1,4,5‐P3. Ins also serves as an organic osmolyte. Active transport of Ins from the extracellular environment is primarily dependent on the sodium/myo‐inositol cotransporter 1, SLC5A3 (SMIT1). To gain insight into the biological role of Ins, and of the SLC5A3 protein, we have generated a knock out mouse model. The Slc5a3 KO newborn mice die post partum, but can be rescued if the pregnant mother is treated with 1% Ins enriched water. Our results show that Ins levels increased in brain and whole KO fetuses following rescue with 1% Ins. Yet, the tissue PtdIns content was normal in the KO with or without Ins treatment. Most importantly, the expression of the mitochondrial ribosomal protein subunit 6 (Mrps6) gene, which shares exon 1 with the Slc5a3 gene, was also not aberrantly expressed in E18.5 KO fetal brain. With or without 1% Ins treatment, selective neuronal immunostaining was normal in the central and peripheral nervous system of E18.5 fetuses and in adult mice, who also had a normal complete necropsy. We conclude that the role of Ins in pathophysiology, and perhaps also in regulation of the SLc5a3/Mrps6 gene(s), which phylogenetically share genomic space since the appearance of vertebrates, requires better delineation, especially in fetal development.
Activation of sphingomyelin synthesis by oxysterol binding protein involves phosphatidylinositol 4‐phosphate kinase activation at the Golgi
Authors: Banerji Sangeeta , Ridgway Neale D
Published: June 2021

Oxysterol binding protein (OSBP) is targeted to the Golgi apparatus after stimulation of cells with 25‐hydroxycholesterol (25OH). OSBP translocation triggers ceramide transfer protein (CERT) translocation leading to increased sphingomyelin (SM) synthesis. The mechanism for CERT translocation is unknown. Our hypothesis is that OSBP translocation leads to increased phosphatidylinositol 4‐phosphate (PI4) synthesis at the Golgi and this promotes translocation of CERT via its PH domain. To test this, CHOK1 cells were treated with 25OH or solvent and isolated Golgi were assayed for PI4 kinase activity. A transient 50% increase in Golgi‐associated PI4 kinase activity was observed after 30 min of stimulation with 25OH that was dependent on OSBP expression. PI4 kinase IIα and IIIβ are localized to the Golgi, and PI4 kinase IIIβ was recently shown to be required for ceramide transport and SM synthesis. However, knockdown of PI4 kinase IIIβ by RNAi reduced PI4 kinase activity of isolated Golgi fractions by only 20%. Moreover, knockdown of PI4 IIIβ did not lead to reduced basal or 25OH‐stimulated SM synthesis in CHOK1 cells, indicating that the kinase was not required for OSBP‐mediated CERT activation. Immunofluorescence experiments further confirmed that OSBP targeting to the Golgi was not affected by PI4 IIIβ knockdown and that the two proteins localized to medial/trans Golgi compartments. In conclusion, SM synthesis is regulated by sterol‐signaling through OSBP leading to PI4 kinase activation at the Golgi. Ongoing experiments suggest that PI4 kinase IIα is required for OSBP‐mediated activation of SM synthesis.
Oxysterol binding protein‐related protein 9 is required for Golgi organization and secretion
Authors: Ngo Michael H , Ridgway Neale
Published: June 2021

Oxysterol binding protein (OSBP)‐related protein 9 (ORP9) is a member of the OSBP gene family implicated in ER/Golgi transport and lipid regulation. Due to alternate promoter start sites, ORP9 is expressed as a full length (ORP9L) and N‐terminal truncated variant (ORP9S). ORP9L localizes to the trans‐Golgi via pleckstrin homology (PH) domain by interaction with phosphatidylinositol‐4‐phosphate (PI‐4P). ORP9L and ORP9S also localize to ER compartments by interaction with vesicle associated‐membrane protein (VAMP)‐associated protein (VAP). Overexpression of ORP9L in CHO cells had no effect on growth, however overexpression of ORP9S caused significant inhibition. Growth inhibition appeared to be related to a seccretory defect. Enforced overexpression of ORP9S and ORP9L depletion by RNAi experiments resulted in fragmentation of the cis and trans elements of the Golgi. VSVG trafficking through the Golgi, measured by an endoH sensitivity, was disrupted by ORP9S expression and ORP9L depletion. Altered expression of ORP9S and ORP9L had no effect on 25‐hydroxycholesterol activated synthesis of sphingomyelin, suggesting that ORP9 does not have a role in OSBP regulation of CERT recruitment to the Golgi. These results indicate that ORP9L plays an important role in maintaining Golgi structure and secretion by interacting with PI‐4P, and ORP9S is a dominant negative regulator of ORP9L.
Suppression of sphingomyelin synthase 1 (SMS1) by small interference RNA (siRNA) is associated with enhanced ceramide production and apoptosis after photodamage
Authors: Separovic Duska , Semaan Louie , Villani Maristella , Bielawski Jacek , Luberto Chiara
Published: June 2021

Rationale:

We have shown that overexpression of SMS1, an enzyme that converts de novo‐generated ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (PDT). To test whether SMS1 overexpression‐related effects after PDT can be reversed, in this study SMS1 was downregulated using siRNA for SMS1.

Methods:

Jurkat T lymphoma/leukemia cells were transfected with SMS 1 siRNA or control (scrambled)‐siRNA. Seventy two‐hours later, cells were treated with PDT, and following 2 h incubation, they were collected and processed for the following analyses: SMS activity was measured using a chemifluorescence assay with subsequent signal detection and quantitation using STORM 860/ImageQuant; sphingolipid mass was determined using high‐performance liquid chromatography‐tandem mass spectrometry; caspase‐3‐like activity (DEVDase) was measured using a spectrofluorimetric assay.

Results:

Compared to control siRNA‐transfectants, in SMS1 siRNA‐transfected cells the activity of SMS at rest was downregulated with concomitant decrease in sphingomyelin mass. In SMS1 siRNA‐transfected cells increases in ceramides were higher than in control siRNA‐transfectants after PDT. Similar findings were obtained for dihydroceramides suggesting the involvement of de novo ceramide pathway. DEVDase activation was enhanced in SMS1 siRNA‐transfected cells compared to their control counterparts.

Conclusions:

The data support the notion that downregulation of SMS1 promotes both de novo ceramide metabolism and apoptosis after photodamage. These findings suggest that SMS1 is a potential novel molecular target that can augment the therapeutic efficacy of PDT.
Characterization of the Biochemical Activities Associated with Splice Variants of Human FATP2
Authors: Melton Elaina Marie , Arias‐Barrau Elsa , DiRusso Concetta C. , Black Paul N.
Published: June 2021

One mechanism governing the transport of exogenous fatty acids (FA) is vectorial acylation whereby the imported FA is converted into acyl CoA upon transport as a consequence of the activities of the fatty acid transport proteins (FATP) and long chain acyl CoA synthetases (Acsl). Splice variants of human FATP2 have been identified that differ in the ATP binding region. FATP2a contains the entire ATP/AMP motif, while FATP2b lacks exon 3 resulting in a protein lacking critical residues within the ATP/AMP motif. Previously we have shown both forms are proficient in FA transport, but only FATP2a functions in very long chain FA activation. These findings demonstrate that these naturally occurring splice variants of FATP2 have distinct and separable biochemical activities. In order to investigate these splice variants further, both were cloned into pcDNA4/TO/myc‐His‐A allowing tetracycline regulated expression of the target protein containing a polyhistidine and c‐myc epitope tag. 293 T‐REx cell lines stably expressing either FATP2a or FATP2b have been generated. Current experimental studies are [1] defining the intracellular localization of both FATP2a and FATP2b, [2] correlating those observations with changes in FA transport and activation activities and [3] addressing the roles of both variants in the trafficking of exogenous FA into lipid metabolic pools. Supported by NIH grant GM56850.
Fatty acids derived from triacylglycerol hydrolysis are a significant source of ligands for peroxisome proliferator‐activated receptor‐α (PPAR‐α) in rat primary hepatocytes
Authors: Sapiro Jessica M. , Mashek Mara T. , Mashek Douglas G.
Published: June 2021

Fatty acids (FAs), especially polyunsaturated fatty acids, have gained a lot of attention for their important roles in regulating transcription factors involved in hepatic energy metabolism. Intracellular FAs are derived from multiple sources including exogenous, de novo lipogenesis, and triacylglycerol (TAG) hydrolysis. However, little is known regarding how FAs from different sources affect activity of transcription factors such as PPAR‐α. Thus, the aim of this study was to determine if FAs generated from TAG hydrolysis influence PPAR‐α activity in rat primary hepatocytes. To manipulate TAG hydrolysis, we overexpressed adipose differentiated‐related protein (ADRP), which resulted in a 50% decrease in TAG turnover. Results from PPAR‐α reporter activity assays demonstrated that 250 μM eicosapentaenoic acid (EPA) alone or in combination with 250 μM oleic acid robustly increased PPAR‐α activity while overexpression of ADRP resulted in an inhibition of TAG hydrolysis and a decline in PPAR‐α activity. These results indicate that FAs generated from TAG hydrolysis are a significant source of ligands for PPAR‐α activation.
In vivo identification of response elements mediating thyroid hormone stimulation of hepatic HMG‐CoA reductase transcription
Authors: Boone Lindsey R , Ness Gene C
Published: June 2021

Hepatic HMG‐CoA reductase (HMGR) is the enzyme that catalyzes the rate‐limiting step in cholesterol biosynthesis. Thyroid hormone‐mediated stimulation of HMGR transcription is decreased in hypothyroid mammals and is increased about 5‐fold by subsequent treatment with triiodothyronine (T3). We have utilized in vivo electroporation to introduce HMGR promoter‐luciferase constructs directly into livers of hypothyroid rats treated ±T3 in an effort to identify the region(s) of the HMGR promoter responsible for this T3‐mediated regulation. A linear relationship between HMGR promoter activity and serum T3 levels was observed. The promoter was analyzed by in vivo electroporation out to −3190 bp. The functional T3 response was localized to the proximal 325 bp, which exhibited a 5‐fold response to T3. Within this segment of the promoter, two response elements were identified: the SRE at −165 to −158 and the sequence AGGTCA at −316 to −321. EMSAs with liver nuclear extracts from T3−treated rats showed strong shifted bands with probes to both of these elements, which were not present in assays with liver nuclear extracts from hypothyroid rats. Antibody supershift assays revealed that SREBP‐2 bound to the SRE and that FTF/SHP, but not TRβ‐1 or LRH‐1, binds to the AGGTCA element at −316. These two regions appear to mediate the thyroid hormone response. (Supported in part by grant 04 TSP‐03 from the Florida Department of Health.)
The Anti‐Lipolytic Effects of Acute AICAR‐Induced AMPK Activation in Visceral and Subcutaneous Rat Adipose Tissue is Independent of HSL Phosphorylation on Serine 563 and 660 Residues
Authors: Anthony Nicole Marie , Gaidhu Mandeep Pinky , Ceddia Rolando Bacis
Published: June 2021

AICAR‐induced AMP‐activated protein kinase (AMPK) activation suppresses basal and epinephrine‐stimulated lipolysis in adipocytes. Our goal was to investigate whether the anti‐lipolytic effects of AICAR‐induced AMPK activation are mediated via inhibition of hormone sensitive lipase (HSL). Isolated rat epididymal (Epi), retroperitoneal (Ret), and subcutaneous (Sc) adipocytes were acutely (1h) incubated either in the absence or presence of AICAR (10 – 500μM), under basal and epinephrine (10μM) stimulated conditions. The lipolytic response and the phosphorylation status of HSL on Ser563 and Ser660 were determined. AICAR treatment significantly inhibited basal (77%, 31%, and 80%) and epinephrine stimulated (84%, 68%, and 86%) lipolysis in Epi, Ret, and Sc adipocytes, respectively. HSL phosphorylation on both serine residues was unaffected by AICAR under basal and epinephrine stimulated conditions. Interestingly, pharmacological inhibition of AMPK increased basal and epinephrine‐stimulated lipolysis and also reversed the anti‐lipolytic effect of low (10–100μM) AICAR concentrations. Even though lipolysis was increased by the AMPK inhibitor; HSL phosphorylation was not modified. These data indicate that the anti‐lipolytic effects of AICAR‐induced AMPK activation is independent of HSL phosphorylation, and appears to occur downstream of this enzyme. Funding was provided by NSERC, CIHR and CDA.
Regulation of the yeast PAH1 gene encoding Mg2+‐dependent phosphatidate phosphatase
Authors: Pascual Florencia , Carman George M.
Published: June 2021

The PAH1 gene in Saccharomyces cerevisiae encodes Mg2+‐dependent phosphatidate (PA) phosphatase. PA phosphatase catalyzes the dephosphorylation of PA yielding diacylglycerol and Pi. This enzyme generates the diacylglycerol used for the synthesis of triacylglycerol, and may generate the diacylglycerol used for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. The PAH1‐encoded enzyme also controls the cellular concentration of its substrate PA, which is the precursor for phospholipids that are synthesized via the CDP‐diacylglycerol pathway. In this work, we examined the regulation of PAH1 expression in response to inositol supplementation and growth phase by utilization of a PPAH1‐lacZ reporter gene plasmid transformed into wild type cells. In the absence of inositol, the β‐galactosidase activity driven by the reporter gene was 3‐fold higher in stationary phase cells when compared with exponential phase cells. In the presence of inositol, the activity in stationary phase cells was 10‐fold higher than that found in exponential phase cells. In stationary phase cells, inositol supplementation resulted in a 2‐fold increase in β‐galactosidase activity. Inositol did not affect the expression of PAH1 in the exponential phase of growth. The transcription factors involved are being examined. Supported by NIH grant GM 28140.
The G80R mutation in the yeast lipin homolog Pah1p abolishes phosphatidate phosphatase activity
Authors: Han Gil‐Soo , Carman George M.
Published: June 2021

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate (PA) phosphatase enzyme. Gly80 that is found in the NLIP domain of Pah1p is conserved in mammalian lipin proteins. A spontaneous G84R mutation in lipin 1 causes lipodystrophy and fatty liver in the fld2J (fatty liver dystrophy) mouse at birth. To examine the importance of Gly80 to the enzymatic function of yeast Pah1p, we constructed a mutant PAH1HA allele that contained arginine in place of the conserved glycine residue. While the G80R mutation did not affect the expression of Pah1p in yeast (as determined by immunoblot analysis using anti‐HA antibodies), the mutation had a major effect on its phosphatidate phosphatase activity. The overexpression of the G80R mutant protein did not have a significant effect on the level of PA phosphatase activity in pah1Δ mutant cells when compared with cells that overexpressed the wild type PAH1HA allele. Moreover, an immune complex containing the G80R mutant protein that was isolated from pah1Δ cells expressing the G80R PAH1HA allele had essentially no PA phosphatase activity. A His6‐tagged G80R mutant protein was expressed and purified from E. coli. The Vmax of the G80R mutant enzyme was 56‐fold lower than that of the wild type enzyme. The G80R mutation also caused a 2.5‐fold increase in the cooperativity respect to PA, but little effect on the Km value for PA. Supported by NIH grant GM 28140.
Phosphorylation of the yeast lipin homolog Pah1p by Cdc28p kinase
Authors: Morgan Jeanelle M. , Carman George M.
Published: June 2021

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate phosphatase enzyme that catalyzes the dephosphorylation of phosphatidate to diacylglycerol and Pi. Pah1p is responsible for de novo lipid synthesis, its reaction being the committed step for the synthesis of the storage lipid triacylglycerol, which is formed from phosphatidate via diacylglycerol. The diacylglycerol generated in the phosphatidate phosphatase reaction is also utilized in the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy Pathway. Previous studies indicate that Pah1p is phosphorylated by cyclin‐dependent Cdc28p kinase and dephosphorylated by the Nem1p‐Spo7p phosphatase complex. A phosphorylation‐deficient Pah1p mutant exhibits elevated phosphatidate phosphatase activity suggesting that phosphorylation inhibits activity. In this work, we examined the phosphorylation of Pah1p by Cdc28p kinase in vitro. Using purified Escherichia coli‐expressed Pah1p as a substrate, Cdc28p kinase activity was dependent on the time of phosphorylation and the concentration of the kinase. In addition, Cdc28p kinase activity was dependent on the concentration of Pah1p and ATP. Serine and threonine residues were targets for phosphorylation. The phosphorylation of Pah1p resulted in the loss of its phosphatidate phosphatase activity. Supported by NIH grant GM‐28140.
Role of Triacylglycerol Hydrolase and Apolipoprotein E in Hepatic Lipid Droplet Metabolism
Authors: Wang Huajin , Lehner Richard
Published: June 2021

The objective of the study was to address the roles of triacylglycerol hydrolase (TGH) and apolipoprotein E (apoE) in the mobilization of hepatic triacylglycerol (TG) stores for very‐low density lipoprotein (VLDL) assembly.

TGH and apoE associate with the same pool of hepatic luminal lipid droplets (LLD) and potentially interact with each other. Lack of TGH or apoE expression alters hepatic TG storage in both the cytosolic lipid droplets (CLD) and in the luminal lipid droplets (LLD), suggesting a crosstalk between the two lipid storage pools. Metabolic labeling studies revealed that ectopic expression of TGH channels the cytosolic rather than the luminal pool of TG for VLDL secretion. Accordingly, it is also observed by confocal imaging that the absence of TGH or apoE alters CLD morphology and distribution in hepatocytes. Apo E deficiency results in redistribution of LLD‐associated proteins, suggesting a role for apoE in altering LLD protein composition. ApoE is also found to translocate to CLD in TGH deficient hepatocytes, indicating correlated roles of TGH and apoE. It is concluded that TGH, in concert with apoE, channels mainly CLD‐TG for VLDL secretion and plays a critical role in regulating lipid droplet formation, morphology and protein composition.

Supported by the a grant from the Canadian Institutes of Health Research
Serum paraoxonase PON1 and its interactions with HDL
Authors: Gaidukov Leonid A. , Aharoni Amir , Khersonsky Olga , Tawfik Dan S.
Published: June 2021

Serum paraoxonase (PON1) is a high density lipoprotein (HDL)‐associated enzyme that exhibits a wide range of hydrolytic activities and important anti‐atherogenic properties. We have generated recombinant PON1 variants by directed evolution. These enabled the bacterial expression of highly purified PON1, the genetic engineering of new PON1 variants, and the determination of PON1's 3D structure, the mode of its HDL anchoring, and catalytic mechanism. Our structure‐reactivity studies and laboratory evolution experiments indicated that PON1's native activity is lactonase. Impairing the lactonase activity through mutations of PON1's catalytic dyad diminished its ability to prevent lipid oxidation and stimulate macrophage cholesterol efflux. Studies of PON1's interaction with reconstituted HDL particles indicated that PON1 is an interfacially activated enzyme. Indeed, apoA‐I‐HDL binds PON1 with high affinity (Kd<10−9 M), induces a single highly stable conformation of the enzyme, and selectively stimulates its lactonase activity. These findings enabled the development of new sera tests for the levels and stability of PON1‐HDL complex and its lipo‐lactonase activity, and opened the road for the engineering of recombinant PON1‐HDL particles with therapeutic potential. We acknowledge financial support from Minerva Foundation.
Apolipoprotein‐lipid binding: importance of lysine ε‐amino groups
Authors: Abdullahi Gezman Ezedine , Weers Paul M.M.
Published: June 2021

Apolipophorin III (apoLp‐III) from Locusta migratoria is an 18 kDa model apolipoprotein that plays an integral role in lipid transport via association with circulating lipoproteins. Prior studies have indicated the importance of ionic forces in lipid‐protein interactions. In the present study, the possibility of electrostatic interactions between lysine residues on the surface of apoLp‐III and phospholipid head groups was investigated. The 7 lysine groups of apoLp‐III were chemically modified with the introduction of acetyl (Ac) groups. Guanidine‐HCl denaturation studies of modified and unmodified apoLp‐III showed minimal changes in denaturation profiles, with 0.62 M and 0.56 M denaturation midpoints, respectively. Circular dichroism spectroscopy revealed a 46% α‐helical content for Ac‐apoLp‐III, compared to 60% for unmodified apoLp‐III. Lipid binding was assessed using dimyristoylphosphatidylcholine small unilamellar vesicles (SUVs). No changes were observed for either type of apoLp‐III in their ability to solubilize SUVs and conversion into nanodiscs. When using negatively charged dimyristoylphosphatidylglycerol, Ac‐apoLp‐III displayed a decreased ability in SUV solubilization. The results indicate the retention of secondary structure in Ac‐apoLp‐III and provide evidence for the role of lysine residues in association with anionic but not zwitterionic phospholipids.