bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the first two ER/ERGIC‐based lysine acetyltransferases that are involved in the post‐translational regulation of nascent BACE1
Authors: Puglielli Luigi , Ko Mi Hee
Published: June 2021

Folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is enhanced by enzymes that modify, either temporally or definitively, the protein. Our group has shown that the lipid second messenger ceramide regulates the molecular stability of the β‐site APP cleaving enzyme 1 (BACE1) by affecting a transient form of lysine acetylation in the ER/Golgi system (Biochem J 2007; EMBO J 2006; J Biol Chem 2003). Specifically, nascent BACE1 is initially acetylated in the lumen of the ER and then deacetylated by a Golgi‐based deacetylase. Here we report the identification of the first two ER/ERGIC‐based lysine acetyltransferases (ATase1 and ATase2). Transfection of ATase1 or ATase2 into CHO cells increased the acetyl‐CoA:lysine acetyltransferase activity recovered from cell lysates. Subcellular fractionation studies showed an ER/ERGIC localization, overlapping with the compartments where the acetylation of BACE1 occurs. When compared to control, ER/ERGIC fractions from ATase1 and ATase2 cells showed increased acetyltransferase activity. Affinity‐purified ATase1 and ATase2 were both able to acetylate BACE1 in vitro and in vivo. Finally, overexpression of ATase1 and/or ATase2 resulted in stabilization and increased steady‐state levels of BACE1. In conclusion, we have identified the first two ER/ERGIC‐based acetyltransferases involved in the molecular stabilization of nascent BACE1.
SPFH1 and SPFH2 form a high molecular weight complex in the ER membrane that mediates the ER‐associated degradation of inositol 1,4,5‐trisphosphate receptors and other substrates in mammalian cells
Authors: Pearce Margaret M.P. , Wang Yuan , Wilkens Stephan , Wojcikiewicz Richard J.H.
Published: June 2021

The endoplasmic reticulum (ER)‐associated degradation (ERAD) pathway is responsible for the degradation of aberrant proteins in the ER and the regulated degradation of several ER resident proteins. Inositol 1,4,5‐trisphosphate receptors (IP3Rs) form tetrameric Ca2+ channels in the ER membrane and are converted from stable proteins into ERAD substrates following their activation by IP3 and Ca2+. Using activated, endogenous IP3Rs as a tool, we have recently identified two novel mediators of the mammalian ERAD pathway—SPFH1 and SPFH2 (SPFH domain‐containing protein, members 1 and 2). SPFH1 and SPFH2 associate with IP3Rs immediately after their activation but prior to their polyubiquitination, and depletion of either SPFH1 or SPFH2 by RNAi inhibited the ERAD of IP3Rs and other mammalian ERAD substrates. SPFH1 and SPFH2, both ER membrane proteins that protrude into the ER lumen, were found to exist exclusively as an ∼1MDa complex by native PAGE, and TEM of purified, endogenous SPFH1/2 complex indicates that it assembles into ring‐like structures. These results suggest that SPFH1 and SPFH2 form a high molecular weight, ring‐shaped complex in the ER membrane that plays a key role in the early steps of mammalian ERAD pathway, perhaps as a factor involved in substrate recognition or retrotranslocation.

Sources of funding: NIH, PhRMA Foundation
Novel supercomplexes of the proteasome in regulation of protein biosynthesis
Authors: Acs Klara , Perreard Laurent , Amir Omar , Groisman Regina , Gerber Scott , Kisselev Alexei
Published: June 2021

26S proteasomes are large (1.6–2.5 MDa) multi‐functional proteolytic complexes that degrade the majority of proteins in mammalian cells. Proteasome inhibitors selectively induce apoptosis of malignant cells; one compound of this class, Velcade, is being used for treatment of multiple myeloma and is in clinical trials for the treatment of other malignancies. We have recently discovered that the majority of proteasomes in mammalian cells exist in much larger complexes. We have purified these supercomplexes and analyze their composition by mass spectrometry. This proteomic analysis has identified ribosomes and multi‐subunit translation‐initiation factor eIF3, and several RNA‐binding proteins, as principal components of these complexes, along with proteasomes. Hence, we hypothesize that proteasomes play a role in the initiation of translation, most likely by destroying translation inhibitor proteins and/or RNA‐binding proteins that protect mRNA from degradation by nucleases but also act as translational repressors. We introduce in this work the proteasome involvement in translation initiation and the interaction of proteasome‐eIF3 supercomplexes. These findings reveal a novel and unexpected link between protein synthesis and degradation and indicate that connection between these processes is tighter than previously thought.

This work was supported by NCCC 203959 to AK.
Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/PARC chemokine induction
Authors: Syrovets Tatiana , Li Xuehua , Paskas Svetlana , Laumonnier Yves , Simmet Thomas
Published: June 2021

Monocyte‐derived immature dendritic cells (DC) do not express protease‐activated receptors (PARs). However, upon maturation with LPS, but not with TNF‐α or CD40L, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of matured DC with thrombin, PAR1‐ or PAR3‐activating peptides elicits actin polymerization and concentration‐dependent chemotactic responses in LPS‐, but not in TNF‐α‐matured DC. Stimulation of PARs with thrombin or the respective receptor‐activating peptide activates ERK1/2 and Rho kinase 1 (ROCK1) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (MLC2). The ERK1/2‐ and ROCK1‐mediated phosphorylation of MLC2 was indispensable for the PAR‐mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho‐dependent release of the CC chemokine CCL18/PARC, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83+ DC with CCL18 in human atherosclerotic plaques as revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin and its receptors in DC biology.

This work was supported by the Deutsche Forschungsgemeinschaft.
Neutrophil elastase cleaves VEGF to generate a VEGF fragment with altered activity
Authors: Kurtagic Elma , Nugent Matthew A
Published: June 2021

Vascular Endothelial Growth Factor (VEGF) and neutrophil elastase (NE) are implicated in the progression of emphysema. We investigated the potential connection between VEGF and NE injury. 125I‐VEGF was treated with NE, and the product visualized by SDS‐PAGE. We found that NE cleaves VEGF165 to a ∼34kDa disulfide linked fragment. VEGF interacts with cell surface receptors and components of the extracellular matrix, such as fibronectin and heparan sulfate. Hence, we examined the binding of the NE generated VEGF fragment (VEGFf) to VEGFR(1 and 2)‐Fc constructs, and found that VEGFf retained the ability to bind VEGFR1 but lost the ability to bind VEGFR2. We also examined VEGFf binding to fibronectin ± heparin and found that, unlike intact VEGF165, VEGFf did not bind fibronectin. VEGFf bound heparin, albeit with a reduced affinity compared to intact VEGF165. We assessed the activity of VEGFf in endothelial cells, and found that it did not activate ERK1/2 alone yet it enhanced VEGF165 activation of ERK1/2. We performed migration assays using macrophage RAW264.7 cells that express VEGFR1, and found that VEGFf and VEGF stimulated migration to a similar extent. VEGFf also activated Akt in endothelial and RAW264.7 cells. Our data indicate that NE generated VEGFf has altered activity, which might lead to functional changes within the injured lung.

Supported by HL56200 and HL46920.
Oncostatin M (OSM) Mediated Alterations in Matrix Metalloproteinase (MMP) ‐ 2 is Regulated by Specific Signal Transduction Pathways in H‐ras Transformed Cells
Authors: Francis Eddie Joesph , Hurta Robert A.R
Published: June 2021

Oncostatin M (OSM) is a pleiotropic cytokine that has been shown to exert both stimulatory and inhibitory effects on tissue invasion in various cell lines. Tissue invasion is a key marker of metastasis and can be monitored by examining matrix metalloproteinase (MMP) activity. OSM has been shown to up‐regulate MMP‐2 protein expression and this regulation may involve the JAK‐STAT pathway for signal transduction. This present study elucidates a link between OSM, MMP‐2 protein expression and specific signal transduction pathways in NR3 cells (H‐ras transformed fibroblasts capable of benign tumor formation). The mitogen activated protein kinase (MAPK) pathway and the protein kinase C (PKC) pathway are involved in the OSM mediated alterations in MMP‐2 protein expression in NR3 cells. The P‐I‐3 kinase pathway and the AKT pathway are apparently not involved in this OSM mediated signaling mechanism in NR3 cells. Treatment of NR3 cells with OSM also resulted in the activation of specific members of the JAK‐STAT signal transduction pathway. Protein expression levels of JAK 1,2,3 and STAT 1,2,3,4 in NR3 cells were determined by Western blot analysis following treatment of NR3 cells with 50 ng/ml OSM for 24 hr. JAK1,2,3, STAT‐2, and STAT‐3 were involved in NR3's MMP‐2 response to OSM but STAT‐1 and STAT‐4 showed no apparent involvement in the signaling pathway [N.S.E.R.C. (R.A.R.H) and Canadian Cancer Society (P.E.I. Division)].
ATP hydrolysis in rho: identifying the residues and their roles
Authors: Balasubramanian Krithika , Stitt Barbara
Published: June 2021

Escherichia coli transcription termination factor rho is an RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of rho are located at the interfaces of adjoining C‐terminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002).

Available rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identity of active site residues predicted to mediate ATP hydrolysis is uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ‐phosphoryl group, and coordinate the magnesium ion of MgATP, we are carrying out site‐specific mutagenesis on candidate residues which are conserved across bacterial species, and characterizing the relevant properties of the mutant proteins.

Support: Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health
Remedial antioxidant action of Withania somnifera on restraint stress‐induced oxidative damage
Authors: Al‐Qirim Tariq Musbah , Zafir Ayesha , Banu Naheed
Published: June 2021

Restraint stress is known to produce chronic physical and emotional stress, with impaired antioxidant defenses playing a key role at the intracellular level. Oxidative stress has been implicated in the complex pathology of stress‐related depression. Withania somnifera (L.) is an evergreen shrub categorized as an adaptogen in the traditional Indian Ayurvedic system of medicine. Thus, in the present study the aqueous solution of a root extract of Withania somnifera (WS) was evaluated for its effect on restraint stress‐induced pro‐oxidant status. Two different groups of rats were orally administered WS root powder (250 mg/kg) either alone, or after exposure to 4 hours of restraint stress. Control animals and stress alone‐treated rats were handled simultaneously. Restraint stress induced a decrease in circulating antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione‐s‐transferease (GST) as well as glucose levels. WS administration to stressed animals was observed to be effective in increasing these values towards their respective controls. Therefore, (WS) can be used as an effective antioxidant to combat stress‐induced oxidative damage and provide protection in a diverse range of stress‐related physical and mental disorders. This research is supported by Al‐Zaytoonah University and A.M. University.
Investigation of Menaquinone Biosynthesis in Mycobacterium Tuberculosis: Catalytic Mechanism and Inhibition Studies of MenB
Authors: Zhang Huaning , Tonge Peter
Published: June 2021

The lipid soluble redox cofactor menaquinone is an essential component of the mycobacterial respiratory chain and selective inhibitors of menaquinone biosynthesis are promising lead compounds for the treatment of latent tuberculosis (TB) infections. Menaquinone is synthesized from chorismate by the action of at least 7 enzymes including 1,4‐dihydroxynaphthoyl‐CoA synthase (MenB), which catalyzes an intramolecular Claisen (Dieckmann) condensation to form 1,4‐dihydroxynaphthoyl‐CoA from O‐succinylbenzoyl‐CoA. Currently we are comparing and contrasting the mechanism of the TB MenB enzyme with that of MenB from E. coli as well as with other members of the crotonase superfamily including 2‐ketocyclohexanecarboxyl‐CoA hydrolase (BadI), which catalyzes a retroDieckmann reaction. Crystallographic and kinetic data indicate that conserved catalytic residues play similar roles in the reactions catalyzed by MenB and BadI. However, the studies also suggest subtle differences in the mechanisms of the reactions catalyzed by the TB and E. coli MenB enzymes. In separate studies we recently identified several μM inhibitors of MenB using high‐throughput screening at the NRPB. These compounds are promising leads for developing novel inhibitors of menaquinone biosynthesis.
Probing the Mechanism of Cysteine Dioxygenase through Substitution of Conserved Active Site Residues
Authors: Imsand Erin , Ellis Holly R
Published: June 2021

Cysteine dioxygenase (CDO) catalyzes the oxidation of L‐cysteine to L‐cysteine sulfinic acid. This reaction is the rate‐limiting step in the eukaryotic taurine biosynthetic pathway, and taurine levels are directly correlated to cysteine dioxygenase activity. Taurine is an important antioxidant and low levels of taurine have been linked to a variety of cardiac, neural, and autoimmune disorders.

Catalytic mechanisms for CDO have been proposed based on recently published three‐dimensional crystal structures, although little direct spectroscopic or kinetic evidence is available to verify these proposals. To probe the mechanism, site‐directed mutagenesis was performed on conserved residues located in the active site of CDO adjacent to the iron center. Substitutions were made for Tyr58, Arg60, Ser153, and His155, replacing each residue with Ala. Kinetic parameters were established and compared to wild‐type to determine if any of the substitutions plays a role in catalysis. Several spectroscopic probes were used to characterize the metal coordination environment. Electron paramagnetic resonance (EPR) spectroscopy in combination with nitric oxide binding studies were performed to simulate the binding of dioxygen with or without the cysteine substrate. Amino acid residues critical to catalysis have been identified in these studies, and a mechanism for cysteine oxidation by CDO is proposed.
Mechanistic investigations of Lacticin 481 synthetase and its application as a general kinase
Authors: You Young‐Ok
Published: June 2021

Lantibiotics are a group of peptide‐based antimicrobial agents with unique structural characteristics which include unsaturated amino acids and thio‐ether rings. A bifunctional enzyme, lacticin 481 synthetase (LctM) is responsible for the maturation of a precursor peptide (LctA) to the lantibiotic lacticin 481. Experiments using MALDI‐TOF MS and Site‐directed mutagenesis revealed important residues of LctM which are responsible for dehydration of Ser and Thr residues in LctA. Briefly, Asp242 or Asp259 may be the base that accepts a proton from Ser/Thr of the substrate. Lys159 we suggest as a critical residue for phosphoryl transfer reaction and also aid phosphate elimination step as a general acid. Study also showed that two LctM mutants, Arg399Met and Thr405Ala, trapped phosphorylated Ser/Thr intermediates of LctA. We think that Arg399 or Thr405 work either as active site base which deprotonates the [alpha]‐carbon of a phosphate intermediate or activates another residue that is the actual base for deprotonation. We tested Arg399Met or Thr405Ala LctM for phosphorylation of Ser/Thr in peptides of various sequences and lengths. Based upon these results the Arg399Met and Thr405Ala mutants can be classified as general Ser/Thr kinase
Defining the Role of Active Site Residues in the Activity of Glyoxysomal Malate Dehydrogenase
Authors: Jean Sophonie , Bell Ellis
Published: June 2021

Although the structure of Malate Dehydrogenase has long been known and a mechanism proposed involving a his‐asp pair in the active site, little is known about the roles that residues further from the active site may play. Analysis of the active site cavity, both visually and by QM‐MM approaches, led to the construction of active site mutants and second sphere mutants [involving residues located 5–10A from the site of catalysis]. Using site directed mutagenesis, kinetics studies and x ray crystallography the roles of both residues directly in the active site and those further afield have been examined. H220 and D193 play well characterized roles in proton abstraction, however the pH dependence of the D193N mutant suggest other factors may be involved. Second sphere residues, N124, T152, T186, A183, G180, V164, N165, S166 and H90 have been mutated, expressed and studied by kinetics and stability studies. N165D has similar Vmax values at pH 7 & 8 but a marked increase in substrate inhibition at pH 7 and a reversal of the pH effects on Km relative to the native protein or S166A. The N165D and the S166A mutants have been crystallized and 3D structure determination is in progress. It is clear that a number of residues within this second sphere contribute to protein stability and aid in the fine tuning of the regulatory effects of substrate inhibition and pH Dependence.

Supported by NF Grant MCB 0448905 to Ellis Bell
Effects of pCMPS Modification of an Intramembrane Cysteine and Oxidative Crosslinking of Transmembrane Domains on the Human NTPDase 2 Activity
Authors: Chiang Wei‐Chieh , Knowles Aileen F.
Published: June 2021

Human NTPDase 2 is a cell surface glycoprotein that is anchored to the membrane by two transmembrane domains (TMDs) near the N‐ and C‐termini, while the active site resides in the extracellular domain. The enzyme is inactivated by low concentrations of detergents, higher temperatures, and its substrate, and is inhibited by p‐chloromercuripheylsulfonate (pCMPS). The mutant in which the only free cysteine residue is replaced by serine (C26S) is not inhibited by pCMPS, indicating that C26 is the target of pCMPS modification. Reaction of pCMPS with C26, which resides in the N‐terminal TMD, may disrupt TMD interaction that has been shown to regulate NTPDase 2 activity. We also generated mutants containing a single cysteine residue in the C‐terminal TMD with or without C26 to investigate the effects of cross‐linking on enzyme activity. Inter‐ and intramolecular oxidative cross‐linking of the cysteine residues in the TMDs result in significant decrease of the human NTPDase 2 activity. Therefore both TMD interaction and mobility are critical for maximal enzyme activity. However, immobilization of the TMD by intramolecular disulfide bond formation reduces the inactivation of the residual activity of human NTPDase 2 by detergent, higher temperatures and substrate. (Supported by the California Metabolic Research Foundation)
Cardiomyocyte‐restricted PPARδ deletion in PPARα null mice leads to cardiac hypertrophy and heart failure
Authors: Liu Jian , Wang Peiyong , Cheng Lihong , Brako Lawrence A. , Yang Qinglin
Published: June 2021

Mice with cardiac PPARδ deficiency exhibit basal fatty acid oxidation (FAO) and bioenergetic deficits, leading to cardiac hypertrophy and heart failure. While PPARα also regulates cardiac FAO gene expression, PPARα null mice display no basal cardiac phenotype. We crossed cardiomyocyte‐restricted the PPARδ−/− (CR‐PPARδ−/−) mice with the PPARα null mice to generate a mouse line that PPARδ is eliminated specifically from cardiomyocytes of the PPARα mice (DKO). QPCR revealed that transcript expression of FAO genes in PPARδ null hearts was depressed similarly to those in PPARα null hearts. Double knockout of both PPARδ and PPARα did not lead to further depression of selected FAO gene expression compared with those from PPARδ and PPARα single knockout hearts. The protein abundance of these FAO enzymes was similarly depressed among these mice. There was no overt basal pathophysiological change in PPARα null hearts. In contrast, pathophysiological changes in the DKO hearts were similar to those of CR‐PPARδ−/− hearts. DKO mice exhibited cardiac dysfunction, mitochondria abnormality and cardiac hypertrophy after 4–5 month of their ages. However, it is interesting that DKO mice exhibited generally delayed dilated cardiomyopathy with lipid overload when compared with the CR‐PPARδ−/− hearts. We conclude that PPARδ plays a more essential role than PPARα does in the heart at basal condition.
Manipulation of Dietary Essential Amino Acids Prevents High‐Fat Induced Non‐Alcoholic Fatty Liver in Mice
Authors: Noguchi Yasushi , Hara Yoshiko , Shikata Nahoko , Aleman Jose O. , Young Jamey D. , Yoshida Shintaro , Mori Masato , Kelleher Joanne K. , Takahashi Michio , Stephanopoulos Gregory
Published: June 2021

We examined the effect of dietary essential amino acids (EAA) on high fat‐induced fat accumulation and hepatic steatosis in C57BL6 mice fed a high‐fat diet (HFD) which normally leads to obesity, hyperlipidemia and hyperinsulinemia. We designed a novel EAA supplemented high‐fat diet (HFED) in which dietary amino acids in the high‐fat diet, were partially replaced by EAA mixtures increasing the amounts of Lys, Thr, Leu, Ile and Val, without altering dietary protein‐amino acid (19%) or fat (52%) content. Mice were fed either HFD or HFED for 2 months. Results: Caloric intake was not different for the two diets, yet a significant reduction of fat mass, postprandial insulin, leptin and cholesterol occurred in HFED. Moreover, for HFED mice, hepatic triglyceride and cholesterol were greatly reduced, and plasma lipoproteins were normalized. The underlying mechanism was investigated measuring gene expression and metabolic flux at 8 weeks. HFED reduced hepatic gene expression for SREBP‐1c and its downstream genes including glucokinase and lipogenic genes. Metabolic flux analysis using deuterated water indicated that HFED repressed both de novo lipogenesis and desaturase flux compared to HFD. In summary, this study demonstrates that EAA supplementation prevents high‐fat induced hepatic steatosis and supports a mechanism by which EAA supplementation suppresses lipogenesis.
Alterations of the triglyceride/fatty acid cycle contribute to the development of insulin resistance.
Authors: Croniger Colleen Marie , Hsieh Chang‐Wen , DeSantis David , Gornicka Aga , Millward Carrie
Published: June 2021

The triglyceride/fatty acid cycle is regulated by the phosphoenolpyruvate carboxykinase (PEPCK‐C) gene. PEPCK‐C gene is expressed primarily in the liver, kidney, adipose tissue as well as the mammary gland. The role of PEPCK‐C in the mammary gland, however, is not clearly understood. Analysis of epithelial cells from the mammary gland indicated that PEPCK‐C expression is induced by progesterone and is required for synthesis of triglyceride through the glyceroneogenesis pathway. Using a mouse model with a deletion of the PPAR binding site in the endogenous PEPCK‐C gene promoter (PPARE−/− mice), we have ablated the PEPCK‐C gene expression in the white adipose tissue (WAT), the mammary gland tissue and reduced PEPCK‐C expression in the brown adipose tissue (BAT). The PPARE−/− females are fertile and able to produce milk for their young; however, the triglyceride content of the milk from the PPARE−/− mothers contains 40% less than the wild type mice. The reduced triglyceride content in the milk has metabolic consequences. Pups from PPARE−/− mothers exhibit insulin resistance as early as 9‐days after birth and this insulin resistance persisted into adulthood. Analysis of the adult PPARE−/− mice by glucose clamps and 2‐deoxyglucose uptake have shown that the PPARE−/− mice have reduced glucose uptake and reduced AKT‐Thr308 phosphorylation. These data suggest that the glyceroneogenesis pathway in the mammary gland and adipose tissues contributes to overall development of insulin resistance.
Mitochondrial proteome of the pressure‐overload hypertrophied rat heart
Authors: Brownsey Roger William , Parsons Hannah L. , Kulpa Jerzy E. , Allard Michael F.
Published: June 2021

Cardiac hypertrophy induced by prolonged hemodynamic overload, leads to changes in myocardial energy metabolism including an acceleration of glycolysis and limitation in oxidation of long‐chain fatty acids and pyruvate. The current study tested the hypothesis that alterations in expression of mitochondrial proteins account for defective oxidative metabolism in hypertrophied hearts. Compensated cardiac hypertrophy was produced in rats by aortic constriction and the mitochondrial proteome was quantitated using amine‐reactive, isobaric tagging (iTRAQ®) reagents and tandem mass spectrometry. 250–300 proteins were identified; the largest functional groups being those involved in electron transport (50%), fatty acid metabolism (21%), the TCA cycle (14%) and amino acid metabolism (13%). The expression of 15 mitochondrial proteins was significantly increased in hypertrophied hearts. Among the proteins up‐regulated in response to hypertrophy were several involved in mediating or responding to oxidative stress. It is concluded that changes in expression of metabolic proteins do not directly account for alterations in substrate utilization in non‐failing hypertrophied hearts. Oxidative stress, induced by pressure overload, may be an important mediator of metabolic adaptation to pressure‐overload hypertrophy. Studies were supported by grants from the Canadian Institutes for Health Research.
Laser Capture Microdissection: Combining LCM with IHC to investigate NMDA receptor subunit composition
Authors: Curry John Aloysius , Brinkmann Hannah , Richard Virgile , Rigby Mike
Published: June 2021

The Neurokinin 1 receptor is a member of the tachykinin receptor sub‐family. It is distributed throughout the CNS, although to a lesser extent than its natural agonist, Substance P.

There is much evidence to implicate the NK1 receptor within the physiology of acute pain, such as the anatomical localisation of Substance P and NK1 receptors within lamina I of the spinal cord, as well as correlative increases in Substance P production and NK1 receptor internalisation in response to increasing noxious stimuli. Reproduction of in vivo efficacy in the clinic has, however, been unsuccessful.

Selective NR2 subunit mediated NMDA antagonism has also shown in vivo efficacy, expression of this subunit within the superficial lamina of the spinal cord adding anatomical evidence to support this mechanism. However, co‐expression of the NK1 receptor and the NMDA receptor has not been reported.

We have developed a methodology to allow the isolation of high quality RNA, via Laser Capture Microdissection, following the immunolabelling of specific proteins of interest.

Using this technology, we validated methodology via PCR of dissected samples, as well as looking at differential expression of the NR2A and NR2B receptor subunits on NK1 +ve and NK1 –ve samples taken from the rat striatum.
Positioning the cell division plane in fission yeast
Authors: Chang Fred , Padte Neal , Howard Martin
Published: June 2021

Both positive and negative spatial cues may ensure the proper placement of the cell division plane in both eukaryotic and prokaryotic cells. In Schizosaccharomyces pombe, the medial positioning of contractile ring assembly is determined by the position of the pre‐divisional nucleus. The nucleus signals to the cortex through mid1p, a protein required for ring placement. Mid1p is a peripheral membrane protein that forms a cortical band of dots overlying the nucleus in interphase and recruits other contractile ring factors in early mitosis. The position of the nucleus provides a positive spatial cue to direct mid1p localization. One negative spatial cue is pom1p, a DYRK‐class of protein kinase, which localizes to a gradient emanating from cell ends. However, pom1p is needed for inhibition only from non‐growing cell ends. We are now characterizing a second inhibitory mechanism responsible for inhibiting mid1p accumulation at the growing cell end. Time‐lapse imaging suggests that this mechanism involves both the disappearance of dots as well as the physical transport of dots away from the growing cell tip. These directed movements are intriguing, as they are much slower than known cytoskeletal‐based movements and are independent of actin cables and microtubules. These studies thus begin to reveal how at multiple systems in distinct cellular locations operate together to position mid1p and the cell division plane.
SCD1 deficiency decreases hepatic lipogenesis and improves insulin sensitivity in obese mice in the presence of leptin
Authors: Sampath Harini , Miyazaki Makoto , Liu Xueqing , Flowers Matthew T , Chu Kiki , Dobrzyn Agnieszka , Ntambi James M
Published: June 2021

Obesity greatly increases the risk for secondary conditions such as insulin resistance. Mice deficient in the enzyme stearoyl CoA desaturase‐1 (SCD1) are lean and protected from diet‐induced obesity as well as insulin resistance, despite marked hyperphagia. In order to determine if SCD1 deficiency ameliorates obesity‐induced insulin resistance, we introduced the SCD1 mutation into three mouse models of obesity:1) leptin‐deficient ob/ob, 2) leptin‐resistant Agouti (Ay/a) and 3) high‐fat diet‐induced obese (DIO) mice. SCD1 deficiency resulted in significantly lower body weight and adiposity in all three mouse models. Hepatic lipid accumulation and lipogenic gene expression were dramatically reduced by SCD1 deficiency in all mouse models of obesity. While these changes in body mass and composition translated to increased glucose tolerance and insulin sensitivity in Ay/a and DIO mice, SCD1−/−;ob/ob mice remained hyperglycemic and hyperinsulinemic, despite significantly decreased body weight, adiposity and hepatic lipid accumulation. Upon subcutaneous leptin infusion, SCD1 deficiency potentiated weight loss in both Ay/a and DIO mice without any changes in food intake. These results uncouple the effects of SCD1 deficiency on weight loss from those on insulin sensitivity and point to increased peripheral leptin sensitivity as a potential mechanism for increased insulin signaling in SCD1−/− mice.
RhlA diverts fatty acid biosynthetic intermediates to rhamnolipid formation
Authors: Zhu Kun , Rock Charles O.
Published: June 2021

Pseudomonas aeruginosa secretes rhamnolipid (RL) that functions in hydrocarbon uptake, motility and pathogenesis. The acyl moieties in RL are derived from type II fatty acid synthesis (FASII), but how intermediates are diverted to RL is unclear. We show that RhlA is the first step in RL synthesis and competes with the dehydratases of FASII for the β‐hydroxyacyl‐ACP. Purified RhlA forms one molecule of β‐hydroxydecanoyl‐decanoate from two molecules of β‐hydroxydecanoyl‐ACP and is the only enzyme required to form the lipid component of RL. More than 95% of the acyl groups in RL are β‐hydroxydecanoyl, thus RhlA functions as a molecular ruler to selectively extract 10‐carbon intermediates. Eliminating either FabA or FabI activity increases RL production in P. aeruginosa showing that slowing FASII allows RhlA to more effectively compete for intermediate. In E. coli, the rate of fatty acid synthesis increases 30% when RhlA is expressed to ensure continued formation of fatty acids destined for phospholipid. Previous studies have placed a ketoreductase (RhlG) before RhlA in RL pathway, however, our genetic, biochemical and structural studies show that RhlG has no role in RL formation. We conclude that RhlA and RhlB are both necessary and sufficient for RL formation and the carbon flux through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid. (supported by GM34496 and ALSAC)
Phosphatidylcholine Biosynthesis and Neuronal Differentiation
Authors: Banchio Claudia , Jackowski Suzanne
Published: June 2021

Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. Changes in gene expression mediate the metabolic redirection leading to neurite formation and function. Acceleration of membrane phospholipid synthesis is associated with neurite elongation and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. Our results indicate that membrane biogenesis is driven by the elevated expression of a subset of lipid biosynthetic genes following retinoic acid (RA)‐induced differentiation of Neuro2A cells. The transcription of genes encoding key enzymes in the CDP‐choline pathway of phospholipid biosynthesis is stimulated, including the Chka gene for choline kinase (CK) and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT). The promoter regions for both genes indicated common DNA sequences which are proposed to be binding sites for transcription factors (TFs) that coordinately activate expression in a RA‐dependent manner. Alteration of CK or CCT expression regulated PtdCho synthesis and neurite extension following RA treatment. These results indicate that PtdCho synthesis is important for RA‐dependent differentiation of Neuro2A cells.
Adipose‐specific knockout of acetyl‐CoA carboxylase 1 causes growth retardation and reduced de novo fatty acid synthesis in fat tissues
Authors: Mao Jianqiang , Yang Tao , Gu Ziwei , Finegold Milton J. , Lee Brendan , Wakil Salih J.
Published: June 2021

Fatty acid synthesis is essential to maintain energy homeostasis in animals. Adipose tissue is one of the major sites for de novo fatty acid synthesis and lipid storage. We generated mice with adipose‐specific inactivation of acetyl‐CoA carboxylase 1 (ACC1), the rate‐limiting enzyme in de novo fatty acid synthesis. The adipose (fat)‐specific ACC1 knockout (FACC1KO) mice were growth retarded in utero and grew more slowly than wild type (WT) mice until weaning, after which they gained weight in parallel with WT mice on a normal diet. Under lipogenic conditions, lipid accumulation in the adipose tissues of four‐month‐old FACC1KO mice was significantly decreased compared to WT mice, which is consistent with 50∼66% reduction in the ACC activity levels in these tissues. The activities of ACC and fatty acid synthase in the liver were not increased, indicating that hepatic de novo fatty acid synthesis was not changed to compensate for the reduction of ACC1 levels in the adipose tissues. Inactivation of ACC1 leads to ∼30% lower level of serum insulin‐like growth factor I (IGF‐I) compared to WT mice, which was accompanied by decreased chondrocyte proliferation in the growth plate, lower bone mineral density, and overall skeletal growth retardation of FACC1KO mice. Serum leptin level in FACC1KO mice was decreased ∼50% compared to WT mice; however, it did not counteract the osteopenic effects of IGF‐I on the bone. Although FACC1KO mice weigh less under normal diet, a high‐fat/high carbohydrate diet makes FACC1KO mice obese, just as WT mice.
Transcriptional regulation of the proximal promoter of the murine pyruvate carboxylase gene in liver cells
Authors: Jitrapakdee Sarawut , Rojvirat Pinnara , Boonsaen Thirajit , Sunyakumthorn Piyanate , Wallace John C
Published: June 2021

Pyruvate carboxylase (PC) plays a crucial role in intermediary metabolism i.e. gluconeogenesis, lipogenesis and insulin secretion. Mammalian PC genes are regulated by 2 promoters, the proximal (P1) and the distal (P2) promoters which mediate alternative transcription in tissue‐specific manner. P1 promoter is active in liver and adipocytes while the P2 is active in pancreatic islets. In islets, P2 promoter is regulated by Sp1/Sp3, USF1, 2 and Foxa2 while P1 promoter is regulated by the PPARgamma. P1 is active in both liver and adipocytes, suggesting that this promoter is regulated by the distinct sets of liver and adipocyte transcription factors. To investigate this, we identify the liver‐specific transcription factors that regulate PC expression. We generate a series of 5’‐nested deletion mutants of the 2.3 kb P1 promoter of the mouse PC gene followed by transient transfection in HepG2 cells. This result showed the presence of 3 regulatory regions, located in regions A (−166/−80), B (−166/−256) and C (−335/−603). Region A contains 3 sites for Sp1/Sp3 while the region C contains a putative binding site for the liver‐enrich nuclear factor, HNF4alpha which could potentially bind to its responsive element identical to the PPRE. We are investigating the molecular interaction between HNF4 alpha to this site as well as identifying the nuclear proteins that bind region B of the P1 promoter.
Alteration of NK Cell Signaling Pathways by Exposure of Human Natural Killer Cells to Dibutyltin
Authors: Odman‐Ghazi Sabah O. , Isom Erica Taylor , Whalen Margaret M.
Published: June 2021

Dibutyltin (DBT) is a widespread environmental contaminant. Detectable levels have been found in human blood. Natural killer (NK) cells are able to lyse tumor cells and virally infected cells. Previous studies have shown that DBT decreases the lytic function of NK cells. In this study we assessed the effects of those concentrations of DBT that decrease NK lytic function on the status of protein tyrosine kinases (PTKs) (Syk, Zap‐70, Src and Pyk), and Mitogen‐activated protein kinases (MAPKs) (p38, p44/42, JNK). Such studies will help elucidate the mechanism by which DBT decreases NK lytic function. NK cells that were exposed to (10, 5, 2.5, 1, 0.5 μM) DBT for 10 min did not show any significant changes in the levels or activation state of Syk, Pyk, or Src. Exposure of NK cells to 10 μM DBT for 10 min resulted in a significant increase in the phosphorylation of p38 (1.8 fold) and p44/42 (1.7 fold). A 6 h exposure to 10 μM DBT caused a significant increase in phosphorylation of p44/42 but no significant change in p38, phospho‐p38, p44/42, phospho‐JNK, or JNK levels. These data indicate that in vitro exposure to DBT, caused no changes in the levels or activation state of PTKs but caused significant activation the MAPKs, p38 and p44/42 after a 10min exposure to 10 μM DBT. This activation of p44/42 maintains out to 6 h. Supported by NIH grant 2S06GM0809228.
Pref‐1 Activates The MEK/ERK Pathway To Inhibit Adipocyte Differentiation
Authors: wang yuhui , kim kyung‐ah , kim jung‐hyun , sul hei‐sook
Published: June 2021

Pref‐1, an EGF‐repeat containing protein, is found in preadipocytes but absent in adipocytes. Pref‐1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref‐1 inhibition of adipognesis has been well studied in vitro and in vivo, the signaling pathway for Pref‐1 is not known. By using purified soluble Pref‐1 in mouse embryo fibroblasts (MEF), we investigated the mechanism of Pref‐1 in the inhibition of adipocyte differentiation. Pref‐1 increases MEK/ERK phosphorylation in a time‐ and dose‐dependent manner. Compared to wild‐type MEF, differentiation of Pref‐1 null MEF into adipocytes is enhanced. During adipocyte differentiation, both wild‐type and Pref‐1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. However, wild‐type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref‐1 level, is absent during differentiation of Pref‐1 null MEF. Prevention of this second increase in ERK phosphorylation by the MEK inhibitor PD98059 or by knockdown of ERK via siRNA enhances adipocyte differentiation. Conversely, treatment of Pref‐1 null MEF with Pref‐1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation. Our data indicate that Pref‐1 activation of MEK/ERK signaling is required for Pref‐1 inhibition of adipogenesis.
Comparative analysis of wild‐type and cAMP‐deathless S49 lymphoma cells identifies genes and pathways of cAMP/PKA‐promoted apoptosis
Authors: Zhang Lingzhi , Zambon Alexander C , Vranizan Karen , Pothula Kanishka , Conklin Bruce R , Insel Paul A
Published: June 2021

The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA‐promoted apoptosis by comparing wild‐type (WT) S49 lymphoma cells and the S49 variant, D‐ (cAMP‐deathless), which lacks cAMP‐promoted apoptosis but that has WT levels of PKA activity as well as cAMP‐promoted G1 growth arrest. Treatment of WT, but not D‐, S49 cells with 8‐CPT‐cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase‐3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D‐ cells incubated with 8‐CPT‐cAMP have similar, but non‐identical, extents of cAMP‐regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D‐ cells, respectively. WT and D‐cells differed in CREB phosphorylation, expression of the transcriptional inhibitor Icer and cAMP‐regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. We conclude that cAMP/PKA‐promoted apoptosis of lymphoid cells occurs via mitochondrial‐mediated events and that such apoptosis likely involves gene networks in multiple biochemical pathways. (Supported by the Leukemia and Lymphoma Society and the NIH).
Sodium butyrate induction of apoptosis in human fibroblasts in culture
Authors: Henry Amanda J , Wagner Rebecca l , Birckbichler Paul J
Published: June 2021

Tissue transglutaminase (tTg) is a multifunctional enzyme possessing two catalytic sites. One is a calcium‐dependent reaction that leads to formation of isodipeptide cross‐linkages. The cross‐linkages lead to the formation of a protein scaffold in cells undergoing apoptosis. GTP, a negative regulator for this cross‐linking activity, shifts the enzyme's catalytic activity to G‐protein activity.

Human embryonic fibroblast cells (WI‐38) and their simian virus‐transformed counterparts (VA13A) grow adherent in vitro. Quantification of the amount of tTg using a fibronectin binding assay showed that VA13A cells contained 0.04493 ng tTg/uL sample in the supernatant and a non‐detectable amount in the pellet fraction. This amount increased to 8.2622ng tTg/uL sample in the supernatant fraction after the VA13A cells were exposed to sodium butyrate. These results were confirmed by indirect immunofluorescence microscopy, electrophoresis and Western blotting.

Sodium butyrate induced apoptosis in VA13A cultures, but had no effect on apoptosis in WI‐38 cultures. Preliminary studies with sodium butyrate alone and in combination with calcium ionophore showed not only an increase in apoptosis but also an increased transglutaminase expression. Additionally, sodium butyrate treatment induced growth arrest in both cell lines and reverted the VA13A cells to a more “normal” morphology. These results taken collectively are indicative of a dual role of sodium butyrate; one involving growth arrest and the other apoptosis.
DNA aptamer as molecular recognition probe to selectively differentiate tumor cells
Authors: Sefah Kwame , Li Ying , Tang Zhiwen , Tan Weihong
Published: June 2021

Cell surface proteins play important roles in cancer pathogenesis and most of these receptors can serve as primary targets for the diagnosis and therapy of cancer. The identification of specific biomolecules as molecular signatures of tumor cells is critical and can be strategically used for clinical applications as well as for research. We recently have developed a novel cell‐based aptamer selection method (Cell‐SELEX) to specifically select a group of aptamers that can distinguish tumor cell lines derived from different cancer types. Molecular aptamers are DNA/RNA sequences that can recognize proteins, peptides and other small molecules with high affinity and specificity. Aptamers are selected from a large pool of oligonucleotide sequences by Systematic Evolution of Ligand by Exponential enrichment (SELEX). In this study, we report the development of group of DNA aptamers that identify different myeloid leukemia cell lines and colorectal cancer cell lines by binding to membrane surface proteins with dissociation constants in the nanomolar range. This approach has the potential to identify disease associated biomarkers, which may enable development of improved diagnostics as well as identification of new molecular signatures for drug development. It may also further the understanding of the molecular aspects of disease progression in various cancers.
Gene 1.7 of Bacteriophage T7 Confers Sensitivity of Phage Growth to Dideoxythymidine
Authors: Tran Ngoc Quang , Tabor Stanley , Rezende Lisa F. , Qimron Udi , Richardson Charles Clifton
Published: June 2021

Bacteriophage T7 has long been used as a model for studying DNA replication. One of the interesting features of this system is the ability of T7 DNA polymerase to efficiently incorporate dideoxynucleotides into DNA, resulting in chain termination. We show that dideoxythymidine (ddT) present in the media at levels not toxic to the host Escherichia coli results in inhibition of phage T7 growth. We have isolated 100 T7 phage that are resistant to ddT. Surprisingly, all contain a mutation in T7 gene 1.7, a nonessential gene of unknown function. When T7 gene 1.7 is expressed from a plasmid, suppressor T7 phage that are resistant to ddT still arise; analysis of 36 of these mutants reveals that all have mutations in gene 5, that encodes the T7 DNA polymerase. The strongest suppressors change tyrosine 526 of gene 5 to phenylalanine. This mutation is known to dramatically increase the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. These results suggest that in the presence of the T7 gene 1.7 protein, ddT is inhibitory to phage T7 because it acts as a chain terminator for DNA synthesis. In support of this, whereas DNA synthesis in E. coli infected with wild‐type T7 is inhibited by ddT, no inhibition is observed in the absence of gene 1.7. Overexpression of gene 1.7 from a plasmid renders E. coli cells sensitive to ddT in the absence of T7 phage, indicating that gene 1.7 protein does not require other T7 proteins to confer sensitivity to ddT. We propose that gene 1.7 protein plays a critical role in the conversion of ddT to ddTTP.
Peptides Inhibitors of F11R/JAM‐A Adhesion Molecules
Authors: Clement Cristina Corina , Babinska Anna , Kornecki Elizabeth , Ehrlich Yigal H , Philipp Manfred
Published: June 2021

F11‐R receptor was characterized as an adhesion protein, called also aka JAM‐A (or JAM‐1), which under normal physiological conditions is expressed constitutively on the surface of the platelets and localized within tight junctions of endothelial cells (EC). The utilization of specific F11R/JAM‐A peptide antagonists and recombinant proteins supported the role of F11R/JAM‐A in the process of platelets adhesion to inflamed endothelial cells and identified plaque formation leading to inflammatory thrombosis and atherosclerosis where the platelets have a critical influence in the progression and development of the cardiovascular disease. Thus, the development of new drugs antagonizing the F11R/JAM‐A function could evolve as an effective strategy for the treatment of atherosclerosis, heart attacks and stroke. We present one of the first trials toward development of peptide‐based inhibitors of F11R/JAM‐A function. Among many trials, the peptide D‐Lys‐Ser‐Val‐Ser‐D‐Arg‐Glu‐Asp‐Thr‐Gly‐Thr‐Tyr‐Thr‐Cys‐CONH2 proved to be a potent inhibitor of human platelets aggregation in vitro. Further molecular docking experiments showed that this peptide makes favorable hydrophobic and electrostatic interactions within the proposed binding site of JAM‐1 (X‐Ray structure 1nbq.pdb was used as template).