bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatase regulation of cellular motility in the tumor microenvironment
Authors: Walsh Jarrett E , Young M. Rita I
Published: June 2021

An increasing number of anti‐tumor therapies have focused on angiogenesis pathways. Targeting various aspects of angiogenesis has been shown to disrupt tumor growth and reduce tumor progression and recurrence. Our studies show that treatment of microvascular endothelial cells with tumor‐conditioned media, and TGF‐β specifically, leads to alterations in the activity of protein phosphatases PP1 and PP‐2A. We hypothesize that dephosphorylation of focal adhesion complex targets lead to an increase in TGF‐β –induced endothelial motility. Specifically, our studies have shown that paxillin, a crucial focal adhesion adapter protein is a novel PP1 binding partner. Paxillin phosphorylation has been shown to induce both focal adhesion stability and turnover, therefore site‐specific phosphatase activity is a key regulatory mechanism. Elucidation of mechanisms of phosphatase regulation and interaction in the tumor microenvironment will provide insight into mechanisms of angiogenesis and novel targets for therapeutics. This work is supported by Dept. of Veteran's Affairs and NIH funding.
Expression of NUANCE, a potential novel oncogene, is inhibited by nonsteroidal anti‐inflammatory drugs (NSAIDs) in human colorectal cancer cells
Authors: Liggett Jason Lee , Choi Chang Kyoung , Donnell Robert L , English Anthony E , Kihm Kenneth David , Baek Seung Joon
Published: June 2021

NSAIDs are well known for treating inflammatory disease and have been reported to have an anti‐tumorigenic affect. Our lab has previously shown that NSAIDs also induce other anti‐tumorigenic genes including NSAID activated gene (NAG‐1). This makes NSAIDs a prime candidate for further study. HCT‐116 cells treated with sulindac sulfide (SS) showed dramatic morphological changes under differential interference contrast microscopy as well as weakened cellular adhesion as measured by micro‐impedance. Microarray analysis was performed using HCT‐116 cells treated with SS, and NUANCE (NUA), a novel connection between the actin skeleton and the nuclear envelope, was selected based on its novelty in relation to cell adhesion and biogenesis. SS diminished NUA mRNA expression by one third and various other NSAIDs also showed inhibition of NUA. Additionally, immunohistochemistry showed higher levels of NUA in tumors of many tissues. Further micro‐impedance experiments on cells with shRNA NUA showed a reduction in cellular adhesion. Thus, our data suggest that NUA may be a potential novel oncogene in human colorectal cancer cells and NSAIDs could decrease its expression.

ACS RSG‐06‐124‐01
Cold adaptation in the yeast Saccharomyces cerevisiae
Authors: Moore Peter , Loertscher Jennifer
Published: June 2021

Although the ability of the yeast Saccharomyces cerevisiae to adapt to environmental stresses has been widely studied, relatively little is know about adaptation to cold temperatures. Previous studies by Loertscher and co‐workers have shown that proteins required for endoplasmic reticulum‐associated degradation (ERAD), such as Ubc7, are also required for cold adaptation and may work by affecting membrane sterol composition. Using both solid and liquid growth media, a panel of deletion mutants lacking endoplasmic reticulum‐related proteins was tested for sensitivity to cold temperatures. Deletion mutant orm2Δ exhibited notable sensitivity to cold when grown on either solid or liquid medium as well as impaired growth at normal temperatures after being grown in cold temperatures for an extended period of time. Orm2 is an ER‐localized protein and may be involved in the cellular response to unfolded proteins. We hypothesize that Orm2 may have a role similar to Ubc7 in helping cells adapt to cold temperature through direct or indirect modification of membrane sterol content. Examining the membrane sterol content of orm2Δ mutants as compared to wild‐type will help determine the role of Orm2 in cold adaptation.
Genetic Contribution to Alcoholic Steatohepatitis
Authors: DeSantis David Andrew , Pisano Sorana , Millward Carrie A. , Gornicka Aga , Soda Pamela , McMullen Megan R. , Pritchard Michelle T. , Nagy Laura E. , Croniger Colleen M.
Published: June 2021

Chronic excessive alcohol intake is associated with multiple liver defects ranging from mild fatty liver (steatosis) to advanced cirrhosis. The mechanism by which chronic ethanol intake causes fatty liver appears to be a complex and multifactorial process. To better understand the contribution of genetics we have studied the response to alcohol in two inbred strains of mice, C57BL/6J and A/J. These mice were fed alcohol using the Lieber De‐Carli high‐fat control and ethanol containing diet. The C57BL/6J mice develop steatosis while the A/J mice are protected. We analyzed the expression of the transcription factor CCAAT/enhancer binding protein â (C/EBPâ) that has been implicated in the development of fatty liver and required for expression of alcohol dehydrogenase gene (ADH). In both mouse strains C/EBP â gene expression was increased with alcohol consumption but in the A/J mice the liver inhibitor protein (LIP) was increased while the C57BL/6J mice had an increase of the liver‐enhanced activator protein (LAP). Markers of liver damage, alanine aminotransferase (ALT), and CYP2E1 were elevated in both C57BL/6J and A/J mice. This suggests that there is a genetic contribution for the development of steatosis and that the elevation of ALT levels is decoupled from development of steatosis in the A/J mice. Further analysis is necessary for better understanding the mechanism of liver injury during alcohol consumption.
Prevention of HIV protease inhibitor‐induced inflammatory response and ER stress by berberine in macrophages
Authors: Liang Guang , Chen Li , Wu Xudong , Gurley Emily , Studer Elaine , Wang Xuan , Hylemon Phillip B , Pandak William M , Li Xiaokun , Zhang Luyong , Zhou Huiping
Published: June 2021

Complementary and alternative medicines are becoming increasingly attractive approaches for the treatment of HIV infection and the complications associated with highly active anti‐retroviral therapy (HAART). Our previous studies suggest that HIV PI‐induced ER stress and inflammatory response in macrophages represents an important cell signaling mechanism of HIV PI‐induced dyslipdemia and atherosclerosis. Berberine (BBR), an isoquinoline alkaloid isolated from many medicinal herbs, has various beneficial effects on the cardiovascular system. The aim of the present study was to determine if BBR was able to prevent HIV PI‐induced inflammatory response, ER stress and lipid accumulation in macrophages. J774A.1 cells and primary mouse peritoneal macrophages were used in this study. Cells were pre‐treated with BBR for 4h, and then treated with individual HIV PI. The mRNA and protein levels of TNF‐á and IL‐6 were measured by real‐time RT‐PCR and ELISA, respectively. Activation of ER stress was detected by Western blot. Intracellular free cholesterol and lipid were detected using filipin and oil‐red O staining. Results: BBR significantly inhibited HIV PI‐induced TNF‐á and IL‐6 expression and ER stress response. HIV PI‐induced free cholesterol and lipid accumulation was also markedly inhibited by BBR. Conclusions: BBR has a potential therapeutic effect on HIV PI‐induced dyslipdemia and atherosclerosis.
Appearance of the Ser‐His‐Asp catalytic triad at the putative active site domain of the acyl coenzyme A:cholesterol acyltransferase (ACAT) enzymes
Authors: Das Akash , Davis Matthew A , Rudel Lawrence L
Published: June 2021

To understand the molecular mechanism of cholesterol esterification reaction we sought to determine the active site residues of the ACAT enzymes using chemical modification and site directed mutagenesis approaches. The ACAT enzymes are 56% similar after N‐terminal 100 amino acids (AA). The sequence similarity within ACAT1 AA 386 to 462 and ACAT2 AA 364 to 440 is 86% and this region contains two conserved motifs FYXDWWN and HEY. We suspect this region has the putative active site domain of the enzymes due its high sequence conservation from yeast to humans. As these enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of S‐H‐D residues. Our results show that in ACAT1, S456, H460 and D400 are essential for activity of the enzyme. In contrast, in ACAT2, only H438 was necessary for enzymatic activity. The A2S434A mutant retained about 20% of the wild type activity whereas A2D378 mutants were not expressed after mutagenesis. Based on these data, we propose the active site may be different among the two ACAT enzymes. In ACAT1, a S‐H‐D catalytic triad serves as the active site whereas in ACAT2 there may be a H‐D catalytic diad in which the esterification reaction occurs in a substrate assisted manner with the 3β OH group of cholesterol acting as the nucleophile. (NIH:HL‐49373)
Statins Induce Plasma Levels of Proprotein Convertase Subtilisin/Kexin Type 9
Authors: Lagace Thomas A. , Bashmakov Yuriy , Anderson Norma N. , McNutt Markey C. , Grefhorst Aldo , Ho Y. K. , Horton Jay D.
Published: June 2021

LDL receptor (LDLR) number is regulated transcriptionally by sterol regulatory element‐binding protein (SREBP)‐2. Studies have shown that inhibitors of HMG‐CoA reductase (statins) used to treat hypercholesterolemia trigger a regulatory response in cells leading to elevated levels of active SREBP‐2, thereby increasing LDLR expression. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a SREBP‐2 regulated protein secreted by the liver, binds to the LDLR and mediates its degradation. Thus, induction of PCSK9 may counter‐balance the effect of statins on liver LDLR protein levels. We demonstrate that simvastatin‐fed rats exhibit elevated levels of nuclear SREBP‐2 in liver and increased transcription of SREBP‐2 target genes, including PCSK9. This was associated with a >3‐fold increase in circulating PCSK9 levels compared to control rats. In human studies, 13 subjects with moderate to slightly elevated plasma LDL‐cholesterol levels (127 – 238 mg/dl) were administered a statin for 7 days (Lipitor ‐ 40 mg/day), which resulted in a 25% decrease in plasma cholesterol levels. ELISA measurements of circulating PCSK9 concentrations showed a mean increase of 35% in these subjects. These studies demonstrate that circulating PCSK9 levels are reflective of liver SREBP‐2 activity and the induction of PCSK9 by statins may partially offset their efficacy in lowering plasma LDL‐cholesterol levels. Supported by CIHR.
Membrane targeting by the GRP1 PH domain
Authors: He Ju , Vora Mohsin , Stahelin Robert , Zhang Honglu , Prestwich Glenn , Kutateladze Tatiana G.
Published: June 2021

Pleckstrin homology (PH) domains regulate a wide range of biological processes including cell proliferation and survival, signal transduction, membrane trafficking and cytoskeletal rearrangement through binding to phosphoinositide lipids. The PH domain of GRP1 (general receptor for phosphoinositides) binds PI(3,4,5)P3 with high affinity and specificity, however the overall molecular mechanism of membrane recruitment and docking by GRP1 remains unclear. Here we show that the membrane anchoring of the GRP1 PH domain involves the specific recognition of PI(3,4,5)P3, which is facilitated by non‐specific electrostatic interactions with acidic lipids and accompanied by a hydrophobic insertion into the bilayer. Our data indicate that the association of the GRP1 PH domain with membranes is regulated by the acidic cellular environment. The membrane insertion interfaces, binding parameters and regulation of the PI(3,4,5)P3 binding by pH are investigated by NMR, liposome binding, monolayer surface tension, and mutagenesis experiments. The functional significance of the pH sensitivity and the effect of acidic and basic media on the phosphoinositide recognition, hydrophobic insertion and non‐specific electrostatic contacts are established.
The Subcellular Localization of VACM‐1/CUL 5 and NEDD8 proteins in Endothelial Cells in vitro
Authors: Dabney Lida R , Burnatowska‐Hledin Maria A
Published: June 2021

VACM‐1, also known as cul‐5, attenuates cellular growth by down‐regulating the production of cAMP and decreasing phosphorylation of mitogen‐activated protein kinase. Cellular localization of VACM‐1 is cell cycle‐dependent; during G1/0 phase it localizes to the membranes, it disappears during S phase, and during M phase it localizes to the nuclear region and to the cytosol. The subcellular localization and biological activity of VACM‐1 may depend on its modification by a ubiquitin‐like protein, NEDD8. Whether NEDD8 expression is cell cycle dependent or if it co‐localizes with VACM‐1 has not been studied. This study aimed to determine whether NEDD8 expression is cell cycle dependent and if in cells transfected with VACM‐1 cDNA its expression co‐localizes with that of the VACM‐1 protein. Double‐immunofluorescent staining experiments were performed using antibodies directed against the VACM‐1 and NEDD8 proteins. Our results indicate that unlike VACM‐1 protein, NEDD8 is present through the cell cycle and co‐localizes with VACM‐1 during the M phase. These results also suggest that NEDD8 may also modify other proteins. Supported by NCI (R15CA104014), Dept of Chemistry and NSF‐URC grant No. 0629174.
Inhibition of phosducin‐like protein–catalyzed G protein βγ dimer assembly impedes the translation of nascent Gβ and Gγ polypeptides
Authors: Sono‐Koree Nana Kwasi , White David W. , Willardson Barry M.
Published: June 2021

Phosducin‐like protein, a ubiquitously expressed member of the phosducin gene family, is a positive regulator of G protein signaling by virtue of its ability to help assemble Gβ and Gγ subunits. Our lab has shown that PhLP mediates the release of nascent Gβ from the cytosolic chaperonin CCT and the subsequent interaction of Gβ with Gγ. Further work on the mechanism of Gβγ assembly has uncovered an intriguing observation. When PhLP is inhibited by RNA interference or a dominant negative variant, not only is Gβγ assembly blocked but de novo synthesis of both Gβ and Gγ is also decreased significantly. This decrease is not a result of changes in the mRNA levels of Gβ and Gγ, nor is it a result of increased degradation of their nascent polypeptides. Thus, it must come from a decrease in translation. The effect is specific because there is no change in actin translation when PhLP is inhibited. We are currently investigating the mechanism by which PhLP controls Gβ and Gγ translation. It is possible that a feedback mechanism exists so that when Gβγ assembly is blocked, the synthesis of the two subunits is reduced to avoid producing unstable free Gβ and Gγ polypeptides.
Regulation of TGF‐beta signaling by RGS3
Authors: Yau Douglas M. , Sethakorn Nan , Taurin Sebastien , Kregel Steven , Dulin Nickolai
Published: June 2021

Regulator of G protein signaling RGS3 belongs to a family of RGS proteins that contain the homologous RGS domain which binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. Through this mechanism RGS3 regulates the signaling mediated by a variety of Gq and Gi coupled receptors. In this study, we found that RGS3 interacts with the novel partners, Smad2, Smad3 and Smad4 ‐ the transcription factors that are activated through a transforming growth factor‐beta (TGF‐beta) receptor signaling. This interaction is mediated by Smad's Mad homology domain 2 (MH2) and by the region of RGS3 outside of the RGS domain. Overexpression of RGS3 results in inhibition of Smad‐mediated gene transcription. RGS3 does not affect TGF‐beta ‐ induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for Smad's transcriptional activity. Functionally, this translates to inhibition of TGF‐beta – induced myofibroblast differentiation by adenovirus‐mediated overexpression of RGS3. In conclusion, this study identifies a novel, non‐canonical role of RGS3 in regulation of TGF‐beta signaling through its interaction with Smad transcription factors.
A Conserved Phosphatase Cascade that Regulates Nuclear Membrane Biogenesis
Authors: Kim Youngjun
Published: June 2021

A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contain the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus. (Satow R., et al (2002) Biochem. Biophys. Res. Commun. 295, 85–91). Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains, they show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1∆ yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p (Santos‐Rosa et al (2005) EMBO J 24, 1931–41) and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis and that this cascade is conserved from yeast to mammals
Phosphorylation Analysis of the Embryo in Germinating Maize Seed
Authors: Lu TianCong , Meng LingBo , Shen Zhuo , Wang BaiChen
Published: June 2021

Protein phosphorylation reactions are an integral part of the regulation of enzymatic activity in diverse biological systems. Relatively little is currently known about such regulatory control mechanisms occurring during plant seed germination. In the present study there is reported a total of 197 protein phosphorylation sites in 144 proteins identified in the embryo of germinating Maize using LC‐MS/MS following phosphoprotein enrichment on passage through a strong cation exchange resin column. Using a shotgun technique a total of 1,700 unique proteins were successfully identified. This included: 90 protein kinases (through searching in Maize and Rice databases and 27 phosphatases (through searching in the same two databases). The transcript level of the protein kinases and phosphatases were assayed with quantitative real time PCR at different time points (including dry seeds, 24 hrs. after seed imbibition and during seed germination. 14 protein kinases and 5 phosphatases were found to be dramatically increased during this period, suggesting these enzymes in phosphorylated form must be involved in the regulation of seed germination processes. Supported by a governmental research grant to Northeast Forestry University, Harbin, Heilongjiang, PR China.
Autophosphorylation of Protein Kinase C Facilitates its Ubiquitination and Down‐regulation
Authors: Gould Christine , Newton Alexandra
Published: June 2021

Protein kinase C (PKC) is a Ser/Thr kinase that tranduces signals generated from the second messengers, diacylglycerol and calcium, the products of receptor‐mediated lipid hydrolysis. PKC has three modes of regulation: phosphorylation, cofactor binding, and localization. Phosphorylation of PKC regulates its maturation, activation, and signaling properties. Autophosphorylation at two conserved sites in the carboxyl‐terminus of PKC is a major step in the generation of catalytically competent enzyme. PKC has been show to readily autophosphorylate at additional sites in vitro, and their physiological role is unknown. Chronic activation of PKC that occurs with phorbol esters, functional analogues of diacylglycerol, results in the degradation and “downregulation” of the enzyme. Here, we examine the role of autophosphorylation at two sites preceding the autoinhibitory pseudosubstrate sequence in the phorbol ester‐mediated downregulation of PKC βII. We find that mutating the in vitro‐identified autophosphorylation residues, Ser16 and Thr17, to generate a phospho‐mimic (S16E/T17D) increases the rate of PKC degradation following phorbol ester stimulation. This mutant was more rapidly dephosphorylated and ubiquitinated in comparison to wildtype PKC. In contrast to its altered degradation, the phospho‐mimic was processed by phosphorylation similarly to wildtype and had similar activity. Generation of a phospho‐specific antibody to Thr17 indicated that PKC autophosphorylates in vitro as well as in vivo in response to phorbol esters in both an overexpression system as well as with endogenous PKC in the K562 lymphona cell line. Based on these data, we propose that PKC is additionally autophosphorylated following activation by phorbol esters, an event that promotes the downregulation of the enzyme.

This work was supported by NIHGM43154.
The Function of Nudix Hydrolase Ysa1 in Saccharomyces cerevisiae
Authors: Tong Lei , Denu John
Published: June 2021

The silent information regulator 2 (Sir2) family catalyzes NAD+‐dependent protein deacetylation and functions in the regulation of gene silencing, chromatin structure, and longevity. O‐acetyl‐ribose (OAADPr) is a unique product of the Sir2 reaction. In Saccharomyces cerevisiae, we discovered three activities that metabolize OAADPr, an esterase, an acetyl transferase, and a Nudix hydrolase, Ysa1. In vitro, Ysa1 possesses comparable activity towards ADP‐ribose (ADPr) and OAADPr. We compared ADPr/OAADPr metabolizing activities among the wild type, Ysa1 deletion, and Ysa1 over expression strains. These data suggest that Ysa1 is a major ADPr/OAADPr metabolizing enzyme in vivo. N‐acetyl–ADPr analogs were utilized to indicate that OAADPr is directly hydrolyzed by Ysa1, and not through a pathway that first involves conversion of OAADPr to ADPr by the esterase. Using a TAP‐Ysa1 strain and cell fractionation, we localized the TAP signal to mitochondria. We propose that Ysa1 controls ADPr/OAADPr levels and is important for maintaining mitochrondrial function during cellular stress. Research supported by NIH.
Investigation of the Interactions of Polyhedral Borane Anions with Serum Albumins
Authors: Blass Devin , Matthews Barrett , Booth Rachell E , Feakes Debra A
Published: June 2021

Boron neutron capture therapy (BNCT) is a type of radiation therapy used in the treatment of cancerous tumors such as gliboblastoma multiforme and metastatic melanoma. BNCT is dependent on the site specific delivery and retention of boron‐10 atoms within the tumor. The ability of polyhedral borane anions to form covalent bonds with biochemical macromolecules appears to be critical for boron retention within the tumor cell. The polyhedral borane anions [B20H17OH]4−, [B20H18]2−, and [B20H17SH]4− were allowed to react with serum albumins, and the reaction products were investigated using polyacrylamide gel electrophoresis (Native‐PAGE), matrix assisted laser desorption ionization time of fight (MALDI‐TOF), electrospray ionization and nuclear magnetic resonance (NMR). The formation of disulfide linkage between [B20H17SH]4− and the free cysteine residue in the albumin was observed while [B20H17OH]4− and [B20H18]2− showed no evidence of covalent bonding. The understanding of covalent binding of polyhedral borane anions will aid in the synthesis and design of agents used in BNCT.
Antibacterial Activity of New Coumarins
Authors: Govori Sevdije R. , Spahiu Sabrije , Haziri Arben
Published: June 2021

4‐(4‐Bromo‐thiazol‐2‐ylamino)‐3‐coumarincarbaldehyde (1), 4‐(4‐Methoxy‐benzothiazol‐2‐ylamino)‐3‐coumarincarbaldehyde (2) and 4‐(2‐Chloro‐6,7‐dimethoxy‐quinazolin‐4‐ylamino)‐3‐coumarincarbaldehyde (3) are synthesized in very good yields, by nucleophilic substitution with aminoheteroaryl derivatives on 4‐chloro‐3‐carbaldehyde. The antibacterial activity of coumarin was tested against: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853.

The compounds showed strong to moderate activity in reducing the microbial growth. The inhibitory effects of compounds were affected by their substitution patterns. All compounds showed the most effective antibacterial activity against Escherichia coli ATCC 25922.
Genistein reduced invasive activity of human MCF‐7 and MDA‐MB 231 breast carcinoma cells through decreased tight junction permeability and regulation of related tight junction proteins
Authors: Kim Sung Ok , Lee Insong James , Kim Mee‐Kyung , Choe Won Kyung , Choi Yung Hyun
Published: June 2021

We investigated whether overexpressed claudin‐3 and ‐4, a component of tight junctions (TJs), repressed invasion activity in MCF‐7 and MDA‐MB 231, when treated with genistein. Genistein inhibited the cancer cell growth and tightened TJs in these cells as demonstrated increase in transepithelial electrical resistance and decreased in paracellular permeability of mannitol. The activities of MMP‐2 and ‐9 in cancer cells were decreased by treatment with genistein and occurred in a time dependent manner. Those of MMP‐2 were observed strong compared with those of MMP‐9 in both cell lines and activities and protein levels of MMP‐2 in MDA‐MB 231 were higher than those of MCF‐7. Concurrently, genistein repressed the levels of claudin‐3 and ‐4 proteins and also decreased metastasis related gene expressions, such as IGF‐l and snail and, reversely, increased that of TSP‐1 and E‐cadherin in cancer cells. The inhibitory effect of genistein on invasive activity was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in invasion assay. And also we validated with using claudin‐3 siRNA on invasion activity of breast cancer calls. The demonstration of ability of genistein to tighten TJs and repression of claudin‐3 and ‐4 protein levels points to a novel mechanism by which genistein and possibly other phytochemicals may prevent cancer and metastasis.
Prevention of palmitic acid‐induced lipotoxicity by Morinda citrifolia (noni)
Authors: Nerurkar Pratibha Vivek , Eck Philip
Published: June 2021

Lipotoxicity, or accumulation of lipids in non adipose tissue, has been implicated in the pathogenesis of obesity‐associated insulin resistance. Lipotoxicity‐associated mitochondrial and/or ER stress are key events leading to defects in insulin signaling and glucose transport. Morinda citrifolia (noni), has been traditionally used to treat diabetes, infections and cancer. Preliminary studies from our laboratory indicate that noni juice improves high fat diet‐induced plasma fatty acid (FA) levels and insulin resistance in mice. We therefore tested the hypothesis that noni juice will alleviate FA‐mediated inhibition of glucose transport and toxicity in skeletal muscle. In brief, mouse muscle cells, C2C12, were treated with palmitic acid (PA) and noni juice at varying concentrations and analyzed for cell proliferation, free fatty acid and glucose uptake, production of reactive oxygen species (ROS) and cell death. Our results indicate that noni inhibits PA‐induced lipotoxicity by inhibiting ROS production and mitochondrial damage. Such studies are significant as they have potential as prevention strategies, and use as nutraceuticals to promote optimal health. [This work was supported by USDA CSREES (2004‐34135‐15182) and RCMI Program, NIH/ NCRR (G12 RR003061)].
Functional role of human natural killer cell receptor 2B4 (CD244) isoforms
Authors: Mathew Stephen O , Rao Krithi K , Bambard Nowland D , Mathew Porunelloor A
Published: June 2021

Natural killer (NK) cells are a subpopulation of lymphocytes that represent the first line of defense against stressed, virally infected and malignant cells. NK cell functions are controlled by a balance between positive and negative signals through various receptors. We have identified, cloned and characterized the 2B4 (CD244) receptor in mice and human. 2B4 interaction with its ligand CD48 regulates NK, T and B cell functions and thus plays an important role in various immune responses. Human NK cells express two isoforms of 2B4, h2B4‐A and h2B4‐B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4‐A and h2B4‐B. Our data demonstrate that these two isoforms differ in their binding affinity for CD48 and this result in differential cytotoxic activity by NK cells against tumor targets. h2B4‐A was able to mediate natural cytotoxicity against CD48 expressing K562 target cells confirming that h2B4‐A is an activating receptor, whereas h2B4‐B showed no effects. Analysis of the predicted 3‐D structure of the two isoforms showed conformational differences which may be responsible for their differences in binding affinity to CD48. Thus differential expression of 2B4 isoforms by NK cells may regulate immune response mediated through 2B4‐CD48 interactions. This work was supported by the NIH grant CA85753.
Itk regulates T cell signaling through localization of active Cdc42
Authors: Singleton Kentner L. , Fowell Deborah J. , Wuelfing Christoph
Published: June 2021

The tyrosine kinase Itk can regulate actin accumulation at the T cell/APC interface, elevation of the T cell intracellular calcium concentration, and IL‐4 secretion. The cellular mechanism Itk uses to control these diverse functions is unresolved. Here we have identified Itk as a general regulator of the spatiotemporal organization of T cell activation: Itk controls the localization of the TCR, PLCγ, Cdc42, and Actin. Cdc42 localization is of particular interest, as not the cellular activity of Cdc42 but only its localization to the center of the T cell/APC interface was dependent on Itk. This control of the localization of active Cdc42 is a central part of the cellular mechanism of Itk function: Providing active Cdc42 selectively at the center of the T cell/APC interface in Itk‐deficient T cells could restore Itk‐dependent aspects of T cell activation. Providing Cdc42 without such targeting as a means to raise Cdc42 activity in the entire T cell could not. These data are of wider relevance, as they prove that the control of the localization of a signaling intermediate without affecting its activity can be a central part of the regulation of cellular activation.

Supported by the NIH.
Phosphorylation status of Polypyrimidine Tract‐Binding Protein (PTB) during a time course of T cell activation and its role in regulating the post‐transcriptional stability of CD40L mRNA
Authors: Vavassori Stefano , Covey Lori
Published: June 2021

CD40L is an essential accessory molecule for regulating a balanced immune response. Its transient expression on CD4+ T cells is regulated both at the transcriptional and post‐transcriptional level. Our previous work has shown the existence of a protein complex (Complex I) capable of binding to cis‐acting elements within the 3′ untranslated region (3′UTR) of CD40L mRNA. PTB and nucleolin were found to be major components of Complex I, which bound to a region of CD40L 3′UTR coincident with an increase in CD40L mRNA stability. To confirm the role of Complex I in stabilizing the CD40L mRNA we used an inducible shRNA system to knock‐down PTB and found that a decrease in PTB caused a drop in the CD40L mRNA half life. Further experiments using a CD4+ TH0 cell clone, SPB.21, revealed that Complex I binds to and stabilizes CD40L mRNA at later times of TCR engagement although PTB is present in the cytoplasm of both resting and activated cells. We therefore tested whether differential PTB phosphorylation could dictate Complex I activity and found that an increase in phosphorylation of one of the two PTB isoforms takes place concomitant with complex binding to the CD40L mRNA. These experiments confirm that differential phosphorylation of cytoplasmic PTB is a key event regulating CD40L mRNA stability in activated T cells. Experiments to identify kinases regulating this process are ongoing. Support by PO1‐AI57596 and The American Heart Assoc.
Modulation of T Cell Activation by DRAK2
Authors: Newton Ryan Henry , Walsh Craig M.
Published: June 2021

As a negative regulator of T cell activation, the serine/threonine kinase DRAK2 has been shown to play a prominent role in dampening signals emanating from the T cell receptor. The presence of DRAK2 in the mature T cell compartment contributes to the requirement of adequate costimulation for further differentiation. Currently, the mechanism of DRAK2 activation is unknown. Likewise, those molecules downstream of DRAK2, presumably substrate targets of this kinase, also remain unidentified. The aim of this project is to determine which signaling pathways DRAK2 functions within; that is, to determine those events required in distal TCR signaling for DRAK2 relocalization, autocatalysis and substrate interaction resulting in the modulation of calcium flux and overall degree of T cell activation. The data presented here indicate that DRAK2 activity is dependent on calcium mobilization, eliciting a feedback regulatory mechanism to limit the extent of the influx of extracellular calcium. Elucidating the pathway DRAK2 functions within will reveal novel therapeutic targets that contribute to the pathology seen in a murine model of multiple sclerosis, the induction of which is significantly thwarted in mice deficient in DRAK2.
Cell‐to‐cell transfer of the G protein Ras at the immunological synapse
Authors: Rechavi Oded , Vernitsky Helly , Rotblat Barak , Kloog Yoel , Goldstein Itamar
Published: June 2021

Immune cells establish dynamic adhesive cell–cell interactions at a specific contact region, termed the immunological synapse (IS). Important features of the IS are the formation of cell‐cell membrane bridges and the exchange of selected cell surface proteins between conjugated cells. In this study we investigated whether upon IS formation, inner plasma membrane associated G‐proteins can also transfer from target cells to lymphocytes and transmit signals across cell boundaries. Here we show by flow cytometry and confocal microscopy that human NK and T cells rapidly acquire from the cells they scan, in a contact dependent manner, the intracellular G‐protein Ras: a signal transducing protein vital for common lymphocyte functions and a prominent participant in human cancer. Importantly, we also show that the transfer of the constitutively active oncogenic mutant of H‐Ras, H‐RasG12V, from 721.221 transfectants to NK cells had significant biological effects. The transfer correlated with augmented ERK phosphorylation and interferon‐γ production in the adopting NK cells as well as increased target cell killing. These findings reveal a hitherto undescribed mode of cell‐to‐cell communication that enhances the capacity of lymphocytes to probe and react to the proteome of the cells they survey, and that may moreover play an important role in natural tumor immunity.
Proinflammatory signaling via the neurokinin 1 receptor promotes dendritic cell survival resulting in robust cellular immune responses
Authors: Janelsins Brian Mark , Mathers Alicia R , Tkacheva Olga A , Erdos Geza , Larregina Adriana T
Published: June 2021

Inflammatory and immune responses are regulated by the secretion of cytokines and neuropeptides released in immune‐competent organs. Substance P, the prototype proinflammatory neuropeptide secreted by nerve fibers, promotes T cell proliferation by binding the neurokinin 1 receptor (NK1R). The initiation of T cell responses requires the activation and survival of dendritic cells (DC). Since DC home strategically to richly innervated organs, we hypothesized that signaling via the NK1R will promote DC survival resulting in robust DC‐T cell stimulatory functions. Using a model of murine DC generated from bone marrow precursors we investigated the: i) expression of the NK1R, ii) ability of the NK1R agonist [Sar9Met(02)11]SP to activate intracellular pathways involved in DC survival and iii) in vivo DC stimulatory function and survival of NK1R‐signaled DC homing to local draining lymph nodes. Our data show that DC expressed functional NK1R and agonistic signaling prevented DC apoptosis via PI3K and ERK signaling pathways and mobilization of intracellular Ca2+. Adoptive transfer of NK1R‐signaled DC favored lymph node homing and survival of DC which resulted in enhanced and prolonged Ag specific DTH responses. We conclude that signaling DC with a NK1R agonist enhances DC survival and their ability to stimulate cellular immunity.

This work was supported by NIH grant R01CA100893 to ATL
SHIP1 associates with DAP12 and negatively regulates TREM2/DAP12 signaling
Authors: Peng Qisheng , Humphrey Mary Beth
Published: June 2021

TREM2‐DAP12 stimulation of macrophages and pre‐osteoclasts (OC) leads to enhanced in vitro osteoclastogenesis. To further define the regulation of TREM2‐DAP12 signals, we studied proteins that associated with DAP12 following TREM2 stimulation. We found that TREM2 crosslinking of RAW264.7 cells leads to a novel association of Src homology region 2(SH2)‐containing inositol phosphatase (SHIP1) with DAP12 at 1 and 5 min, which was not seen after stimulation with isotype control Ab. Recombinant GST‐SHIP1‐SH2 protein, but not mutated GST‐SH2(R34G) protein, could bind phosphorylated DAP12 indicating that the SHIP1‐DAP12 interaction is mediated via the SH2 domain of SHIP1. Biotinylated‐DAP12 ITAM phosphopeptide, but not un‐phosphorylated or Y65/76F mutated peptides, interacted with SHIP1. SHIP1 co‐immunoprecipitated with DAP12 in OC generated from C57BL6 bone marrow macrophages (BMM) treated with RANKL and M‐CSF. To determine the functional significance of this interaction, we evaluated TREM2 stimulation of SHIP1 deficient OC precursors during osteoclastogenesis. Crosslinking with TREM2 monoclonal Ab but not control Ab lead to a 4 fold increase in OC generated from SHIP1−/− BMM compared to wild type BMM. Together these data reveal a novel association of SHIP1 with DAP12 that is dependent on the DAP12 ITAM and the SH2 domain of SHIP1 and this association inhibits DAP12 signaling during osteoclastogenesis.
In vivo requirement for autophagy in antigen presentation by dendritic cells
Authors: LEE HEUNG KYU , Lee Yun Hee , Chervonsky Alexander , Mizushima Noboru , Iwasaki Akiko
Published: June 2021

CD4 T cells recognize peptides loaded in the context of class II major histocompatibility complex (MHC). Classically, the peptides found on MHC II complex are generated in the acidified endocytic vesicles upon degradation of antigens derived from intracellular vesicles and extracellular sources. Recent evidence from in vitro experiments indicates that certain cytosolic antigens could also be loaded onto MHC II molecules following autophagy. However, the role of autophagy in antigen presentation in vivo is unknown. Here we examined the in vivo requirement of autophagy in antigen presentation to CD4 T cells using mice deficient in a key component of autophagosome, Atg5. Dendritic cell's ability to induce CD4 T cell responses was measured in Atg5−/− → WT fetal liver chimeric mice and in CD11c‐Cre‐Atgflox/floxconditional knockout mice in which DCs lack Atg5. Despite their intact ability to capture antigens, migrate, mature and produce cytokines, Atg5‐deficient dendritic cells (DCs) were impaired in their capacity to process and present certain forms of antigens to CD4 T cells. These results revealed a critical in vivo requirement for autophagy in processing and presentation of antigens for MHC II by DCs, leading to the activation of CD4 T cell responses.
Adjuvanting of Fluzone® with JVRS‐100 in Mice, Rabbits and Non‐Human Primates Demonstrates Increased Immunogenicity and Dose‐Sparing
Authors: Callejo Bernadette , Lay Marla , Carroll Timothy D. , Matzinger Shannon , Fritts Linda , Miller Christopher J. , Chang Stella , Warner John F. , Fairman Jeffery
Published: June 2021

JVRS‐100 (lipid‐DNA complexes) is a unique and promising adjuvant for vaccine applications that require high levels of antibody and T‐cell immunity. The JVRS‐100 adjuvant was mixed with a split influenza vaccine (Fluzone®‐sanofi pasteur) and administered parenterally to mice, rabbits and non‐human primates. Vaccination with JVRS‐100‐Fluzone® resulted in a significant increase in total IgG, IgG1 and IgG2a influenza antibodies. Further hemagglutination inhibiting (HAI) antibody were higher compared with Fluzone® alone. Administration of decreasing amounts of Fluzone® mixed with JVRS‐100 resulted in a ∼50‐fold dose‐sparing effect based on HAI titer. In vitro stimulation of splenocytes from JVRS‐100‐Fluzone® vaccinated mice with Fluzone® demonstrated increased antigen‐specific T cell responses (IFN‐gamma production) compared with Fluzone® alone. Splenocytes from JVRS‐100‐Fluzone® vaccinated mice responded to unmatched H1N1, or H3N2, and influenza B viruses, suggested induction of cross‐reactive T cell responses to conserved viral antigens. These results suggest the JVRS‐100 adjuvant enhanced immune responsiveness and reduced antigen doses needed for strong immune responses to a licensed flu vaccine. The JVRS‐100 adjuvant has also been shown to potentiate immune responses to multiple viral and bacterial antigens, and could be a broadly applicable adjuvant for human and veterinary vaccines.
Inverse relationship between innate and acquired immune responsiveness in mice initially dosed with cationic lipid DNA complexes (JVRS‐100)
Authors: Lay Marla , Callejo Bernadette , Giffon Thierry , Lewis David , Chang Stella , Fairman Jeffery
Published: June 2021

Hyporesponsiveness to excessive innate immune stimulation (i.e., lipopolysaccharide, lipoteichoic acid, and CpG oligonucleotides) has been studied extensively in vitro and documented in clinical treatment of sepsis patients with low inflammatory cytokine production, reduced leukocyte expression of HLA‐DR, and decreased antigen presentation. Closely spaced intravenous (IV) administration of JVRS‐100 has been shown to induce a short (7–10 day) refractory period for innate immune activation, with 50% of responsiveness restored after three days and 100% by 10–14 days. This refractory period was observed in interferon‐gamma and IFN‐gamma receptor knockout mice as well as mice pre‐dosed with IFN‐gamma or depleted of NK cells or plasmacytoid dendritic cells. In contrast to reduced capacity for antigen presentation (sepsis patients), IV administration of JVRS‐100, one day prior to vaccination with HKx31 heat‐inactivated influenza virus adjuvanted with JVRS‐100 significantly enhanced the influenza‐specific humoral immune response. Enhancement of the adaptive response to vaccination was greatest at the timepoints showing the maximum refractory period, suggesting an inverse relationship between cytokine production and immune activation. These findings may have implications for development of future vaccines, in which single or limited vaccination strategies are critical.
A role for major histocompatibility complex (MHC) class II molecules in MHC class I presentation and CD8 T cell priming
Authors: Belizaire Roger , Rohrs Henry , Suri Anish , Unanue Emil
Published: June 2021

The CD4 T cell response against hen egg lysozyme (HEL) in non‐obese diabetic (NOD) mice comprises a majority of T cells reactive against HEL 11–25 despite the presence of another epitope, HEL 20–35, that binds to Ag7 with comparable affinity. To investigate further the relationship between peptide affinity and the T cell response, we used ELISPOT to measure cytokine production after priming with HEL. As expected, HEL immunization of NOD mice generated HEL 11–25‐reactive CD4 T cells that secreted interleukin‐2 (IL2) and interferon‐γ (IFNγ). Surprisingly, HEL immunization also gave rise to a robust CD8 T cell response specific for HEL 23–31 presented by Db. Another interesting aspect of these studies was the finding that mice expressing Db but lacking Ag7 showed a reduced or absent CD8 T cell response after HEL immunization. A lack of CD4 T cell help could not explain this result, as depletion of CD4 T cells prior to immunization with HEL had a minimal effect on CD8 T cell priming. To test the global effect of MHC class II molecules on MHC class I peptide selection, we have initiated mass spectrometry studies on peptides bound to Kd in the presence or absence of Ag7. Initial results indicate a substantial difference in the Kd peptide repertoire between cell lines with and without Ag7 expression. Altogether, these data suggest a cooperativity between MHC class I and II that affects T cell priming and peptide selection.
Regulation of the TRAP Promoter by IL‐4
Authors: Yu Minjun , Moreno Jose L. , Stains Joseph P. , Keegan Achsah D.
Published: June 2021

IL‐4 inhibits RANKL‐induced osteoclast formation, functional activity, and expression of tartrate‐resistant acid phosphatase (TRAP), a key osteoclast marker, in a STAT6‐dependent manner. Since the TRAP promoter contains a STAT6‐binding motif, we proposed that STAT6 directly regulates TRAP transcription. To test this, RAW264.7 cells were transfected with a TRAP promoter‐luciferase reporter. IL‐4 suppressed the ability of RANKL to upregulate TRAP‐luc activity after 72 hours, suggesting that IL‐4 represses RANKL‐induced transcription of the TRAP promoter. Similarly, we found that IL‐4 reduced the association of RNA polymerase II with the TRAP gene by chromatin immunoprecipitation (ChIP). Further, we found that STAT6 binds to the endogenous TRAP promoter within the first 24 hours of IL‐4 treatment; this binding was not observed at later times. To determine the impact of this STAT6 binding, we transfected cells with STAT6VT, an active STAT6 mutant. STAT6VT alone slightly upregulated TRAP‐luc activity, and did not inhibit the upregulation of TRAP promoter activity caused by NFATc1, PU.1 and MITF overexpression C downstream targets of M‐CSF and RANKL. These results suggest that STAT6 binds the TRAP promoter transiently after IL‐4 treatment enhancing transcription. However, STAT6 binding may not be directly responsible for the suppression of RANKL‐induced TRAP promoter activity by IL‐4.
Augmenting CD8+ T cell responses following infection with IL‐15/sIL‐15Rá complexes.
Authors: Rubinstein Mark P , Lind Nicholas A. , Filippou Pauline , Best J. Adam , McGhee Patrick A. , Goldrath Ananda W
Published: June 2021

Administration of IL‐15 has shown efficacy in augmenting CD8+ T cell responses following vaccination. However, soluble IL‐15 is limited in vivo by poor half life and limited biological activity. Recent studies have demonstrated that the biological activity of soluble IL‐15 could be significantly improved by association with its soluble IL‐15 receptor alpha subunit (sIL‐15Rα). After injection, soluble IL‐15/IL‐15Rα complexes rapidly induced strong and selective expansion of memory CD8+ cells and natural killer cells. We now find that injection of IL‐15/IL‐15Rα complexes following viral or bacterial infection can dramatically increase the number of responding antigen‐specific CD8+ T cells. Importantly, IL‐15/IL‐15Rα complexes not only augmented the response of donor CD8+ T cells but also endogenous CD8+ T cells present at low precursor frequencies. Our results suggest that administration of IL‐15/IL‐15Rα complexes may have utility for augmenting vaccine strategies against infectious disease or cancer.