bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the conserved distal heme asparagine in coral allene oxide synthase and human catalase
Authors: Gao Benlian , Boeglin William E. , Brash Alan R.
Published: June 2021

Allene oxide synthase (AOS) metabolizes a specific fatty acid hydroperoxide to an unstable allene epoxide, a biosynthetic precursor of cyclopentanones such as jasmonic acid and marine clavulones. Plant AOS is a cytochrome P450 (CYP74A), whereas coral AOS (cAOS) is structurally related to catalase. The striking similarity of catalase and cAOS revealed by the X‐ray structures opens new questions on the structure‐function of both enzymes. Here we addressed the role of the conserved distal heme Asn in H2O2 breakdown in catalase and in metabolism of the natural hydroperoxide substrate 8R‐HPETE by cAOS. Point mutations were introduced by site directed mutagenesis and the proteins expressed in E. coli. Activities were assayed by UV spectrophotometry and cAOS products were identified by HPLC‐UV. In human catalase, the point mutations N148A, N148S, or N148D reduced Vmax and Km, with little net effect on kcat/Km. In cAOS, the mutations N137A, N137Q, N137S, N137D, and N137H drastically reduced the rate of reaction (to 0.8–4%), yet the mutants all formed the allene oxide as product. This is remarkable because there are many potential heme‐catalyzed transformations of fatty acid hydroperoxides. The distal heme Asn is not essential in either catalase or cAOS. Its conservation throughout evolution may relate to a role in enzyme stabilization and fine‐tuning of catalysis.

Supported by NIH grant GM‐074888
Tautomerism of histidine 64 associated with proton transfer in catalysis of Carbonic Anhydrase
Authors: Shimahara Hideto , Yoshida Takuya , Sugimori Kimiichi , Sugiyama Ayumu , Mizukami Taku , Nagao Hidemi , Nakazawa Takashi , Ohkubo Tadayasu , Kobayashi Yuji
Published: June 2021

The imidazole 15N signals of histidine 64 (His‐64), associated with the proton‐transfer function of human carbonic anhydrase II (hCAII), were observed [1]. Using the pH‐dependency of ring 15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His‐64 is 1.0. This unique value characterizes the nitrogen atoms of the His‐64 ring as both a general acid and base. Based on this feature, we suggest that the tautomerization of His‐64 can mediate the transfer of protons at a neutral pH with high efficiency. Comparing the KT of His‐64 with that of the histidine analog compounds shows that the imidazole group intrinsically tends to be the Nε2‐H tautomer in aqueous solution, unless hydrogen bond interacts with δ1‐nitrogen. This clarifies the structure ‐specifically, the hydrogen bonding pattern of His‐64. As a result, we propose that the formation and decay of hydrogen bonds should occur in the water bridge between His‐64 and zinc‐bound water during hydration and dehydration reactions in catalysis. This provides a new insight into the proton‐transfer mechanism in hCAII, i.e., a reorganization or flow of water molecules occurs in the water bridge when His‐64 is the Nε2‐H tautomer. During this conference, extensively low field shifts of 1H signals and theoretical consistency will be also discussed.
Exploring the Mechanism of a Class D β‐lactamase Through Site‐Saturation Mutagenesis of OXA‐1 at the Valine 117 Position
Authors: Buchman Jennifer S. , Barthel Lea , Leonard David A.
Published: June 2021

OXA‐1 is a class D lactamase that confers resistance to penicillins such as ampicillin and oxacillin. Site saturation mutagenesis of OXA‐1 at the valine 117 position was completed using the overlap extension method of the polymerase chain reaction. All nineteen mutant PCR products were ligated into expression vector pOXA‐1, and transformed into DH10B E. coli cells. Minimum inhibitory concentration of each mutant was established using a range of antibiotics including ampicillin, cefepime, cefotaxime, ceftazidime, and imipenem. Substitution of valine 117 with threonine, isoleucine, leucine, arginine or lysine resulted in lower but significant resistance to ampicillin, while other substitutions resulted in much lower levels of resistance. The Val117Asn variant was purified and shown to have a reduced kcat but a similar Km compared to the wildtype enzyme.
A PGC‐1α:O‐GlcNAc Transferase Complex Regulates Foxo1a Activation in Response to Glucose
Authors: Housley Michael Patrick , Rodgers Joseph T , Udeshi Namrata D , Hunt Donald F , Puigserver Pere , Hart Gerald W
Published: June 2021

Paradoxically, hepatic gluconeogenesis is elevated in diabetes contributing to fasting hyperglycemia. Expression of the gluconeogenic enzymes, Pepck and Glc‐6‐Phosphatase is regulated by Foxo1a and the co‐activator Pgc‐1α. Insulin signaling results in nuclear exclusion of Foxo1a but post‐translational regulation within the nucleus also occurs. O‐GlcNAc modification of Ser/Thr residues serves as a nutrient and stress sensor. Foxo1a is GlcNAcylated, as shown by anti‐O‐GlcNAc antibodies, lectins and glycotransferase labeling. In rats, STZ‐induced diabetes increases UDP‐GlcNAc (donor for O‐GlcNAc), elevates GlcNAcylation of Foxo1a, and concomitantly increases Foxo1a target gene expression (Pepck and Glc‐6‐Pase). Hyperglycemia activates Foxo1a via the UDP‐GlcNAc synthesis pathway in a luciferase reporter assay. 4 O‐GlcNAc sites were mapped by ETD‐MS. Mutation of one of these sites reduces Foxo1a activation by glucose. Pgc‐1α interacts with and targets OGT to Foxo1a, increasing Foxo1a GlcNAcylation. We conclude that hyperglycemia increases GlcNAcylation of Foxo1a via a PGC‐1α:OGT complex, resulting in elevated hepatic gluconeogenesis, exacerbating glucose toxicity in diabetes. Supp. by NIH DK61671, HD13563 (GWH) and GM37537 (DFH). GWH receives royalties on CTD110.6. Mgd. by JHU.
Activation of toll‐like receptor‐4 modulates skeletal muscle substrate metabolism in a manner that mimics the metabolic phenotype observed in skeletal muscle of obese humans
Authors: Hulver Matthew W , Frisard Madlyn I. , McMillan Ryan P , Marchand Julie , Wu Yaru , Voelker Kevin
Published: June 2021

Obesity is associated with chronic inflammation and the development of type 2 diabetes. Skeletal muscle from obese humans is resistant to insulin and characterized by abnormal substrate metabolism, i.e. increased glycolytic activity, reduced oxidative capacity, and elevated lipid accumulation. Toll‐like receptors (TLRs) are transmembrane receptors that are important in the induction of inflammatory responses. TLRs have been linked to lipid‐induced skeletal muscle insulin resistance and may contribute to reduced oxidative capacity and skeletal muscle lipid accumulation associated with obesity. In the current study, we demonstrate that the TLR4 signaling pathway is up regulated in skeletal muscle of obese humans and regulates substrate metabolism in a manner that reproduces the metabolic phenotype of skeletal muscle observed with obesity. TLR4 gene expression is increased in skeletal muscle of obese humans (p<0.001) and significantly related to BMI (r=0.92, p<0.001) and intramyocellular triglyceride synthesis (r=0.86, p<0.006). In addition, treatment of skeletal muscle cells with Lipopolysaccharide results in a 15% reduction in fatty acid oxidation (p<0.002), a 30% increase in glucose oxidation (p=0.04), and a 33% increase in triglyceride synthesis (p<0.002). These results provide a potential mechanism linking innate immunity with abnormal substrate metabolism associated with obesity.

Supported by the American Diabetes Association (Junior Faculty Award, MWH) and the National Institutes of Health (DK078765‐01, MWH).
The effect of methyl acceptors on betaine metabolism and the fate of betaine‐derived methyl groups in rat hepatocytes
Authors: Treberg Jason Robert , da Silva Robin P. , Brosnan Margaret E. , Brosnan John T.
Published: June 2021

Despite well articulated metabolic pathways, little is known of how betaine metabolism is regulated or what controls its catabolism and the fate of the three methyl groups of the trimethylamine moiety. Using rat primary hepatocytes we investigated the metabolism of 3‐[14CH3]‐labeled betaine. This study, for the first time, explores the fate of the betaine methyl groups in isolated liver cells. Approximately 50% of the metabolised methyl groups are found in an acid soluble fraction that, based on ion exchange chromatography, is consistent with the products of betaine:homocysteine methyltransferase, methionine and dimethylglycine. The remaining methyl groups were metabolised to volatile compounds, likely CO2 or formate. Betaine concentration did not affect the relative proportion of label found in the volatile fraction. Total product formation from betaine has an apparent Km of 0.41 mM, somewhat above arterial plasma values (∼ 0.1–0.2 mM). The addition of homocysteine as a methyl acceptor had no effect on betaine metabolism; however, glycine (methyl acceptor and precursor of serine) markedly decreases the relative amount of volatile product formation. This work was funded by CIHR.
Comparative effects of palmitic acid and ω‐3 PUFA on insulin regulation of glucose metabolism in rat skeletal muscle
Authors: Fiamoncini Jarlei , Hirabara Sandro Massao , Lambertucci Rafael Herling , Curi Rui
Published: June 2021

The effect of palmitic and ω‐3 polyunsaturated fatty acids (PUFA) on glucose metabolism stimulated by insulin in incubated rat soleus muscle was compared. The effect of palmitic acid on insulin signaling was also examined. Rat soleus muscles were incubated in the presence of palmitic, α‐linolenic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (800 or 400 μM) for 5 hours in the presence of insulin (100 or 10000 μUI). 2‐Deoxi‐[2,6‐3H]‐D‐glucose (2‐d‐G) uptake, [14C]‐glycogen synthesis and [U‐14C]‐D‐glucose oxidation were measured. Tyrosine phosphorylation of insulin receptor substrate 1/2 (IRS 1/2), protein kinase B (AKT), glycogen synthase kinase 3 (GSK‐3) and MAP kinases p44 e p42 was evaluated. Palmitic acid inhibited the increase in 2‐d‐G uptake, glucose oxidation and [14C]‐glycogen synthesis stimulated by insulin. The two last effects were also induced by α‐linolenic acid. Palmitate effects were preceded by a reduction in the phosphorylation state of IRS‐1/2 (30%), AKT (51%), GSK‐3 (33%) and MAP kinases p44 e p42 (88%). Palmitate induced insulin resistance in 5h‐incubated soleus muscle. It was noteworthy that EPA and DHA did not impair glucose metabolism. The divergent effects of DHA and EPA with that of α‐linolenic acid indicates that the effects of a specific FA are not necessary mimicked by all FA of the same family. Financial Support: FAPESP, CNPq and CAPES.
G Protein‐Activated Inward Rectifier Potassium Channel 4 is a Regulateable Protein; Its Role in Autonomic Neuropathy in Type I Diabetes
Authors: Zhang Yali , Park Ho‐Jin , Madias Christopher , Aronovitz Mark J , Karas Richard H , Galper Jonas B
Published: June 2021

Parasympathetic stimulation decreases heart rate and force of contraction and involves acetylcholine binding to M2, activation of the G protein‐activated inward rectifier K+ channel (GIRK1)2/(GIRK4)2. Although GIRK4 is required for the generation of IkAch, no data on the regulation of its expression are available. In a model for lipid lowering, culture of atrial myocytes in lipoprotein depleted serum (LPDS) increased GIRK4 promoter activity (GIRK4‐Luc) and protein compared to cells grown in FBS. LPDS stimulated phosphorylation of Akt while LPDS‐stimulated GIRK4 promoter was inhibited by the p‐Akt inhibitor, LY294002. LPDS increased SREBP1 and SREBP1 increased GIRK4 promoter in myocytes. Insulin treatment of myocytes increased SREBP‐1, GIRK4 promoter, GIRK4 protein and p‐Akt. LY inhibited insulin‐stimulated GIRK4 expression. Diabetic autonomic neuropathy is associated with parasympathetic dysfunction. In the type 1 diabetic Akita mice, which demonstrate parasympathetic dysfunction, expression of GIRK4 and SREBP1 in the atrium was decreased compared to WT. Insulin treatment of Akita increased level of SREBP1 and GIRK4 to that in WT and reversed parasympathetic dysfunction. Given that insulin has been shown to regulate SREBP, these data suggest that hypoinsulinemia in type 1 diabetes might result in parasympathetic dysfunction due to decreased SREBP and GIRK4 and Akt might play a role in this pathway.
Rutin Metabolites as Powerful Inhibitors of Advanced Glycation End Products
Authors: Laurean Daniel Cervantes , Pashikanti Srinath
Published: June 2021

Glycation is a non‐enzymatic reaction between reducing sugars and protein‐amino groups that through ketoamines leads to advanced glycation end products (AGEs) such as fluorescent and non‐fluorescent (Nε‐carboxymethyllysine, CML) protein adducts. Glycation is implicated in diabetic complications involving kidney, eye and neuron damage. Aminoguanidine (AG), a powerful AGE inhibitor, induced critical side effects when tested as anti‐diabetic drug in clinical trials. This showed a need for new and improved AGE inhibitors. Histone H1 and ADP‐ribose were used as a glycation model since it allows to distinguish true AGE inhibitors from general antioxidants. Rutin derivatives were tested as AGE inhibitors since rutin, a dietary flavonoid contained in plant‐derived products, isn't absorbed intact due to gut microflora degradation. Our data showed that 3,4‐dihydroxyphenylacetic acid and 3,4‐dihydroxytoluene were equal or better inhibitors of fluorescence, and CML‐protein adducts than AG. In contrast, homovanillic acid and 3‐hydroxyphenylacetic acid, weren't as effective AGE inhibitors. Methylglyoxal and glyoxal were used as glycating agents of histone H1 to elucidate the mechanism of AGE‐inhibition and effective AGE inhibition was observed with rutin metabolites and AG. These results present alternative effective AGE inhibitors to AG that might be recommended as supplements in the prevention of diabetes complications.
Metabolic and Genetic Risk Factors Associated With Development Of Complications In Type 2 Diabetes Mellitus (DM)
Authors: Hemimi Neveen Salah El Din , Elwahab Mahmoud M Abd
Published: June 2021

Background: Glutathione S‐transferases (GSTs) are a family of multifunctional enzymes playing an important role in the cellular detoxification. Therefore, diminished expression of GSTs may result in a reduced body defense against oxidative stress, followed by development of diabetic complications.

Aim: To test the hypothesis that the loss of activity of the GSTs enzyme due to deletion polymorphism in GSTT1 and/or GSTM1 may affect the risk of developing complications in DM and to study the other risk factors for complications in DM.

Methods: 150 non‐complicated type 2 diabetic patients and 150 complicated type 2 diabetic patients were enrolled in the study. The GSTM1 and GSTT1 genotypes were identified by polymerase chain reaction of peripheral blood DNA samples. Analysis of data was done by using SPSS program.

Results: The frequencies of null GSTM1 and GSTT1 genotypes were 53.3% (80/150) and 40% (60/150) respectively in non‐complicated DM group. The frequencies of null GSTM1 and GSTT1 genotype in complicated DM group were 57.3% (86/150) and 58% (87/150) respectively. The null GSTT1 genotype showed statistically significance higher rate in complicated DM compared with non‐complicated DM.

Conclusion: Our data provide evidence that diabetics with null GSTT1 genotypes are substantially at higher risk for developing complications.
RGS/G‐protein control of liver carbohydrate and fatty acid metabolism
Authors: Wilkie Tom , Pashkov Victor , Esser Victoria , Uyeda Kosaku , Huang Jie
Published: June 2021

G protein coupled receptor (GPCR) pathways regulate glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein Signaling (RGS) proteins are Gα‐GAPs that control the intensity and duration of G protein signaling. One RGS gene, Rgs16, is up regulated 20‐fold in liver during fasting and rapidly down regulated by feeding. Rgs16 mRNA expression is restricted to the periportal hepatocytes which preferentially oxidize fatty acids and produce glucose during fasting. Interestingly, dietary saccharides induce Rgs16 mRNA up to 300‐fold and expression expands to all hepatocytes. The bHLH transcription factor ChREBP is required for glucose‐dependent induction of Rgs16 transcription in mice and cultured liver slices. Transgenic mice that express Rgs16 protein specifically in liver have reduced expression of hepatic genes that promote fatty acid oxidation, lower rates of fatty acid oxidation in liver extracts, and lower plasma β‐ketone levels compared to wild type littermates. By contrast, Rgs16 knockout mice exhibit the reciprocal phenotypes. In summary, Rgs16 is a glucose‐responsive inhibitor of fatty acid oxidation in liver. We are investigating broader roles of Rgs16 in glucose and fatty acid metabolism in healthy, obese and diabetic conditions. Research support: NIH & Welch Foundation.
Mathematical modeling and systems biology approach to study biomaterial composition‐mediated inflammatory signals in endothelial cells
Authors: Shah Miti , Kelso Bret G. , Caplan Michael R.
Published: June 2021

Understanding of intracellular signaling in response to biomaterials is crucial for rational design of bioactive materials that elicit desired cellular behavior. To understand correlation of signaling to recruitment of inflammation by endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured on substrates prepared by adsorbing nine different extracellular matrix components to tissue culture plates in addition to a non‐adsorbed plate. Activation of kinases (ERK1/2, JNK‐1, Akt and IKK) was quantified by high‐throughput kinase assays for HUVECs exposed to these surfaces for various times. Principal component analysis (PCA), a well‐known matrix analysis technique, indicates that principal component 1 (PC1), captured the most information, is positively correlated to JNK activity, gelatin, polystyrene and negatively to chondroitin sulfate. PC2 correlated positively to ERK1/2 activity, vitronectin and negatively to Akt activity, chondroitin sulfate and heparin sulfate. PC3 correlated mostly with IKK activity, and PC3 is high for matrigel and laminin but low for vitronectin and heparan sulfate. Correlating these observations with ICAM‐1/E‐selectin expression and leukocyte adhesion will provide further insight into biomaterial composition‐mediated inflammatory signals in endothelial cells.

Source of research support: K22 DE014846 (NIDCR), R21 EB004386 (NIBIB)
Quantitative proteomics analyses of the effect of insulin on the human visceral adipose tissue secretome using a metabolic labeling approach
Authors: Vonk Roel J , Roelofsen Han , Weening Desiree , de Vries Marcel , Hoek Annemieke
Published: June 2021

We previously characterized the visceral adipose tissue secretome (MCP 2007, 6, 589). In the present study we determined incorporation rates of 13C‐labeled lysine into newly synthesized secreted proteins. Comparison of the C13/C12 ratios in the absence and presence of insulin should offer quantitative information on the effect of insulin on the production of secreted proteins. To prove this concept, human visceral adipose tissue was cultured for 72 hrs in 13C‐Lys containing medium in the absence or presence of 60 nM insulin. Subsequently, media were collected, concentrated and fractionated by SDS‐PAGE, followed by in‐gel digestion and LC‐MS/MS analyses. The datasets shared 274 proteins of which 224 proteins contained label. After removing contaminating intracellular proteins and applying a threshold C13/C12 ratio of 0.5, 82 proteins remained. In the absence of insulin, highest label incorporation was found for Zinc‐alpha‐2‐glycoprotein. In the presence of insulin, CRISP‐11 showed the highest label incorporation. By comparing C13/C12 ratios in the absence and presence of insulin we found eight proteins that were up‐regulated by insulin, four proteins were down‐regulated and 70 showed no change. The highest increase (4.7 fold) was found for CRISP‐11. We developed a new proteomics method which allows quantitative assessment of effects of insulin or other agents on protein secretion by tissues in culture.
Quantum Dots as FRET Donors in Oligonucleotide Hairpin Probes for Gene Expression
Authors: Davydenko Olga , Cohen Brian
Published: June 2021

Quantum dots (QD) are fluorescent semiconductor nanostructures that have the ability to emit light through the visible and infrared regions. Due to various coatings of quantum dots, they can be conjugated to macromolecules such as peptides, DNA or RNA. Oligonucleotides with dyes can be used for such conjugations to achieve fluorescence resonance energy transfer (FRET) between the QD and the dye when excited by UV light; this is due to the hairpin sequences encoded into the nucleotides, which bring the dye within the Foster radius of the QD. In order to utilize these hairpin probes as gene expression monitors, reversibility of FRET is necessary. This was achieved by incubation of the probes with oligonucleotides of complimentary sequence and allowing the strands to denature fully and anneal. An increase in the distance between the fluorescent dye and the quantum dot in the probes hybridized to complimentary oligonucleotides brought the dye out of the Foster radius and resulted in FRET reversal. FRET reversal was observed using steady state fluorescence analysis and fluorescent lifetime measurement. As a result of this study, a successful hairpin probe conjugation protocol was established and reversible FRET was achieved through complementation.
Site‐directed mutagenesis of bovine deoxyhypusine hydroxylase at the hinge loop area
Authors: Murtaugh Megan L , Wang Yinglu , Smith Joshua L , Soustek Meghan S , Appidi Vidya , Huang Jenq‐Kuen , Wen Lisa
Published: June 2021

Deoxyhypusine hydroxylase (DOHH) is the second of the two enzymes that catalyze the maturation of eukaryotic initiation factor 5A (eIF5A). It has been well established that hypusinated eIF5A is essential for eukaryotic cell proliferation and survival and that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy.

We have recently cloned bovine DOHH cDNA. The deduced amino acid sequence shared 88% identity with human DOHH. The dual function of human DOHH has been reported: the Fe containing holoenzyme exhibited hydroxylase activity while the apoenzyme showed molecular chaperone activity (Kang et al., The FASEB Journal. 2007;21:lb122). The structure transition between the holoenzyme and the apoenzyme is believed to take place around the variable hinge loop area. In this work, we prepare three amino acid substitution mutants at the loop area: P143S (Pro at the position 143 to Ser), D148A (Asp at 148 to Ala), and E154V (Glu at 154 to Val) of the bovine DOHH. The mutant DNA has been created by site‐directed mutagenesis using PCR primed with mutagenic primers followed by overlap extension. The mutant DNA is subcloned into pGEX‐2T, overexpressed, and characterized. We will compare the hydroxylase and chaperone activities of the mutant proteins to the wild‐type DOHH.

Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.
Post Translational Modifications by PAK6 and CARM1 Regulate p54nrb Function
Authors: Sarma Jagarlapudi Srinath Venkateshwara , Sewer Marion B
Published: June 2021

In the human adrenal cortex, transcription of CYP17 is regulated by adrenocorticotropin (ACTH), which activates a cAMP/cAMP‐dependent protein kinase (PKA) signal transduction pathway. ACTH/cAMP triggers a series of events, including reciprocal presence of coactivator and corepressor complexes on the promoter of CYP17. One of the proteins that binds to the CYP17 gene is the splicing/transcription factor p54nrb, a ubiquitously expressed 54‐kDa, non‐POU domain‐containing protein that activates CYP17 transcription by forming a trimeric complex with steroidogenic factor 1 (SF‐1) and polypyrimidine‐tract‐binding protein‐associated splicing factor (PSF). p54nrb is known to couple transcription and splicing by interacting with both RNA Polymerase II (Pol II) and splicing factors. In this study, we show that phosphorylation and methylation by p‐21 activated kinase (PAK) 6 and coactivator associated arginine methyltransferase (CARM1), respectively, control the coactivator potential of p54nrb by modulating interactions with Pol II and SF‐1. We show that PAK6 and CARM1 are essential for transcription of CYP17 and identify them as key effectors in ACTH/cAMP‐stimulated CYP17 gene expression.
Cell Division Autoantigen 1 Modulates p53 Stability and Smad3 Activation in TGF‐β Signaling Leading to Transcriptional Regulation of p21Waf1/Cip1
Authors: Chai Zhonglin , Tu Yugang , Pham Yen , Wu Tieqiao , Cao Zemin , Toh Ban‐Hock , Cooper Mark E.
Published: June 2021

We have previously reported that Cell Division Autoantigen 1 (CDA1) arrests cell growth via stabilization of p53, which subsequently upregulates cyclin‐dependent kinase (CDK) inhibitor p21Waf1/Cip1 (p21) resulting in inactivation of various CDKs. p21 and many other genes involved in cell proliferation, differentiation and apoptosis are under dual regulation of p53 and TGF‐β. Here we report our recent findings regarding the involvement of CDA1 in intracellular TGF‐β signaling. Firstly, we found that overexpression of CDA1 resulted in increased phosphorylation of Smad3 and its transcriptional activity as shown by the increased luciferase activities of two TGF‐β responsive reporter constructs, 3TP‐Lux and (CAGA)12‐Luc. Secondly, CDA1 knockdown markedly attenuates TGF‐β stimulated Smad3 phosphorylation and TGF‐β target gene expression. Thirdly, CDA1‐dependent induction of p21 was attenuated by the TGF‐β type I receptor inhibitor SB431542. These new data implicate a requirement for CDA1 in TGF‐β signaling involving Smad3, and that CDA1 alone is sufficient to activate the intracellular signaling downstream of TGF‐β. We conclude that CDA1 is not only an upstream regulator of p53 as reported previously, but is also able to activate the Smad signaling pathway, thereby leading to a synergistic regulation of target genes under dual control of p53 and TGF‐β.
Mechanism by which CA150 inhibits the activities of C/EBPα
Authors: Banman Shanna L. , Roesler William J.
Published: June 2021

Co‐Activator of 150 kDa (CA150) is a transcription elongation factor, but has also recently been shown by our lab to act as a co‐repressor for CCAAT/enhancer binding protein α (C/EBPα) transcriptional activity. Interestingly, CA150 also inhibits the growth arrest activity of C/EBPα. In this study, we examined domains within CA150 that mediated its inhibitory activity towards C/EBPa and the mechanism whereby this effect occurs. Our structure/function analysis using mutants of CA150 suggest that the inhibitory activity lies within the N‐terminal domain. Additionally, laser‐scanning confocal microscopy was used to visualize the subnuclear localization of fluorescent‐tagged CA150 and C/EBPα. Results show that co‐expression of CA150 influences the intranuclear organization of C/EBPα, suggesting a possible mechanism whereby CA150 inhibits the growth arrest and transcriptional activities of C/EBPα. Funding for this project has been provided by Canadian Institutes of Health Research, Saskatchewan Health, and University of Saskatchewan.
Probing the role of the potent microtubule disrupting agent disorazole C1 in premature cellular senescence induction
Authors: Kitchens Carolyn A , Tierno Marni Brisson , Petrik Bethany , Graham Thomas H , Wipf Peter , Raccor Brianne S , Balachandran Raghavan , Day Billy W , Reese Celeste E , Lazo John S
Published: June 2021

Cellular senescence is a permanent state of cell cycle arrest that prevents cell proliferation but allows the cells to be metabolically active. Premature cellular senescence is stimulated by a variety of extracellular and intracellular stimuli including oxidative damage, supraphysiological mitogenic signaling, transforming growth factor‐β, retinoids, oncogenes, DNA damage and some cancer chemotherapeutic agents. Disorazoles are macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum that are potent inhibitors of tubulin polymerization. We synthesized disorazole C1 and found it inhibited in vitro tubulin polymerization and competed with vinblastine and dolastatin 10 for binding to purified tubulin. Treatment with disorazole C1 caused profound inhibition of cell proliferation in a wide variety of cancer cell lines. In cells surviving 7 days of treatment, disorazole C1 induced a senescent phenotype at concentrations equal to or below the IC50 for growth inhibition. Additionally, we observed that a variety of other inhibitors of tubulin polymerization also induced a senescent phenotype indicating that, like other mechanisms of cellular stress, microtubule disruption can induce premature cellular senescence. This research was funded in part by USPH grant CA78039.
Secondary Alkyl Hydroperoxides as Inhibitors and Alternate Substrates for Lipoxygenase
Authors: Decker Ilka , Waller Tiffany , Frisch Jonathan , Funk Max
Published: June 2021

In polyunsaturated fatty acid metabolism, lipoxygenase catalyses the reaction of membrane‐derived lipids with molecular oxygen to yield hydroperoxides. Lipoxygenase needs to be activated by its own peroxide products via a redox mechanism that converts the enzyme's non‐heme iron co‐factor from an iron (II) state is to its active iron (III) form. To better understand the activation mechanism, the reaction of soybean lipoxygenase‐1 with secondary alkyl hydroperoxides was analyzed. The small molecule peroxides mimicked the hydrophobic end of the hydroperoxide product. While the reaction rates were found to follow the same kinetic mechanism as the hydroperoxide products, the secondary alkyl hydroperoxides inhibited the enzyme especially at high substrate concentration. Molecules with greater chain length were more effective inhibitors and proved to be more efficient in oxidation of the iron during EPR measurements. Isothermal titration calorimetry experiments showed a constant heat output in the reaction of lipoxygenase‐1 with the hydroperoxides. In this reaction, the hydroperoxides were converted to the corresponding carbonyl compounds via homolytic dehydration. We will also report on the characterization of the newly discovered reaction.
Lipin 2 is a Liver‐enriched Coactivator of Peroxisome Proliferator‐activated Receptor γ (PPARγ) Coactivator‐1α (PGC‐1α) and Regulates Fatty Acid Metabolism in Hepatocytes
Authors: Gropler Matthew Channing , Chen Zhouji , Finck Brian Neal
Published: June 2021

Lipin 1 acts in the nucleus to regulate gene expression via interactions with DNA‐bound nuclear receptor transcription factors and the PGC‐1α coactivator. Interestingly, lipin 1 also functions as an enzyme, catalyzing the penultimate step in triglyceride (TG) synthesis. However, little is known about the second lipin family member (lipin 2). We found that the expression of lipin 2 mRNA and protein was quite high in murine and human liver, but low in adipose tissue and skeletal muscle, where lipin 1 was enriched. The hepatic expression of lipin 1 and 2 was induced by fasting, but unlike lipin 1, the increase in lipin 2 was preserved in mice lacking PGC‐1α. Moreover, adenoviral‐driven PGC‐1α overexpression increased the expression of lipin 1, but not lipin 2, in hepatocytes. Nevertheless, lipin 2 protein physically interacted with and coactivated PPARα and PGC‐1α. Lipin 2 overexpression also augmented palmitate oxidation rates via increased expression of mitochondrial fatty acid oxidation enzymes. Rates of TG synthesis were surprisingly unchanged by overexpression of either lipin 1 or 2 in hepatocytes. These data indicate that lipin 2 is a liver‐enriched and fasting‐induced PGC‐1α activator. Furthermore, although the effects of lipin 1 and 2 on hepatic fatty acid metabolism significantly overlap, the expression of these two lipin genes is under the control of distinct regulatory circuits.
Protein Kinase C‐dependent Manganese Superoxide Dismutase (MnSOD) Regulation in A549 cells
Authors: Chung Youn Wook , Yim Moon Bin , Chock P. Boon
Published: June 2021

MnSOD protein was induced by TPA and TNF‐α but not by hydrogen peroxide in mitochondria. The amount of ROS generated by TPA treatment was very small, however it was blocked by DPI, a NADPH oxidase and flavoprotein inhibitor. Small interfering RNA (siRNA) experiment showed that all components of NADPH oxidase in A549 cells were required to MnSOD induction by TPA. PKC‐α, ‐βI, ‐δ, ‐ε and ‐μ were also translocated to subcellular membrane when the cell was treated with TPA but none of these PKC isoforms were affected by hydrogen peroxide. The TPA‐induced MnSOD expression was down regulated by PKC inhibitors in a PKC‐α, ‐βI and ‐δ dependent manner and also was reduced by knock down of PKC‐α, ‐βI and ‐δ. A small enhancer region in intron 2 of SOD2 (MnSOD) gene (I2E), which is responsive to tumor necrosis factor‐α (TNF) and interleukin‐1β (IL‐1), was added to the previous construct pSODLUC‐3340 for more physiological effect. TPA‐mediated induction of SOD2 reporter gene was down regulated by PKC inhibitors in PKC‐α, ‐βI and ‐δ dependent manner. In addition, site‐specific mutations of FOXO3a, CREB and NF‐κB reduced the TPA‐responsive MnSOD induction and inhibition of CREB and FOXO3a transcriptions with siRNA also led to a reduction in the TPA‐induced MnSOD gene expression. Finally, siRNA and PKC inhibitors studies revealed that the phosphorylations of CREB and FOXO3a are PKC‐α dependent in MnSOD induction pathway. These results suggested that the expression of MnSOD might be tightly regulated by complex signal transductions including PKC‐α and PKC‐δ pathways.
PKA‐RSK1 Interaction Modulates RSK1 Activity and Cellular Apoptosis
Authors: Chaturvedi Deepti , Cohen Michael S. , Taunton Jack , Patel Tarun B.
Published: June 2021

Cyclic AMP dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. Previously, we showed novel interactions between RSK1 and the subunits of the PKA (Chaturvedi et al., Mol. Cell. Biol. 2006, Vol. 26, 4586–4600) that regulate the activity of PKA and cellular distribution of RSK1 as well as phosphorylation of RSK1 substrates. Using small inhibitory RNA (siRNA) against subunits of PKA (RI and PKAc) we have examined the role of PKA‐RSK1 interactions in regulation of RSK1 activity and apoptosis. In mouse lung fibroblast (B82L) cells, silencing of the RI subunit of PKA increases the phosphorylation of RSK1 whereas, silencing of PKAc results in decrease in phosphorylation of RSK1 in response to EGF. These changes in phosphorylation status of RSK1 are accompanied by the changes in kinase activity of the enzyme. Further, silencing of either the regulatory or catalytic subunit of PKA decreases the nuclear accumulation of active RSK1 and increases its cytosolic content in response to EGF. These changes in subcellular redistribution of active RSK1 were accompanied by decreased cellular proliferation and increased phosphorylation of its cytosolic substrate BAD with a resultant increase in the anti‐apoptotic actions of EGF. Our data also show that silencing of RI subunit increases the activity of PKA with a resultant increase in phosphorylation of PKA substrates including BAD (at Ser 155) and thus contributing in the protection of cells against apoptosis under basal conditions. Thus, we conclude that RSK1 interactions with the subunits of PKA play an important role in the regulation of RSK1 activity and its subsequent biological actions.

Supported by NIH grants GM 079226
Understanding Lupus and Auto‐Immune Diseases
Authors: Asermily Laurie Ellen , Anolik Jennifer
Published: June 2021

The immune system is a complex and dynamic subject. It can be challenging to understand our bodies’ responses to stimulation by foreign proteins, yet alone what would prompt an attack against ourselves. The following curriculum reviews the key concepts of the immune system and introduces an autoimmune disease: Lupus. This curriculum addresses different learning styles by engaging high school students in varied activities. Students begin by participating in a Jeopardy game to reinforce comprehension of previously taught vocabulary. Then, classes learn what specific effects Lupus has on the body, how it is detected, and how to apply this knowledge to explore other auto‐immune diseases. The laboratory portion formally begins with an investigation utilizing an interactive Immunology Lab web site to obtain familiarity with techniques and procedures necessary for antibody detection. Next, students review the scientific principles behind enzyme linked immunosorbent assay (ELISA) testing in order to perform an actual ELISA test. At the completion of the activities, students present an individual research poster on a specific auto‐immune disease.
“Can You Hear Me Now?” Cell Communication
Authors: Martell Dawn Marie , Cruikshank William Walter
Published: June 2021

In the immune system cell communication is required to make sure that the appropriate cells are recruited to sites of inflammation. Communication usually occurs through secretion of specific proteins called chemokines and cytokines, which attract specific cell types. A series of laboratory investigation provide students with the understanding of how the body reacts to foreign invaders by recruiting these appropriate cell types. Students will understand how chemokines and cytokines as proteins that are secreted by macrophages, dendritic cells and helper T‐cells act as signals that induce other immune cells to perform functions that help to regulate an inflammation response. The first step of the laboratory protocol consists of students separating an “unknown” simulated blood sample by column chromatography. Once the red blood cells are separated from the plasma, the elution from the column, representing the immune cell fraction, is collected for use in the next step. This part of the protocol is called the Ouchterlony technique; it determines the interaction of chemoattractant‐interleukine 16 (IL‐16) and macrophage inflammatory proteins (MIP) in the migration of CD4 Helper T cells (which become Th1 and Th2 cells), cheek cells and an unknown. If a chemical reaction occurs between the cytokine, helper T cell, cheek cells and the unknown, a precipitation band will form demonstrating cell‐specific chemokine to cell migration therefore cell signaling. Students will analyze these results to determine if a migratory response has occurred, how these cells are communicating and also determine which chemokine attracts the migration of the unknown.
The role of PKC η in T cell development and activation
Authors: Fu Guo , Niederberger Nathalie , Gascoigne Nicholas RJ
Published: June 2021

Protein kinase C (PKC) is a family of serine‐ and threonine‐specific protein kinases. PKC family members phosphorylate a wide variety of proteins and are involved in diverse cellular signaling pathways. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. Among the PKC isoforms expressed in T cells (e.g.α, β, δ, η,ζ), only PKC θ has been shown to be recruited to the immunological synapse. PKC θ knockout mice showed a profound peripheral T cell defects due to loss of TCR‐initiated NFκB activation, but normal T cell development. These data suggest a redundant role of PKC θ in T cell development, and hence there are possibly other PKC isoforms replacing the role of PKCθ. We found that PKC η is upregulated after TCR ligation in developing thymocytes, and is expressed at an earlier stage of thymocyte development in the absence of PKCθ. Also, like PKCθ, PKC η is rapidly recruited to the immunological synapse during antigen‐recognition, unlike other PKCs. To examine the effect of PKC ηon thymocyte development, we transduced PKC θ−/&minus bone marrow cells with retroviruses expressing PKC η specific shRNA cassette and performed bone marrow reconstitution assay. Preliminary data showed that knock‐down of PKC η, in the absence of PKC θ, led to a decrease of SP cells (both CD4+ and CD8+), and an increase DP cells (CD4+CD8+). Taken together, these results suggest that PKC η may play an important non‐redundant role in T cell development and activation. In order to more thoroughly investigate the biological functions of PKC η, we had made a PKC η conditional knock‐out mice and are currently working on them. This study is supported by NIH grant 5R01GM048002‐14.
AP‐1 inhibition enhances the activity of IL‐17 producing cells
Authors: Betz Briana C , Jordan Kimberly L , Thieu Vivian , Kaplan Mark , Taparowsky Elizabeth J
Published: June 2021

The activator protein‐1 (AP‐1) transcription factor complex plays a role in the growth, differentiation, and apoptotic responses of mammalian cells. AP‐1 is comprised of basic leucine zipper (bZIP) protein dimers that bind specific DNA sequences to regulate gene transcription. Studies have shown that AP‐1 dimers containing the bZIP protein BATF function as transcriptional inhibitors. In the mouse, endogenous BATF expression is modulated during discrete stages of T cell development, suggesting that genes differentially regulated by BATF are critical to growth decisions in this lineage. Using transgenic mice that overexpress BATF throughout all stages of T cell development (CD2‐HA‐BATF), we show that persistent AP‐1 inhibition leads to a reduced number of natural killer T (NKT) cells. In contrast, we have observed that the Th17 lineage is over‐represented in these same animals. Flow cytometry and ELISA experiments reveal an increase in cell numbers as well as an increase IL‐17 secretion when compared to controls. Real time PCR experiments reveal that transgenic splenocytes show an increase in IL‐17 and IL‐23R mRNA expression, suggesting that naïve CD4+ cells are biased toward the Th17 lineage. Current work is focused on identifying the AP‐1 target genes whose expression is influenced by BATF to generate these intriguing, yet opposing, T cell phenotypes. This work is supported by PHS Awards CA78264 and CA114381.
Alpha‐melanocyte stimulating hormone (α‐MSH) induction of Treg cell differentiation
Authors: Taylor Andrew W. , Li Dayu
Published: June 2021

Alpha‐MSH is a potent anti‐inflammatory neuropeptide. We previously reported our discovery that mouse CD4+ T cells primed to function as Th1 cells are suppressed in their expected Th1 functionality when treated with α‐MSH as they were restimulated through their T cell receptor (Tcr). Also, we discovered that the re‐stimulated α‐MSH‐treated primed CD4+ T cells are now TGF‐β‐producing CD25+ Treg cells. We asked whether these α‐MSH‐induced Treg cells express markers and FoxP3 in common with other characterized Treg cells. In addition, we examined the possibility that α‐MSH expands the natural CD25+ Treg cells in the primed CD4+ T cell population. The α‐MSH‐treated Tcr‐stimulated primed CD25+ CD4+ T cells had high expression of CD44 and CTLA‐4, and low expression of CD62L and GITR. The FoxP3 mRNA levels in the CD4+ T cells were 2‐fold enhanced by α‐MSH‐treatment with a parallel decline in high CD127 expression. The depletion of CD25+ cells from the primed CD4+ T cell population before restimulation prevented activation of Th1 cell functionality, but did not change α‐MSH‐induced TGF‐β production. Therefore, α‐MSH‐induces Treg cells that are found in the CD25+ CD4+ T cell population that are not derived from natural CD25+ CD4+ Treg cells. The results suggest that α‐MSH is a potential Treg cell differentiation factor with implications on α‐MSH activity at the resolution of immune responses and in immune homeostasis.

Work supported by PHS grant EY10752
Distinctive tyrosine phosphorylation pattern of CD19 during BCR and CD40 signaling
Authors: Fujimoto Manabu , Ishiura Nobuko , Adachi Takahiro , Tsubata Takeshi , Tedder Thomas F. , Tamaki Kunihiko
Published: June 2021

CD19 regulates constitutive and B cell antigen receptor (BCR)‐induced signaling through its cytoplasmic domain that contains nine conserved tyrosine residues. Of nine tyrosine residues, studies have clarified that three tyrosines, CD19‐Y513, CD19‐Y482, and CD19‐Y391, have central roles. Herein, we demonstrate distinctive patterns of tyrosine phophorylation of CD19 following various stimulations using antibodies reactive with each phosphorylated tyrosine residue. BCR ligation caused tyrosine phophorylation of Y513, followed by Y391 phosphorylation that preceded Y482 phosphorylation. IgG‐BCR induced more rapid phosphorylation of Y513 and more intense phosphorylation of Y482 and Y391 when compared with IgM‐BCR. Furthermore, simultaneous CD40 stimulation with BCR ligation resulted in much enhanced and prolonged phosphorylation of the three tyrosine residues. Phosphorylated tyrosine residues of CD19 also exhibited unique distribution: majority of phosphorylated Y513 located within the lipid rafts, while Y482 and Y391 were phosphorylated mainly out of the lipid rafts following the BCR ligation. Collectively, these observations contribute to better understanding of CD19 functions.
Mast cells are critical for the degranulation‐induced shift from Th1 to Th2 immune responses in vivo
Authors: Meyer‐Manlapat Anna Katrina , Segal David M
Published: June 2021

The involvement of mast cells on the generation of Th2 responses has been suggested; however, it is still unclear whether mast cells are critical for the degranulation‐induced shift from Th1 to Th2 responses. We hypothesize that mast cell degranulation would influence the Th1/Th2 balance in vivo and that this effect will not be observed in mice lacking mast cells. In this study, we show that when ovalbumin (OVA)‐specific CD4+ T cells adoptively transferred into normal mice are challenged with OVA in the presence of degranulation stimulus, these T cells proliferate in response to OVA but exhibit decreased IFNγ and increased IL‐4 production compared to control T cells primed in the absence of degranulation stimulus. In addition, when OVA‐specific CD4+ T cells are adoptively transferred into mast cell‐deficient mice and are challenged with OVA in the presence of degranulation stimulus, the decrease in IFNγ and increase in IL‐4 production are not observed. Moreover, when mast cell‐deficient mice are reconstituted with bone marrow‐derived mast cells, the adoptively transferred OVA‐specific CD4+ T cells proliferate in response to OVA and exhibit decreased IFNγ and increased IL‐4 production in the presence of degranulation stimulus as observed in normal mice. Altogether, these findings suggest that mast cells are essential for the degranulation‐induced shift from Th1 to Th2 responses.