bluuebunny/crossref_metadata_2025 huggingface dataset:
Interesting effects including apoptosis induced by protein aggregation of Gts1p with polyQ‐tail in yeast
Authors: Kuroda Kouichi , Sanada Mitsuaki , Ueda Mitsuyoshi
Published: June 2021

The induction of Saccharomyces cerevisiae GTS1 gene produces pleiotropic effects such as cell aggregation, heat tolerance, unbudding period, drug resistance, cell size, and oscillation of energy metabolism. We have demonstrated that Gts1p inhibits the function of Tup1‐Cyc8 repressor to activate 19 genes including the genes involved in cell aggregation. Gts1p has the polyglutamine (polyQ) sequence at the C‐terminus, and this sequence plays an important role in nuclear localization and Gts1p‐metiated cell aggregation. In this study, the clarification of molecular mechanisms underlying the pleiotropic phenotypes by GTS1 induction was attempted by examining Gts1p‐mediated protein aggregation. The degrees of protein aggregation mediated by Gts1p with and without polyQ were investigated by agarose gel electrophoresis. Gts1p‐mediated protein aggregates were purified by immunoprecipitation, and the trapped proteins were identified by mass spectrometry. As a result, it was suggested that proteins trapped in Gts1p‐mediated protein aggregations affect cellular machineries such as apoptosis and heat tolerance (1–3).
The neuroendocrine peptide 7B2 prevents neurodegenerative disease‐related protein aggregation
Authors: Helwig Michael , Hoshino Akina , Lee Sang-Nam , Lorenzen Nikolai , Otzen Daniel Erik , Lindberg Iris
Published: June 2021

Neurodegenerative diseases like Alzheimer's (AD) and Parkinson's (PD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ), tau (in AD) and α‐synuclein (in PD). In healthy cells, the correct folding of proteins is controlled by ubiquitously expressed molecular chaperones, which prevent the accumulation of harmful protein species. Our understanding of this quality control machinery and its involvement in the pathogenesis of neurodegenerative diseases is, however, very limited. We here describe a novel chaperone‐like function for the neuroendocrine protein 7B2, a highly conserved protein that is widely expressed in the nervous and endocrine systems. In vitro, 7B2 efficiently prevented the fibrillation of Aβ(1–42), Aβ(1–40), tau and α‐synuclein at a molar ratio of 1:10. This anti‐aggregation effect was dose‐dependent and most effective when 7B2 was present as the full‐length (27 kDa) form. Both exogenous application of 7B2 into the medium of Neuro2A cells, as well as endogenous adenoviral‐mediated overexpression of 7B2 blocked the neurocytotoxic effect of Aβ(1–42) and significantly increased cell viability. In the substantia nigra of a human PD‐affected brain, 7B2 was highly colocalized with α‐synuclein, indicating a physiological association. Taken together our results suggest that 7B2 may play a role in the etiology of neurodegenerative diseases.
Glycosylation and phosphorylation induce alternative structural conformations in tau's proline‐rich domain
Authors: Brister Michael Angelo , Bielska Agata A , Zondlo Neal J
Published: June 2021

The microtubule‐associated protein tau is the primary constituent of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease. In its diseased state, tau is phosphorylated on over 30 residues, many of which are alternatively modified by glycosylation by N‐acetylglucosamine (O‐GlcNAc) in tau's native state. We hypothesized that glycosylation of threonine and serine residues on tau peptides may induce a structural effect different from that of phosphorylation at these locations. We have employed a scheme for the synthesis of glycopeptides incorporating O‐GlcNAc and isolated O‐GlcNAcylated peptides derived from tau's proline‐rich domain. Furthermore, we have developed a functional group to mimic O‐GlcNAc modification (pseudo‐glycosylation). Phosphorylated, glycosylated, pseudo‐glycosylated and unmodified tau peptides were structurally characterized using circular dichroism and NMR. While phosphorylated sequences exhibited high propensity to form a type II polyproline helix, glycosylated and pseudo‐glycosylated peptides exhibited more flexible secondary structures. These findings suggest a structural role for tau modification by O‐GlcNAc. This project was supported by the Howard Hughes Medical Institute.
Human RMND5 Proteins Function as E3 Ubiquitin Ligases in Prostate Cancer Cells
Authors: Louw Alison , Harvey Jennet , Bentel Jacqueline
Published: June 2021

Required for Meiotic Nuclear Division 5A (RMND5A) and RMND5B are uncharacterised proteins that contain a putative Really Interesting New Gene (RING) domain, typically found in E3 ubiquitin ligases. To investigate their function, in vitro ubiquitin assays were performed indicating that RMND5A associates with UbcH2, UbcH5b and UbcH5c, while RMND5B interacts with UbcH5b and UbcH5c to mediate ubiquitin transfer. Supporting this, RMND5 proteins were found to associate with ubiquitinated proteins in vivo. Although target proteins of RMND5 ubiquitination are unknown, RMND5B interacts with the prostatic tumour suppressor NKX3.1, and overexpression of either RMND5A or RMND5B in LNCaP prostate cancer cells resulted in a dose‐dependent decrease in NKX3.1 levels, an effect that was partially reversed by proteasome inhibition. Finally in vivo ubiquitin assays established that NKX3.1 ubiquitination was increased following RMND5 protein overexpression. These studies demonstrate that RMND5 proteins exhibit E3 ubiquitin ligase activity and that NKX3.1 is a target of their ubiquitination. The findings suggest that the frequent disruption of the RMND5A/RMND5B gene loci in cancers will alter the turnover of important cellular proteins. (Research Support: RPH Medical Research Foundation, University of Western Australia).
Mutational and kinetic analysis of the endosome‐associated deubiquitinating enzyme, AMSH
Authors: Davies Christopher Williamson , Kim Myung-Il , Paul Lake N , Das Chittaranjan
Published: June 2021

The endosomal‐sorting complexes required for transport (ESCRT) is a ubiquitin‐mediated process that is implicated in endosomal sorting, trafficking, and viral budding. The ESCRT machinery regulates signaling events by internalizing cell surface receptors and targeted them for degradation by the lysosome. Two deubiquitinating enzymes (DUBs) regulate the activity of the ESCRT complex, STAM‐binding protein (AMSH), and ubiquitin specific protease Y (UBPY), by counteracting the ubiquitination of the cargo. Recently, we published two crystal structures of the catalytic domain of AMSH, and biochemical and modeling studies revealed that AMSH is significantly different from the ESCRT‐independent, AMSH‐like protein (AMSH‐LP) in its stability and ubiquitin recognition.. In the present study, we sought to understand AMSH's mode of substrate recognition, through mutational analysis. Surprisingly, we have discovered a point mutant that shows higher catalytic activity than the wild‐type enzyme. These results support the idea that AMSH is conformationally plastic, which may be biologically relevant. The mechanism of activation and its implication in the functional role played by AMSH within ESCRT complex will be discussed. Results from our studies could provide further insight into understanding the role of AMSH within the ESCRT complex.

Support: NIH (1R01RR026273) and the Indiana Space Grant Consortium
Label Transfer Reagents for the Study of Protein Kinase Complexes
Authors: Andrews Simeon , Perera Gayani , Ranjitkar Pratistha , Maly Dustin
Published: June 2021

Protein kinases are heavily involved in the regulation of many intracellular processes. The specificity of these kinases is often regulated by their interactions with other proteins. Consequently, we have sought to develop new tools to elucidate the identities and spatial relationships of these protein kinase complexes. We proposed a novel label transfer reagent (LTR) which has three components: a protein kinase inhibitor to direct the LTR to the kinase of interest; a photocrosslinker; and a label to use as a chemical handle for visualization or purification of labeled proteins. We have synthesized and tested several LTRs. They retain the specificity and potency of the kinase inhibitor portion, and effectively label known activators and substrates of the kinases. They are highly selective, with no discernible labeling of other highly abundant proteins. Moreover, we can use these LTRs to study kinase complexes expressed in mammalian cells, expanding the use of the LTRs beyond proteins expressible in bacteria. These LTRs promise to be useful tools for validating proposed kinase binding partners and for identifying the spatial relationships of protein kinase complex members.
Combinatorial biosynthesis of unnatural polyketides using a type III polyketide synthase from Oryza sativa
Authors: Chow Jeng Yeong , Wongsantichon Jantana , Robinson Robert , Gan Yunn Hwen , Yew Wen Shan
Published: June 2021

Polyketides are a class of secondary metabolites found in many bacteria and fungi that display broad range biological and pharmacological activities. Biological syntheses of these molecules by type III polyketides synthases are particularly interesting, due to their ability to catalyze the complete synthesis of the polyketide molecule within the active site of the enzyme. Studies have also shown that these enzymes are promiscuous, allowing them to accept various unnatural substrates as starter molecules to generate novel polyketides. In this study, a recombinant chalcone synthase from Oryza sativa (OsCHS) was purified and characterized. Like most other chalcone synthases, OsCHS is able to utilize ρ‐coumaroyl‐CoA as the starter molecule and catalyze the addition of three acetate units to make naringenin. We also further demonstrate that precursor‐directed combinatorial biosynthesis with unnatural substrates and rational engineering of mutant enzymes can be adopted as strategies to alter the product specificity of OsCHS. Coupling these approaches to highthroughput screening methods will allow us to generate and identify novel polyketides with interesting biological activities.
The Study of a Catalytic Intermediate of Methylenetetrahydrofolate Reductase
Authors: De la Rosa Metzere Bierlein , Trimmer Elizabeth
Published: June 2021

The flavoprotein Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in the methionine biosynthesis pathway. It catalyzes the reduction of 5,10 methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate), which acts as a methyl donor to homocysteine to form methionine. It has recently been discovered that there is at least one, uncharacterized intermediate in the folate oxidative half‐reaction of MTHFR. Stopped‐flow experiments show the formation of a species at 337 nm which is decayed by one second. We believe this intermediate to represent either an Ered*5‐iminium cation complex or an Ered* CH2‐H4folate complex in which either enzyme or substrate has undergone a conformational change. We have gathered preliminary data on the putative intermediate by performing a series of anaerobic titrations with the enzyme and a variety of substrates. Additionally, two folates have been characterized by fluorescence in the absence and presence of enzyme. We are currently gathering quench‐flow samples in an attempt to analyze the intermediate by LC‐MS. Research was supported by HHMI grant #52006298 to Grinnell College.
Fluorescence lifetimes as a monitor of conformational distributions in NOS
Authors: Umejiego Ezigbobiara N , Ghosh Dipak K , Hopper Benjamin L , Enweani Chioma M , Lester Army , Salerno John C
Published: June 2021

The effects of calmodulin activation on the fluorescence of nitric oxide synthases are well established and parallel the activation of the enzyme by stimulation of electron transfer from NADPH to heme via the flavin containing domains. During this process, the FMN binding domain undergoes obligatory large scale conformational movements, resulting in the production of states in which the fluorescence lifetime of FMN, typically ~4.5 ns, is modified by interactions with FAD or heme, producing shorter lived states with lifetimes of ~90 ps or 1 ns.

Changes in fluorescence have been observed by previous workers in response to calmodulin binding, but salt and chaotrope effects have also been observed. The enzyme has a pronounced dependence on ionic strength, characteristic of shuttle mechanisms, and some activities are greatly enhanced by chaotropes. Ionic strength has a relatively small effect on the distribution of lifetime states in the absence of calmodulin, consistent with the inability of salt to initiate NO synthesis or greatly enhance cytochrome c reduction in eNOS or nNOS. Chaotropes including guanidinium and urea, on the other hand, are effective at promoting the long lifetime states assigned to the free FMN domain (~4.3 ns) and the FMN‐oxygenase complex (1ns) at the expense of the short lifetime ‘input’ state in which FMN is in close association with FAD. The temperature dependence of the fluorescence decay indicates that increasing the temperature from 4C to 37C progressively favors the input state, in which the FMN binding domain has a large surface area of interaction with the FAD and NADPH binding domains. This suggests an entropy driven association in which reduction in size of a shell of ordered water provides the free energy.
PKA‐induced Interaction of Phosphorylated PDE3A (pPDE3A) and BIG1 (Brefeldin A‐inhibited guanine nucleotide exchange protein 1) in Cytosolic Fractions from Human Myocardium may be Important in Regulation of BIG1 Function
Authors: Khan Faiyaz Ahmad , Szabo-Fresnais Nicolas , Krall Judy , Moss Joel , Vaughan Martha , Movsesian Matthew , Manganiello Vincent
Published: June 2021

BIG1 and BIG2 proteins, which contain A‐kinase anchoring protein (AKAP) motifs, catalyze activation of class I ARFs (ADP‐ribosylation factors) by accelerating exchange of bound GDP for GTP (GEF activity). PKA‐catalyzed phosphorylation of BIG inhibits GEF activity. In Hela cells, PDE3A interacts with BIG proteins (PNAS 106, 6158, 2009). Three PDE3A isoforms (PDE3A1‐3), with identical sequences except for N‐terminal deletions of different lengths, are present in human myocardium. PDE3A1 is found exclusively in membranes; PDE3A2‐3, in both membrane and cytosolic fractions. Non‐phosphorylated cytosolic PDE3A2‐3 eluted from Superose 6 (S6) in LMW (low molecular weight) peaks(~800 kD). Incubation of cytosol with PKA and ATP induced assembly of macromolecular signalosomes that eluted from S6 as HMW (high molecular weight) (>3000 kD) complexes and contained pPDE3A (predominantly pPDE3A2) and proteins involved in cAMP signaling (i.e., PKA‐RII, PP2A, 14‐3‐3, BIG1). These molecules co‐immunoprecipitated with pPDE3A in HMW, but not with non‐phosphorylated PDE3A in LMW fractions. These data suggest that, in cardiomyocytes, pPDE3A is incorporated into BIG1‐organized signalosomes, and in these spatially restricted signalosomes, pPDE3A may modulate compartmentalized cAMP/PKA signaling and thereby contribute to regulation of BIG1 activity and ARF function.
Improvement of cell survival by ammonium ions in glutamine‐deprived cells
Authors: Abusneina Abdelmuhsen , Gauthier Eric R.
Published: June 2021

The amino acid L‐glutamine (Gln) is essential to the survival of proliferating cells. How Gln is mechanistically linked to cell survival remains unknown. We wished to determine whether ammonium (NH4+) ions play a role in the survival of Gln‐deprived Sp2/0‐Ag14 (Sp2/0) cells, which undergo apoptosis within minutes of Gln starvation. Supplementation of Gln‐deprived Sp2/0 cultures with NH4+ chloride led to a significant, concentration‐dependent increase in viability over PBS‐treated control. The addition of 5 mM NH4+ acetate was more effective in improving viability than 5 mM NH4+ chloride or 5 mM sodium acetate, suggesting an additive effect between NH4+ and the organic moiety. Interestingly, the combination of methyl‐pyruvate and NH4+ chloride did not improve cell viability over NH4+ chloride alone, suggesting that specific metabolic pathways cooperate with NH4+ to promote viability. Neither NH4+ acetate nor NH4+ chloride prevented caspase‐3 activation or the cytosolic release of cytochrome c, indicating that NH4+ ions promote survival by interfering with late apoptotic events. This study uncovers an unexpected role for NH4+ ions in the modulation of cell survival. Funded by NSERC‐Canada.
Transforming growth factor‐beta and Vascular Endothelial Growth Factor Expressions in the Kidney of Fabry Mouse
Authors: Lee Mi Hee , Choi Eun Nam , Jung Sung-Chul
Published: June 2021

Fabry disease is a lysosomal storage disorder caused by deficiency of α‐galactosidase A, which results in deposition of globotriaosylceramide (Gb3) in lysosomes. Patients develop painful neuropathy, vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Since the level of Gb3 and clinical manifestations of Fabry disease have not been correlated, we investigated altered gene expressions in Fabry mouse kidney. Microarray analysis revealed that renal Tsps (Thrombospondins) were significantly up‐regulated in Fabry mouse kidney. Tsp‐1 and Tsp‐2 act as inhibitors of angiogenesis that is an extracellular matrix protein involved in cell adhesion, proliferation and survival. Tsp‐1 suppresses tumor growth through transforming growth factor‐β (TGF‐β) activation. Fibroblast growth factor‐2 (FGF‐2) induces vascular endothelial growth factor (VEGF) expression. Up‐regulation of Tsp‐1, VEGF, TGF‐β1 and FGF‐2 are associated to vascular and renal pathology in Fabry mouse kidney by western blot analysis. These findings suggested that relation between angiogenic factor and anti‐angiogenic factor leads to vascular dysfunction in the kidney of Fabry disease.
Analysis of ethanol metabolic enzymes in primary human mammary epithelial cells and MCF‐10A cells: possible participation of CYP2E1 in ethanol‐induced oxidative stress and epidermal growth factor receptor (EGFR) activation
Authors: Rodríguez-Fragoso Lourdes , Buitimea Angel Leon , Lauer Fredine , Thompson Todd , Bowels Harmony , Burchiel Scott
Published: June 2021

Ethanol (EtOH) consumption is a well‐established risk factor for breast cancer in women. However, the mechanism by which EtOH exerts its carcinogenic activity in breast tissue remains known. In the present work, we analyze the ethanol metabolic enzymes in primary human mammary epithelial cells (HMEC) and MCF10A cells and to study the role of those enzymes in ethanol‐ induced oxidative stress and EGFR activation. Our results show that EtOH decreased CYP2E1 protein expression at 10, 30 and 100 mM when cells were treated every 12 h for 3 days. We also found that 30 and 100 mM EtOH increased ROS levels after 2 h treatment in CYP2E1over‐expressing 4.1.2E1 cells. Additionally, we found that in CYP2E1over‐expressing 4.1.2E1 cells increased the level of pY1086 EGFR phosphorylation after 18 h ethanol treatment. These studies suggest that ethanol can transactivate EGFR and activate downstream cell signaling in MCF10A cells, mediated by oxidative stress. These studies were supported in part by NIH RO1 ES‐07259 and by CONACYT ‐Mexico a Fellowship to Angel León‐Buitimea number 212849.
Excess Protein O‐GlcNAcylation and the Progression of Diabetic Cardiomyopathy
Authors: Fricovsky Eduardo Santiago , Suarez Jorge , Ihm Sang-Hyun , Scott Brian T. , Suarez-Ramirez Jorge A. , Villarreal Francisco J. , Dillmann Wolfgang H.
Published: June 2021

We examined the hypothesis that enzymatic protein O‐GlcNAcylation plays a role in the development of diabetic cardiomyopathy (DC) in a mouse model of type 2 diabetes (DM2). Mice injected with low‐dose STZ and fed high‐fat diet developed hyperglycemia and obesity. After 1 month of treatment, DM2 mice showed increased BW, impaired FBG and glucose tolerance, and hyperinsulinemia. Echo evaluation revealed LV diastolic dysfunction by 2 months which was associated with increased overall cardiac protein and nuclear transcription factor Sp1 O‐GlcNAcylation. By 4 months systolic dysfunction was observed and SERCA2a expression decreased by 50%. Levels of the rate limiting enzyme of the hexosamine pathway GFAT were increased as early as 1 month. Fatty acids, which are elevated in DM2, were linked to excessive protein O‐GlcNAcylation levels as cultured cardiac myocytes treated with oleic acid showed increased O‐GlcNAcylation and GFAT levels. These data indicate that the early onset of diastolic dysfunction followed by the loss of systolic function is associated with increased cardiac protein O‐GlcNAcylation and increased O‐GlcNAcylation levels of key calcium handling proteins. Furthermore, fatty acids play a role in stimulating excess O‐GlcNAcylation. The nature and time course of changes observed in cardiac function suggest that protein O‐GlcNAcylation plays a mechanistic role in the triggering of DC in DM2.
H2S and L‐Cysteine Increase the Glucose Utilization via the PIP3 Mediated Insulin Dependent (PI3K/AKT) and the Insulin Independent (SIRT1/AMPK/PPAR gamma) Signaling Cascades in U937 Human Monocytes Exposed to High Glucose (HG)
Authors: Manna Prasenjit , Jain Sushil K
Published: June 2021

Diabetic patients have lower blood levels of hydrogen sulfide (H2S) and L‐cysteine (LC). Using U937 human monocytes, this study examined the role of H2S and LC in the impaired glucose metabolism and insulin signaling cascades in diabetes. Monocytes were treated with HG (25 mM,20 h) in presence or absence of LC (100, 500, 1000 μM) or H2S (10, 100 μM). Both LC and H2S treatment caused PI3K activation, PTEN inhibition, increase in intracellular PIP3 (phosphatidylinositol‐3,4,5‐trisphosphate), AKT phosphorylation (Ser 473) and glucose utilization in HG‐treated cells. The effect of LC on PIP3 and glucose utilization was prevented by propargylglycine, an inhibitor of cystathionine‐γ‐lyase that catalyzes H2S formation from LC. This demonstrates that H2S mediates the effect of LC on increased PIP3 and glucose utilization. Treatment with PIP3, H2S and LC caused activation of SIRT1, AMPK phosphorylation (Thr 172) and PPARγ and inhibition of GSK3β (Ser 9) in HG treated cells. Thus, LC/H2S supplementation increases the glucose utilization via the PIP3 mediated activation of SIRT1, AMPK and PPARγ and inhibition of GSK3β. This study reports a novel molecular mechanism by which H2S or LC can up‐regulate the insulin stimulated (PI3K/AKT) and insulin independent (SIRT1/AMPK/PPARγ) glucose metabolism pathways essential for maintenance of glucose homeostasis. (Supported by NIH RO1 DK072433 and Malcolm Feist Chair in Diabetes)
IRS‐1/2 Mediated Regulation of Hepatic OGT and OGA Expression
Authors: Sundararaj Kamala , Alqalam Abdelmohsen M , Robinson Katherine A , Buse Maria G , Ball Lauren E
Published: June 2021

To investigate the role of hepatic insulin signaling in the postprandial regulation of O‐GlcNAc transferase (OGT) and OGlcNAcase (OGA) expression, C57BL/6 male mice with a conditional knock out of IRS‐1/2 in liver (LKO) were fed a chow or high fat diet (HFD) for 16 weeks and then either fasted for 18hrs or fasted and refed ad libitum for 4hrs. In control animals that were fasted or refed, qRT‐PCR revealed a postprandial increase in hepatic OGT expression (146% ± 12.8, n=5) and a reduction in OGA transcript levels (65.7% ± 6.1, n=5). The postprandial increase in hepatic OGT expression was attenuated in LKO mice. In control mice, high fat feeding attenuated both the postprandial rise in hepatic OGT expression (123% ± 5.0, n=5) and the suppression of OGA expression (77.2% ± 4.6, n=5). In LKO mice in the refed state, OGT mRNA expression was reduced by 68% ± 1.5 (n=5) after 16 weeks HFD as compared to animals fed a chow diet. Furthermore with 16 weeks HFD, elevated expression of OGA was observed in hepatic (170% ± 7.5) and adipose (316.5% ± 21.6, n=5) tissue as compared to chow diet. These studies reveal that the postprandial changes in hepatic expression of OGA and OGT are regulated, in part, by IRS‐1/2 mediated insulin signaling in vivo and that the suppression of OGA expression in fat fed animals is lost in the absence of hepatic IRS‐1/2. Supported by T32 HL07260 and RO1DK002001.
Fatty acid elongase‐5 (Elovl5) regulates the mTORC2‐FoxO1 pathway in obese‐diabetic C57BL/6J mice
Authors: Tripathy Sasmita , Jump Donald B
Published: June 2021

Elevated fatty acid elongase‐5 (Elovl5) activity in livers of high fat diet induced obese‐ diabetic C57BL/6J mice corrects fasting hyperglycemia by suppressing gluconeogenesis (GNG) through the regulation of Akt2 and FoxO1 phosphorylation status. This report examines Elovl5 regulated signaling mechanisms controlling Akt2 & FoxO1 phosphorylation. Elevated hepatic Elovl5 activity in obese‐diabetic mice and in HepG2 cells increased Akt2‐Serine473 (an mTORC2 site) phosphorylation. Increased Akt2‐Serine473 correlates with increased FoxO1 phosphorylation and suppressed Pck1 gene expression. In HepG2 cells, Akt inhibitor (Akt1/2i) blocked Elovl5 mediated FoxO1 phosphorylation while the PP2A inhibitor (mycrocystin‐LR) augmented FoxO1 phosphorylation. The mTORC1 and mTORC2 inhibitor (PP242), but not the mTORC1 inhibitor (rapamycin), blocked Elovl5 regulated Akt2‐Serine473 and FoxO1 phosphorylation. In vivo, high fat diets suppressed raptor (a mTORC1 component), but induced rictor (a mTORC2 component). Induction of hepatic Elovl5 activity augmented rictor protein abundance ~2‐fold and stimulated rictor‐mTor interaction. These studies establish rictor as a target for Elovl5 mediated suppression of GNG by regulating Akt2‐Serine473 and FoxO1 phosphorylation.

Research support: NIH (DK43220) and NIFA/USDA (2009‐65200‐05846).
Analysis Of The Metabolic Changes Occurring During The Development Of Metabolic Disorder Induced By A High‐Fat Diet
Authors: De la Rosa Medina Angelica Viviana , Gutierrez Irais Sanchez , Reynoso Rosalía , Salgado Luis Miguel
Published: June 2021

The high incidence of obesity and metabolic disorders are caused, principally, by a high fat diet (HFD) and a high‐sweetened beverage consume. The characterization of the metabolic changes that appear during the development of metabolic syndrome are subjected to numerous studies but they are not completed. It is important to investigate these biochemical and metabolic changes, those results would identify metabolites that could be used as diagnostic and prognostic biomarkers. For our studies, we used an animal model, the obesity was induced by feeding a commercial diet (nu3Lab 22‐5) supplemented with 15% saturated fat and 15% fructose. Physical and biochemical data were registered monthly and every three months blood samples were withdrawn. The serum from the samples was analyzed by HPLC using a non‐focused metabolomic analysis. Since the six months, it was observed that the animals had a considerable gain of body weight with a high relationship with different metabolic alterations, such as an increased level of basal glucose, an extended glucose tolerance curve and high amount of serum triglycerides. We also observed differences in the metabolic profile for the animal model. It is expected to identify through a focused metabolomic analysis, the metabolites that could be possible obesity and metabolic disorders biomarkers. (A.V.R. and I.S. received a CONACYT and COFAA‐IPN scholarships; the financial support came from SIP‐IPN and QRO‐2008‐CO2‐10090, CONACYT, Mexico).
Evaluation of Anti‐diabetics and Cardiovascular Effects of a Nigerian poly herbal Extract (Picralima nitida seed, Acacia nilotica pod and Aristolochia ringens root) on Postprandial and Alloxan Induced Diabetic Albino Rats
Authors: Ani Anthony Amaechi , Ogbonnia Steve O. , Okosun Anthonia
Published: June 2021

Twenty Wister strain albino rats were randomly assigned to four groups; A,B, C, and D with each consisting of five animals received extract as follows: 500mg/kg body weight‐bwt; 250mg/kg bwt; 100mg/kg bwt; 0.5mL(2%) acacia solution serve as control. After 30 minutes the animals were administered orally with 40 % (w/v) glucose at a dose 1mL/100 g bwt. Blood glucose level was monitored at 30, 60 and 120 minutes intervals and reports as the average glucose level of each group. Another set of twenty rats were randomly distributed into four groups of five animals each, while the additional fifth group consisting of five normal rats, positive control. Group I: Control rats received 0.5% Acacia suspension;group II: Diabetic rats treated with glibenclamide 600μg/kg bwt; group III: Diabetic rats treated with 250mg extract/kg; group IV: Diabetic rats treated with 500mg extract/kg ; group V: untreated diabetic rats. There was significant reduction (P<0.05) in the plasma glucose level and low density lipoprotein (LDL)‐cholesterol levels, and significant increase in high density lipoprotein (HDL)‐cholesterol levels in the treated diabetic rats compared to the negative control. This results showed that the poly herbal formulation had both good hypoglycemic activities and beneficial effects on cardiovascular risk factors.
Vascular endothelial cells as a source of multipotent stem‐like cells
Authors: Olsen Bjorn R
Published: June 2021

During development, cartilage‐ and bone‐forming stem cells collaborate with vascular, nerve and hematopoietic cells to produce skeletal elements and the joints that link them together. When activated during postnatal repair, stem cells in the periosteal regions or recruited from bone marrow via the circulation are fully capable of healing most fractures. However, regeneration of skeletal tissues fails in patients with loss of articular cartilage (osteoarthritis), large, localized defects in bone (when large tumors or necrotic bones are removed) or generalized bone loss (osteoporosis). Bone marrow‐derived mesenchymal stem cells, long thought of as promising for cartilage, bone and skeletal muscle repair/regeneration, appear to be more important for the growth factors they produce than for their in vivo ability to differentiate into specific cell lineages. This talk will discuss the discovery that differentiated vascular endothelial cells can undergo a conversion into mesenchymal stem‐like cells (endothelial‐mesenchymal transition; EndMT); these stem‐like cells can in turn differentiate into chondrocytes, osteoblasts or adipocytes in vitro and in vivo. Thus, blood vessels may not only bring blood supply and oxygen to tissues undergoing repair but may also provide mesenchymal stem cells for cell differentiation. EndMT is regulated via TGF_/BMP receptor signaling. In patients with Fibrodysplasia ossificans progressiva (FOP), where skeletal muscle and associated soft tissues progressively turn into bone, endothelial cells contribute significantly to the chondrocytic and osteoblastic cells that form the ectopic bone. FOP patients are heterozygous for activating mutations in the BMP type I receptor ALK2. However, EndMT can also be induced in endothelial cells from individuals without FOP when BMP receptor signaling is activated in the absence of inhibitors of EndMT. One of these inhibitors, Vascular Endothelial Growth Factor‐A (VEGF), is critical at skeletal tissue repair sites by stimulating angiogenesis and osteoblast differentiation. New insights into mechanisms by which VEGF regulates stem cell differentiation suggest that it may be possible to block the negative effect of VEGF on EndMT without affecting its positive effect on osteoblast differentiation.
The Effects of the O‐xylosyltransferase (oxt) Mutation on Wg, Hh, and Dpp Signaling
Authors: Liu Alicia
Published: June 2021

The o‐xylosyltransferase (oxt) encodes a glycosyltransferase needed for the first step of proteoglycan glycosaminoglycan (GAG) biosynthesis. Disruptions in heparan sulfate proteoglycan (HSPG) biosynthesis change the extracellular environment, which has been shown to block Wingless (Wg)/Wnt, Hedgehog (Hh), and Decapentaplegic (Dpp)/TGF‐β intercellular signaling. Therefore, mutations in oxt are predicted to disrupt normal Wg, Hh, and Dpp signaling due to the absence of heparan sulfate (HS) and chondroitan sulfate (CS) modified extracellular proteoglycans. Characterization of the oxt mutant in Drosophila wing discs shows this mutation has an unexpectedly mild phenotype as compared with other mutants acting further down in the HS biosynthetic pathway, such as sulfateless (sfl), which is responsible for specific downstream sulfation and activation of only HS chains. The double mutant oxt‐sfl was made to address why the oxt phenotype is less severe. Clonal analysis shows that the double mutant looks more like sfl, suggesting the oxt null mutation does not yield a fully penetrant phenotype, allowing some xylose addition to the core proteoglycan upon which sfl can act. Because the oxt mutation is a nonsense mutation that eliminates its active site, the lack of penetrance can be attributed to either perdurance of residual Oxt protein or mRNA in mutant cells.

2P20RR01645 NIH/NCRR Administrative Supplement University of Delaware Summer Undergraduate Research Fellowship
Characterization of the EMT response in Crybb2Phil mutants
Authors: Roop Victoria Helen
Published: June 2021

The most abundant protein in the adult lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene cause cataracts in mice and humans. A twelve nucleotide deletion in mouse lens epithelial cells (LEC), known as the Crybb2Phil mutation, leads to cataracts associated with lens fiber cell defects as well as epithelial to mesenchymal transition (EMT) of the LEC in adulthood. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to characterize the EMT response caused by the Crybb2Phil mutation.

Tissues obtained from Crybb2Phil mutants were immunostained for alpha and beta integrin subunits, phospo‐SMAD3 (p‐SMAD3), transforming growth factor β‐induced (BIGH3), and EMT the markers E‐cadherin, vimentin, follistatin, and α‐smooth muscle actin (α‐SMA).

Compared to the wildtype lens, β1, αV, and α5 integrins, vimentin, α‐SMA, and p‐SMAD3 were upregulated in the Crybb2Phil mice. No changes were seen in α2, α3, and α6 integrin subunits or follistatin. Consistent with EMT, E‐cadherin was downregulated in the Crybb2Phil mice.

Upregulation of α‐SMA signifies that Crybb2Phil mice undergo EMT around two months of age. Furthermore, over expression of β1 and αV integrins along with p‐SMAD3 in the Crybb2Phil mutant suggests that the EMT seen in Crybb2Phil mice may be occurring via the TGFβ‐SMAD interacting pathway. This project is funded by National Eye Institute grant EY015279.
Silent scaffolds: inhibition of JNK3 activity in living cells by a dominant‐negative arrestin‐3 mutant
Authors: Gurevich Vsevolod V , Breitman Maya , Gimenez Luis E , Kook Seunghyi , Lizama Britney N , Palazzo Maria C , Gurevich Eugenia V
Published: June 2021

We established a new in vivo arrestin‐3‐JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3‐luciferase and Venus‐arrestins. We tested the ability of WT arrestin‐3 and its 3A mutant, both of which readily bind the β2‐adrenergic receptor, as well as two mutants impaired in receptor binding, Δ7 and KNC, to interact with JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding‐deficient mutants bind JNK3 significantly better than WT and 3A arrestin‐3. WT arrestin‐3 and the Δ7 mutant robustly promoted JNK3 activation, whereas the 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct functions of arrestins that can be independently manipulated by appropriate mutations. We found that the KNC mutant, which tightly binds JNK3 without facilitating its phosphorylation, functions as a dominant‐negative, competitively decreasing JNK3 activation by WT arrestin‐3. Thus, KNC is a silent scaffold, a novel type of molecular tool for selective suppression of MAPK signaling. These data provide the first demonstration that this mechanism of MAPK regulation can be used in living cells.

NIH grants GM081756, GM077561, and EY011500 (VVG).
TCTP as a Regulator of Rheb: Impact on mTOR Signaling
Authors: Grabiak Mary , Cortez Jessica , Parmar Nitika
Published: June 2021

TCTP (Translationally Controlled Tumor Protein) is a highly conserved protein that has been suggested as a tumor associated antigen as shown by its deregulation in many different types of cancers. TCTP has been shown to interact with Rheb, a key player of the PI3 Kinase/mTOR signal transduction cascade. This pathway regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. In this study the contribution of TCTP toward the mTOR pathway was investigated by over‐expression and gene silencing of TCTP in HEK293 and HeLa cells. The role of TCTP as a guanine nucleotide exchange factor (GEF) for Rheb has been controversial and this study highlights potential contribution of TCTP as a regulator of Rheb. Random mutant forms of TCTP were made and over‐expressed in cell lines. Additionally, cells were treated with multiple TCTP siRNAs in a dose and time dependent manner. The effects of TCTP over‐expression and silencing on mTOR signaling were monitored via analysis of S6 phosphorylation and cell viability assays. Results were compared to wild‐type TCTP expression and treatment with non‐specific siRNAs respectively. The impact of TCTP expression on cell viability under serum starved conditions was also explored.

This work was supported by a Faculty Development Minigrant to Nitika Parmar.
Endothelin‐1 (ET‐1) induces Ca2+‐Calmodulin‐dependent Protein Kinase II α (CaMKII‐α) expression in an ERK1/2‐dependent pathway in vascular smooth muscle cells (VSMC)
Authors: Cheyou Estelle Rolande Simo , Srivastava Ashok K
Published: June 2021

ET‐1 is implicated in the pathogenesis of vascular abnormalities, such as hypertension and atherosclerosis, through the hyperactivation of growth promoting signaling pathways such as ERK1/2 and PKB. We have recently shown that CaMKII‐α, a multifunctional protein kinase plays a critical role in mediating ET‐1‐induced ERK1/2 and PKB signaling as well as proliferative and hypertrophic responses in VSMC. We have also shown that CaMKII‐α level is upregulated in VSMC from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY) rats. Since elevated levels of ET‐1 have been reported in hypertension, we wished to determine if there was a role of ET‐1 in the heightened expression of CaMKII‐α observed in SHR. Therefore, we examined the effect of ET‐1 on CaMKII‐α expression in VMSC. ET‐1 (100nM) increased CaMKII‐α expression in VSMC in a time dependent fashion, peaking at 90 minutes. Pretreatment with actinomycin D and cycloheximide, respective inhibitors of mRNA transcription and protein synthesis, potently inhibited ET‐1‐induced effect on CaMKII‐α. Furthermore, BQ123 and PD98059, two selective pharmacological inhibitors of ETA receptor and MEK pathway, respectively, attenuated ET‐1‐induced CaMKII‐α expression. These data demonstrate that ET‐1 induces CaMKII‐ α expression in VSMC via an ETA receptor‐mediated activation of ERK1/2 pathway in VSMC. (Supported by CIHR grants).
Cellular localization of tenomodulin isoforms
Authors: Qi Jie , Dmochowski Jack , Banes Ashely , Tsuzaki Mari , Bynum Donald , Gomez Shawn , banes Albert J
Published: June 2021

Tenomodulin, as a major tendon biomarker, TNMD is highly expressed in tendons. However, the functions of TNMD in tendons are not clear although it was suggested to play a role in anti‐angiogenesis in eye and its single nucleotide polymorphisms are associated with obesity. To investigate the function of TNMD in tendons, we performed studies on TNMD isoforms. Data mining showed that three different transcripts exist in human tissues, with deduced molecular weights of 37.1, 20.3 and 25.4 kDa. To clone TNMD isoforms, the cDNAs of human TNMD isoforms were amplified from human total RNA isolated from human tendon specimen and cloned into pEGFP‐C1 vector. GFP‐fused TNMDs were expressed in human and porcine tenocytes. Knockdown of TNMD expression was performed with a RNA interference technique. In the present study, we showed the existence of multiple Tnmd isoforms in tendons with Real time PCR and western blot. Knockdown of all TNMD isoforms with siRNA reduced cell proliferation. Each TNMD isoform showed different localization in tenocytes. Prediction of each isoform in I‐TASSER gave different functions, TNMD I was predicted to be a cytosine‐specific methyltransferase, TNMD II was classified as a SUMO protease, SENP1, and TNMD III was similar to á‐syntrophin. These results suggest that each Tnmd isoform may play distinct roles in tendons.

This study was supported by Flexcell International Corp.
Bone morphogenetic protein 2 and casein kinase 2 regulate the differentiation of osteoblasts
Authors: D'Angelo Alex Michael , Bragdon Beth , Bonor Jeremy , Nohe Anja
Published: June 2021

The objective of this research is to study the shuttling and localization of BMP2 receptors on the cell membrane during signaling leading to osteoblast differentiation. Signaling is initiated by the binding of bone morphogenetic protein 2 (BMP2) to transmembrane BMP receptors type Ia (BMPRIa) and type II (BMPRII). Intracellularly, the protein casein kinase II (CK2) is associated with BMPRIa at three binding sites, denoted 2.1, 2.2, and 2.3. CK2 is a negative regulator of BMP2 signaling. Its dissociation, in response to BMP2 binding or blockage of CK2 binding sites, leads to signal transduction. As a location of BMP receptors, the membrane domains caveolae are known to be involved in signaling. To study the localization of BMP receptors in response to BMP2 and blockage of CK2 binding sites with blocking peptides, mesenchymal stem cells of the mouse cell line C2C12 were labeled for caveolin 1 (marker for caveolae), BMPRIa, and CK2 and imaged. The technique of image cross correlation spectroscopy determined the spatial similarity (colocalization) of the proteins to establish where BMP receptors were located on the membrane. The results showed that BMP2 stimulation and blockage of CK2 binding site 2.3, led to increased colocalization of BMPRIa with caveolin 1. This indicates that BMP receptors are localizing to caveolae with these particular treatments. Funding provided through the Howard Hughes Medical Institute (HHMI)
Evaluating multiple signaling activities in LPS induced human peripheral blood mononuclear cells
Authors: McGivern Meghan , Wiewel Kurstin , Borkovec Ashley , Jett Marti , Mendis Chanaka
Published: June 2021

Lipopolysaccharide (LPS) is an endotoxin that provokes the secretion of pro‐inflammatory cytokines in many cells; this can induce septic shock in humans. The intent of this project is to develop a rapid diagnostic method to reduce the effects of LPS by applying the functions of signal transduction pathways induced by LPS in Peripheral Blood Mononuclear Cells (PBMC) as a prototype module. After identifying a set of common signaling pathways induced by LPS through micro array analysis we blocked key components such as JNK and p38 of these pathways that function as pathway inter‐connectors and then evaluated the extent of alterations to the previously identified gene expression patterns. We also extended our investigation to include other signaling pathway components such as DHRS9, HSPH1, and MOCS3. Our main objective was to investigate the effect of two inhibitors (SP600125 and SB203580) on the time dependent gene expression pattern and then the impact on components that are indirectly associated with the inhibited signaling pathways. In addition, we also evaluated the specific inhibitors by correlating the observed gene expression pattern to the protein expression using ELISA. We believe the study will allow us to better understand the complex interactions of multiple signal transduction pathways induced by LPS, and how to combat the effects.
Differential effect of chronic opioid exposure on signaling and translational pathways in rat brain
Authors: Korneeva Nadejda L , Schrott Lisa M
Published: June 2021

There have been dramatic increases in the nonmedical use of prescription opioids, such as oxycodone and hydrocodone. While there has been significant research on the use of these drugs for short‐term analgesic use, much less is known about the consequences of chronic use. In this study we have investigated molecular biosignatures of chronic oxycodone treatment of adult rats. We have analyzed three brain regions: brainstem, nucleus accumbens and cortex. We observed that oxycodone activates ERK1/2, p38 and mTOR pathways in brainstem and nucleus accumbens suggesting pro‐survival and cytoprotective action. In agreement with these data, the level of phosphorylated 4E‐BP1 and eIF4E increased in brainstem and nucleus accumbens suggesting regulation of translation initiation rate in these tissues. Changes in the rate of translation may also indicate that oxycodone affects the spectrum of mRNAs translated. Polysomal analyses suggested translational down‐regulation of MOR‐1, Bcl‐2, and cFos mRNAs and up‐regulation of DOR mRNA by oxycodone in brainstem. Western blotting confirmed that levels of MOR‐1 and cFos proteins indeed decreased in brainstem lysates from oxycodone‐treated rats. Down‐expression of MOR‐1 maybe a marker of tolerance and dependence, while and upregulation of DOR following chronic oxycodone may be a marker for altered pain and analgesia sensitivity in these rats. Supported by Department of Emergency Medicine and Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center; Shreveport.
in vitro and in vivo investigation of modulators of hyperactivated ion channel induced necrosis in C. elegans
Authors: Kamat Shaunak , Bianchi Laura , Yeola Shrutika , Driscoll Monica
Published: June 2021

Exposure of tissue to extreme temperature, corrosive chemicals, ischemia or other noxious stimuli can induce necrotic degeneration that is characterized by cellular swelling, loss of organellar integrity, accumulation of reactive oxygen species, activation of proteases and ultimately cell lysis. Necrosis can be genetically induced in C. elegans neurons by expression of mutant hyperactive ion channels. Mutant DEG/ENaC channels cause entry of excess calcium into the cell, triggering release of ER calcium stores and subsequent activation of calpain proteases. Due to the role of calcium in the induction and progression of this type of necrotic cell death we screened genes that encode calcium binding proteins and assayed for potential modulation of the cell death phenotype. RNAi and deletion mutant data indicates the involvement of EF hand containing proteins localized to ER and mitochondria. We will report on progress in deciphering their roles in neuronal necrosis.

To extend mechanistic dissection, we have also been studying properties of hyperactivated ion channel‐induced necrosis in C. elegans embryonic cell culture and show that cultured neurons behave similarly to their counterparts in vivo. We have been pioneering the use of total internal reflection fluorescence microscopy to measure the MEC‐4/MEC‐10 channel at the neuron surface. We find changes in the surface expression and distribution of mechanotransduction complex proteins when necrosis is induced using a temperature‐sensitive mec‐4(d) allele. We are also studying the effects of mutant DEG/ENaC channels on mitochondrial morphology by imaging mitochondrially localized GFP in vitro and in vivo, as data suggest a relationship between maintenance of mitochondrial integrity and susceptibility to necrotic stimuli.