bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium induces electron transfer in NADPH oxidase 5
Authors: Wei Chin‐Chuan , Johnson Tremylla , Bux Stephen M , Hamilton Lindsey N , Stuehr Dennis J
Published: June 2021

Superoxide generated by non‐phagocyte NADPH oxidases (NOXs), such as NOX5 and DUOX, is of growing importance for vascular physiology and pathology. NOX5 and DUOX enzymes consist of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. They appear to be regulated by self‐contained Ca2+ binding domains (EFD). The basis for their catalytic profiles and regulation is unclear. Here, we demonstrated that Ca2+ gates heme reduction NOX5 in anaerobic conditions. To better understand this interaction, we monitored its Ca2+ and terbium (Tb3+) bindings by fluorescent spectroscopy using wild type and mutants of the recombinant EFD. The titration of EFD yielded a [Ca2+]0.5 of 4.5 μM, which is similar to published results, but the Ca2+ bindings appears to be tighter and occur in a cooperative manner. About 10 μM Ca2+ is required to fully saturate EFD and completely activate NOX5 superoxide production. EFD binds terbium with a [Tb3+]0.5 of 20 μM. The site‐directed labeling on Cys107 of EFD with IAEDNS had the same response to the Ca2+ level as the non‐labeled EFD. It reveals an anisotropy value of 0.11 and will be used to gather the structural information of the EFD‐flavoprotein complex using FRET. NOX5 contains a consensus peptide sequence that interacts with Ca2+‐bound calmodulin (CaM), and it has been demonstrated that this CaM/Ca2+ binding increases its sensitivity for free Ca2+. We used Troponin C (TnC)‐CaM chimeras to pinpoint the structural‐functional relationship to understand how individual EF‐hands motifs in CaM affect the electron transfer in NOX5. We are currently performing similar studies in DUOX. This work was supported by Beginner‐Grant‐in‐Aid from the American Heart Association
MafA sumoylation and its function in pancreatic beta cells
Authors: Shao Chunli , Cobb Melanie
Published: June 2021

Insulin is an essential hormone for maintaining glucose homeostasis. Insulin gene expression is regulated by several transcription factors. Among them, MafA is the beta cell specific activator, which binds to the RIPE3b/C1 element. It is also known that MafA is required for both the development and maintenance of mature insulin producing pancreatic beta cells. In the present study, we provide the first evidence that MafA can be modified by SUMO1/2 (Small Ubiquitin‐related modifier) at lysine 32 in pancreatic beta cells. Starvation with low concentration of glucose and oxidative stress greatly enhance MafA sumoylation levels. Furthermore, we show that sumoylated MafA reduces MafA transcriptional activity on both insulin and CHOP‐10 (C/EBP homologous protein 10) gene promoters. However, MafA sumoylation has no effect on its nuclear localization. Additionally, MafA sumoylation does not prevent ubiquitin‐dependent degradation. Thus, the modification of MafA by SUMO affects insulin gene transcription and beta cell function.
The alpha2 catalytic subunit of AMP‐activated protein kinase (AMPK) is O‐GlcNAc modified
Authors: Bullen John William , Hart Gerald
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a highly dynamic Ser/Thr specific protein modification often occurring at the same or adjacent sites as phosphorylation. Increased flux through the hexosamine biosynthetic pathway (HBP) elevates protein O‐GlcNAcylation and causes insulin resistance. Recent studies in adipocytes showed that chronic hexosamine flux through the HBP activates fatty‐acid oxidation through AMP‐activated kinase (AMPK), a vital sensor and regulator of whole body and cellular energy homeostasis. They further suggest that AMPK, or a binding partner, may be O‐GlcNAc modified in adipocytes. Using O‐GlcNAcylated protein specific immunoblotting and enzymatic radioactive labeling experiments, we conclusively show that immunoprecipitated AMPKα2 is itself O‐GlcNAc modified in two different cell types. Furthermore, global elevation of O‐GlcNAcylated protein in C2C12 muscle cells increases AMPKα2 protein levels, but in contrast to adipocytes, decreases phosphorylation of its primary downstream target, acetyl‐CoA carboxylase (ACC). We are currently site mapping O‐GlcNAcylated sites on AMPKα2, as well as investigating further the role O‐GlcNAc may have on tissue‐specific regulation of AMPK activity. (Supported by NIH grant R01 DK61671. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU).
Rosiglitazone protects against beta cell destruction in ZDF (Type 2) diabetic rats
Authors: Wijekoon Enoka P. , Brosnan Margaret E , Lhoták Šárka , Austin Richard C , Brosnan John T
Published: June 2021

The progression of type 2 diabetes involves insulin resistance, impairment in insulin secretion, and eventual loss of β‐cell mass. Current therapies largely address insulin sensitivity and secretion but it is also important that therapies be found which protect against β‐cell loss. Finegood et al (2000) have reported that rosiglitazone (RSG) treatment largely prevents loss of β‐cell mass. We have confirmed this observation and present new information on islet morphology during this process. Examination of pancreas sections from 6 week‐old ZDF fa/fa rats, by means of double immunofluorescence staining for insulin and glucagon, revealed the normal oval morphology of islets with a mantle of α‐cells at the periphery and β‐cells in the central region. Six weeks of treatment with RSG (10 μmol/kg BW/day) maintained this structure whereas, in untreated rats, the islets had lost their normal architecture. After another 6 weeks, atrophy of the islets, fibrosis and loss of insulin‐positive cells was observed in the untreated rats whereas in the treated group, the islets were larger with numerous insulin‐positive cells. However, at this time point, islets from both groups showed diffusion into exocrine tissue and loss of the α‐cell mantle. The appearance of signs of endoplasmic reticulum stress during the loss of islet integrity was also protected against by RSG. (Supported by CIHR and Canadian Diabetes Association).
site mapping and quantitation of O‐GlcNAcylated proteins in diabetic erythrocytes
Authors: Park Kyoungsook , Wang Zihao , Saudek Chtistopher D. , Hart Gerald W.
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a ubiquitous post‐translational modification found on nucleocytoplasmic proteins. This modification is very dynamic and sensitive to cellular nutrients and stress. Cycling of O‐GlcNAc on many proteins can modulate diverse events in cells. Elevated global O‐GlcNAcylation is associated with development of many diseases including neurodegenerative diseases, cancer and diabetes. In diabetic patients, prolonged exposure to high glucose may result in global changes of O‐GlcNAcylation and altered occupancy of O‐GlcNAc on certain proteins.

We examined the changes of O‐GlcNAcylation in diabetic red blood cells and site‐mapped O‐GlcNAc on proteins using semi‐quantitative mass spectrometry method. We modified beta‐elimination/Michael addition detection (BEMAD)/mass spectrometry method by adding enrichment steps for O‐GlcNAcylated peptides. This step improved the efficiency of O‐GlcNAc detection. We have identified multiple O‐GlcNAc sites on erythrocytic proteins such as catalase and carbonic anhydrase I and observed site‐specific differential occupancy of O‐GlcNAc. Significant changes of O‐GlcNAcylation of certain proteins/sites will be used for further study to develop a diagnostic tool for diabetes.
Investigation of the effects of 2‐naphthylamine on the protein expression of Pseudomonas putida KT2440
Authors: Filkins Laura Marie , Tubbs Laura Ellen , Craig Paul , Kenney MaryEd , Mallonga Jamie Lou
Published: June 2021

Two‐dimensional gel electrophoresis can be used in proteomics to study changes in protein expression. Currently, this tool is being employed to determine the impact of 2‐Naphthylamine, a known carcinogen, on the growth and protein expression of Pseudomonas putida KT2440. In order to grow strong cells and to increase gel resolution, modifications were made to growth media concentrations and protein isolation methods. Following this adapted procedure, a control was produced using Succinic Acid in Hutner's Growth media and new two‐dimensional control gels were analyzed. The optimal concentration at which to grow the bacteria in the presence of 2‐Naphthylamine was determined to be 0.25mM. The proteins from the 2‐Naphthylamine variable are similarly separated using two‐dimensional gel electrophoresis and the gels are analyzed using the PDQuest Software. A comparison is made between the 2‐napthylamine gels and the Succinic Acid control gels to look for changes in the bacteria's protein expression. Changes in protein expression in the 2‐Naphthylamine gels (when compared to the master control gel) can be new proteins, missing proteins, and under or over‐expression of proteins. Financial support by the Rochester Institute of Technology College of Science Dean's Office and the Department of Chemistry.
Presenilin 1 (PS1) levels may indirectly affect amyloid precursor protein (APP) processing and impair memory
Authors: Kumar Vijaya Buddhiraju , Franko Mark , Banks William A , Farr Susan A , Morley John E
Published: June 2021

The P8 line of the senescence accelerated (SAMP8) mice exhibits early loss of memory and premature aging. This loss of memory could be partially reversed by reducing the levels of amyloid precursor protein (APP), a ubiquitous membrane protein that is physiologically processedby site‐specific proteolysis by the endo peptidases called α, β and γ secretases. Cleavage by α‐or β‐secretases followed by γ‐secretase at multiple sites inthe transmembrane region of APP results inthe release of small peptides, Aβ1–40 and Aβ1–42. γ‐secretase is a high molecular weight complex which is composed of presenilin 1 (PS1), mature nicastrin, APH‐1, and Pen‐2. As PS1 has been shown to play a critical role in facilitating γ‐secretase activity as one of its integral components, and mutations in this protein are associated with Familial Alzheimer's Disease (FAD), for this investigation we have cloned it from SAMP8 mouse hippocampus and compared its sequence with other species. Further, changes in the expression of PS1 with age in the hippocampal tissue of SAMP8 were studied. The results showed that PS1 cDNA sequence of SAMP8 is identical to that of normal mouse. However, its expression in the hippocampal tissues of SAMP8 exhibited a decrease when normalized to actin, while CD‐1 mice, a strain that des not exhibit premature memory loss, showed either no loss or a slight increase with age. This reduced amount or mutation(s) in PS1, which may alter the stoichometric balance of the γ‐secretase complex, may be the cause for aberrant or increased processing of APP resulting in Aβ accumulation consequently causing early loss of memory.
Differential and non‐labeling quantitative peptidomics reveals the circulating peptide biomarkers for the disease in the blood
Authors: Uchida Kazuhiko , Suzuki Hideaki , Kohji Meno , Ishii Takashi , Fujimoto Hirotaka , Tanano Jun , Asashima Makoto , Nagashima Renpei , Shiraki Katsuya
Published: June 2021

The low‐molecular‐weight proteome, peptidome, is a source of potential biomarkers for the disease. However, almost all detected peptides in previous reports are fragments originating from highly abundant plasma proteins. In addition, the major plasma proteins in blood mask the presence of circulating peptide biomarkers indicative of the disease state, making the discovery of such biomarkers challenging. Here we have established the methodology for differential and quantitative serum peptidome profiling that is efficient in the discovery of these biomarkers. By using non‐labeling and quantitative 2D‐LC‐MALDI‐TOF MS, all of the MS spectra from 1,152 fractions per sample were analyzed using a total of about 170 serum samples consisting of healthy individuals and patients with liver disease, i.e., chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Upon such analysis, we were able to identify circulating peptides useful as diagnostic biomarkers, including a glycosylated fragment and cellular proteins that are present at low concentrations in serum. These results indicate that circulating peptides at low abundance, particularly those of a cellular origin, are potential biomarkers for the virus‐related chronic liver disease and may contribute to molecular diagnostics and early intervention of virus‐related hepatocellular carcinoma.
Mechanism of redox‐ and CO‐sensing by the heme protein, CooA: Cryoradiolysis studies
Authors: Clark Robert W. , Kalafut Deanna F.
Published: June 2021

Gas‐sensing heme proteins participate in a variety of signal‐transduction mechanisms across a broad range of biological systems. This class of proteins functions by undergoing an allosteric conformational change in response to the binding of a small gas molecule to a heme group. CooA, which is a redox‐ and CO‐sensing transcription factor that is found in several bacteria, regulates gene expression that enables growth on CO as a sole energy source. In the current study, we have employed the technique of gamma‐irradiation / cryoreduction spectroscopy (cryoradiolysis) to investigate facile changes that occur to the heme coordination structure of CooA during activation. Specifically, we conducted cryoradiolysis experiments to elucidate the chemical steps of an unusual ligand switch that occurs rapidly when the CooA hemes are reduced from the Fe(III) to Fe(II) state. Presented are our protocols for the preparation of samples suitable for cryoradiolysis experiments, as well as results we have obtained that provide evidence for the isolation of a highly‐reactive Fe(II)‐Cys/Pro species upon gamma irradiation at 77 K. Finally, we present initial results of annealing experiments that seek to identify the chemical details of the redox‐mediated ligand switching mechanism utilized by CooA from Rhodospirillum rubrum.

The authors wish to thank Valparaiso University, the Hope College Chemistry Department, and the Riechel Fund for financial support.
Direct visualization of triplex DNA molecular dynamics by fluorescence resonance energy transfer and atomic force microscopy measurements
Authors: Chang Chia‐Ching , Lin Po‐Yen , Chang Chia‐Seng , Kan Lou‐Sing
Published: June 2021

We have detected the dynamics of 17‐mer DNA‐triplex dissociation mechanism at the molecular level. Fluorescence resonance energy transfer (FRET) was used as an indicator of intermolecular interaction in nanometer range, whereas atomic force microscopy (AFM) was employed to address single molecule with sub‐angstrom precision. The maximum rupture force of DNA‐triplex was found at pH 4.65, consistent with nuclear magnetic resonance (NMR) observations. These results indicated that the FRET together with an AFM detection system could be used to reveal the DNA‐triplex interaction in nanometer scale unambiguously. We also find that the single molecule dynamic of bio‐macromolecule may represent the statistical reaction of macromolecule system.
Purification of Strep(II)‐tagged proteins using new prepacked columns
Authors: Heijbel Anna , Bergh Ann , Carlsson Marianne , Hjellstrom‐Nilsson Ulrika , Karlsson Anneli , Nilsson Martina
Published: June 2021

The Strep(II)‐tag is an ideal tag due to the small size (8 aa, 1 kDa), often making removal of the tag unnecessary. The prepacked StrepTrap™ HP 1 ml & 5 ml columns have been specially developed for high specificity towards the Strep(II)‐tag facilitating fast and convenient purification. Purification is done under physiological conditions and mild elution preserves the activity of the target protein. The 34 um bead size of the resin allows elution in narrow peaks, minimizing the need for further concentration steps.

In this work dual‐tagged fluorescent protein, (His)6‐mCherry‐Strep(II) expressed in E. coli, was purified in a purification procedure including two affinity columns, StrepTrap HP followed by HisTrap™ HP. The second affinity column was necessary for removal of a truncated variant of the target protein. The multi‐step purification was performed fully automatically using ÄKTAxpress™. Another dual‐tagged protein expressed in insect cells was purified to high purity in a single step using StrepTrap HP. The final purity was above 95% according to SDS‐PAGE. Finally, six repeated purifications of a Strep(II)‐tagged protein expressed in E. coli were performed on the same StrepTrap HP column with 0.5 M NaOH regeneration between the runs. The final purity and yield of the target protein remained almost unchanged, no tendency of decreasing values, demonstrating the high reproducibility.
Secondary Alcohol Dehydrogenase in Micrococcus luteus (WIUJH‐20)
Authors: Youngblut Nicholas David , Huang Jenq‐Kuen , Wen Lisa
Published: June 2021

In our efforts to screen microorganisms that can convert agricultural commodities to value‐added products, we isolated a strain of Micrococcus luteus (WIUJH‐20) that effectively converted 10‐ or 12‐ hydroxysteric acid to their corresponding keto‐derivatives. The results suggested that this strain is an excellent source for isolation of a secondary alcohol dehydrogenase. The secondary alcohol dehydrogenase has been purified to homogeneity by several chromatographic steps. The native secondary alcohol dehydrogenase appears to be a homotetramer with a molecular weight of about 160 kDa. The isoelectric point of the enzyme is about 4.8 as determined by two‐dimensional gel electrophoresis. N‐terminal amino acid sequencing revealed that it is quite different from that of cytosolic secondary alcohol dehydrogenases. Very likely, the secondary alcohol dehydrogenase we are dealing with belongs to the membrane‐bound secondary alcohol dehydrogenase for the fact that both the substrate (10‐hydro‐ or 12‐hydroxysteric acids) and the product (10‐oxo‐ or 12‐oxo derivatives) are solid at room temperature preventing translocation across the membrane. We are doing peptide mapping and N‐terminal amino acid sequencing of the internal peptide fragments to design primers to amplify the gene by PCR for further study. This research is supported by the Western Illinois University Centennial Honors College.
NF90/NF45 complexes regulate cell division
Authors: Pe'ery Tsafi , Guan Deyu , Altan‐Bonnet Nihal , Parrott Andrew M , Arrigo Cindy J. , Lee Chee‐Gun , Mathews Michael B.
Published: June 2021

Nuclear factor 90 (NF90) and its C‐terminally extended isoform, NF110, belong to a family of nucleic acid binding proteins. NF90/110 are isolated with the less‐studied protein NF45, as complexes interacting with highly‐structured and double‐stranded RNAs. They have been implicated in the regulation of cellular and viral genes at several levels but it remains unknown whether NF90 and NF110 form functionally distinct complexes. We studied the composition of the complexes in vivo and used siRNA strategies to elucidate their function in cells.

NF90 and NF110 form heterodimeric core complexes with NF45, and large in vivo complexes in which multiple NF90/110 isoforms interact with each other. NF45 knock‐down by RNA interference led to decreased expression of NF90 and NF110. This co‐regulation is at the protein level: the stability of NF90 family proteins is dependent on binding to their NF45 partner. Reciprocally, NF90 knock‐down reduced the expression of NF45 as a result of its accelerated degradation. Cell growth is retarded and giant multi‐nucleated cells accumulate when the expression of NF45 or NF90, but not NF110, is reduced in HeLa cells. The defect leaves nuclei attached by constrictions. Thus, NF90 family members form complexes inside cells whose stability is dependent on their binding to NF45. NF90 and NF45 are essential for cell growth and their depletion results in defects in nuclei division and cytokinesis.

Support: NIH AI034552
Down‐regulation of Mitofusin2, a target gene of PPARδ, disrupts mitochondria network in the hearts of cardiomyocyte‐restricted PPARδ knockout mice
Authors: Li Yuquan , Yin Ran , Broke Lawrence , Floyd Donna , Yang Qinglin
Published: June 2021

Cardiomyocyte‐restricted PPARδ knockout in mice (CR‐PPARδ−/−) leads to depression of myocardial FAO and bioenergetics in the heart. A substantial loss of mitochondria network is one of the ultrastructural features in CR‐PPARδ−/− hearts. We hypothesized that mitofusin 2, a protein essential for mitochondrial networking is responsible for the loss of mitochondrial network in the CR‐PPARδ−/− hearts. Quantitative real‐time PCR revealed that mitofusin 2 transcript levels in the CR‐PPARδ−/− hearts were about 50 % of those in controls (n=4, P<0.05). Mitofusin 2 protein levels in CR‐PPARδ−/− hearts were also about 30% less than those in controlled hearts (n=4, P<0.05). Protein levels of mitochondrila marker proteins such as MTCO‐1 in the CR‐PPARδ−/− hearts were not changed. Since expression of PGC‐1 is not changed in CR‐PPARδ−/− hearts, we further assessed if PPARδ can directly regulate mitofusin 2 expression. We discovered a putative PPRE consensus sequence located at around −550 bp upstream of the mouse mitofusin 2 promoter. Using a LightShift chemiluminescent EMSA kit, we confirmed that the probe derived from a fragment of this putative −550 bp PPRE was strongly bound in nuclear protein. Therefore, we conclude that mitofusin 2 is a direct PPAR target gene. Its deficiency due to PPARδ knockout in cardiomyocyte contributes to the mitochondria abnormalities found in CR‐PPARδ−/− hearts.
Pyrroloquinoline Quinone: CREB Phosphorylation and PGC‐1alpha Expression
Authors: Chowanadisai Winyoo , Handley R , Tchaparian E , Bauerly K , Rucker R
Published: June 2021

Pyrroloquinoline quinone (PQQ) is found in numerous foods. Oxidative metabolism can be depressed when diets fed to mice and rats are devoid of PQQ. Gene array and QRTPCR studies indicate that neonatal and perinatal PQQ deficiency reduces mitochondria amount and mitochondrial‐related gene expression. Exposure to PQQ in hepatocyte culture stimulates mitochondrial biogenesis and activates nuclear respiratory factors by unknown mechanisms. We determined that PQQ mediates mitochondrial biogenesis in Hepa1‐6 hepatocytes by increasing PGC‐1alpha mRNA and protein expression, measured by QRTPCR and immunoblotting, respectively. PQQ stimulated PGC‐1alpha expression through cAMP response element binding protein (CREB) activation, since PQQ stimulated a PGC‐1alpha promoter reporter vector, but PQQ stimulation was absent in PGC‐1alpha promoter constructs with a mutated CRE site. PQQ stimulated the phosphorylation of CREB at serine 133, determined by immunoblotting using phospho‐specific antibodies. This suggests that the ability of PQQ to regulate mitochondrial biogenesis occurs through the stimulation of the CREB‐PGC‐1alpha pathway and provides evidence of how a minor dietary constituent can improve mitochondrial function. (Support: Mitsubishi Chem. Inc.; CHNR, UC Davis)
Chronic exposure to palmitate impairs fatty acid oxidation and insulin‐stimulated glucose uptake despite activation of AMPK in skeletal muscle cells
Authors: Pimenta Aurelio da Silva , Gaidhu Mandeep Pinky , Habib Sarah , So Mandy , Fediuc Sergiu , Curi Rui , Ceddia Rolando Bacis
Published: June 2021

The aim of this study was to investigate the chronic effects of palmitate on fatty acid oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8h to 200, 400, 600, and 800uM of palmitate reduced fatty acid oxidation by ∼26.5%, ∼43.5%, ∼50%, and ∼47%, respectively. Interestingly, this occurred despite significant increases in AMPK (∼2.5‐fold) and ACC (∼3‐fold) phosphorylation and in malonyl‐CoA decarboxylase activity (∼38% – 60%). Low concentrations of palmitate (50–100uM) caused an increase (∼30%) in CPT‐1 activity. However, as the concentration of palmitate increased, CPT‐1 activity decreased by ∼32% after exposure for 8h to 800uM of palmitate. Although fatty acid uptake was reduced (∼35%) in cells exposed to increasing palmitate concentrations, intracellular lipid accumulation increased in a dose‐dependent manner, reaching values ∼2.3‐, ∼3‐, and 4‐fold higher than control in muscle cells exposed to 400, 600, and 800uM palmitate, respectively. This was accompanied by a progressive and time‐dependent inhibition of glucose uptake (∼65%) in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose metabolism in skeletal muscle under conditions of chronically elevated levels of NEFAs, such as in obesity and type 2 diabetes.
FOXO transcription factors are required for DNA damage‐induced growth arrest checkpoints in hematopoietic cells
Authors: lei hong , Quelle Frederick W.
Published: June 2021

Proliferation of hematopoietic cells is governed by cytokine‐regulated signaling pathways. We have previously shown that a cytokine‐induced PI3K/AKT signaling pathway promotes CDK2 activation and overrides DNA damage‐induced growth arrest in hematopoietic cells. In the present work, we have assessed the role of FOXO transcription factors as a potential intermediate in this signaling pathway. In this regard, overexpression of FOXO3a activity was found to reduce proliferation and CDK2 activity in cytokine‐dependent hematopoietic cell lines. Moreover, enhanced FOXO activity potentiated the growth arrests that were induced by DNA damage and PI3K/AKT inhibition. By contrast, shRNA‐knockdown of endogenous FOXO proteins attenuated the DNA damage‐induced growth arrest in cells lacking PI3K/AKT activity. We also show that the ability of FOXO3a to inhibit cytokine‐induced proliferation was dependent on expression of the CDK‐inhibitor, p27. Together, these observations suggest that FOXO‐mediated expression of p27 contributes to DNA damage‐induced growth arrest, and that inhibition of this activity by cytokine‐induced AKT can promote proliferation and override DNA damage checkpoints in hematopoietic cells. NIH Grant R01 CA79889
Calcium dependent p21 cip1/waf1 stabilizer cyclin I (Ccni).
Authors: Chu Hsueh‐Liang , Chen Tzu‐Hsuan , Yeh Yi‐Chen , Tsai Chuan‐Mei , Wu Chang‐You , Svanidze Anna , Chang Chia‐Ching
Published: June 2021

Cyclin I (Ccni), a new member of cyclin family which contains a typical cyclin box at N‐terminal and PEST sequences at its C‐terminal. Unlike other cyclins, the mRNA level of cyclin I is high in post‐mitotic tissues and expresses constantly during cell cycle progression. However, the function of cyclin I remains unknown until this study. In this study, we have cloned, expressed and refolded the recombinatant human cyclin I from E. coli system. Spectroscopy analysis by circular dichroism spectra and thermal stability analysis by differential scanning micro‐calorimeter indicated that cyclin I is a native disorder protein but with helical secondary structure. Both far western blot analysis and reaction heat measurement by isothermal titration calorimeter showed that cyclin I can bind with p21cip1/waf1directly only at the condition with calcium ion present. These results suggested that cyclin I prolongs the life time of p21 by calcium regulation at the early stage of apoptosis.
Oxysterol‐ and cholesterol‐dependent translocation of CTP:phosphocholine Cytidylyltransferase to the nuclear envelope
Authors: Gehrig Karsten , Lagace Thomas A , Ridgway Neale D
Published: June 2021

Oxysterols potently regulate cholesterol, phospholipid and sphingomyelin synthesis. Here, we investigated how 22‐, 25‐, and 26‐hydroxycholesterol (22OH, 25OH, and 26OH, respectively) stimulate the CDP‐choline pathway for phosphatidylcholine (PtdCho) synthesis using CHOK1 and CHO cells that express a sterol‐resistant mutant of sterol regulatory element binding‐protein (SREBP)‐cleavage activating protein (CHOSCAP D443N). Analysis of [3H]choline‐labelled metabolites showed that oxysterols activated CTP:phosphocholine cytidylyltransferase (CCTα) and increased PtdCho synthesis. Oxysterols also promoted complete localization of CCTα to the nuclear envelope and intranuclear tubules in both CHO‐SCAP D443N and CHOK1 cells. Cholesterol was required for this effect since depletion with cyclodextrin or lovastatin resulted in reduced translocation and activation of PtdCho synthesis. In vitro, CCTα activity was stimulated by PtdCho vesicles containing oxysterols. Synergistic activation of CCTα by vesicles with 25OH and cholesterol suggested that the membrane environment created by the combination of these two sterols has unique stimulatory properties that could be reproduced in CHO‐SCAP‐D443N cells. Thus 22, 25, and 26‐OH promotes translocation and activation of CCTα at the nuclear envelope in a cholesterol‐dependent manner. (Supported by CIHR and IWK)
Cyclooxygenase‐2 and RhoA modulates E‐cadherin expression through the transcriptional repressor ZEB1
Authors: Bean Ronald R. , Jakobi Rolf , Chang Yu‐Wen E.
Published: June 2021

ZEB1 is a potent transcriptional repressor of the adherens junction (AJ) core protein, E‐cadherin. Previously it has been shown that cyclooxygenase‐2 (COX‐2) activates the small GTPase RhoA which in turn down‐regulates E‐cadherin to disrupt AJ and increase motility of colon carcinoma cells. In this study we examine the possibility that an increase in RhoA activity decreases E‐cadherin expression levels utilizing the ZEB1 pathway in two human colon carcinoma cell lines, HT‐29 and HCT‐15. Total protein and mRNA were isolated from both cell lines either expressing constitutively active (CA) or dominant negative (DN) RhoA. Using qRT‐PCR, both E‐cadherin and ZEB1 mRNA levels were monitored, whereas E‐cadherin protein levels were determined by western blot analysis. The same experiments were also conducted on parental HT‐29 and HCT‐15 cells as well as cells overexpressing COX‐2. In all experiments, RhoA‐CA or COX‐2 increased ZEB1 expression and decreased E‐cadherin expression by at least two‐fold. Expression levels of both ZEB1 and E‐cadherin were indistinguishable from wild type in those cells expressing RhoA‐DN. Also, inhibition of RhoA restored both ZEB1 and E‐cadherin expression levels to those of wild type. In conclusion, COX‐2 and RhoA mediates AJ formation by down‐regulating E‐cadherin expression through the ZEB1 transcriptional repressor pathway.
Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta‐arrestin 2 dependent ERK/MAPK‐mediated pathways
Authors: Coscia Carmine James , McLennan Gregory P , Kiss Alexi , Miyatake Mayumi , Chambers Kari T , Pozek John J , Mohabbat Yasmin , Belcheva Mariana M
Published: June 2021

Seven‐ transmembrane receptors activate ERK/MAPK via G protein dependent and independent mechnanisms. Opioid receptors (ORs) modulate proliferation of immortalized and primary astrocytes via ERK. We examined κ OR‐induced, ERK‐mediated proliferation of rat immortalized and primary astrocytes to delineate participation of G protein vs. β‐arrestin (β‐arr). κ opioid agonist, U69,593, activated ERK by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation of immortalized and primary astrocytes as measured by bromodeoxyuridine labeling. κ agonist, MOM‐Sal‐B (a salvinorin derivative), induced ERK activation transiently (min) and did not affect proliferation of immortalized astrocytes suggesting that sustained ERK activity is required. In primary astrocytes, MOM‐Sal‐B elicited sustained ERK activity and proliferation. Transfection of immortalized astrocytes with cDNA of a Gβγ scavenger reduced ERK activaty by U69,593. Treatment of immortalized cells with siRNA indicated that ERK activation by U69,593 is β‐arr 2 dependent. The same inhibitors decreased MOM‐Sal‐B activation of ERK in immortalized cells. Gβγ scavenger or β‐arr 2 siRNA abolished proliferation of primary astrocytes induced by both κ opioids. The results suggest that Gβγ and β‐arr 2 dependent ERK activation pathways are required to elicit proliferation of primary astrocytes. Supported by NIH DA 05412
Signaling Events in Nitric Oxide‐Induced Tumor Cell Resistance to Photodynamic Eradication
Authors: Bhowmick Reshma , Girotti Albert W
Published: June 2021

Purpose Photodynamic Therapy (PDT) is an antitumor modality that employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive species that kill tumor/tumor vasculature cells in a site‐specific manner. Nitric oxide (NO) at low levels can inhibit apoptosis and promote angiogenesis and tumor growth. We showed recently that NO from a chemical donor/activated macrophages induced a resistance to photokilling in protoporphyrin IX (PpIX)‐sensitized COH‐BR1 breast tumor cells. Signaling events associated with this hyperresistance was examined in the present study. Methods COH‐BR1 cells were sensitized with PpIX in mitochondria via metabolism of ALA and irradiated in the presence/absence of SPNO‐derived NO. Apoptotic/necrotic photokillling was assessed by fluorescence microscopy using Hoechst/PI staining. Redox signaling associated with MAP kinase phosphorylation‐activation and heme oxygenase‐1 (HO‐1) upregulation was examined by immunoblotting. Results NO delivered during irradiation of ALA‐treated cells protected them against apoptotic death. This was accompanied by a large induction of HO‐1 and an increase in the activation of overall p38 and ERK1/2, but not JNK. Conclusions These findings suggest that antioxidant stress protein, HO‐1 plays a major role in NO's anti‐PDT effects and is associated with activation of the anti‐apoptotic p38 and ERK1/2 signaling pathways.
Metabolic Regulation of Long‐Term Potentiation in the hippocampus: Therapeutic Implications.
Authors: Potter Wyatt Benjamin , O'Riordan Ken , Burger Corinna , Roopra Avtar
Published: June 2021

Energy supply is critical for essential functions in all cells. There is increasing evidence that dietary carbohydrate and caloric restriction moderate cellular responses to damage, alter ageing processes, enhance longevity and may have therapeutic affects in neurological disorders such as epilepsy, Alzheimer's Disease and Huntington's Chorea. Dietary regimes termed ‘ketogenic diets’ which iso‐calorically replaces carbohydrates with fats and protein, can produce significant anticonvulsant effects in patients with epilepsy and have successfully been used therapeutically. The mechanisms behind the beneficial effects of ‘ketogenic diets’ are completely unknown. We show that signaling pathways heavily implicated in learning and memory, LTP and epileptogenesis are controlled by cellular metabolism via the actions of AMPK. We propose that these mechanisms may underlie some of the beneficial effects of ‘ketogenic diets’ in epilepsy.
The function of Foxp3 independent of suppressive activity
Authors: Han Shuhua , Guo Linjie
Published: June 2021

The forkhead family transicrption factor Foxp3 is critically important for the development and function of regulatory T cells (Tregs). Lack of Foxp3 leads to the development of fatal autoimmune lymphoproliferative diseases. Previously studies have shown that ectopic Foxp3 expression can phenotypically convert effector T cells to regulatory T cells. However, we have recently generated Foxp3 transgenic mice under the control of lck distal promoter. Our data showed that enforced Foxp3 expression rendered CD4+CD25– T cells hyporesponsive but conferred neither Treg phenotype nor suppressive function. When Foxp3 transgenic mice were crossed to scurfy mice, the scurfy phenotype was partially rescued but the lymphoproliferative disease was still apparent. Thus, Foxp3 expression, presumably depending on its expression levels, can result in distinct effects on the functions of CD4+ T cells. In addition, we found that Foxp3 transgenic mice exhibit reduced susceptibility to collagen‐induced arthritis (CIA). Therefore, Foxp3 may play important roles in regulating immune responses and inflammation independent of suppressive function.
Antigen‐induced translocation of SLAT to immunological synapse is required for NFAT activation and Th1/Th2 immune responses
Authors: Becart Stephane , Charvet Celine , Canonigo Balancio Ann J. , Altman Amnon
Published: June 2021

SLAT is a novel guanine nucleotide exchange factor for Rho GTPases, which is highly expressed in T cells, but whose physiological function is unclear. SLAT−/− peripheral CD4+ T cells demonstrated impaired TCR/CD28‐induced proliferation and IL‐2 production, which was rescued by the addition of exogenous IL‐2. Importantly, SLAT−/− mice were grossly impaired in their ability to mount Th2 and Th1‐mediated lung inflammatory responses. This defect in mounting Th1/Th2 responses was traced to a severe reduction of Ca2+ mobilization from ER stores, which consequently led to defective TCR/CD28‐induced NFATc1/2 nuclear translocation. Furthermore, TCR engagement induced SLAT translocation to the immunological synapse (IS) via the Lck‐mediated phosphorylation of two tyrosine residues located in its ITAM‐like sequence. Moreover, this antigen‐induced subcellular relocalization of SLAT was coupled to, and necessary for, specific activation of the NFAT pathway. In addition, once translocated to the IS by a proper targeting signal, the Dbl‐homology (DH) domain of SLAT was sufficient to trigger TCR‐mediated NFAT activation and Th1/Th2 differentiation, most likely via its GEF activity. Therefore, tyrosine phosphorylation‐mediated relocalization of SLAT to the site of antigen recognition is required to exert its pivotal role in NFAT‐dependent CD4+ T cell differentiation.
CD27 plus CD28 Cross‐linking Induces Phosphorylation of STAT‐6 and Synthesis of IL‐4 in CD4+CD45RO+ T Cells
Authors: Tamma Seetha M. , Lee H. J. , Dandamudi Durga B.
Published: June 2021

Expression of CD27, a 120 KDa transmembrane protein, is predominantly confined to lymphocytes and equally distributed between CD4 and CD8 lymphocytes. We investigated the role of CD27 in phosphorylation of STAT‐6, and in induction of IL‐4 synthesis in resting and memory CD4+ T cells. We isolated human CD4+CD45RA+ and CD4+CDRO+ subsets using Dynal magnetic beads and cultures were set up by cross‐linking with antibodies as indicated: CD3 + CD28; CD27 alone; CD27 + CD3; CD27 + CD28; CD28 alone and control cells. We report that CD27 plus CD28XL resulted in enhanced and accelerated phosphorylation of STAT‐6 in RO+ cells when compared with that in RA+ cells. CD27 on its own failed to induce STAT‐6 phosphorylation in both populations. We also analyzed the cytokine profiles following stimulation of both populations as indicated and observed that RA+ cells responded by producing TNF‐alpha and RO+ cells responded by producing IL‐4. We conclude that CD27 plus CD28 cross‐linking induces TH2 differentiation in RO+ cells and that augmentation of STAT6 would be one of the underlying mechanisms of induction of IL‐4 synthesis. The data may suggest that CD4+CD45RA+ T cells act as TH1 type and CD4+CD45RO+ cells act as TH2 type.

Grant from C.W. Post Campus Research Committee of Long Island University, New York supported this work.
Tim‐3 Signaling in T cells and Dendritic Cells
Authors: Su Ee W. , Hainline Sarah G. , Phuah Jia Y. , Kane Lawrence P.
Published: June 2021

The T‐Cell Immunoglobulin and Mucin 3 Protein (Tim‐3) is a type I transmembrane protein that plays a role in regulating T helper responses and promoting tolerance. The extracellular portion of Tim‐3 consists of an immunoglobulin‐V and a mucin‐like domain followed by a transmembrane domain and a cytoplasmic tail that has 6 tyrosine residues. While previously thought to be specifically expressed by certain T cell subsets, we have also found Tim‐3 expressed on the surface of dendritic cells. Cross‐linking of Tim‐3 induces apoptosis in TH1 cells but activates dendritic cells. Therefore, our hypothesis is that the signaling pathway of Tim‐3 differs in lymphoid and myeloid cells. Our preliminary data indicate that AE7 (mouse TH1 clone) and D2SC/1 (mouse dendritic cell line) cells stimulated with anti‐Tim‐3 antibody display different tyrosine phosphorylation patterns. We are currently trying to identify specific phospho‐proteins present only in the lysates of Tim‐3 activated AE7 cells but not D2SC/1 cells. In addition, we have generated truncation and point mutants of Tim‐3 to determine which, if any, of the 6 tyrosine residues are required for the signaling capabilities of Tim‐3 in T cells and DC's. With these studies we aim to identify proximal signaling events downstream of Tim‐3 in lymphoid versus myeloid cells.
Cyclophilin A and MIP‐2 synergize to promote greater neutrophil migration
Authors: Heine Shannon Jennifer , Olive Denise S. , Constant Stephanie L.
Published: June 2021

Chemokines induced during inflammatory diseases contribute to the inflammatory response by inducing leukocyte migration and extravasation. Extracellular cyclophilins are an alternative group of proteins with chemokine activity. In previous studies, we have shown that extracellular cyclophilins are highly elevated during inflammatory responses and their inhibition reduces leukocyte infiltration by 40–50%. Such a major contribution by cyclophilins to leukocyte recruitment led us to investigate whether they might function synergistically with other chemokines. Using a mouse model of acute lung inflammation we first established the chemokines present concurrently with cyclophilin A (CypA) in inflamed lung airways and selected two (MIP‐2 and Gro1‐α) for further testing. Using in vitro Boyden chamber chemotaxis assays we determined that MIP‐2 synergizes with CypA to induce augmented neutrophil migration, but that Gro1‐α does not. The observed in vitro synergistic effects were confirmed in vivo by co‐injection of CypA and MIP‐2 into air pouches. These data provide evidence that CypA has the capacity to synergize with other chemokines present during inflammatory responses, potentially explaining the significant reduction in cell recruitment observed when cyclophilin activity is inhibited in vivo.

This work was funded by NIH AI067254.
Islets of Langerhans are the portal of entry for activated diabetogenic T cells mediated by resident islet dendritic cells
Authors: Calderon Boris , Unanue Emil R.
Published: June 2021

We evaluated the features of dendritic cells (DC) within the islets of Langerhans and the entry of T cells directed against beta cell‐derived peptides. We used various mouse strains, including mice expressing hen egg white lysozyme under the insulin promoter and NOD.rag‐2−/− mice. Islets from normal mouse strains and lymphocyte‐deficient mice contained 8–10 DC per islet. Most DC were found next to blood vessels, expressed peptide‐MHC complexes derived from beta cell proteins and presented them to CD4 T cells ex vivo and in vivo. Islets were shown to be the portal of entry for activated diabetogenic CD4 T cells but not unactivated T cells. The first stage of T cell homing into the islets was dependent on DC presenting beta cell‐peptide‐MHC complexes. Administration of blocking antibody for MHC II decreased the early entry of T cells into the islets. Furthermore, activated diabetogenic CD4 T cells had the ability to bring non‐specific activated T cells into the islets. The percentage of infiltrated islets with non‐specific T cells was dependent on the number of diabetogenic T cells injected. We conclude that the mechanisms of entry and recognition of the activated T cell depends on peptide‐MHC complexes from islet DC. Experiments in progress are analyzing other molecules that could participate in the specific and non‐specific migration. Understanding these issues will set the base for new mechanisms of diabetogenesis.
Using HLA DRB1*1501 transgenic mice to study the HLA‐linked autoimmune Goodpasture's disease
Authors: Ooi Joshua Daniel , Fugger Lars , Holdsworth Stephen R. , Kitching A. Richard
Published: June 2021

Goodpasture's disease is characterised by destruction of glomerular and alveolar basement membranes due to an autoimmune response against the non‐collagenous domain of the alpha 3 chain of type IV collagen [α3(IV)NC1]. It is strongly associated with HLA DRB1*1501 (OR: 8.5) and negatively associated with HLA DRB1*0101 and *0701. This study sought to determine if MHCII−/− HLA DRB1*1501 transgenic (*1501 tg) mice can be used to model human Goodpasture's disease and if the T‐cell autoepitopes are similar to that found in human disease. *1501 tg mice immunized with human α3(IV)NC1 developed autoantibodies specific for mouse α3(IV)NC1 (ELISA) and developed linear intraglomerular IgG binding (immunohistochemistry). Autoantibody binding was stronger when compared to C57BL/6J wildtype mice. *1501 tg mice also developed elevated urinary protein levels and significant structural abnormalities. T‐cell autoepitope studies performed by immunization with overlapping peptides of the α3(IV)NC1 reveal that immunogenic peptides (IFNγ‐ELISPOT) correlate with autoepitope studies with PBMCs from HLA DRB1*150x positive Goodpasture's patients; in particular a key candidate epitope which was not immunogenic in wildtype C57BL/6J or COL4A3 KO mice. The data suggest that *1501 tg mice are a promising tool in understanding the HLA‐linked susceptibility in Goodpasture's disease. This work supported by the Australian NHMRC.
Drak2 is critical for the survival of autoreactive T cells
Authors: McGargill Maureen A. , Choy Carmen , Wen Ben G. , Hedrick Stephen M.
Published: June 2021

Drak2 is a serine/threonine kinase expressed in T cells and B cells. In the absence of Drak2, mice are remarkably resistant to experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We tested if the absence of Drak2 affected other mouse models of autoimmune disease and found that Drak2−/− mice were completely resistant to type 1 diabetes when crossed to the NOD background. However, Drak2−/− mice were still susceptible to other models of autoimmunity including the T cell transfer model of colitis, collagen‐induced arthritis, and Systemic Lupus Erythematosus. In order to understand how Drak2 affects certain autoimmune diseases, we investigated the mechanism for the resistance to EAE and found that it is intrinsic to the T cells. There was a similar number of MOG‐specific T cells in the lymphoid organs of wildtype and Drak2−/− mice following immunization with MOG peptide. Surprisingly, Drak2−/− T cells entered the central nervous system, however the cells did not accumulate and cause damage like wildtype T cells. A higher proportion of the Drak2−/− T cells in the central nervous system were undergoing apoptosis compared to the wildtype T cells, which indicates that Drak2 is important for the survival of these autoreactive T cells. Moreover, in the NOD model, islet‐reactive T cells were more susceptible to death in the pancreatic draining lymph nodes in the absence of Drak2. Together, these data suggest that Drak2 is important for the survival of activated T cells, and the absence of Drak2 inhibits autoimmune diseases in which the presence of the T cells in the target organ is required to perpetuate disease by decreasing the survival of the autoreactive T cells. This work is supported by the Juvenile Diabetes Research Foundation.