bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of protein oligomerization and assembly of a DNA translocation channel
Authors: Swerdlow Kyle Justin , Mancuso Gianna R , Berkmen Melanie Barker
Published: June 2021

Mating, or conjugation, is the process by which a cell horizontally passes genetic information to another cell through a multi‐protein DNA translocation channel. ConE is a key ATPase of the mating machinery of ICEBs1, the Integrative and Conjugative element of Bacillus subtilis. Characterized homologs form a variety of oligomers in solution and crystallize as hexamers. Previously, we showed that purified His6‐ConE is a mixture of dimers, trimers, and higher‐order oligomers via Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE). To more thoroughly understand the interactions between ConE and other players in the mating machinery, we have employed a bacterial two‐hybrid system. We investigated the interaction between ConE fused to the T18 and T25 domains of Bordetella pertussis adenylate cyclase. Using β‐galactosidase assay to indirectly report cyclic‐AMP levels, we found that T25‐ConE and T18‐ConE interact. We hope to use this technique to measure possible interactions between ConE and other ICEBs1 proteins that make up the DNA translocation channel. This research was funded by an NSF‐RUI grant to Melanie B. Berkmen.
The hZIP4 transporter has two binding affinities and transports multiple transition metal ions.
Authors: Antala Sagar , Dempski Robert E.
Published: June 2021

Cells possess numerous membrane proteins to transport essential metal ions across cellular organelles. hZIP4 (human ZIP4) is one member of the ZIP (Zrt‐ and Irt‐like proteins) family of proteins that imports zinc to the cytoplasm. Autosomal mutations in hZIP4 have been associated with the zinc deficient genetic disorder Acrodermatitis Enteropathica. hZIP4 is expressed in small intestine, stomach, colon, cecum, kidney and pancreas. Recently, hZIP4 overexpression has also been associated with the pancreatic cancer. hZIP4 has 8 transmembrane domains with extracellular N‐ and C‐termini. Furthermore, the histidine rich non‐conserved N‐terminus and intracellular domain between transmembrane‐III and IV are believed to be important for Zn2+ coordination.

We have established a radio‐isotope uptake experiment in Xenopus leavis oocytes to study the selectivity and kinetics of hZIP4 cation transport. An analysis of our experimental results demonstrate that hZIP4 transports Ni2+ and Cu2+ in addition to Zn2+. Our data also shows that hZIP4 has two binding affinities where one is in the μM range and the other one is in nM concentration. Subsequent experiments have also identified possible zinc coordination amino acid residues.
Structures and allosteric motions of Ryanodine Receptor Domains
Authors: Van Petegem Filip , Tung Ching‐Chieh , Kimlicka Lynn , Yuchi Michael , Lobo Paolo A , Lau Kelvin
Published: June 2021

Ryanodine Receptors (RyRs) form large (>;2.2 MDa) ion channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. They are thus involved in many calcium‐dependent events, including the contraction of skeletal and cardiac muscle. Mutation in the ryr genes can cause severe skeletal muscle disorders and cardiac arrhythmias. Here we present our recent insights in the structure‐function of these membrane protein giants. We have solved crystal structures of multiple domains in the N‐terminal and central regions and mapped the positions of ~100 disease‐causing mutations. By creating pseudo‐atomic models, we analyzed the motions these domains undergo during opening and closing of the pore region.

Channel opening includes major motions in the central rim. Incidentally, this area contains a hot spot for disease mutations (>;80 total). In particular, channel opening disrupts an intersubunit interface that contains the largest cluster of mutations. By solving structures of several mutant forms of the domains, we observe that mutations can alter relative domain orientations. They thus mimic the effect of binding of ligands to the related IP3 receptor. Overall, we present a model whereby the N‐terminal region of the RyR serves as a gating ring that forms a hub for allosteric control.
Identification of Accessory Proteins Critical for the Epithelial Sodium Channel (ENaC)
Authors: Horn Daniel , Booth Rachell E
Published: June 2021

The epithelial sodium channel (ENaC) is rate‐limiting for the reabsorption of sodium in the distal nephron of the kidneys. Genetic mutations in ENaC can cause diseases such as Liddle's syndrome and pseudohypoaldosteronism type 1, causing severe hypertension or hypotension, respectively. Accessory proteins including Nedd4, ubiquitin protein‐ligase, and a serum and glucocorticoid‐regulated kinase have been shown to regulate ENaC and additional proteins are likely to play an essential role in its proper function. In an effort to identify proteins necessary for proper assembly, localization, and function of ENaC, BY4742 yeast deletion strains were transformed with αENaC, which induces a salt‐sensitive phenotype, and yeast dilution pronging assays were performed. While five deletion strains showed no effect on ENaC function in yeast, three strains lacking scj1, jem1, or lhs1 showed loss of ENaC function while deletion of ypk1 seemed to enhance ENaC function. Localization studies of αENaC in these deletion strains were used to elucidate the role of each gene on ENaC surface expression. Funded by NIH (R15GM086798) and Welch Foundation (Dept Grant A10048).
VAMP Usage In Regulated Platelet Secretion
Authors: Joshi Smita , Whiteheart Sidney
Published: June 2021

Platelets play important roles in hemostasis and thrombosis through secretion of their granular content. The releasate mediates activation of other platelets, clot formation, wound healing, angiogenesis and inflammation. Granule‐plasma membrane fusion requires Soluble N‐ethylmaleimide Sensitive Factor Attachment Protein Receptors (SNAREs) proteins from the granule (v‐SNAREs) and plasma membrane (t‐SNARE). The SNARE proteins form a trans‐bilayer complex that is essential for fusion. We showed that syntaxin‐11 and SNAP‐23 combine to form the functionally relevant t‐SNARE heterodimer in platelets. VAMP‐8 functions as the primary v‐SNARE though residual release occurs in VAMP‐8−/− platelets. This residual release is inhibited by treatment of permeabilized VAMP‐8−/− platelets with tetanus toxin, implying a secondary role for VAMP‐2 or ‐3. To address this differential usage of platelet v‐SNAREs, we have created a mouse strain lacking all brevin family VAMPs and one that lacks VAMP‐2 and ‐3. By crossing a RC::PFtox strain, containing a genomic insertion encoding the catalytic subunit of tetanus toxin which is controlled by a Cre‐recombinase‐excisable element with a platelet/megakaryocyte‐specific PF4‐promotor‐driven Cre strain, we created platelets that lacked VAMP‐2 and ‐3. These mice were crossed to VAMP‐8−/− mice to create VAMP‐2,‐3,‐8 null platelets. Secretion assays measuring the release of cargo from platelets derived from these strains are being used to evaluate the differential roles of these v‐SNAREs in platelet secretion. Upon completion of this study we will have a better understanding of molecular nature of platelet secretion. This work was supported by Grants HL56652 and HL091893 from the National Institutes of Health.
VAMP7 differentially regulates biosynthetic trafficking of apical proteins
Authors: Szalinski Christina Mae , Raghavan Venkatesan , Bruns Jennifer R. , Weisz Ora A.
Published: June 2021

Maintaining kidney function requires continual delivery of newly synthesized proteins to the apical and basolateral surface domains of polarized cells. SNARE proteins are required for fusion of vesicles carrying newly synthesized and recycling proteins. Newly synthesized proteins use several pathways to reach the apical surface of kidney cells and the post‐Golgi R‐SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 is involved in a subset of apical protein trafficking pathways in intestinal and thyroid cells. We hypothesized that knockdown of VAMP7 would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. Apical delivery of cargoes was examined using pulse‐chase experiments and microscopy upon VAMP7 knockdown. Surprisingly, knockdown of VAMP7 slightly stimulated apical delivery kinetics of a subset of apical proteins while having no effect on other cargoes. VAMP7 is unique among VAMPs in having a longin domain that inhibits SNARE complex formation. Depletion of VAMP7 may enhance the ability of redundant VAMPs that lack inhibitory constraints to promote vesicle fusion. Alternatively, cargo in VAMP7‐depleted cells may have access a more efficient delivery pathway. Experiments are underway to dissect the role of the VAMP7 longin domain in exocytosis. Research is supported by NIH R01 DK064613.
Involvement of Phospholipase C‐related but catalytically inactive protein in regulated exocytosis through the pleckstrin homology and the C2 domains
Authors: Takeuchi Hiroshi , Zhang Zhao , Gao Jing , Wang DaGuang , Hirata Masato
Published: June 2021

Phospholipase C‐related but catalytically inactive protein (PRIP) is a scaffolding protein identified in our laboratory. We have recently found that the secretion of various peptide hormones from multiple organs were upregulated in PRIP knock‐out mice, indicating that PRIP regulates the exocytic event common to dense‐core vesicles in a negative manner. In this study, we investigated the molecular mechanism underlying the inhibitory role of PRIP in exocytosis.

We examined the effect of exogenously expressed various mutants of PRIP on noradrenaline (NA) secretion using rat pheochromocytoma cell line, PC12. Expression of the wild‐type PRIP suppressed High‐K+ induced NA release from PC12 cells, while the mutant of which Arg‐134 in the pleckstrin homology (PH) domain was replaced with Glu (R134Q) to loose the binding to membrane phospholipid PtdIns(4,5)P2, little suppressed NA release. The mutant lacking the C2 domain was also less effective than the wild‐type PRIP to suppress exocytosis. We found that the C2 domain of PRIP interacts with the components of SNARE complex including syntaxin 1A and synaptosomal‐associated protein of 25 kDa (SNAP‐25) and inhibited the formation of stable SDS‐resistant SNARE complexes. The results suggest that the direct bindings of the PH domain to PtdIns(4,5)P2 and the C2 domain to SNARE proteins are both required for PRIP to negatively regulate exocytosis.
Truncated O ‐ glycans Enhance Tumorigenicity of Pancreatic Tumors
Authors: Radhakrishnan Prakash , Grunkemeyer James A , Clausen Henrik , Hollingsworth Michael A
Published: June 2021

The interaction between cancer cell surface mucins and growth factor receptors enhances the tumorigenesis of pancreatic adenocarcinoma (PDAC). The truncated O‐glycans (Tn and sialyl Tn) on mucin type glycoproteins enhances the malignant properties of cancer cells, in part by altering ligand affinities for receptors and constitutively activating oncogenic signaling pathways. Truncated O‐glycans on the cancer cell surface are often due to lack of core 3 synthase or core 1 synthase activity and/or defects in the Core 1 synthase specific chaperone ‐ Cosmc. We investigated the contribution of Cosmc to the tumorigenicity of PDAC. A human pancreatic cancer (PC) cell line was genetically targeted to knockout Cosmc with Zinc Finger Nucleases and subsequently used for evaluation of tumorigenicity. The knockout of Cosmc in PC cells resulted in activation of ErbB2 signaling pathway through increased interactions with MUC4, Tn on MUC4 and MUC16. This leads to activated PI3/AKT downstream signaling pathways and altered oncogenic gene expression. Cosmc knockout cells showed significantly increased in vitro migration, invasion and in vivo tumor growth as compared to Cosmc expressing cells. These results demonstrate that aberrant expressions of Tn or STn epitopes on glycoproteins enhance the tumorigenecity of PC cells by activating oncogenic signaling through cell surface receptors. Supported by NCI U01CA111294, U01CA128437, P50CA127297.
Enzymatic Production of Heparin Anticoagulants for Human Use
Authors: Joice April Coley , Mencio Caitlin , Raman Karthik , Painter Chelsea , Tran Vy , Kalita Mausam , Kuberan Balagurunathan
Published: June 2021

Currently, the pharmaceutical heparin being used as an anticoagulant is isolated from bovine lungs or porcine intestines. The primary problem resulting from heparin isolation from animal sources is that this process leaves the heparin open to contamination from animal pathogens that may also be infectious to humans. Enzymatic production of heparin anticoagulant is an attractive alternative to animal derived heparin as it removes the potential for contamination with animal pathogens and also potentially reduces molecular heterogeneity. Heparosan, produced in bacteria, has a structure similar to the backbone structure of heparin and therefore is used as a precursor in the enzymatic production of heparin anticoagulant using various recombinant sulfotransferases. After production, the synthetic heparin was assessed for anticoagulant activity. This approach offers a great promise for human use in the coming years.

Supported by NIHLBI grant HL107152.
Maturation and trafficking of a HMW sucrase‐isomaltase species expressed via maltose sensing
Authors: Chegeni Mohammad , Amiri Mahdi , Naim Hassan Y , Hamaker Bruce R
Published: June 2021

As the primary product of starch digestion by pancreatic α‐amylase, maltose and maltooligosaccharides, are the dominant substrates for α‐glucosidases at the intestinal brush border. Here, immunoblotting of SI from Caco‐2 cells showed maltose induce the formation of a higher molecular weight (HMW) SI species. As this finding is potentially indicative of a maltose sensing ability by enterocytes, our goal is to investigate the molecular mechanisms associated with the maturation and trafficking of this HMW SI. Biosynthetic labeling using [35S]‐methionine revealed a higher rate for early trafficking and maturation of the HMW SI species. Endoglycosidase treatment of the immunoprecipitated SI showed the increased molecular weight of SI is a consequence of altered N‐ and O‐glycosylation of this enzyme. In comparison to the control, the HMW SI was found to be more associated with Triton X‐100 lipid rafts which results in higher apical sorting of these species, as defined by isolation and characterization of the brush border membrane of Caco‐2 cells. Thus, maltose sensing ability of enterocytes influences the intracellular processing of the glycoproteins, speculatively to enhance their digestive property.
Quantitative phosphoproteomics implicates clusters of proteins involved in cell‐cell adhesion and transcriptional regulation in the vasopressin signaling network
Authors: Bolger Steven J. , Hurtado Patricia A Gonzales , Hoffert Jason D , Saeed Fahad , Pisitkun Trairak , Knepper Mark A
Published: June 2021

Vasopressin regulates transport in the renal collecting duct in part by modifying the phosphorylation of transcriptional regulators present in the nucleus. To assess changes in the nuclear phosphoproteome of vasopressin‐sensitive mpkCCD cells in response to dDAVP, a vasopressin V2 receptor analog, we employed stable isotope labeling and mass spectrometry to quantify changes in the abundance of phosphorylated peptides in nuclear fractions. Of the 1,251 phosphorylation sites identified, 39 sites changed significantly in response to dDAVP. Network analysis showed that the regulated proteins fell into two major clusters: cell‐cell adhesion and transcriptional regulation. The hubs of these two clusters were the transcriptional coactivator beta‐catenin and the transcription factor c‐Jun respectively. Phosphorylation of beta‐catenin at Ser552, a known target of protein kinase A or Akt in the collecting duct, was increased in response to dDAVP (log2(dDAVP/vehicle) = 1.79). Phosphorylation of c‐Jun at Ser73, a known canonical target for the MAP kinase Jnk2 in the collecting duct, was decreased (log2(dDAVP/vehicle) = −0.53). The data suggest that vasopressin‐mediated control of transcription in the renal collecting duct involves selective changes in the nuclear phosphoproteome.
Influence of Rho and Rac1 proteins on functions of rat brain mitochondria
Authors: García‐Berumen Claudia I. , Saavedra‐Molina Alfredo , Meléndez‐Herrera Esperanza , Manzo‐Avalos Salvador
Published: June 2021

The brain coordinates vital functions necessary for survival of organisms. Because it is a metabolically active organ requires a lot of energy, which is provided by the mitochondria. We have evidence about of presence of RhoA, RhoB and Rac1 proteins in rat brain mitochondria but the function in this organelle is unknown. The aim of this work was to assess the influence of Rho and Rac proteins over mitochondrial oxygen consumption and ATP synthesis. The inhibition of Rho and Rac proteins with C3 exoenzyme from C. botulinum and NSC23766 compound, respectively, showed a significant decrease in oxygen consumption principally in state 3 of mitochondrial respiration, and also a significant decrease in ATP synthesis compared to mitochondria control. This data suggest that Rho and Rac proteins could play an important role in regulation of ATP synthesis.
Fluorescent protein and epitope tagging within the helical domain of G12 subfamily proteins
Authors: Brawley Douglas N. , Meigs Thomas E.
Published: June 2021

Epitope and fluorescent tagging of trimeric G protein α subunits poses challenges due to potential disruption of functional regions, including the receptor‐interacting N‐ and C‐termini. In previous work, we inserted green fluorescent protein (GFP) and a Myc tag into a specific loop within the helical domain of Gα12, flanking each adduct with the sextet ser‐gly‐gly‐gly‐gly‐ser, and showed these tagged Gα12 variants to retain interaction with downstream target proteins, stimulate serum response factor, and exhibit conformational activation. Recently, we employed a similar approach for tagging Gα13, which shares 67% identity with Gα12 but has significant sequence divergence in the helical domain. Using molecular visualization software (PyMol) to superimpose the crystal structures of Gα12 and Gα13, we identified Gα13 residues corresponding to the site of tag insertion in Gα12, and made key substitutions of Gα12‐specific residues into Gα13. GFP‐tagged Gα13 is detectable in transfected cells by epifluorescence microscopy and immunoblotting, and shows robust activation of serum response factor. We are utilizing tagged Gα13 in binding assays to determine whether mutations in Gα13 disrupt binding to effectors. Furthermore, these tagged constructs are facilitating side‐by‐side comparisons of Gα12 and Gα13 in their relative affinity for common downstream effector proteins, such as Rho‐specific guanine nucleotide exchange factors, cadherins, and other targets. We acknowledge support from the North Carolina Biotechnology Center (DNB) and the Lineberger Comprehensive Cancer Center (TEM).
The A2B adenosine receptor controls a fork in the road of mesenchymal stem cell differentiation
Authors: Carroll Shannon H , Ravid Katya
Published: June 2021

The A2B adenosine receptor (A2BAR) is a G‐protein coupled receptor that signals by increasing cAMP and/or activating PKC signaling. The A2BAR is expressed on mesenchymal stem cells (MSCs), which give rise to osteoblast, chondrocyte and adipocyte lineages. We recently reported that activation of the A2BAR upregulates RUNX2, an inducer of osteogenesis, and increases the differentiation of MSCs to osteoblasts. Additionally, A2BAR null mice have impaired bone formation at baseline and after injury, and increased cartilage. To investigate whether the A2BAR is regulating fate determination of the MSC, we chose to study another lineage, the chondrocyte. We considered the possibility that the A2BAR upregulating effect on RUNX2 leads to differentiation of chondrocytes by a default pathway, involving RUNX2‐mediated downregulation of Sox9. Here, we report that A2BAR activation inhibits differentiation to chondrocytes by causing the downregulation of the key transcription factor Sox9, prior to any observed effect on RUNX2 transcription. This effect is at least partially due to cAMP signaling, as direct activation of adenylyl cyclase mimics the effect. These results support the contention that A2BAR‐induced cAMP elevation influences the lineage determination of the MSC via a direct stimulatory or inhibitory effect on key regulators of the osteoblastic or chrondrocytic lineage, respectively.

This work was supported, in whole or in part, by National Institutes of Health Grant HL93149
Signal Regulation of Mouse Hematopoietic Stem Cell Proliferation Derived from Pluripotent Embryonic Stem Cells
Authors: Liou Jun‐Yang
Published: June 2021

As increase cell proliferation of hematopoietic stem cells (HS) cells has a significant impact on clinical application, to investigate the molecular mechanism underlying the regulation of HS cell expansion is thus important. This study aims to investigate the molecular signaling involves in regulating cell proliferation of mouse HS cells derived from pluripotent embryonic stem (ES) cells. We have first established the protocol to differentiate mouse ES cells into HS cells and performed the gene expression profile by microarray analysis. Among the genes expression altered during the hematopoietic differentiation, we found a significantly reduction of 14–3‐3σ in mouse ES derived HS cells. Re‐introduced 14–3‐3σ overexpression significantly increased HS cell growth and number of hematopoietic forming colonies. In addition, suppression of GSK‐3β with inhibitor enhanced, but PI‐3K inhibitor reduced HS cell proliferation. Taken together, 14–3‐3σ combined with PI‐3K/Akt and GSK‐3β/β‐catenin signals are considered as critical targets for inducing HS cell expansion.
The effect of IL‐17 on the extracellular matrix expression in dermal fibroblasts
Authors: Jinnin Masatoshi , Nakashima Taiji , Fukushima Satoshi , Ihn Hironobu
Published: June 2021

Among interleukin (IL)‐17 family, IL‐17A and IL‐17F share amino acid sequence similarity and IL‐17 receptor type A. IL‐17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix (ECM) expression remains to be elucidated. We found that dermal fibroblasts express type A receptor for IL‐17. We then determined whether IL‐17 affects ECM expression in dermal fibroblasts.

The cDNA array of ECM genes and Immunoblot showed IL‐17A, not IL‐17F, reduced the expression of connective tissue growth factor (CTGF) as well as α1(I) collagen. miR‐129–5p increased by IL‐17A and mediated the α1(I) collagen reduction. These results suggest that IL‐17A signaling, not IL‐17F, has an anti‐fibrogenic effect via the up‐regulation of miR‐129–5p and the down‐regulation of CTGF and α1(I) collagen. Expression of IL‐17 receptor in cultured dermal fibroblasts derived from patients with scleroderma, one of the fibrotic skin diseases, was significantly down‐regulated, which may contribute to the tissue fibrosis.

IL‐17A may play important roles in tissue remodeling of the skin. Clarifying the novel regulatory mechanisms of tissue remodeling by IL‐17A signal may lead to a new therapeutic approach against various skin diseases with connective tissue abnormalities.
SREBP‐1 Mediates Angiotensin II‐induced TGFβ Upregulation in Kidney Mesangial Cells
Authors: Wang Tony , Gao Bo , Uttarwar Lalita , Krepinsky Joan C
Published: June 2021

Angiotensin II (Ang II) is a key determinant of the progression of kidney disease of diverse etiology. Mesangial cells (MC) contribute importantly to the accumulation of glomerular matrix proteins and hence glomerular sclerosis. We recently demonstrated a role for the transcription factor sterol regulatory element binding protein (SREBP‐1), best known for its regulation of lipid metabolism, in high glucose fibrotic responses in MC. Here we assessed whether it mediated Ang II profibrotic signaling. We found that Ang II induced generation of the mature transcription factor (mSREBP‐1) in a dose‐ and time‐dependent manner in primary MC. Transcriptional activation was confirmed. The chaperone SCAP and protease S1P, as well as signaling through the Ang II type 1 receptor and PI3K/Akt were required for SREBP‐1 activation. Endoplasmic reticulum (ER) stress was a central mediator of Ang II‐induced PI3K/Akt and SREBP‐1 activation. In uninephrectomized C57BL/6 mice, Ang II infusion for 1 week upregulated expression of glomerular SREBP‐1 and the ER stress chaperone GRP78. The profibrotic cytokine transforming growth factor beta (TGFβ) is a central mediator of glomerular sclerosis. Inhibition of either ER stress or SREBP‐1 in MC prevented Ang II‐induced TGFβ upregulation. SREBP‐1 is thus an important mediator of Ang II‐induced profibrotic signaling through its effects on TGFβ upregulation.
Human Prolactin Receptor: Impact of subdomain D1 of the short form variant for its inhibitory action on prolactin induced function of the long form of the receptor
Authors: Kang J‐H , Tsai‐Morris C. H. , Hassan S.A. , Dufau M. L.
Published: June 2021

The prolactin receptor (PRLR) long form (LF) mediates diverse cellular actions of prolactin (Prl) in target tissues. Short forms (SF) through heterodimerization inhibit the LF function. Reduced SF/LF ratio in breast cancer could contribute to tumor development. Lack of dominant negative action of SF by disruption of the intra‐molecular Cys‐Cys of S1b indicated a pivotal role of the D1 domain in SF function. Mutation of E69 in the D1 (S1bX) increased homodimer formation of the expressed protein. S1bX did not inhibit Prl induced LF function on β‐casein promoter activity. Mutation of adjacent amino acids showed a similar loss of function. In contrast, SF mutants in the D2 domain at the D2/D2 interface displayed inhibitory action as the WT SF. BRET analysis showed decreased affinity in LF/S1bX heterodimers and increased S1bX/S1bX homodimers in transfected cells stably expressing LF. This in turn favors LF/LF homodimerization and Prl mediated signaling. However, the affinity of LF/SF or SF/SF was unchanged in SF with the D2 domain mutated. Molecular dynamics indicate a role of long‐range D1‐D1 electrostatic interactions and structural changes propagating down the D2 dimers. These results have demonstrated the essential role of the D1 domain on the configuration of SF for its inhibitory action on the LF mediated function. Variations in the SF D1 domain in tumor tissues could provide insights of its role in cancer development.
Gene Expression Analysis to Evaluate the Effect of p38 Specific Inhibitor SB203580 on SEB‐Induced Apoptosis Related Pathways
Authors: Sondgeroth Casey , Hesse Lisa , Allbaugh Alyssa , Mendis Chanaka , Jett Marti
Published: June 2021

Staphylococcal enterotoxin B (SEB) is an enterotoxin that has been thoroughly investigated. However, little is known about the cascades of signaling events that explain the pathomechanism of SEB. This research involves the pathogenic nature of Staphylococcal aureus, the bacterium responsible for the production of the superantigen SEB which can cause death in human peripheral blood mononuclear cells (PBMC). Since SEB is known to induce multiple signal transduction pathways, we have identified a crucial pathway inter‐connector (p38) that may have an inhibitory effect in unwanted, SEB induced cellular activities. This study is focused on obstructing signaling pathways with the use of the inhibitor 4‐[5‐(4‐Fluorophenyl)‐2‐[4‐(methylsulfonyl)phenyl]‐ 1‐H‐imidazol‐4‐yl]pyridine (SB203580), followed by analyzing alterations to known expression patterns of genes associated with apoptosis. Some of these genes include Caspase 1, 3, 6–10, Heparanase precursor (HEP), genes associated with retinoid‐interferon‐ induced mortality (GRIM)‐19, and CASP2 and RIPK1 domain containing adaptor with death domain (CRADD). These effects will be further confirmed through a set of protein expression analyses. As our experimental design specifically targets pathway inter‐connector p38, we believe the target is unique and has the ability to sustain longer lasting inhibitory effects. We are also confident that inhibiting such a component will have a maximum effect on a cell module, terminating the “leaking effect,” which may benefit a number of experimental modules focused on disease prevention and rapid diagnostics.
p53 is a major component of the transcriptional and apoptotic program regulated by PI 3‐kinase/Akt/GSK3 signaling
Authors: NAYAK GAURI , COOPER GEOFFREY
Published: June 2021

The phosphatidylinositol (PI) 3‐kinase/Akt signaling pathway plays a prominent role in cell survival and proliferation, in part by regulating gene expression at the transcriptional level. Global expression profiling combined with computational and experimental analysis previously identified FOXOs and the E‐box‐binding transcription factors MITF and USF1 as key targets that lead to the induction of proapoptotic and cell cycle arrest genes in response to inhibition of PI 3‐kinase. In this study, we investigated the role of p53 downstream of PI 3‐kinase signaling by analyzing the effects of PI 3‐kinase inhibition in Rat‐1 cells and Rat‐1 cells expressing dominant‐negative p53 mutant. Dominant‐negative p53 expression conferred partial resistance to apoptosis induced by PI 3‐kinase inhibition and p53, along with FOXO, MITF and USF1, contributed to gene induction in response to PI 3‐kinase inhibition. Activation of p53 was mediated by phosphorylation of the histone acetyltransferase Tip60 by glycogen synthase kinase (GSK) 3, leading to activation of p53 by acetylation. Many of the genes targeted by p53 were also targeted by FOXO and the E‐box‐binding transcription factors, indicating that p53 functions coordinately with these factors to regulate gene expression. Current studies are focused on analyzing the effect of PI‐3 kinase inhibition on transformed cell lines with hyperactive PI‐3 kinase activity.
Yes‐associated protein, YAP, functions as a positive regulator of androgen receptor signaling in prostate cancer cells
Authors: Cinar Bekir , Alptekin Ahmet , Abali Gamze Kuser
Published: June 2021

Androgen receptor (AR) plays a central role in prostate tumor biology. Here, we studied the impact of yes‐associated protein (YAP) oncogene, a nuclear effector of the hippo tumor suppressor network, on AR signaling. We first showed that native YAP formed protein complexes with endogenous AR, and the complex formation that mainly occurred in cell nuclei was enhanced by androgen, as revealed by immunoprecipitation (IP) and western blots. Our promoter reporter analysis demonstrated that enforced YAP expression increased the activation of AR‐dependent promoter reporter gene, and that the carboxyl‐terminal site of YAP, which consists of WW and coiled‐coiled domains, appeared to be responsible for AR transactivation. Chromatin‐IP experiment demonstrated that YAP interacted with the DNA regions where AR also binds, and that androgen enhanced the YAP and chromatin DNA complexes. We also provide evidence that YAP induction promoted an androgen‐dependent and ‐independent cell growth, and that YAP knockdown had the opposite effects. Our data indicate that YAP is a binding partner and physiologic positive regulator of AR and suggest that altered Hippo‐YAP signaling could play a critical role in prostate tumor progression in humans.
TCTP as a Regulator of mTOR Signaling
Authors: Milbrandt Laura , 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 is thought to be a guanine nucleotide exchange factor (GEF) for Rheb, a key player of the mTOR signal transduction cascade. In this study, the contribution of TCTP toward the mTOR pathway was investigated by over‐expression, gene silencing, and mutational analysis of TCTP in HEK293 cells. Specifically, the overall effects of treatment were studied by performing cell proliferation assays and mTOR signaling was monitored via the phosphorylation status of the protein S6, the downstream effector which gets phosphorylated upon mTOR activation. Overexpression was achieved using a recombinant human TCTP construct and silencing was achieved using specific siRNAs. We constructed site‐directed TCTP mutants at three serine residues at positions 46, 53 and 64 based on their ability to be phosphorylated to determine if these serines have an effect on TCTPs activity. Our results from over‐expression, silencing and mutational analysis of TCTP indicate that silencing of TCTP influences cell viability and mTOR signaling in a manner contrary to what has been reported previously using over‐expression studies and provide interesting insights into TCTP's role in regulating the mTOR pathway.
Signaling Mechanisms and Physical Structure of Biofilm Growth in Synechocystis sp. Strain PCC 6803
Authors: Fishbein Jennifer , Huang Jean , Christianson Rebecca , Allen Mary
Published: June 2021

Chemical signaling has been found in the past to play a central role in bacterial biofilm formation, and may be important for the cyanobacterium Synechocystis sp. Strain PCC 6803. We investigate the cell‐cell communication system of this species and whether it involves acyl homoserine lactone (AHL) signaling molecules. We use the biosensor, Chromobacterium violaceum CV026, which produces the violacein pigment in the presence of AHLs (McClean, et al., 1997). This biosensor was found to be a sensitive and quantitative method of detecting a variety of AHL chain lengths. Optimal growth temperature is 29°C and growth is not affected at low concentrations of acid, which is required for AHL stability. The physical structure of the biofilms of a super motile cyanobacterial mutant is also being analyzed using confocal microscopy. The biofilms begin as pillars 20–30um tall, grow to 50um deep and spread across the surface. The motile cyanobacterial cells exhibit unique biofilm formation and structure, and we are determining whether the process involves AHL signaling. This research is supported by the Wellesley College Biological Sciences Department and a Jerome A. Schiff Fellowship.
Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples of menopausal women
Authors: Conde Sandro José , Luvizotto Renata de Azevedo M. , de Síbio Maria Teresa , Nogueira Célia Regina
Published: June 2021

We investigated thyroid hormone levels in menopausal BrC patients, and verified the action of triiodothyronine (T3) on genes regulated by estrogen (E2) and by T3 itself in BrC tissues. We selected 15 postmenopausal BrC patients and a control group of 18 postmenopausal women without BrC. We measured serum thyroid peroxidase antibody, TSH, free thyroxine, and estradiol, before and after surgery, and used immunohistochemistry to examine E2 and progesterone receptors. BrC primary tissue cultures received the following treatments: ethanol, T3, T3+tamoxifen (TAM), TAM, E2, or E2+TAM. Genes regulated by E2 (TGFA, TGFB1, PGR) and by T3 (TNFRSF9, BMP‐6, THRA) in vitro were evaluated in each patient. Three BrC patients presented with clinical hyperthyroidism. Thyroxine levels of BrC patients were statistically higher than controls (1.78±0.20 vs. 0.95±0.16 ng/dl). TGFA and TGFB1 were upregulated and downregulated, respectively, after E2 and T3 treatment. T3 treatment increased PGR expression, however TAM did not block T3 action on PGR expression. TAM, alone or associated with T3, modulated gene expression of TNFRSF9, BMP‐6 and THRA, similar to T3 treatment, showing that TAM can interfere with gene expression modulated by T3. Our results reinforce that the thyroid hormone status of BrC patients can influence E2‐controlled processes, even after TAM intervention and/or in the absence of circulating E2 postmenopause.
Association of lipocalin‐2 in colorectal cancer metastasis to the liver
Authors: Elamparo Fides , Zhang Yu , Hughes Daniel , Pena Maria
Published: June 2021

Metastasis of colorectal cancer (CRC) to the liver reduces the five‐year survival rate to less than 12% and is the major cause of death from CRC. Metastasis occurs due to productive collaborations between tumor cells and host‐derived cells in the tumor microenvironment. A pre‐metastatic niche is created to prime for cancer cell invasion into the target organ. In Balb/c mice bearing cecal tumors derived from a highly metastatic CRC cell line, microarray analysis revealed that lipocalin‐2 (LCN2) is the most highly expressed protein in the liver prior to metastasis. Western blot analysis and examination of blood serum by ELISA illustrated increased levels of LCN2 as tumors progressed into metastasis. RT‐PCR analysis showed that greater levels of LCN2 mRNA were found in highly metastatic cells as compared to less metastatic CRC cells. Our results show that LCN2 is highly associated with the development of colorectal cancer metastasis to the liver, with increased levels indicating advanced stages of metastatic progression.

Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina
Hypoxia and the sodium hydrogen exchanger in non small cell lung cancer.
Authors: Novak Alexander , Berthelsen Nick , Provost Joseph , Wallert Mark
Published: June 2021

The challenge of hypoxia in solid tumors induces several physiological changes in the cancer cell microenvironment including increased extracellular acidity. While proton exchangers including the sodium hydrogen exchanger (NHE1) support tumor cell invasion and metastasis, the relationship between hypoxia and increased acidity on migration/proliferation remains unclear. In this study we investigate the relationship between the sodium hydrogen exchanger isoform 1 (NHE1) and hypoxia inducible factor 1 (HIF‐1α) expression in H1299. HIF‐1α expression was observable after 24 hours of either 1% oxygen or chemically induced hypoxia and was maximally expressed after 72 hours of hypoxia. In parallel we found that NHE1 expression slightly increased after 72 hours in hypoxic conditions. We also show that hypoxia decreased cell proliferation in H1299 non small lung cancer cells. In addition, the impact of NHE1 inhibition on hypoxia induced MMP‐9 activity was altered. Hypoxia induced changes in cell motility and alkalization of intracellular pH was also observed. These results point to a relationship between hypoxia and NHE1 that influences cancer cell behavior. NIH Grant 1 R15 HL074924–01A1
Scientific discovery in a fast‐forward mode: getting personal and creative with virtual viruses
Authors: Marintcheva Boriana
Published: June 2021

Virtual virus is a semester–long interdisciplinary project modeling the process of scientific discovery, offered as a part of the BIOL450/Virology course at Bridgewater State University. Students are challenged to apply molecular biology and biochemistry concepts to “synthesize” their own virus in a series of hands‐on sessions and mini‐projects. Each student built a physical model and developed virtual scenario how their virus propagates while interacting with cellular machinery and the host organism as a whole. The project results were communicated via class presentation and a report in the format of scientific review article. Project outcomes demonstrated high level of student engagement as identified by the personal dimensions of the developed virtual viruses, the breath of the explored biochemical and molecular biology processes and the diversity of virtual hosts. Analysis of student work demonstrates that the Virtual virus project is a successful model for capstone experience in biology combining active learning by design, creativity and problem solving to assemble a virtual scientific puzzle, thus modeling in fast‐forward mode the process of scientific discovery. This project was generously supported by a course‐embedded research grant from the Office of Undergraduate Research at Bridgewater State University.
Isothermal Titration Calorimetry in College Physical and Biochemical Chemistry Laboratory Courses
Authors: Wei Chin‐Chuan , O'Brien Leah C , Reynolds Nicole , Tatro Allison , Jensen Drake , Eder Douglas
Published: June 2021

Isothermal titration calorimetry (ITC) is a relatively new technique that is used to determine thermodynamic parameters for a reaction. ITC has become an essential tool in research and in the pharmaceutical industry. However, such an ultra‐sensitive calorimetry technique is absent in most undergraduate curriculums. Our institute implemented ITC in both Physical and Biochemistry laboratory courses in a sequential way. Students were first introduced to ITC and studied the reactions of EDTA to Mg2+ in the Fall semester. This exercise strengthened their understanding of the relationship among pKa of functional groups, pH of buffer, and protonation states of protein, which are commonly found in enzymatic catalysis. When paired with inferential questions, this exercise brought about effective critical thinking. In the following Spring semester, students performed ITC to determine the binding affinity of lysozyme to NAG3 and extended the measurement to a much weaker inhibitor, NAG, by a competitive binding assay. The enthalpy difference obtained from ITC was correlated to the number of hydrogen bonds formed, visualized by PyMol software. The assessment indicated that the successive ITC experiments in the Physical and Biochemistry labs served as an effective education tool. This work is supported in part by National Science Foundation and SIUE's Excellent in Undergraduate Education.
Exploration of the diffusion of discipline‐based education research in published introductory science laboratory exercises
Authors: DeChenne Sue Ellen , Stains Marilyne , Carew Jenna
Published: June 2021

This study explores the diffusion and adoption of discipline‐based education research (DBER) related to inquiry in introductory science laboratories at the undergraduate level. Two hundred and nine introductory science laboratories from four DBER journals which target instructors were scored for level of inquiry (limited, structured, guided and open). Results suggest a weak adoption of open and guided inquiry, which are practices supported by science education research (19% of the laboratories) and a low rate of reference to science education research literature (23% of the laboratories). Interestingly, citation of education research correlated with type of inquiry. Even though DBER has dramatically improved its reach over the last decade, there was no significant change in the level of inquiry in published laboratory experiments over the 2001 to 2010 decade. Despite evidence that inquiry process skills are important for student's content knowledge and research skills, this study indicates that DBER is having little impact on early undergraduate laboratory experiences. Funding provided by Department of Chemistry, University of Nebraska – Lincoln.
Novel LC/MS/MS‐based Proteomics Approach for the Identification of S‐Glutathionylated Proteins: Analysis of Redox Regulation of Metabolic Priming in Monocytes
Authors: Ullevig Sarah Lynn , Asmis Reto
Published: June 2021

We reported previously that metabolic stress primes monocytes for increased responsiveness to chemokines, a process mediated by Nox‐4‐derived H2O2 and protein‐S‐glutathionylation. Established proteomics approaches have identified only a limited number of S‐glutathionylated proteins. We therefore developed a novel technique using glutaredoxin to selectively reduce S‐glutathionylated proteins, followed by labeling with a thiol‐reactive probe (iodoacetamide‐desthiobiotin), and purification using streptavidin‐coated agarose resin. We compared untreated THP‐1 monocytes with metabolically stressed (glucose, 25 mM; LDL, 100 μg/ml), or oxidatively stressed (H2O2; 1 mM) monocytes. Using LC/MS/MS, we identified over 200 S‐glutathionylated proteins. We also provide evidence that S‐glutathionylation targets specific proteins for proteasome‐dependent degradation. Applying Ingenuity Systems IPA software analysis, S‐glutathionylated proteins were associated with several cellular functions, including, protein folding and synthesis, cell motility, and cell death. In summary, we developed a novel sensitive proteomic technique for the identification of S‐glutathionylated proteins, and provided new insights into the role of protein‐S‐glutathionylation in redox regulation and monocyte function in the context of metabolic disorders. Funded by NIH RO1 HL070963 & HL115858 (RA).
Dual Routes for Bacillus halodurans Asparaginyl‐tRNA Formation
Authors: Nair Nilendra , Sheppard Kelly
Published: June 2021

Two evolutionary distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis, are found in nature. In the first, asparaginyl‐tRNA synthetase directly ligates Asn to tRNAAsn in an ATP‐dependent manner. In the second pathway, a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS) first ligates Asp to tRNAAsn and GatCAB then modifies the tRNA‐bound Asp to Asn by transamidating the amino acid. Our bioinformatics analysis of the Gram‐positive Bacillus halodurans suggests it may encode the indirect pathway in addition to the direct one. Encoding the indirect Asn‐tRNAAsn biosynthetic pathway requires an ND‐AspRS. We are testing whether the B. halodurans AspRS is non‐discriminating (i.e., uses tRNAAsn as a substrate along with tRNAAsp). Using the E. coli JF448 system, we have determined the B. halodurans AspRS is non‐discriminating and works in concert with GatCAB to synthesize Asn in a tRNA‐dependent manner. Further verification of the nature of the AspRS using the E. coli TrpA34 system and in vitro studies are underway. We speculate retention of both pathways for Asn‐tRNAAsn synthesis in B. halodurans may be vestigial and that a number of other bacteria may also maintain both routes.
Dual Routes for S. aureus Asparaginyl‐tRNA Formation
Authors: Mladenova Stefani , Sheppard Kelly
Published: June 2021

Two routes have evolved for attaching the amino acid asparagine (Asn) to its correct transfer RNA (tRNAAsn), an essential step in protein synthesis. The direct pathway involves aminoacylation of tRNAAsn by asparaginyl‐tRNA synthetase (AsnRS) when free Asn is available. In the indirect pathway, a non‐discriminating aspartyl‐tRNA snthetase (ND‐AspRS) attaches Asp to tRNAAsn, and the tRNA‐bound Asp is then amidated to Asn by GatCAB. Bioinformatics suggests the human pathogen Staphylococccus aureus may encode the indirect pathway in addition to the direct one. We set out to determine if, S. aureus does in fact encode an ND‐AspRS and GatCAB for tRNA‐dependent Asn synthesis. Using the E. coli JF448 system, we demonstrate that the concerted activities of S. aureus AspRS and GatCAB can form Asn on tRNA. In addition, using the E. coli TrpA34 system, we are verifying the S. aureus AspRS enzyme is non‐discriminating supported by in vitro studies with the enzyme. We speculate encoding dual routes for Asn‐tRNAAsn formation enables S. aureus to take advantage of situations when Asn is plentiful.