bluuebunny/crossref_metadata_2025 huggingface dataset:
17β‐Estradiol inhibits isoproterenol activated lipolysis in murine adipocytes
Authors: Tagawa Noriko , Mitsumasu Satoshi , Yuda Ryosuke , Kobayashi Yoshiharu
Published: June 2021

Stress stimulates sympatho‐adrenomedullary activity and lipolysis in adipocytes. Increase in plasma free fatty acids has been demonstrated to be a major link between obesity and insulin resistance. However, there are many reports describing the anti‐obesity effect of estrogen. This study investigated the effect of estrogen on isoproterenol (ISO) activated lipolysis in murine adipocytes. ISO (5 μM) and 17β‐Estradiol (E2) was added to mature 3T3‐L1 adipocytes in 6 well plates. After 2 hr incubation at 37 °C in 5% CO2/95% air, the glycerol concentration in DMEM medium was measured. Effects of β2‐adrenoceptor inhibitor (yohimbine, 50 μM), phosphodiesterase inhibitor (cilostamide, 2 μM) and estrogen receptor blocker (tamoxifen, 10 μM) on the inhibitory effect of lipolysis of E2 were also tested. Further, effect of E2 on cAMP production and hormone sensitive lipase (HSL) activity in 3T3‐L1 adipocytes were investigated. E2 (5–50 μM) inhibited ISO activated lipolysis in 3T3‐L1 adipocytes dose‐dependently. Yohimbine, cilostamide and tamoxifen did not abolish the inhibitory effect of E2 on ISO activated lipolysis. E2 inhibited HSL activity, whereas E2 did not reverse the increased cAMP level induced by ISO stimulation. These results suggest that E2 exerts its inhibitory effect of ISO activated lipolysis by inhibition somewhere down‐stream of cAMP production but up‐stream of the HSL signaling pathway.
Fatty Acid Uptake in African Trypanosomes
Authors: Paul Kimberly Sue , Vigueira Patrick , Winston Brette , McCall Barbara , Cain Sarah , Ligon Marianne , McCallister Jennifer
Published: June 2021

Trypanosoma brucei is a parasite that causes lethal disease African Sleeping Sickness in humans. T. brucei has two options to supply itself with fatty acids: synthesis via an ER‐localized elongase pathway, or uptake from the host. We used fluorescence‐ and radioactive‐based assays to characterize fatty acid uptake in T. brucei. Uptake of fluorescent BODIPY‐C12 fatty acid was concentration‐ and time‐dependent and appeared to be saturable. Uptake of BODIPY‐C5 was inefficient compared to BODIPY‐C12 and ‐C16, suggesting chain‐length specificity. Surface proteolysis with trypsin reduced BODIPY‐C12 uptake in a concentration‐dependent manner, suggesting a surface protein may be involved. Treatment with 2‐deoxyglucose or RNA interference of hexokinase reduced BODIPY‐C12 uptake, suggesting partial dependence on glycolysis (and perhaps ATP). Uptake of [3H]fatty acids complexed with BSA exhibited 10‐fold less uptake compared to uncomplexed fatty acids. Finally, RNA interference targeting a tandem array of 4 acyl‐CoA synthetases resulted in reduced BODIPY‐C12 and [3H]fatty acid uptake and subsequent cell death, suggesting at least one ACS gene is involved in uptake. Taken together, our data indicate that T. brucei use multiple pathways to meditate their robust fatty acid uptake. Elucidation of these mechanisms will enhance our understanding of the host‐parasite relationship and may lead to novel therapies.
Arachidonoyl substrate specificity of diacylglycerol kinases
Authors: Shulga Yulia , Topham Matthew K. , Epand Richard M.
Published: June 2021

Diacylglycerol kinases (DGKs) are an important class of lipid signaling enzymes. DGKε is unique among DGK isoforms in having specificity for DAG substrates with an arachidonate moiety. This contributes to the enrichment of lipid intermediates in the PI‐cycle with arachidonate. The importance of DGKε in neuronal function has been demonstrated in studies with knockout mice. We have shown that removal of 58 residues from the amino terminus of this protein, including the membrane‐inserting segment, had a negligible effect on substrate specificity. It is intriguing that the domain, responsible for DGKε specificity for DAG substrates with an arachidonate moiety, still is unknown.

We have now identified a region of DGKε containing four conserved residues, very similar to those found responsible for recognition of arachidonic acid in lipoxygenases. We demonstrated that several mutations within this region of DGKε significantly impact enzyme activity, decreasing it to less than 2% of the activity of wild‐type DGKε. Moreover, the mutants showed a lower ratio of the enzyme activity with a DAG substrate containing an arachidonate moiety to substrates without an arachidonate moiety. The relationship of this finding to the substrate specificity of other isoforms of DGK is also explored. (Supported by the Natural Sciences and Engineering Research Council of Canada, grant 9848)
Biochemical and molecular confirmation of candidate Endosome and Vacuole interface (env) mutants derived from a genomic screen and characterization of four novel env deletion mutants: env6 ‐ env9
Authors: Chhun Surya , Serranilla Maribeth , Olson Daniel , Ricarte Florante , Soreta Tattika , Gharakhanian Editte
Published: June 2021

The vacuole of the fungus Saccharomyces cerevisiae has been a seminal model for lysosomal trafficking, biogenesis, and function. Our laboratory uncovered 15 deletion mutants associated with these processes utilizing a genome‐wide immunodetection screen of a MAT‐α deletion strain library. This study focuses on confirmation of the l5 mutants for defects at the endosome & vacuole interface (env mutants) and characterization of four uncharacterized env mutants : env6Δ, env7Δ, env8Δ, and env9Δ. Microscopic, biochemical, and bioinformatics techniques were used to characterize the four mutants with respect to vacuole morphology, growth under various conditions, and gene homology. env6Δ has fragmented vacuoles with acidification defects, exhibits severe growth sensitivities, and the deletion is a dubious open reading frame (ORF). env7Δ exhibits normal vacuolar morphology, no severe growth sensitivities, and ENV7 encodes a putative serine/threonine kinase with 29% identity to human STK16 kinase. env8Δ has fragmented vacuoles, no severe growth sensitivities, and the ORF encodes a highly conserved protein with possible involvement in membrane insertion of tail‐anchored proteins. env9Δ has vacuoles with MVBs, and exhibits no severe growth sensitivities. Complementation studies of the four mutants following reintroduction of each ORF are currently in progress. Funding was provided by NSF‐RUI and NIH‐AREA grants.
Characterization of apical and basolateral PAR‐2
Authors: Lau Chang Shun
Published: June 2021

Studies suggest there are two distinct pools of protease‐activated receptor‐2 (PAR‐2) present in colonic epithelial cells: an apical pool exposed to the lumen, and a basolateral pool exposed to bloodstream. Further, studies demonstrate introduction of PAR‐2 agonists to the lumen activate apical PAR‐2, while it has been predicted mast cells and recruited leukocytes release proteases and activate basolateral PAR‐2. However, whether both pools of PAR‐2 are capable of signaling, and the possible distinction between apical and basolateral PAR‐2 induced responses have yet been addressed. Here we demonstrate presence of apical and basolateral pools of PAR‐2 in cultured CACO‐2 monolayers and mouse intestinal mucosa. We show that the signals generated by the two receptor pools differ temporally and mechanistically. Apically administered PAR‐2 agonist promotes rapid, G‐protein dependent ERK1/2 activation, while basolaterally administered PAR‐2 agonist promotes prolonged G‐protein and β‐arrestin‐dependent ERK1/2 and cofilin activation. Furthermore, basolaterally administered PAR‐2 agonist increase Isc and decreases transepithelial resistance while apically administered PAR‐2 agonist does not. These results suggest that the outcome of PAR‐2 activation is dependent upon the specific receptor pool that is accessible to a given agonist, allowing for fine‐tuning of the physiological response to different serine proteases.
Does Presynaptic GSK‐3Beta Signaling Play a Role in Bipolar Disorder: Studies of on Dopamine Transporter (DAT) Coding Variants
Authors: Miller Leah , Blakely Randy
Published: June 2021

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission and has been implicated in several neuropsychiatric disorders, including bipolar disorder (BPD). The protein kinase B (Akt)/glycogen synthase kinase (GSK)‐3β pathway has been implicated in the control of DAT‐mediated trafficking/efflux. We reasoned that a direct connection between DAT and DAT regulation via Akt/GSK‐3b pathways could help link genetic associations and the pharmacotherapy of BPD, e.g. with the GSK‐3β inhibitor Lithium. The current study examines the regulation of wildtype DAT and two bipolar associated‐DAT coding variants, A559V and E602G, by the AKT‐dependent GSK3β signaling pathway. Initial studies with the GSK‐3β inhibitor SB216763 (0.1uM‐10uM) to human embryonic kidney 293 cells stably expressing wildtype, epitope‐tagged hDAT induced an increase in DAT trafficking as well as increased [3H] DA uptake, as assessed by biotinylation studies and transport assays. Treatment with BIO, a GSK3β inhibitor, also led to increased DA uptake in A559V transfected cells but decreased DA uptake in E602G transfected cells. Current studies are aimed at a further examination of mutation‐induced differences in wildtype and mutant DAT regulation through the Akt/GSK‐3β pathway and their structural basis. Supported by NIH grant #T32NS06120106.
Measuring arrestin binding to G Protein Coupled Receptors: BRET and recruitment assays tell different stories.
Authors: Gimenez Luis E , Digby Gregory J , Weaver Charles D. , Conn P. Jefffrey , Gurevich Vsevolod V.
Published: June 2021

In mammals, visual (Arr1) and cone arrestin (Arr4) target activated photoreceptors, whereas ubiquitous arrestin2 and 3 (Arr2 and Arr3) regulate non‐visual GPCRs. We explored the interaction of Arr1, 2 and 3 with the M1–M2 muscarinic acetylcholine receptors (M1/M2AChR) and the β2‐adrenergic receptor (β2AR) by direct visualization of arrestin recruitment and bioluminescence resonance energy transfer (BRET). Venus‐tagged arrestins were expressed in HEK‐293 cells and imaged after agonist stimulation of co‐transfected M1AChR, M2AChR or β2AR. The formation of puncta corresponding to the receptor‐arrestin complexes recruited to coated pits was observed for M1AChR and β2AR with Arr3, but not Arr1. For BRET assays, the same receptors fused with Renilla luciferase 8 (RLuc8) were co‐expressed in COS‐7 cells with increasing amounts of Venus‐tagged arrestins. In contrast to recruitment assays, all three arrestins yielded saturable BRET signals with near‐identical profiles. Considering that measured affinity of Arr2 and Arr3 for M2AChR or β2AR is much higher than that of Arr1, our results suggest that BRET is less sensitive to differences in receptor preference of arrestins than recruitment assays.

NIH grants GM077561, GM081756 (VVG), MH073676 (PJC), and F32 MH087039 (GJD)
Binding‐folding induced regulation of the glucocorticoid receptor AF1 transactivation domain by a cofactor that binds to its DNA binding domain
Authors: Khan Shagufta H , Garza Anna S , Moure Carmen M , Edwards Dean P , Kumar Raj
Published: June 2021

Intrinsically disordered (ID) regions commonly exist within the N‐terminal AF1 activation domains of steroid receptors (SRs). The mechanisms by which SRs pass signals from a steroid hormone to control gene expression remain a central unresolved problem. Role of ID AF1 in this process is of immense importance as it provides platforms (through conditional folding) for interactions of specific co‐regulatory proteins, and thereby dictates the final outcome SR‐regulated target gene expression. However, the means by which AF1 acquires functionally folded conformations is not well understood. It has been suggested that under physiological conditions, inter‐ and intra‐ molecular interactions lead to structure formation in AF1. In this study, we tested whether binding of jun dimerization protein 2 (JDP2) within the DNA binding domain (DBD) of the glucocorticoid receptor (GR) leads to acquisition of an active conformation in AF1. Our results show that signals mediated from GR DBD:JDP2 interactions in a two domain GR fragment, consisting of the N‐terminal domain and DBD (GR500), significantly increased secondary/tertiary structure formation of AF1. This induced structure facilitated AF1’s interaction with specific co‐regulatory proteins, and subsequent GRE‐mediated AF1 activity. These results support the hypothesis that inter‐ and intra‐ molecular signals give a functionally active structure(s) to AF1.
BS69 negatively regulates the canonical NF‐kappaB activation induced by Epstein‐Barr virus‐derived LMP1
Authors: Ikeda Osamu , Sekine Yuichi , Muromoto Ryuta , Nanbo Asuka , Matsuda Tadashi
Published: June 2021

Epstein‐Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF‐kappaB signaling pathways through the two C‐terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1‐induced c‐Jun N‐terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor‐associated factor 6. In the present study, our experiments manipulating of BS69 expression clearly indicated that BS69 negatively regulated LMP1‐mediated NF‐kappaB activation including up‐regulated IL‐6 mRNA expression and IkappaB degradation. For the mechanisms, our immunoprecipitation experiments proposed that BS69 decreased complex formation between LMP1 and TNFR‐associated death domain protein (TRADD).
Deuterium exchange mass spectrometry reveals structural dynamics of glucan phosphatases
Authors: Sherwood Amanda , Gentry Matthew
Published: June 2021

Laforin and Starch Excess 4 (Sex4) are unique phosphatases from humans and plants, respectively, that dephosphorylate phosphoglucans, making them glucan phosphatases. Each protein contains a carbohydrate binding module (CBM) and a dual specificity phosphatase domain (DSP). Mutations in the laforin gene lead to a neurodegenerative disease called Lafora disease (LD). A hallmark of the disease is the presence of hyperphosphorylated, sparsely branched, and thus insoluble glucan accumulations in the cytoplasm. The loss of SEX4 in plants yields a similar accumulation of insoluble glucans. The mechanism by which laforin and SEX4 are believed to dephosphorylate phosphoglucans is unknown, and there is no crystal structure of these phosphatases. We utilized hydrogen‐deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. Our results suggest that the protein undergoes minimal structural rearrangement upon glucan binding. As expected, the CBM undergoes protection from deuteration upon substrate binding, and surprisingly regions of the DSP also undergo protection. The three regions of the DSP that undergo protection likely participate in substrate presentation to the active site. Collectively, these data provide the first structural analyses and mode of action for this unique class of phosphatases. NIH 5R00NS061803 and 5P20RR020171.
REGULATION OF HUMAN CYTIDINE TRIPHOSPHATE SYNTHETASE 2 BY PHOSPHORYLATION
Authors: Au Da Ryung , Kassel Karen M , Hines Maria , Graves Lee M
Published: June 2021

Cytidine triphosphate synthetase (CTPS) is the rate‐limiting enzyme that catalyzes the de novo synthesis of CTP from UTP although little is known about the regulatory mechanisms that control the activity of this enzyme. In addition to UTP, CTPS requires the substrates ATP, GTP, and glutamine. Previous studies determined that an increase in phosphorylation of the carboxyl‐terminal regulatory domain (CRD) decreased CTPS1 activity. Therefore, the purpose of this study was to identify the major phosphorylation sites in the CRD of human CTPS2 and to determine their influence on CTPS2 activity. Using mass spectrometry, five potential phosphorylation sites were indentified: S564, Y567, S568, S571, and S574. After mutation of each of these sites, kinetic analysis of both wild‐type and mutated CTPS2 were performed. Analysis of a Ser/Ala mutation at S568 (S568A CTPS2), at various glutamine concentrations, showed much higher enzymatic activity compared to wild type CTPS2 (WT‐CTPS2). In comparison, mutation of the other phosphorylation sites had minimal effects on CTPS2 activity. In conclusion, this study investigated the effects of phosphorylation on CTPS2 activity and identified S568 as a major phosphorylation site in CTPS2.
Defining the Conserved Internal Architecture of a Protein Kinase
Authors: Kornev Alexandr P. , Taylor Susan S
Published: June 2021

Protein kinases constitute a large protein family of important regulators in all eukaryotic cells. All of the protein kinases have a similar bilobal fold, and their key structural features have been well studied. However, the recent discovery of non‐contiguous hydrophobic ensembles inside the protein kinase core shed new light on the internal organization of these molecules. Two hydrophobic "spines" traverse both lobes of the protein kinase molecule, providing a firm but flexible connection between its key elements. The spine model introduces a useful framework for analysis of intramolecular communications, molecular dynamics, and drug design.
Investigation of Rho associated kinase phosphorylation of carboxyl terminus of the Sodium Hydrogen Exchanger Isoform 1
Authors: Hammes Daniel J , Slusser Andrea , Shabb John B , Muhonen Wallace W , Wallert Mark A , Provost Joseph J
Published: June 2021

Rho associated kinase p160 ROCK (Rock) and Ribosome S‐6 Kinase (RSK) both phosphorylate the Na+/H+ exchanger isoform 1 (NHE1) regulating ion transport, cell migration and binding to cytoskeletal linker proteins, ezrin, radixin, and moesin (ERM). NHE1 is an integral membrane protein involved in the regulation of intracellular pH, extracellular acidification, cellular migration, and cytoskeletal organization. Phosphorylation of NHE1 induces an intracellular alkalynization which promotes cellular proliferation and coordinates cellular migration through ERM binding and the accompanying organization of actin filaments. Using the Rock consensus sequence of RXXS/T and RXS/T and MS analysis of Rock phosphorylated NHE1, we have identified potential phosphorylation sites and created a series of site directed mutants. With the phosphorylation sites identified we have confirmed phosphorylation using in vitro labeling and generated stable cell lines expressing single and double Ser‐Ala mutations of the Rock and RSK sites to study changes in NHE regulation of ion translocation and cell motility events. This study intends to identify the Rock phosphorylation site on NHE1 and provide a new understanding for how transporters function to direct cell motility. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01
Structure of D‐AKAP2‐PKA RI isoform complex: Insights into AKAP specificity and selectivity
Authors: Sarma Ganapathy N , Kinderman Francis S , von Daake Sventja , Taylor Susan S
Published: June 2021

cAMP‐dependent protein kinase (PKA) regulates a variety of biological activities by phosphorylating a broad range of target proteins. Spatial and temporal specificity of PKA activity is, in turn, achieved by a family of scaffolding proteins called as the A‐kinase anchoring proteins (AKAPs). One of these, dual‐specific AKAP 2 (D‐AKAP2) binds to both the Regulatory (R) isoforms (RI and RII) of PKA. Specifically, the dimerization and docking (D/D) domain at the N‐terminus of the R subunits forms a four‐helix bundle that provides an interface for AKAP binding.

In order to understand the basis of D‐AKAP2 binding to the RIα subunit and understand the molecular determinants that make D‐AKAP2 dual‐specific, we solved the structures of the RIα D/D domain with and without the D‐AKAP2 peptide. The structure of the apo crystal form was solved using the anomalous signal from inherently present sulfur atoms. The structures highlight the importance of redox‐sensitive disulfides in affecting AKAP binding. An unexpected shift in the helical register of D‐AKAP2 compared to RIIα:D‐AKAP2 complex structure makes the mode of binding to RIα, novel. Further, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity. This work was supported by HHMI, American Heart Association Postdoctoral Fellowship to G.N.S, and NIH grants GM34921 and DK54441 to S.S.T.
Heterologous expression of microbial flavohemoglobin can modulate the effects of nitric oxide in mammalian cells
Authors: Eyler Christine Elissa , Forrester Michael T. , Hjelmeland Anita B. , Rich Jeremy N.
Published: June 2021

Nitric oxide (NO) contributes to a wide variety of intracellular signaling and cell stress pathways in mammalian cells. Experimental manipulation of NO is frequently achieved by inhibition or activation of the NO synthase (NOS) family of enzymes responsible for intracellular NO production, or through employment of relatively nonspecific NO‐reactive compounds or antioxidants. Here we introduce a novel methodology to manipulate NO levels in mammalian systems via heterologous expression of the prokaryotic NO‐consuming flavohemoglobin (FlavoHb). This enzyme is an NO‐specific dioxygenase that converts NO to inert nitrate under aerobic conditions, thus functioning to consume NO and protect microbes from nitrosative stress. We demonstrate that FlavoHb can be employed in a similar fashion in mammalian cells to inhibit both NO‐dependent signal transduction and nitrosative stress. This strategy provides a highly specific and novel method for modulating intracellular NO levels and represents an alternative or complimentary strategy to utilization of NOS inhibitors or promiscuous antioxidants. Importantly, FlavoHb expression is well tolerated by mammalian cells with no evidence of NO‐independent toxicity. Future studies of NO in mammalian systems could employ FlavoHb as either a primary experimental or a confirmatory tool for probing functional and molecular consequences of NO within cells.
Insulin Coordinately Regulates Nitric Oxide Synthase and Argininosuccinate Synthase to Maintain Vascular Endothelial Function
Authors: Pendleton Laura C , Thompson Ricci J , Corbin Karen D , Meininger Cynthia J , Eichler Duane C
Published: June 2021

The pathogenesis of diabetes includes impairment of vascular endothelial cell nitric oxide (NO) production. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate‐limiting step of the citrulline nitric oxide cycle. First, using serum starved, cultured vascular endothelial cells, we show that insulin coordinately up‐regulates both eNOS and AS expression, supporting NO production. Second, using a rat model, we show that diminished eNOS and AS expression in rats with streptozotocin‐induced diabetes is reversed by insulin treatment, delivered via slow release pellets. Impaired endothelium‐dependent vasorelaxation is also reversed with insulin treatment. Thus, insulin supports endothelial function not only through regulation of eNOS, but also through the coordinate regulation of AS. (Supported by the James and Esther King Biomedical Research Program, DOH Florida; NIH RO1 HL083153‐01A2).
Processing of various sizes of (CAG)n/(CTG)n hairpins in human nuclear extracts
Authors: Zhang Tianyi , Huang Jian , Gu Liya , Li Guo‐Min
Published: June 2021

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have shown that human cells possess a repair system that can remove DNA hairpins containing a sized of 20–25 CAG/CTG repeats. The repair system specifically targets the repeat tracts for incisions in the nicked strand in a nick‐directed and error‐free manner. To determine the substrate spectrum of the hairpin repair system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies slightly differ among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand, a pattern consistent with the repair of 25 repeat‐containing hairpins. Analysis of the repair intermediates allowed us to precisely determine the incision site for each substrate. These results suggest that the hairpin repair system in human cells prevents CAG/CTG stability by processing various sizes of CAG/CTG hairpin structures during DNA metabolism.
Evidence that nucleosomes inhibit mismatch repair in eukaryotic cells
Authors: Li Feng , Tian Lei , Gu Liya , Li GuoMin
Published: June 2021

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSalpha and a mismatch located within a nucleosome or between two nucleosomes. The results show that whereas MutSalpha specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSalpha displays reduced ATPase and ADP binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP‐induced MutSalpha sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells.
Influence of Werner helicase deficiency on nonhomologous end‐joining in human cells
Authors: Smith Krissy , Waldman Alan S.
Published: June 2021

Mutation of Werner Helicase (WRN) causes Werner Syndrome, a rare disorder associated with symptoms of premature aging and cancer predisposition. WRN has been implicated in DNA double‐strand break (DSB) repair via nonhomologous end‐joining (NHEJ). Plasmid‐based assays suggested that WRN deficiency results in NHEJ that produces large deletions. To our knowledge, the influence of WRN on NHEJ as it occurs in human chromosomes has not been investigated. We stably transfected WRN‐deficient human fibroblast cell line AG11395 with pTNeo99‐7 which contains a thymidine kinase (tk)‐neo fusion gene disrupted by the 18 bp recognition site for endonuclease I‐SceI. A DSB was introduced in the tk‐neo gene by expression of I‐SceI and repair events were recovered by selection for G418‐resistant clones. From AG11395‐derived cell lines, 95 G418‐resistant clones were analyzed and 45 were found to have arisen via simple NHEJ. 39 NHEJ events displayed deletions, while six NHEJ events displayed sequence insertions. Deletions ranged from 1 bp to 619 bp, with a median size of 7 bp. NHEJ in normal human fibroblasts displayed a median deletion size of 37 bp, indicating that WRN deficiency did not increase deletion size. However, nearly half of the DSB repair events recovered from WRN‐deficient cells showed evidence of gross chromosomal rearrangements, suggesting corruption of DSB metabolism. Supported by NIH grant R01GM081472 to ASW.
Role of Sororin in sister chromatid cohesion and separation in vertebrates
Authors: Zhang Nenggang , Panigrahi Anil K , Jiang Yunyun , Rao Pulivarthi H , Pati Debananda
Published: June 2021

Sororin, a cohesin associated protein, is conserved in vertebrates but has no apparent homolog in lower eukaryotes. It is expressed in S phase and degraded in early G1 phase. Co‐expression analysis indicates that Sororin gene, CdcA5, is co‐regulated with known cell cycle genes, such as Cdk1, Cyclin B, Bub1, Cdc20 etc. High‐throughput protein phosphorylation analysis indicates that Sororin is phosphorylated during CDK‐driven phase of cell cycle. Sororin interacts with cohesin complex and is required for sister chromatid cohesion. Here we report that Sororin mediates the maintenance and removal of sister chromatid cohesion. Co‐immunoprecipitation and in vitro binding assay show that Sororin interacts with cohesin complex via cohesin subunit SA1/2. Sororin also interacts with Plk1 via a PBD binding motif. Mutation on PBD binding motif of Sororin or PBD of Plk1 inhibits the interaction of Sororin and Plk1. Since phosphorylation of SA2 by Plk1 is required for the removal of sister chromatid cohesion on chromosomal arms, it is possible that Sororin acts as a docking protein to recruit Plk1 to phosphorylate SA2 and facilitates the dissolution of cohesin on chromosomal arms.

(Supported by National Cancer Institute grant 1 R01 CA109330)
Fluorescent Based Assay for Group I Ribozymes using an ABI 310 Genetic Analyzer
Authors: Stevens Timothy , Rohlman Christopher
Published: June 2021

Group I introns are catalytic RNAs capable of performing a range of phosphotransesterification reactions including self splicing and RNA cleavage. To better understand their nature, variants of the Tetrahymena, Anabaena, and Twort Group I ribozymes and fluorescently tagged substrates were synthesized. We are currently developing an assay to utilize the capillary electrophoresis (CE) and spectroscopy capabilities found in an ABI 310 Genetic Analyzer. The goal of this assay is to acquire kinetic data for reactions catalyzed by wild‐type and variant Group I introns to better understand their catalytic nature. The ABI 310 was designed to function with fluoroscein based tags and a similar tag was selected for the substrate during these assays. CE will separate the bound ribozyme, unbound ribozyme, and free substrate. When combined with the fluorescent spectroscopy capabilities of the ABI produced quantifiable multiple turnover kinetics data with picomolar levels of ribozyme. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.
An all RNA Hypercyclic Network
Authors: Vaidya Nilesh , Lehman Niles
Published: June 2021

A hypercycle is a cooperative organization of autocatalytic units that are associated in a cyclic linkage in such a way that there is a mutual survival and regulated growth of all the units involved. Compared to non‐coupled self‐replicating units, which can only sustain a limited amount of genetic information, the hypercycle allows the maintenance of large amounts of information through cooperation among otherwise competitive units. As such, this reaction network may have played a significant role in precellular evolution during the origins of life on the Earth. However, hypercycles have never been empirically demonstrated in the absence of cell‐like compartmentalization. In the current work, hypercyclic behavior is demonstrated in the autocatalytic assembly of Azoarcus group I ribozyme. Three different constructs of the Azoarcus ribozyme with different internal guide sequences (IGS) – GUG (canonical), GAG, and GCG – are capable of a minimal amount of self‐assembly when broken into two fragments. Here, self‐assembly depends on a mismatch with non‐complementary sequences, CGU, CAU and CUU, respectively, to be recognized by IGS via autocatalysis. Yet when all three constructs are present in the same reaction vessel, concomitant assembly of all three is enhanced through an interdependent hypercyclic reaction network. Analysis of these reactions indicates that each system is capable of guiding its own reproduction weakly, along with providing enhanced catalytic support for the reproduction of one other construct system through matched IGS‐tag interactions. Also, when co‐incubated with non‐interacting (i.e., selfish) yet efficient self‐assembly systems, the hypercyclic assembly outcompetes the selfish self‐assembly systems, demonstrating the ability of a hypercyclic organization to possess an evolutionary advantage.
Optimizing conditions for cleavage of pre‐tRNA‐Gln(CAA) by Haloferax volcanii tRNA intron endonuclease
Authors: Tellings Kelsey , Salva Corinne , Ginn‐Pease Margaret E. , Hemmingsen Jens M.
Published: June 2021

In the halophilic archaeon Haloferax volcanii, tRNA intron endonuclease is known to cleave precursor tRNAs at a specific bulge‐helix‐bulge (BHB) motif. The enzyme cleaves pre‐tRNA‐Trp into a 103 nucleotide intron and two exons that are subsequently spliced at position 37/38. To explore the substrate specificity of the enzyme, pre‐tRNA‐Gln(CCA) has been subjected to cleavage by tRNA intron endonuclease under various conditions. Pre‐tRNA‐Gln(CCA) contains a shorter intron of 31 nucleotides, but can still adopt a BHB motif. Optimal cleavage conditions appear to be 40 mM Tris‐HCl pH 7.4, 2% glycerol, 20 mM MgCl2 and 2 M KCl. The enzyme activity is not sensitive to moderate pH changes or MgCl2 concentration between 5 mM and 50 mM. Ionic strength is important with increasing activity observed at higher KCl concentrations from 200 mM to 2M KCl and no cleavage observed below 200 mM KCl. These studies will allow for additional exploration of substrate specificity including sequence variation in the RNA substrate. This work will lead to a better understanding of tRNA processing in archaea.
Murine Leukemia Virus Pseudoknot: Structural Analysis of Conformational Changes
Authors: Salguero Carolina , D'Souza Victoria , Durney Michael
Published: June 2021

The Murine Leukemia Virus’ (MLV) pseudoknot is an RNA motif that regulates readtrough translation, which is critical for packaging and maturation of new viral particles. The MLV pseudoknot undergoes a p.H dependent conformational change to a structure that regulates the readtrough mechanism of the retrovirus. However, at low pH NMR signals are too broad due to the fast exchange between the two conformers, and the structural analysis of the triple helix conformation is ambiguous. We postulate that design of a construct that trapped into a single folded conformation will help determine the structure of the triple helix conformation using NMR. In order to test our hypothesis, we analyzed 3 constructs. The first construct has a deleted GC base pair on stem 2 creating an artificial bend of stem. This construct cannot be tested by in vitro translational assays, so a double mutation –GC and replacement of Adenine 38 for Uracil (U38A‐GC) was also designed. In the last construct, Adenine 17 was replaced by a Uracil (A17U) eliminating the protonation of stem 2. Our results indicate that –GC base pair construct has lost pH dependence, and it is in slow exchange between two conformations; spectra of U38A‐GC construct exhibits characteristics of the native pseudoknot at low pH. Finally, spectra from construct A17U indicate that the triple helix is still pH dependant suggesting the need of multiple protonation events.
Fluxome analysis of C13‐pyruvate reveals differential metabolic responses between heart, liver and red blood cells following endotoxemia
Authors: Bateman Ryon M , Nagahata Yoshiko , Ohmura Mitsuyo , Hishiki Takako , Suematsu Makoto
Published: June 2021

The systemic inflammatory response results in a “hypermetabolic” state; yet, metabolic changes are not clearly understood. The objective was to determine glycolytic, TCA and PPP metabolites, amino acids and ATP levels in heart, liver and red blood cells. C57BL/6 mice (30‐35g) were injected intraperitoneally with lipopolysaccharide (LPS, 40mg/kg). Six hours post LPS, C13‐pyruvate (a key intermediate metabolite) was administered subcutaneously. At 20, 40 and 60 minutes, heart, liver and RBCs were collected. Labeled metabolites were measured using capillary electrophoresis ‐ mass spectrometry, quantified by calculating the AUC/t0‐60 and expressed relative to control. The RBC preferentially metabolized pyruvate (9‐fold increase) compared to heart (1.2‐fold increase) or liver (‐2.1‐fold decrease), and was a net lactate source (2.1‐fold increase). Glycolytic intermediates increased in the heart, but decreased in the RBC, while TCA intermediates decreased in the heart. The liver showed increased amino acids. ATP was stable in heart, but decreased in liver and RBC. Endotoxemia induces differential metabolic responses between tissues. Support: Global COE Program for Metabolomics Systems Biology, MEXT, Japan.
Molecular mechanism of RBP4 on the basis of insulin signaling pathway in primary hepatocytes
Authors: Namkung Jun , Kim Moon Young , Woo Mi Kyoung , Sohn Joon Hyung , An Sung Wan , Yeh Byung‐Il
Published: June 2021

Metabolic syndrome is a cluster of symptoms closely associated with diabetes, obesity, and cardiovascular disease such as hypertension. Thought the prevalence is being increased the mechanism is not known so far. In this study, we tried to confirm the molecular mechanism of RBP4 which is regarded one of the critical factors in the pathogenesis of metabolic syndrome.

Recombinant mouse RBP4 was treated to the primary hepatocytes prepared from the ICR mouse and cDNA microarray was performed. We focused insulin signaling pathway and mRNA expressions were measured real‐time PCR. The results are: 1) RBP4 has an undesirable effect for metabolic syndrome by increase of gluconeogenesis and decrease of glycolysis. 2) The increase of gluconeogenesis is due to decreased expression of Akt. 3) The effects of several factors which influences on PIP3 expression, which plays a role as controller at the upper level than PKCα and Akt in the insulin signaling pathway, are not constant by RBP4.

In summary, RBP4 increase gluconeogenesis mediated by Akt and decrease glycolysis in insulin signaling pathway. To get the clue to solve the metabolic syndrome the researches to confirm the phosphoryation of the factors and protein expression analysis are needed.
Effects of 2‐Chloroethyl ethyl sulfide(CEES) on Guinea Pig Brain Morphology
Authors: Gadsden‐Gray Jessica , Mukherjee Shyamali , Ogunkua Olugbemiga , Das Salil K.
Published: June 2021

Mustard gas is a chemical warfare agent used in World Wars I and II, the Gulf War, and on civilian occupants of the Iranian village of Halabja. Inhalation of sulfur mustard is known to cause hemorrhagic inflammation of the tracheobranchial tree in addition to severe pulmonary complications such as adult respiratory distress syndrome (ARDS). Studies of the mustard gas analog 2‐chloroethyl ethyl sulfide (CEES) using the guinea pig model system have indicated that its inhalation not only has the ability to cause significant lung damage but also to cross the blood‐brain barrier as evidenced by down‐regulation of the pro‐opiomelanocortin (POMC) gene. Down‐regulation of the POMC gene also suggests abnormal brain function. CEES is known to decrease antioxidant defense systems and increase lipid peroxidation in the brain, which may occur by microglial activation. Increasing microglial number and migration in the brain can cause inflammatory and NADPH‐derived stress. Activated microglia are also known to play an important role in the development of neurodegenerative diseases such as Parkinson's disease and amylotropic lateral sclerosis(ALS). The purpose of this study was to determine if intratracheal CEES infusion causes abnormality in brain morphology in guinea pigs. Our results show that intratracheal CEES exposure causes changes in brain morphology that result in dose dependent increases in blood cell aggregation and microglial cell number, microglial activation, and brain inflammation. These changes may be mediated by induction of oxidative stress.

This project was supported by grants from the Department of the Army (W81XWH‐06‐2‐0044) and NIH (5T32HL007735‐12).
Myo‐Inositol Synthesis in Drosophila melanogaster Effects Development
Authors: Batugedara Gayani Dinusha , Ho Thach‐Vu , Jackson Natasha A. , Ugarte Arjuna , Carter Ashley J.R. , Eldon Elizabeth D. , Klig Lisa S.
Published: June 2021

Myo‐inositol is an essential metabolite that plays an important role in development, osmoregulation, and disease states such as diabetes, bipolar disorder and spinal chord defects. Inositol is synthesized from glucose‐6‐phosphate by myo‐inositol‐1‐phosphate synthase (MIPS). More than 75% of genes involved in human diseases have orthologs in the model system Drosophila melanogaster. The MIPS gene of D. melanogaster encodes a protein 62.2 kDa (pI=6.17) and is 58.5% identical (72.3% similar) to the human MIPS protein. In silico experiments have revealed regulatory sequences responsive to inositol, osmotic and/or oxidative stress. MIPS RNA has been shown to be highly expressed in the fly head, hindgut, ovary and testis (FlyAtlas, 2007). Preliminary examination of a MIPS mutant D. melanogaster strain revealed reduced fecundity in crosses with homozygous mutant males. Further analysis suggested that reduced fecundity is due to defective sperm. Inositol metabolism was also examined by growing flies on defined media with sucrose or inositol as the energy source. Inositol‐grown flies demonstrated a reduction in eggs laid but progress normally through development. Proteomic analyses revealed many differences between crude lysates of flies grown on sucrose versus inositol.
Regulation of de novo thymidylate biosynthesis by ubiquitination
Authors: Eom Jae Yong , Anderson Donald D , Stover Patrick J
Published: June 2021

Folate‐mediated one‐carbon metabolism is required for the biosyntheses of purines, de novo thymidylate (dTMP), and S‐adenosyl methionine. Deficiencies in folate and SNPs in folate‐utilizing enzymes are associated with developmental anomalies and diseases such as neural tube defects, cardiovascular disease, and cancer. The mechanisms underlying these, however, still remain unestablished. Our lab has shown that Serine hydroxymethyltransferase 1 (SHMT1) regulates the flux of one‐carbon units towards de novo dTMP biosynthesis at the expense of S‐adenosyl methionine biosynthesis. The de novo dTMP biosynthetic pathway, which consists of SHMT1, SHMT2α, TYMS, and DHFR, is localized to the nucleus during S‐phase in a SUMO‐dependent manner. Here we show in SHMT1 SUMO‐site mutants, SHMT1 stability is increased and ubiquitination is inhibited. Furthermore, polyubiquitination of SHMT1 is absent in cytoplasmic fractions, but is greatly increased in nuclear fractions at G2/M‐phase, suggesting the degradation of SHMT1 occurs within the nucleus. These results suggest an antagonistic relationship between SHMT1 SUMOylation and ubiquitination, in which SUMOylation facilitates its nuclear import for de novo dTMP biosynthesis, while ubiquitination leads to degradation within the nucleus to turn off dTMP biosynthesis as cells progress into G1 phase.
Robotic high throughput fatty acid analysis of umbilical cord serum
Authors: Lin Yuhong , Hibbeln Joseph R , Zhou Weiyin , Loewke James , Brown James A , Lands William EM , Wells Ellen M , Goldman Lynn R , Salem Norman
Published: June 2021

Several epidemiology studies show that inadequate dietary intakes of highly unsaturated omega‐3 fatty acids are adversely related to child IQ and aggressive behaviors. As a long term goal, our laboratory is developing several techniques to facilitate large scale quantification of plasma or serum fatty acids. These include the automation of chemical procedures for transmethylation and extraction using a robotic system, analytical analysis using fast gas‐liquid chromatography and automated peak identification and calculation of fatty acid content. The system has been validated as follows: (1) Fatty acid recovery was 97% on average at 3 different concentrations. (2) The CV for the 24 fatty acids over 47 replicates was 3.26±1.2% for concentrations and 2.44±1.3% for fatty acid percentages. A Microsoft Visual Basic program was written to automate data calculation and peak identification. The complete procedure for robotic transmethylation of up to 72 samples currently requires about 6 hours. A total of 300 umbilical cord serum samples were assayed for fatty acids profiles using this system as an initial application of the high throughput methodology.

This project was partially funded by the DICBR, NIAAA, NIH.
Exploring Signaling Events Surrounding Extracellular Amastigote Invasion Processes Of Trypanosoma Cruzi
Authors: Ferreira Eden Ramalho , Silva Erika Alves , Costa Renata Watanabe , Cruz Mario Costa , Fernandes Maria Cecília , Mortara Renato Arruda , Bahia Diana
Published: June 2021

Trypanosoma cruzi is a protozoan pathogen that infects humans and other mammals, producing a pathology called Chagas disease. The disease is endemic in most of central and South America affecting 18 million people, with an increasing number of cases in North America. Amastigotes which are generated by the extracellular differentiation of trypomastigotes are referred to as Extracellular Amastigotes (EA) and are able to invade cultured cells. EA is dependent on host actin filaments polymerization to invade cells. Cortactin has emerged as a key signaling protein in cellular processes such as endocytosis and tumor invasion. The ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Cortactin showed to be a substrate of PKD phosphorylation in vivo. In this study, cells were transfected with cortactin and PKD‐GFP‐vectors, infected with EA and examined for the acquisition of these markers. Cells were previously treated with kinase (Fyn‐Src, PI3K) inhibitors and control cells were left untreated. PKD is recruited to the sites of actin remodeling during EA invasion, which also recruits cortactin. Co‐localization of PKD and cortactin may be an indication that PKD plays a role in cytoskeletal reorganization. The inhibition of PI3K pathway enhances both EA invasion and LAMP‐1 recruitment. Here, we propose a signaling pathway model of EA entry.
Metadoxine, an ion‐pair of pyridoxine and L‐2‐pyrrolidone‐5‐carboxylate, inhibits adipocyte differentiation through its repression of PKA‐dependent CREB activity
Authors: Yang Yoon Mee , Kim Hyun Eun , Ki Sung Hwan , Kim Sang Geon
Published: June 2021

Obesity has become a widespread issue in modern society. It is considered as a hypertrophic disease resulting from an increase in adipogenesis (e.g., increase in fat cell volume and size). Adipogenesis is orchestrated by the expression of master adipogenic regulators, such as protein kinase A (PKA), cAMP response element binding protein (CREB) and CCAAT/enhaner‐binding protein beta (C/EBP beta). Metadoxine, an ion‐pair of pyridoxine and L‐2‐pyrrolidone‐5‐carboxylate, increases the clearance of alcohol and acetaldehyde, reduced the damaging effect of free radicals. This study investigated the effects of metadoxine on the adipogenesis of 3T3‐L1 preadipocytes and its cellular mechanism. Metadoxine has an inhibitory effect during the late stage of hormone‐induced adipogenic differentiation. However, metadoxine did not inhibit mitotic clonal expansion at the early proliferation phase. PKA catalyzes phophorylation of CREB at serine 133 and thereby activates transcription of target genes, resulting in adipogenic maturation. Metadoxine decreased the activity of PKA as well as the level of CREB phosphorylations and binding to the cAMP response element. Thereby, metadoxine repressed C/EBP beta during hormone‐induced adipogenesis. Overall, metadoxine inhibits adipogenic differentiation via the inhibition of CREB/cAMP response element‐dependent C/EBP beta induction in the PKA‐CREB pathway.