bluuebunny/crossref_metadata_2025 huggingface dataset:
A Functional Role for Glutamine 183 in the Folate Half‐Reaction of Methylenetetrahydrofolate Reductase from E. coli
Authors: Trimmer Elizabeth Eloise , Satzer David I. , Jolly Amber L. , Cao Sirui , Nikolova Andriana P.
Published: June 2021

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes a ping‐pong reaction with NADH and 5,10‐methylenetetrahydrofolate (CH2‐H4folate) as substrates. In humans, mutations in MTHFR have been correlated with elevated levels of homocysteine, a risk factor for cardiovascular disease, and with neural‐tube defects in the fetus. We study MTHFR from Escherichia coli as a model for the catalytic domain of the human enzyme. This work focuses on Glutamine 183, an invariant, active site amino acid. X‐ray crystal structures [Biochemistry (2005) 44(34), 11447–11457] indicate that Gln 183 makes key hydrogen‐bonding interactions with both substrates. We have created the variants Gln183Glu and Gln183Ala and have begun characterization of the enzymes by steady‐state and stopped‐flow kinetics and by measurement of the flavin redox potentials. Our data suggest a major role for Gln 183 in folate binding and catalysis. Compared to the wild‐type enzyme, the Gln183Ala variant binds CH2‐H4folate with 20‐fold decreased affinity and catalyzes the folate half‐reaction at a 12‐fold decreased rate. The Gln183Glu mutant appears less impaired in the folate half‐reaction than the Gln183Ala variant. NADH binding and catalysis have not been altered by either mutation. Research supported by ACS‐PRF grant #39599‐GB4, HHMI grants #71100‐503702 and #52006298 to Grinnell College.
Characterization of the Metal Binding in NADPH Oxidase 5 by Fluorescence, Isothermal Titration Calorimetry, and Circular Dichroism
Authors: Palka Christina Diana , Reynolds Nicole , Tatro Allison , Wei Chin-Chuan
Published: June 2021

Superoxide generated by non‐phagocytic NADPH oxidases (NOXs) plays roles in disease development and cancer. The activity of NOX5 appears to be regulated by its self‐contained calcium binding domain (CaBD). To study its calcium binding properties, we isolated the N‐ and C‐terminal halves of CaBD (N‐ / C‐CaBD), and separately studied their metal binding. Isothermal titration calorimetry reveals a positive cooperation for both halves, with differences in enthalpy values ranging from −30 to −40 kJ/mol. Using mutants whose Glu residue (−z) in each EF‐hand was replaced with Gln, we determined the semi‐microscopic binding constants of Ca2+ for the 1st and 2nd EF‐hands; the result indicates a weak Ca2+ binding to the 1st EF‐hand is enhanced by the binding of Ca2+ to its 2nd EF‐hand. No Mg2+ binding was seen in N‐CaBD. The binding of Ca2+ and Mg2+ to C‐CaBD are exothermic, and Ca2+ binding is only 2–3 fold tighter than Mg2+. Our findings suggest that in the resting cells, the 3rd and 4th EF‐hands are Mg2+‐bound; increasing Ca2+ levels affects the EF‐hands of N‐terminal half. Our results imply that Ca2+ binding to the 1st and 2nd EF‐hands is more critical in controlling NOX5 activity. To investigate structural dependency between half domains that affects metal binding, we created CaBD mutants, such as CaBD(E31Q/E64Q) and CaBD(E99Q/E143Q), and determined their metal binding affinities. This work is supported in part by NSF‐CCLI grant.
Regulation of PASK function by mTOR signaling pathway
Authors: Kikani Chintan K , Rutter Jared P
Published: June 2021

PAS domain containing serine/threonine kinase (PASK) is an evolutionary conserved protein kinase implicated in insulin resistance and type II diabetes. Specifically, we have shown a role for role for PASK in regulation hepatic lipogenesis pathways and insulin resistance using PASK knockout mice. It, however, remains unknown how PASK is regulated in cells and its downstream effectors. Here, we demonstrate that PASK is a nutrient responsive kinase. Serum and glucose stimulated the activity of PASK which was significantly suppressed by either rapamycin treatment or mTOR silencing by RNAi. In addition, the in vivo phosphorylation of PASK increases in response to serum and insulin treatment in an mTOR catalytic function dependent manner. Mutation of the phosphorylation sites to alanine suppresses serum stimulated phosphorylation and activation of PASK. Finally, we show that PASK physically associated with mTOR complex 1 in serum dependent manner, suggesting a direct role for mTOR complex 1 in regulating PASK function. Taken together, our findings provide the first evidence for the role of mTOR in regulating PASK activity and function in response to nutritional cues.
Protein Stability of Wild Type and Mutant forms of the RET Tyrosine Kinase
Authors: Vega Quinn , Maslovski Shani
Published: June 2021

The ret proto‐oncogene encodes a trans‐membrane receptor tyrosine kinase that plays a key role in neural differentiation as well as development of the enteric nervous system and kidneys. Mutations of the RET gene have been associated with several cancers such as MEN2A, MEN2B and FMTC; however, the down‐regulation mechanism is largely unknown, especially between these various forms of RET. In order to analyze the effect of the mutated form of RET on signal transduction and down‐regulation, VERO cells were transfected with wild type and activated forms of RET and the effect of RET mutations on protein degradation analyzed. The degradation studies show that MEN2B has a lower level of protein expression and protein degradation, compared to wild type RET. The decrease in RET protein levels inversely correlates with levels of the ubiquitin pathway enzyme, Cbl. The connection between decreased MEN2B down regulation and the loss of Cbl provides a possible explanation for the mutant receptor's oncogenic potential.
Quercertin as a Possible Inhibitor of Glycation in Diabetes
Authors: Maynard Danille , DeBauche Callie , Wing Barbara
Published: June 2021

Previous work has indicated Quercertin inhibits the glycation of proteins in an in vitro system designed to closely simulate interactions between proteins and sugars in diabetic patients. The addition of sugars to proteins and other biological molecules is a major contributor to the development of the complications of diabetes

This experiment compared the effects of the addition of concentrations of 150 μg/ml, 200 μg/ml, and 3000 μg/ml of Quercetin to mixtures of 400μg/ml of albumin and 0.5M glucose Testing was done using spectrophotometry to measure glycoproteins at 530 nm. The hypothesis is that there would be lower absorption levels of the solutions containing Quercetin than those of the albumin/glucose solution. Our results show that high levels of Quercetin, such as the 300 μg/ml concentration does not show inhibition; while the 150 μg/ml, and 200 μg/ml concentrations are variable; indicating that under certain conditions the Quercetin may have some inhibition properties, but the inconsistencie suggest the need for further investigation.

This Project is supported by the Smith Glynn Callaway Medical Foundation.
Circulating Adipokine Profile in Type 2 Diabetes Mellitus in Lagos, Nigeria
Authors: Ebuehi Albert , Oshodi Tope , Udenze Ifeoma , Ebuehi Olufunke
Published: June 2021

Adipokines refer to biologically active substances found in the adipocytes of white adipose tissue, namely, adiponectin, resistin and leptin. The circulating adipokine levels in Type 2 Diabetes mellitus and non‐Diabetes mellitus patients were determined at the Lagos University Teaching Hospital, Lagos, Nigeria. 53 diabetic and 27 non diabetic patients were recruited into the study. Fasting blood glucose (FBS), glycated haemoglobin (Hb A1C), leptin and resistin were assayed. The body mass index (BMI) was also measured. Mean diabetic BMI was not significant (P>0.05) as compared to non‐dibetics ( Diabetics 28.77+ 5.35, non‐diabetics 27.38 + 6.04. Resisitin levels were significantly higher in diabetics, 31.25 + 2.58 compared to the non‐diabetics, 16.61 + 2.16. Leptin level correlated strongly with BMI ( r = 0.620, p < 0.0001 ) and was significantly higher in the female diabetics than in males. The resistin level did not correlate with FBS and Hb A1C levels in the diabetics, while the leptin level strongly correlated with BMI in the female diabetics. Data suggest that adipokine levels may vary with gender and provides relevant information in the management of Type 2 diabetes mellitus.
TGF‐beta activates Akt kinase and induces glomerular mesangial hypertrophy related to diabetic nephropathy through FOG2 inhibition by microRNA‐200b/c
Authors: Park Jung Tak , Kato Mitsuo , Lanting Linda , Putta Sumanth , Castro Nancy , Natarajan Rama
Published: June 2021

Glomerular hypertrophy is a hallmark of diabetic nephropathy (DN). Akt kinase activated by transforming growth factor‐beta1 (TGF‐b) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF‐b are not fully understood. Recently, miR‐200 and its target FOG2 were reported to regulate PI3‐Kinase (the upstream activator of Akt) activity in insulin signaling. Here we show that TGF‐b activates Akt in glomerular mesangial cells (MC) by inducing miR‐200b/c, both of which target FOG2, an inhibitor of Akt activation. FOG2 expression was reduced in diabetic mice glomeruli as well as TGF‐b treated mouse MC (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased protein content/cell ratio suggesting hypertrophy. miR‐200b/c levels increased significantly in diabetic mice glomeruli and TGF‐b stimulated MMC. Transfection of MMC with miR‐200b/c mimics decreased the expression of FOG2. Conversely, TGF‐b induced decrease in FOG2 expression was attenuated by miR‐ 200b/c transfection into MMC. Furthermore, miR‐200b/c mimics increased the protein content/cell ratio, while miR‐200b/c inhibitors abrogated the TGF‐b induced increase in protein content/cell. These data suggest a new mechanism of Akt activation by TGF‐b through FOG2 down regulation by miR‐ 200b/c which can lead to MC hypertrophy in the progression of DN.

(supported by NIH DK08170 and NIH DK058191)
Endothelin‐1 (ET‐1)‐induced Early Growth Response Factor‐1 Expression in Vascular Smooth Muscle Cells (VSMC) requires c‐SRC and ERK1/2 activation
Authors: Youreva Viktoria , Vardatsikos George , Srivastava Ashok K.
Published: June 2021

Egr‐1 transcription factor plays an important role in vascular biology. Following activation, Egr‐1 is expressed in the nucleus and can regulate transcription of several genes implicated in the development of vascular disease (VD). A potential role of aberrant ET‐1 signaling is thought to contribute to the development of VD, such as atherosclerosis, through the hyperactivation of growth promoting pathways, including PI3K/PKB and MAPK pathways, as well as regulation of multiple transcription factors. We have previously shown that several protein tyrosine kinases are upstream regulators of ET‐1‐induced activation of PKB in VSMC, however, a role of ET‐1 on Egr‐1 expression and its modulation by signaling components activated by ET‐1 remains unclear. Therefore, we have investigated the involvement of IGF‐1R, c‐Src and MAPKs in ET‐1‐induced Egr‐1 expression. ET‐1 time‐dependently enhanced Egr‐1 protein levels in VSMC. AG1024 and PP2, selective pharmacological inhibitors of IGF‐1R and c‐Src, respectively, inhibited ET‐1‐induced Egr‐1 expression. PD98059, a selective inhibitor of MEK and ERK1/2, also inhibited ET‐1‐ induced Egr‐1 expression in VSMC, however, SB202190, a highly selective inhibitor of p38mapk, failed to do so. In summary, these data demonstrate that ET‐1 potently induces the expression of Egr‐ 1 through a pathway implicating IGF‐1R and c‐Src, as well as ERK1/2 in VSMC. (Supported by CIHR grants)
Testing the HYPE: Studies of the mammalian Fic protein
Authors: Woolery Andrew Ryan , Hao Yi-Heng , Luong Phi , Orth Kim
Published: June 2021

Objective

To characterize the mammalian Fic protein HYPE in terms of its subcellular localization, tissue specific expression, molecular function and potential substrates

Methods

Click chemistry, qRT‐PCR, stable cell lines, protein expression and purification

Results

Using a variety of tools developed in our lab, we have partially characterized the subcellular localization, glycosylation state and tissue specific expression of the mammalian Fic protein termed HYPE. Several forms of the human and mouse HYPE protein have been successfully expressed and purified for use in a variety of assays to determine its potential substrates, and stable cell lines containing HYPE shRNA knockdown and HYPE mutants are being generated in a variety of cell lines to attempt to determine its cellular function and substrates.

Conclusion

The significance of the Fic domain activity of mammalian HYPE is not currently known, but our current knowledge, including its subcellular localization and tissues of relevance, combined with our ongoing research with tools like click chemistry, recombinant HYPE protein and stable mammalian cells lines should allow us to elucidate the substrates and functions of this enigmatic protein.
Reproductive effects of SirT1 expression in astrocytes
Authors: Choi Irene Sujeen , Rickert Emily , Olefsky Jerry , Webster Nicholas
Published: June 2021

SirT1 modulates gene‐expression in several organs, including the liver, pancreas, adipose tissue, intestine, and brain. Whereas the impact of SirT1 in these tissues has been shown, its effect on reproduction is still elusive. It has been shown that during times of caloric restriction, SirT1 levels increase and reproductive events such as GnRH pulses, LH pulses, and ovulation cease. We hypothesize that the increase of SirT1 levels in the hypothalamus are the cause of decreased reproductive function. To evaluate this, our laboratory has developed conditional overexpression or knockdown of SirT1 using a tamoxifen‐inducible cre recombinase under the control of the GFAP promoter, which expresses in astrocytes. To prevent any effects on mouse reproductive development, tamoxifen treatment will not be started until 12 weeks of age. We will present data on the reproductive changes by monitoring estrous cyclicity, endocrine and steroid hormone levels, as well as examine fertility of these mice. This research is supported by NIH grants U54HD12303 and T32 DK007494.
Impaired cognitive function is associated with changes in serotonin receptor activity following prenatal exposure to Dexamethasone
Authors: Page Kathleen C , Shah Darshan S , Aloyo Vincent J
Published: June 2021

We investigated the effects of prenatal Dexamethasone (Dex) exposure on 5HT1A receptor activity and cognitive function in adult rat offspring. Pregnant rat dams were injected with Dex (sc; 150ug/kg/day) from gestation day 14 through 19. Control dams were treated with saline. Morris Water Maze analysis showed that at both 120 days (Adult) and 365 days (Aged) the Dex‐exposed group exhibited impairments in learning and memory. Control and Dex‐exposed adults were also challenged with an acute stressor (Adult‐Stressed). A second adult group were similarly stressed and allowed to return to baseline (Adult‐Basal). A third group of 365 day‐old control and Dex‐exposed animals were subjected to the same stress paradigm (Aged‐Stressed). Radioligand receptor binding data showed that Kd and Bmax were statistically different in Dex‐exposed hippocampi from basal and stressed adults compared to controls. This difference was not seen in the aged group. Under basal conditions, corticosterone (CORT) was significantly higher in the Dex‐exposed adults. Following stress, CORT levels were significantly lower in Dex‐exposed animals. Leptin, a hormone shown to influence cognitive function, was significantly decreased in Dex‐exposed adult males, but leptin increased in aged animals. Prenatal exposure to Dex is associated with changes in hormonal output and serotonin 5HT1A receptor activity which may impair cognitive functions.
Structural biology of MAPK (p38/ERK) regulation by phosphatases and scaffolding proteins
Authors: Page Rebecca , Peti Wolfgang
Published: June 2021

~70% of all eukaryotic cellular proteins are regulated by phosphorylation. While structural data for kinases and phosphatases are readily available, only a handful of protein:protein complex structures, which are necessary to understand the biological function of the underlying regulation, have been described. Here we report our efforts to close this gap by describing our novel, structural insights into the regulation of MAPKs (p38, ERK2) by the kinase interaction motif family KIM‐PTPs (HePTP, STEP, PTP‐SL). In order to obtain a molecular picture of these large MAPK protein:protein complexes, we are integrating high resolution techniques (NMR/X‐ray) for the analysis of basic components with lower resolution techniques (SAXS) for constraints on the higher order, dynamic, multi‐protein assemblies. This has allowed us to determine the first detailed structures of MAPK:MAPK regulatory protein complexes, which, in combination with a thorough ITC study, has enabled us to identify the molecular determinants that drive MAPK specificity and fidelity. Finally, we are also providing new structural insights into the MAPK spatial control by elucidating the structures of MAPK scaffolding complexes.

Supported by RSG‐08‐067‐01‐LIB from the American Cancer Society.
The endocrine disruptors cadmium chloride and sodium arsenate induce human lung adenocarcinoma cell proliferation by activating the estrogen receptor‐mediated signaling pathway
Authors: Murphy Robert D. , Smith Aaron L. , Todd Sarah L. , Elpers Julia T. , Radde Brandie N. , Klinge Carolyn M. , Huff Mary O.
Published: June 2021

Estrogens have been implemented in non‐small cell lung cancer (NSCLC) risk, but the contribution and possible mechanisms of the endocrine disruptors cadmium chloride and sodium arsenate have not been delineated. Previous studies indicate that 17β‐estradiol (E2) induces lung cancer cell proliferation through activation of EGFR, Src‐kinase, MEK1 and ERK1/2. The purpose of this study is to determine if cadmium chloride and sodium arsenate stimulate NSCLC growth using a similar mechanism. Treatment of human lung adenocarcinoma NCI‐H1793 cells with nanomolar concentrations of these endocrine disruptors stimulate proliferation in a manner similar to E2, and proliferation is reduced when cells were first treated with the estrogen receptor (ER) antagonist ICI 182,780. Further, cadmium chloride and sodium arsenate, like E2, stimulate phosphorylation of ERK1/2 (pERK1/2) within 15 min of treatment, and pERK1/2 is reduced by ICI 182,780 suggesting that activation is mediated through ER. Studies using the MEK1 antagonist PD98059, the Src‐kinase antagonist PP2 and the EGFR antagonist Tyrphostin‐AG1478 indicate that MEK1, Src‐kinase and EGFR activation is required for the cadmium chloride‐ and sodium arsenate‐ induced increase in pERK1/2. These results support the involvement of membrane‐ ER signaling in mediating cellular responses to cadmium chloride and sodium arsenate in human lung adenocarcinoma cells.
A Simple Arg to Lys Mutant of Protein Phosphatase 1 Exhibits Catalytic Efficiencies toward Monoanionic Substrates Superior to Wild Type
Authors: Chu Yuan , Hengge Alvan C. , Williams Nicholas H.
Published: June 2021

The promiscuous phosphatase PP1 is a member of the phosphoprotein phosphatase (PPP) gene family, which is critical for the control of many cellular pathways by antagonizing the effects of protein phosphorylation mediated by kinases. The catalytic promiscuity of PP1 WT and two mutants has been investigated with a set of monoanionic and dianionic phosphoester substrates. The PP1 WT is an efficient catalyst for all substrates, with rate enhancements from 1011 to 1013. The catalytic efficiencies and substrate preferences were evaluated for two active site arginine mutants, R96K and R221K. Both the R96K and R221K mutations have minor effects on KM but major effects on kcat. The R96K mutant is more impaired than the R221K mutant for all substrate classes. For the monoanionic substrates the R221K mutant exhibits a kcat that is about 2–3 fold faster than WT, leading to better catalytic efficiency. This finding lends support to the notion that simple, conservative mutations can result in an altered enzyme with different catalytic capabilities and preferences. (Funding from NIH GM47297)
Identification of Methylation‐Dependent Protein Phosphatase 2A Activity Correlated with Changes in Insulin Secretion and Glucose Tolerance in Mice with Hypomorphic Expression of the LCMT1 Protein Carboxyl Methyltransferase
Authors: MacKay Kennen Burke , Clarke Steven , Young Stephen
Published: June 2021

Protein Phosphatase 2 A (PP2A) is a highly conserved, broad specificity, heterotrimeric serine/threonine phosphatase involved in nearly all cytosolic processes. PP2A control is manifested through subunit composition, small molecule and peptide inhibitors as well as post‐translational modifications. One such modification, methylation of the C‐terminal leucine by LCMT1, regulates PP2A subunit assembly, specifically Bα subunit binding, and thus substrate specificity. In this study we generate a mouse homozygous for an Lcmt1 gene‐trap cassette, yet expressing reduced Lcmt1 transcript and protein. Full length Lcmt1 transcript is achieved through alternative splicing around the genetrap cassette in a tissue specific manner. Reduced LCMT1 correlates with a concomitant reduction of PP2A methylation and an increase in PP2A demethylation confirming LCMT1 as the main PP2A methyltransferase. Lcmt1 homozygous gene‐trapped mice were born in a non‐mendelian ratio suggesting embryonic lethality. Adult Lcmt1 hypomorphic animals display decreased glucose tolerance and increased glucose‐stimulated insulin secretion suggesting an insulin resistance phenotype. Our results indicate that PP2A methylation has a role in propagation of the insulin‐signaling cascade, possibly through altered subunit composition of PP2A.

This work was supported by the National Institutes of Health (GM026020)
The Box C/D sRNP dimeric architecture is conserved across Kingdom Archaea
Authors: Baserga Susan J. , Phipps Kathleen R. , Taylor David W. , Wang Hongwei
Published: June 2021

Site‐directed 2′‐O‐methylation of RNA is catalyzed, in eukaryotes and archaea, by box C/D small (nucleolar) ribonucleoproteins (box C/D s(no) RNPs). Recent structural studies have led to a controversy over whether box C/D sRNPs are monomeric or dimeric macromolecules. Here we examine the role of the omnipresent sRNA loop as a structural determinant of multimeric sRNP assembly. Mutational analysis of the sRNA shows that the conserved loop between boxes C′ and D′ is required for dimeric sRNP formation, and explains how mutations in sRNAs can effect oligomeric architecture. In this study, we present negative‐stain electron microscopy single particle reconstructions of catalytically active dimeric box C/D sRNPs from three distantly related archaeal species, Solfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus, Our results demonstrate that the dimeric architecture of box C/D sRNPs is broadly conserved across the Archaeal Kingdom.
Modeling receptor‐mediated uptake of polymer‐functionalized iron oxide nanoparticles by macrophages
Authors: Lunov Oleg , Zablotskii Vitalii , Syrovets Tatiana , Roecker Carlheinz , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas
Published: June 2021

Although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. Here we have analyzed the uptake of carboxydextran‐coated superparamagnetic iron oxide particles by human macrophages. In vitro knock‐down and pharmacological approaches revealed that the main uptake mechanism proceeds via clathrin‐dependent, scavenger receptor‐mediated endocytosis. We created a mathematical model of the uptake process that permits determination of key parameters of endocytosis, such as the uptake rate, the mean uptake time, the number of particles per cell, and the correlation between the number of internalized particles and the extracellular concentration. The calculated parameters correlate well with experimental data obtained by spinning disc confocal microscopy. The model also provides information on the individual and collective wrapping time of the nanoparticles and describes the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity and the uptake time. Finally, we also gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that governs the collective uptake process. This model provides novel insight into the biophysical processes involved in endocytosis of nanoparticles.

Supported by the DFG, SPP1313.
High throughput transcriptomic analysis of the effects of radiation exposure in a mouse model
Authors: Miller Stacy-Ann , Shupp Jeffrey W , Moffatt Lauren T , Rosenthal Dean S , Nam Jason , Hammamieh Rasha , Jett Marti
Published: June 2021

Early detection of radiation exposure is important in the treatment of radiation injury and appropriate triage of patients. Detecting the cellular response to DNA double‐strand breaks (DSBs) has been thought to be a valid form of biodosimetry. A target biomarker for this is the rapid phosphorylation of H2AX, which is the minor histone H2A variant at serine residue‐139 to produce γ‐H2AX. Although the use of immunofluorescence‐based assays to visualize discrete γ‐H2AX foci has resulted in a sensitive and effective method for detecting DSBs, more sensitive and easy to analyze biomarkers of radiation exposure are desired. Here, a series of C57Bl6 mice were exposed to varying doses of X‐ray radiation and skin biopsies were harvested over a 4‐week time course. Subsequent to nucleic acid isolation, cDNA microarrays were used to study differential gene expression. We found that genes involved in response to stress and cell adhesion are decreased, while genes involved in mitosis, coagulation, lipoprotein metabolism, and protease inhibitor activities were increased following exposure. Further work will be directed at understanding the dose‐response nature of these signature gene responses.
Identification of Proteins O‐GlcNAc Modified During Osteoblastogenesis
Authors: Ball Lauren E , Schilling Michael , Waller Lashanda N , Nagel Alexis
Published: June 2021

O‐GlcNAcylation of key transcription factors critical to adipogenesis and osteoblastogenesis alters the rate of differentiation. To assess the extent of O‐GlcNAcylation during osteoblast differentiation, global protein O‐GlcNAc modification was measured in murine MC3T3‐E1 and primary human osteoblasts (NHOst) 0, 1, 3, 7, 14, and 21 days after induction in osteogenic media. The extent of global protein O‐GlcNAc modification peaked between days 3–7. To identify these proteins, O‐GlcNAcylated peptides were enriched from trypsin digested MC3T3‐E1 protein lysate at day 7 by WGA lectin weak affinity chromatography. Peptides were analyzed by C18 nLC‐MS/MS with CID and ETD fragmentation (LTQ‐XL). ETD MS/MS spectra were searched with Protein Prospector 2 and positive hits were manually confirmed. To date, 23 O‐GlcNAcylated peptides were identified primarily from proteins known to regulate transcription. Ten novel sites of O‐GlcNAc modification and six sites of O‐GlcNAc modification at known phosphorylation sites were identified. New sites of O‐GlcNAcylation were identified in Tab‐1 and Tab‐2, signaling proteins hypothesized to contribute to cell fate determination. Current studies are measuring changes in OGT and OGA expression during osteoblastogenesis and further characterizing the role of O‐GlcNAc modification of proteins important to osteoblastogenesis. Supported by RO1DE020925 and T32DE017551.
Electron Microscopy Reveals Variance in the Assembly of Mutant Qβ Virus‐Like Particles
Authors: Campbell Melody G. , Fiedler Jason D. , Higginson Cody , Kislukhin Alexander , Castillejos Alexandra , Manzenreider Florian , Potter Clinton S. , Carragher Bridget , Finn M. G.
Published: June 2021

Cryo‐electron microscopy (Cryo‐EM) allows for the imaging of biological samples in near native states. Here, we examine the assemblage of coat proteins from Qβ, a virus‐like particle (VLP), which, due to its stability in varying conditions, has been exploited to encapsulate various cargo materials. While within the VLP, proteins can retain properties including fluorescence, luminescence, and enzymatic activity. By introduction of a single coat protein point mutation (L35W) we found that the particles, now deemed “Mini‐Qβ,” were significantly smaller than wild type. Initial analysis though electron microscopy revealed a population with high heterogeneity, providing a unique challenge for VLP classification by conventional methods. Through template‐based particle selection and iterative rounds of two‐dimensional alignment and classification, three distinct particle types, all deviating from the 28 nm in diameter wild type icosahedral particle, emerged. These particles, which are present in unequal percentages in the sample, vary both in symmetry and in size, ranging from 16.5 to 24 nm. These slightly less stable Mini‐Qβ particles exemplify the sensitivity of the assembly process and provide a new vector for use as a therapeutic and building block for materials science.
LL‐37/CpG oligonucleotide complex formation and its effects on prostate cancer progression
Authors: Gupta Angela Satya , Craig Maria Lee , Deeble Paul D
Published: June 2021

LL‐37 is an antimicrobial DNA‐binding peptide that has been implicated in anti‐self DNA and anti‐tumor immune responses. The interaction between LL‐37 and DNA is not well‐characterized. We have used electrophoretic mobility shift assays (EMSA) and western blotting to investigate the interaction between LL‐37 and unmethylated CpG sequences known to activate Toll‐like receptor 9 (TLR9) pathways. TLR9 activation by CpG promotes weak anti‐tumor TH1 immune responses that are enhanced by LL‐37. In addition, we have tested the effects of LL‐37/CpG complexes in a prostate cancer progression model. By EMSA, we observe that LL‐37 is aggregated (has low gel mobility) in the absence of DNA, but that LL‐37/DNA association occurs with increased LL‐37 mobility, suggesting that CpG binding disrupts LL‐37 aggregation. Increases in LL‐37 concentration lead to reduced mobility, indicative of further complexation. We previously found that CpG/LL‐37 increases prostate cancer cell growth (PC‐3 cell line) and invasion through a matrigel matrix (LNCaP cell line). Currently, we are testing the efficacy of CpG/LL‐37 treatments on migration and anchorage‐independent growth in a prostate cancer progression model (PC‐3 cell line) using a cell scratch wound assay and soft agar assay. This work was supported by NIH EARDA grant 5G11HD037064‐08 and a Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowship.
Discovery and purification of AaLpxE, a bi‐functional lipid phosphatase from Aquifex aeolicus
Authors: Gillespie Robert A , York John D , Raetz Christian R H
Published: June 2021

In Gram‐negative bacteria, highly immunogenic lipopolysaccharide is anchored to the outer membrane by the bisphosphorylated saccharolipid lipid A. In some bacterial species, the phosphate at the 1‐position is removed by LpxE, a dedicated, integral membrane lipid A 1‐phosphatase. Removal of the 1‐phosphate from lipid A greatly reduces host inflammatory response and coverts lipid A into a powerful adjuvant. Previous attempts to identify an LpxE ortholog in Aquifex aeolicus have failed. A distant lipid A 1‐phosphatase ortholog was identified in Aquifex aeolicus (AaLpxE) and expressed in E. coli. To date, all characterized LpxE orthologues have been unable to dephosphorylate phosphatidylglycerol‐phosphate (PGP), a major intermediate of phospholipid biosynthesis. AaLpxE is unique among LpxE family members in that it functions as both a lipid A 1‐phosphatase and PGP phosphatase in vitro and in vivo. AaLpxE was purified to homogeneity and its dual activities were studied using highly sensitive in vitro assays. The study of AaLpxE provides a model system for the study of substrate specificity of lipid processing enzymes. This research was supported by NIH grant GM51796 to C.R.H. Raetz.
Investigations into how a lyso‐glycerophospholipid acyltransferase alters headgroup acylated glycerophospholipid levels in Escherichia coli
Authors: Garrett Teresa A.
Published: June 2021

Previously, we demonstrated that, in vitro, the Arabidopsis thaliana enzyme At1g78690, previously identified as an N‐acyl phosphatidylethanolamine synthase, acylates 1‐acyl phosphatidylethanolamine (1‐acyl lysoPE) and 1‐acyl phosphatidylglycerol (1‐acyl lysoPG) at the sn‐2 position to generate phosphatidylethanolamine and phosphatidylglycerol, respectively. Also, expression of At1g78690p in E. coli leads to the accumulation of acylphosphatidylglycerol (acyl‐PG). To understand why the overexpression of a lyso‐glycerophospholipid (GPL) acyltransferase leads to the accumulation of a headgroup acylated GPL we have constructed a N‐terminally histidine tagged version of At1g78690 and are purifying the protein from solubilized membranes using nickel affinity chromatography. Using both solubilized membranes and partially purified At1g78690 we are assessing the ability of At1g78690 to acylate a variety or substrates including glycerol‐3‐phosphate, other lyso GPLs, and three isoforms of bis‐monoacylglycerophosphate (BMP). 2‐acyl lyso GPLs are also being tested as substrates. Preliminary data suggests that At1g78690 has broad substrate specificity; the ability of At1g78690 to acylate BMP may explain why acyl‐PG accumulates in E. coli when the protein is overexpressed in cells. This work is supported by the National Science Foundation Major Research Instrumentation Award #1039659.
Regulation of Yeast Phosphatidate Phosphatase by Fatty Acids
Authors: FAKAS STYLIANOS , CARMAN GEORGE
Published: June 2021

The PAH1‐encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of the storage lipid triacylglycerol (TAG), and simultaneously controls the level of PA used for phospholipid synthesis. PA phosphatase activity is induced by exogenous fatty acids and this induction is critical for protecting the cells from the toxic effects exerted by fatty acids. Indeed, PA phosphatase channels the fatty acids to TAG thereby neutralizing them. In this work, we show that the level of PA phosphatase induction depends on the fatty acid species supplied to the cells, with palmitoleic acid (C16:1) being the most potent inducer. Lipid analysis showed that cells grown in the presence of palmitoleic acid present a massive induction of TAG accumulation, as opposed to cells grown in the presence of either palmitic (C16:0), stearic (C18:0) or oleic (C18:1) acid. This discrepancy could be because palmitoleic acid gets incorporated much more efficiently than the rest of the fatty acids. This notion is supported by the fatty acid profile of lipids in cells supplied with palmitoleic acid where this fatty acid becomes more than 80% (w/w) of the total cellular fatty acids. In contrast, supplementation with either palmitic, stearic or oleic acid causes a less pronounced effect on cellular lipids (i.e. 45% (w/w)). Supported by NIH grant GM‐28140.
Crystal structure of LpxK: The tetraacyldisaccharide 4′‐kinase of lipid A biosynthesis
Authors: Emptage Ryan Paul , Daughtry Kelly D. , Pemble Charles W. , Raetz Christian R. H.
Published: June 2021

The tetraacyldisaccharide 4′‐kinase LpxK catalyzes the sixth step in the biosynthetic pathway of lipid A, the hydrophobic anchor of lipopolysaccharide and an essential membrane component for the vast majority of Gram‐negative bacteria. LpxK is the only known member of the diverse P‐loop containing nucleoside triphosphate hydrolase superfamily to phosphorylate a lipid substrate. Using a single anomalous dispersion dataset collected from selenomethionine‐derivitized protein, the structure of Aquifex aeolicus LpxK was solved in both apo and ADP/Mg2+‐bound forms to a resolution of 1.9 Å and 2.2 Å respectively. LpxK consists of two α/β/α domains connected by a two‐stranded β‐sheet linker which close around ADP/Mg2+ upon binding. The larger N‐terminal domain is responsible for catalysis at the P‐loop and positioning of the disaccharide‐1‐phosphate substrate for phosphoryl transfer while the smaller C‐terminal domain's primary role is nucleoside binding. The structures along with point mutagenesis studies reveal the importance of highly conserved active site functionalities which are necessary for positioning of the β‐phosphate and catalytic Mg2+ ion. This work has led to an increased understanding of the 4′‐kinase's enzymatic mechanism and provided the foundation for structure‐based antimicrobial design. This research was funded by N.I.H. grant GM51310 to C.R.H. Raetz.
How Apaf‐1 relays the death signal in the mitochondrial pathway of apoptosis
Authors: Eschenburg Susanne , Reubold Thomas Frank
Published: June 2021

The apoptotic protease‐activating factor 1 (Apaf‐1) is the key switch in the mitochondrial pathway of apoptosis. Apaf‐1 oligomerizes upon binding of mitochondrially released cytochrome c into the heptameric apoptosome complex, which ignites the downstream cascade of caspases. We have recently solved the 3.0 Å crystal structure of full‐length murine Apaf‐1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its “off” position. By comparing the “off” state with a recent cryo‐EM derived model of Apaf‐1 in its apoptosomal conformation, we depict the molecular events that transform Apaf‐1 from auto‐inhibited monomer to a building block of the caspase‐activating apoptosome. Combining the results from X‐ray crystallography, mutational studies, and small angle scattering experiments we elucidate the role of nucleotide exchange for the release of the autoinhibition of Apaf‐1.
Localization of different conformation forms of Transglutaminase 2 in living cells
Authors: Pavlyukov Marat S , Shakhparonov Mihail I , Shemyakin M.M. , Yu A
Published: June 2021

Transglutaminase (TG2) is a ubiquitously expressed enzyme that has been implicated in a variety of biological disorders ranging from inflammatory and cancer to neurodegenerative diseases. Initially TG2 was described as a Ca2+‐dependent transamidating enzyme (EC 2.3.2.13) that catalyzes protein cross‐linking via formation of an isopeptide bond between a specific glutamyl containing peptide substrate and a ε‐amine group of a protein‐bound lysine residue. Later investigations have showed that TG2 possesses other biochemical functions and participates in several cell processes. However, it is steel a matter of discussion what process requires TG2 crosslinking activity. But it is now clear that activated and inactivated TG2 are present in different conformational states. We have developed a new FRET based method for observing the changes in TG2 conformation in living cells. Using that method we studied localization of different conformation forms of TG2 in cells in «healthy» conditions and after apoptosis induction. We have shown that there are distinct subcellular pools of TG2 that localize in the cytoplasm, perinuclear endosomes and under the cellular membrane. After apoptosis induction rapid activation of cytoplasmic TG2 occurs. Ongoing work further investigates the exact role of different TG2 pools in apoptosis regulation. [Supported by Russian Foundation for Basic Research grant 10‐04‐01206]
Ceramide synthase 6 suppression leads to resistance to photodynamic therapy in human head and neck cancer cells
Authors: Separovic Duska , Breen Paul , Joseph Nicholas , Gudz Tatyana I.
Published: June 2021

Defective apoptosis contributes to resistance to cancer treatment by photodynamic therapy (PDT). Ceramide synthase 6 (CerS6) has been implicated in apoptosis. Here we tested the role of CerS6 downregulation in apoptosis after PDT with the silicone phthalocyanine Pc 4 in human head and neck squamous carcinoma cells. CerS6 knockdown led to reduction in PDT‐induced DEVDase activation, mitochondrial depolarization, apoptosis, and cell killing. CerS6 knockdown also resulted in distinct changes in sphingolipid profile after PDT. Overall, our data show a novel role for CerS6 in regulating resistance to PDT.
Detection and prevention of aggregate formation of cobinamide as an antidote for cyanide poisoning
Authors: Glasheen Megan Elizabeth , Chan Adriano , Boss Gerry , Ali Sameh S.
Published: June 2021

Cobinamide, a vitamin B12 precursor, has a high affinity for cyanide and is an effective antidote for cyanide poisoning in animal models. However, when given as an intramuscular injection at high concentrations, the compound forms aggregates that both inhibit its absorption and damage the muscle tissue. The objective of this study is to investigate aggregate formation in terms of aggregate number and thermodynamics of formation as a means to prevent aggregate formation in the drug formulation. Cobinamide solutions ranging in concentrations from 5μM to 1mM were scanned from 400nm to 600nm at 25°C, 35°C, 45°C, and 65°C using a spectrophotometer. The spectrum within this range was representative of aggregate formation as visible changes were observed with increasing concentrations and temperatures. A variety of compounds were tested for their ability to prevent aggregate formation. We analyzed the effect of temperature on the stability of the aggregated compounds to determine the heat of formation of the complexes. Our results show that cobinamide forms very strong aggregates that can be largely prevented using carboxylic acids.

Supported by the CounterACT Program, Office of Director, NIH and NINDS, Grant #U01NS058030.
Herding cats and academics: Skills for leading a professional organization
Authors: Burr David B
Published: June 2021

Leading a professional organization can be one of the most rewarding opportunities of your professional career. Enter with an agenda you want to accomplish, but remain flexible and listen when not everyone shares your vision. Your responsibility is to collect information, distill it to essential and manageable bits, and present it back to the committee in a digestible form, ready for more thorough discussion. Don't rush the process; be patient and let ideas percolate. This approach allows everyone to feel they have had input to the process and decision. It encourages consensus, and reduces divisive dissent. Although it can be difficult to release control, delegate responsibility. When ideas are too complex, or discussion is likely to overtake the entire agenda, assign it to a subcommittee for consideration. One big challenge in leadership is to encourage participation, but do not allow dominant personalities to dominate. Egos on a committee are deadly. Be constructive and tolerant. Remember: “Any jackass can kick down a barn, but it takes a good carpenter to build one” (Sam Rayburn). The final result of your leadership is to implement your ideas: Take action. You can't plow a field by turning it over in your head. If the idea fails, take responsibility. If it is successful, deflect credit. “The people do not notice the existence of the best leaders. When the work is done, the people say ‘we did it ourselves.’” (Lao Tzu)
Leading a medical school academic unit: Institute vs department
Authors: Jones Kathryn Jane
Published: June 2021

Leadership within an academic institution brings with it much opportunity to advance the scientific and educational mission locally and nationally. Included in this are opportunities for mentoring, enhancing scientific goals, curriculum reform, and fostering bridges across units within the institution that advance the mission. Today, in medical schools, different structures exist in which to contribute to the aforementioned mission. In particular, traditional units such as discipline‐based departments exist in concert with cross‐disciplinary units such as institutes or centers. The goals of departments vs institutes and centers play an important role in the identification of requisite leadership skills and interests. What is the skill set necessary for academic unit leadership? What are the distinct roles and responsibilities for each? How does one build cohesiveness based upon diversity in science? In an era where grant dollars define everything, how does one measure success in effective leadership and set a strategic plan for the future that incorporates the spirit of the mission ‐ while focusing on the bottom line?
Towards the glycoproteome of Mycobacterium tuberculosis
Authors: Hess Sonja , Bell Christina , Smith Geoffrey T , Sweredoski Michael J
Published: June 2021

Tuberculosis continues to be a major threat to public health having one of the highest mortalities of any infectious disease despite the use of antibacterial therapy and a partially effective vaccine. Mycobacterium tuberculosis has a complex relationship with its host that is mediated in part by glycosylated proteins, but knowledge about the glycoproteome of tuberculosis is still lacking. In fact, the glycostructures are currently known only for two glycoproteins (alanine and proline rich secreted protein APA and superoxide dismutase SODC).

To identify potentially glycosylated proteins in M. tuberculosis, we investigated mycobacterial subcellular fractions (cell wall, cell membrane and culture filtrate proteins) using the following lectins: Concanavalin A (ConA), Soybean Agglutinin (SBA), and Wheat Germ Agglutinin (WGA) followed by liquid chromatography‐mass spectrometry approaches and bioinformatic analyses.

As expected, there was some overlap between the different lectins in their ability to bind potential glycoproteins. Interestingly, there was no general trend in that one lectin may be better than another. In fact, ConA identified the most glycoprotein candidates in CFP but the least in cell wall and membrane. SBA identified the most candidates in the cell wall whereas WGA identified the largest number of potential glycoproteins from the cell membrane..

To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation and electron transfer dissociation techniques, novel bioinformatics analyses involving a novel virtual neutral loss algorithm and statistical analysis of enrichment in cell fractions with and without lectin treatment.
Comparison of early sucking dynamics of breastfeeding infants born by either caesarean section and vaginal birth
Authors: Kent Jacqueline Coral , Sakalidis Vanessa , Williams Tracey , Hepworth Anna , Garbin Catherine , Paech Michael J , Hartmann Peter , Geddes Donna
Published: June 2021

This study compared infant sucking and breastfeeding behaviour in infants of mothers who delivered by caesarean section (CS) and received pethidine for post‐caesarean section analgesia, with mothers who delivered vaginally (V), during the period of secretory activation (day 3) and again after the establishment of lactation (follow‐up). Infant sucking was measured with ultrasound images of the infant's intra‐oral cavity, breastfeeding behaviour was assessed by the LATCH‐R tool and the times to first breastfeed and breast fullness were recorded. CS infants displayed significantly more anterior tongue movement on day 3 than at follow‐up (p<0.001), whereas the V group showed a similar amount of movement between assessments. Compared to the V group, in the CS group LATCH‐R scores improved at follow‐up (CS; p=0.02, V: p=0.15), CS suck rates were faster, especially on day 3 (p<0.001), and times to first breastfeed (p=0.01) and breast fullness (p=0.03) were significantly later. These results suggest a small influence of CS birth and/or pethidine on breastfeeding during the critical period of secretory activation. Funding: Medela AG, Switzerland.