@misc{zimmer_barycki_simpson_2022, title={Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars}, volume={322}, ISSN={["1522-1563"]}, DOI={10.1152/ajpcell.00130.2022}, abstractNote={ Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review. }, number={6}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY}, author={Zimmer, Brenna M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2022}, month={Jun}, pages={C1201–C1213} }
 @misc{zimmer_barycki_simpson_2021, title={Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase}, volume={69}, ISSN={["1551-5044"]}, DOI={10.1369/0022155420947500}, abstractNote={Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:}, number={1}, journal={JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY}, author={Zimmer, Brenna M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2021}, month={Jan}, pages={13–23} }
 @article{hengel_bosso-lefevre_grady_szenker-ravi_li_pierce_lebigot_tan_eio_narayanan_et al._2020, title={Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy}, volume={11}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-020-14360-7}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, author={Hengel, Holger and Bosso-Lefevre, Celia and Grady, George and Szenker-Ravi, Emmanuelle and Li, Hankun and Pierce, Sarah and Lebigot, Elise and Tan, Thong-Teck and Eio, Michelle Y. and Narayanan, Gunaseelan and et al.}, year={2020}, month={Jan} }
 @article{nowialis_lopusna_opayska_haney_abraham_sheng_riva_natarajan_guryanova_simpson_et al._2019, title={Catalytically inactive Dnmt3b rescues mouse embryonic development by accessory and repressive functions}, volume={10}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-019-12355-7}, abstractNote={Abstract}, journal={NATURE COMMUNICATIONS}, author={Nowialis, Pawel and Lopusna, Katarina and Opayska, Jana and Haney, Staci L. and Abraham, Ajay and Sheng, Peike and Riva, Alberto and Natarajan, Amarnath and Guryanova, Olga and Simpson, Melanie and et al.}, year={2019}, month={Sep} }
 @article{mcatee_booth_elowsky_zhao_payne_fangman_caplan_henry_simpson_2019, title={Prostate tumor cell exosomes containing hyaluronidase Hyal1 stimulate prostate stromal cell motility by engagement of FAK-mediated integrin signaling}, volume={78-79}, ISSN={["1569-1802"]}, DOI={10.1016/j.matbio.2018.05.002}, abstractNote={The hyaluronidase Hyal1 is clinically and functionally implicated in prostate cancer progression and metastasis. Elevated Hyal1 accelerates vesicular trafficking in prostate tumor cells, thereby enhancing their metastatic potential in an autocrine manner through increased motility and proliferation. In this report, we found Hyal1 protein is a component of exosomes produced by prostate tumor cell lines overexpressing Hyal1. We investigated the role of exosomally shed Hyal1 in modulating tumor cell autonomous functions and in modifying the behavior of prostate stromal cells. Catalytic activity of Hyal1 was necessary for enrichment of Hyal1 in the exosome fraction, which was associated with increased presence of LC3BII, an autophagic marker, in the exosomes. Hyal1-positive exosome contents were internalized from the culture medium by WPMY-1 prostate stromal fibroblasts. Treatment of prostate stromal cells with tumor exosomes did not affect proliferation, but robustly stimulated their migration in a manner dependent on Hyal1 catalytic activity. Increased motility of exosome-treated stromal cells was accompanied by enhanced adhesion to a type IV collagen matrix, as well as increased FAK phosphorylation and integrin engagement through dynamic membrane residence of β1 integrins. The presence of Hyal1 in tumor-derived exosomes and its ability to impact the behavior of stromal cells suggests cell-cell communication via exosomes is a novel mechanism by which elevated Hyal1 promotes prostate cancer progression.}, journal={MATRIX BIOLOGY}, author={McAtee, Caitlin O. and Booth, Christine and Elowsky, Christian and Zhao, Lei and Payne, Jeremy and Fangman, Teresa and Caplan, Steve and Henry, Michael D. and Simpson, Melanie A.}, year={2019}, month={May}, pages={165–179} }
 @article{grady_thelen_albers_ju_guo_barycki_simpson_2016, title={Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking}, volume={55}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/ACS.BIOCHEM.6B00259}, DOI={10.1021/ACS.BIOCHEM.6B00259}, abstractNote={The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.}, number={22}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Grady, George and Thelen, Ashley and Albers, Jaleen and Ju, Tong and Guo, Jiantao and Barycki, Joseph J. and Simpson, Melanie A.}, year={2016}, month={May}, pages={3157–3164} }
 @article{zimmer_howell_wei_ma_romsdahl_loughman_markham_seravalli_barycki_simpson_2016, title={Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential}, volume={7}, ISSN={1868-8497 1868-8500}, url={http://dx.doi.org/10.1007/S12672-016-0268-Z}, DOI={10.1007/S12672-016-0268-Z}, abstractNote={Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen-dependent versus castration-resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration-resistant tumor cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, the magnitude of androgen-activated UGDH and prostate-specific antigen (PSA) expression, as well as the androgen receptor (AR)-dependent repression of UGT2B15 and UGT2B17, was blunted several-fold in these cells. Consistent with these results, the ligand-activated binding of AR to the PSA promoter and subsequent transcriptional activation were also significantly reduced in castration-resistant cells. Analysis of the UDP-sugar pools and flux through pathways downstream of UDP-glucuronate production revealed that these glucuronidation precursor metabolites were channeled through proteoglycan and glycosaminoglycan biosynthetic pathways, leading to increased surface expression of Notch1. Knockdown of UGDH diminished Notch1 and increased glucuronide output. Overall, these results support a model in which the aberrant partitioning of UDP-glucuronate and other UDP-sugars into alternative pathways during androgen deprivation contributes to the loss of prostate tumor cell androgen sensitivity by promoting altered cell surface proteoglycan expression.}, number={4}, journal={Hormones and Cancer}, publisher={Springer Nature}, author={Zimmer, Brenna M . and Howell, Michelle E. and Wei, Qin and Ma, Linlin and Romsdahl, Trevor and Loughman, Eileen G. and Markham, Jonathan E. and Seravalli, Javier and Barycki, Joseph J. and Simpson, Melanie A.}, year={2016}, month={Jun}, pages={260–271} }
 @article{mcatee_berkebile_elowsky_fangman_barycki_wahl_khalimonchuk_naslavsky_caplan_simpson_2015, title={Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking}, volume={290}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M115.647446}, DOI={10.1074/JBC.M115.647446}, abstractNote={Background: Hyal1 is a turnover enzyme for hyaluronan that accelerates metastatic cancer by increasing cell motility. Results: Hyal1-overexpressing cells have a higher rate of endocytosis that impacts cargo internalization and recycling. Conclusion: The higher rate of vesicle trafficking increases motility receptor function and nutrient uptake. Significance: This novel mechanism implicates Hyal1 trafficking in multiple signaling events during tumor progression. Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.}, number={21}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={McAtee, Caitlin O. and Berkebile, Abigail R. and Elowsky, Christian G. and Fangman, Teresa and Barycki, Joseph J. and Wahl, James K., III and Khalimonchuk, Oleh and Naslavsky, Naava and Caplan, Steve and Simpson, Melanie A.}, year={2015}, month={Apr}, pages={13144–13156} }
 @article{haney_hlady_opavska_klinkebiel_pirruccello_dutta_datta_simpson_wu_opavsky_2015, title={Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis}, volume={34}, ISSN={0950-9232 1476-5594}, url={http://dx.doi.org/10.1038/ONC.2014.472}, DOI={10.1038/ONC.2014.472}, abstractNote={DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3aΔ/Δ mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3aΔ/Δ lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3aΔ/Δ mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.}, number={43}, journal={Oncogene}, publisher={Springer Nature}, author={Haney, S L and Hlady, R A and Opavska, J and Klinkebiel, D and Pirruccello, S J and Dutta, S and Datta, K and Simpson, M A and Wu, L and Opavsky, R}, year={2015}, month={Feb}, pages={5436–5446} }
 @article{peters_hlady_opavska_klinkebiel_pirruccello_talmon_sharp_wu_jaenisch_simpson_et al._2014, title={Tumor suppressor functions of Dnmt3a and Dnmt3b in the prevention of malignant mouse lymphopoiesis}, volume={28}, ISSN={0887-6924 1476-5551}, url={http://dx.doi.org/10.1038/LEU.2013.364}, DOI={10.1038/LEU.2013.364}, abstractNote={Tumor suppressor functions of Dnmt3a and Dnmt3b in the prevention of malignant mouse lymphopoiesis}, number={5}, journal={Leukemia}, publisher={Springer Science and Business Media LLC}, author={Peters, S L and Hlady, R A and Opavska, J and Klinkebiel, D and Pirruccello, S J and Talmon, G A and Sharp, J G and Wu, L and Jaenisch, R and Simpson, M A and et al.}, year={2014}, month={May}, pages={1138–1142} }
 @article{kovar_curtis_othman_simpson_michael olive_2013, title={Characterization of IRDye 800CW chlorotoxin as a targeting agent for brain tumors}, volume={440}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/J.AB.2013.05.013}, DOI={10.1016/J.AB.2013.05.013}, abstractNote={Primary brain tumors present significant challenges for surgical resection because of their location and the frequent occurrence of malignant projections extending beyond the primary tumor. Visualization of the tumor margins during surgery is critical for a favorable outcome. We report the use of IRDye 800CW chlorotoxin (CLTX) as a targeted imaging agent for brain tumors in a spontaneous mouse model of medulloblastoma, ND2:SmoA1. Specificity and functionality of the targeted agent were confirmed in cell-based assays. Tumors were detected by magnetic resonance imaging and IRDye 800CW CLTX administered to individual animals for optical imaging at 1-month increments. The integrity of the blood–brain barrier (BBB) was measured by Evan’s Blue perfusion prior to sacrifice. Results show that IRDye 800CW CLTX specifically targeted tumor tissue. The extravasation of Evan’s Blue was observed in all tumors, suggesting that the presence of the tumors can introduce alterations in the permeability of the BBB. Because increased vascular permeability was observed early in the disease model, larger dye-labeled imaging agents that exceed current BBB size restrictions may warrant renewed consideration as candidates for tumor detection and surgical resection. Our study provides data characterizing in vitro and in vivo use of IRDye 800CW CLTX as a broadly applicable tumor imaging agent.}, number={2}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Kovar, Joy L. and Curtis, Evan and Othman, Shadi F. and Simpson, Melanie A. and Michael Olive, D.}, year={2013}, month={Sep}, pages={212–219} }
 @article{hyde_thelen_barycki_simpson_2013, title={UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces}, volume={288}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M113.519090}, DOI={10.1074/JBC.M113.519090}, abstractNote={Background: UDP-glucose dehydrogenase (UGDH) mutants were engineered to perturb hexamer:dimer quaternary structure equilibrium. Results: Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells. Conclusion: Only dynamic UGDH hexamers support robust cellular function. Significance: Manipulation of UGDH activity by hexamer stabilization may offer new therapeutic options in cancer and other pathologies. UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.}, number={49}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Hyde, Annastasia S. and Thelen, Ashley M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2013}, month={Oct}, pages={35049–35057} }
 @article{stephens_saltarrelli jr_balaoing_baggett_nandi_anderson_morrisett_reardon_simpson_weigel_et al._2012, title={Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves}, volume={208}, ISSN={0344-0338}, url={http://dx.doi.org/10.1016/j.prp.2012.08.001}, DOI={10.1016/j.prp.2012.08.001}, abstractNote={The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.}, number={11}, journal={Pathology - Research and Practice}, publisher={Elsevier BV}, author={Stephens, Elizabeth H. and Saltarrelli Jr, Jerome G. and Balaoing, Liezl R. and Baggett, L. Scott and Nandi, Indrajit and Anderson, Kristin M. and Morrisett, Joel D. and Reardon, Michael J. and Simpson, Melanie A. and Weigel, Paul H. and et al.}, year={2012}, month={Nov}, pages={642–650} }
 @article{simpson_weigel_weigel_2012, title={Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer}, volume={131}, ISSN={0020-7136}, url={http://dx.doi.org/10.1002/ijc.27427}, DOI={10.1002/ijc.27427}, abstractNote={Abstract}, number={5}, journal={International Journal of Cancer}, publisher={Wiley}, author={Simpson, Melanie A. and Weigel, Janet A. and Weigel, Paul H.}, year={2012}, month={Feb}, pages={E836–E840} }
 @article{hyde_farmer_easley_van lammeren_christoffels_barycki_bakkers_simpson_2012, title={UDP-glucose Dehydrogenase Polymorphisms from Patients with Congenital Heart Valve Defects Disrupt Enzyme Stability and Quaternary Assembly}, volume={287}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M112.395202}, DOI={10.1074/jbc.M112.395202}, abstractNote={Background: UDP-glucose dehydrogenase (UGDH) polymorphisms were identified in a screen of candidate genes for heart valve defects. Results: Two individual mutants fail to rescue cardiac valve defects in UGDH-deleted zebrafish and have reduced stability in vitro. Conclusion: UGDH loss of function mutations result in a subset of human congenital cardiac valve defects caused by reduced enzyme activity during morphogenesis. Significance: Screening these alleles could predict valve defects. Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.}, number={39}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Hyde, Annastasia S. and Farmer, Erin L. and Easley, Katherine E. and van Lammeren, Kristy and Christoffels, Vincent M. and Barycki, Joseph J. and Bakkers, Jeroen and Simpson, Melanie A.}, year={2012}, month={Jul}, pages={32708–32716} }
 @article{willis_liu_biterova_simpson_kim_lee_barycki_2011, title={Enzymatic Defects Underlying Hereditary Glutamate Cysteine Ligase Deficiency Are Mitigated by Association of the Catalytic and Regulatory Subunits}, volume={50}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi200708w}, DOI={10.1021/bi200708w}, abstractNote={Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.}, number={29}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Willis, Melanie Neely and Liu, Yilin and Biterova, Ekaterina I. and Simpson, Melanie A. and Kim, Heejeong and Lee, Jaekwon and Barycki, Joseph J.}, year={2011}, month={Jul}, pages={6508–6517} }
 @article{bharadwaj_goodrich_mcatee_haferbier_oakley_wahl_simpson_2011, title={Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling}, volume={317}, ISSN={0014-4827}, url={http://dx.doi.org/10.1016/j.yexcr.2011.01.026}, DOI={10.1016/j.yexcr.2011.01.026}, abstractNote={Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.}, number={8}, journal={Experimental Cell Research}, publisher={Elsevier BV}, author={Bharadwaj, Alamelu G. and Goodrich, Nathaniel P. and McAtee, Caitlin O. and Haferbier, Katie and Oakley, Gregory G. and Wahl, James K., III and Simpson, Melanie A.}, year={2011}, month={May}, pages={1214–1225} }
 @article{kovar_xu_draney_cupp_simpson_olive_2011, title={Near-infrared-labeled tetracycline derivative is an effective marker of bone deposition in mice}, volume={416}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/j.ab.2011.05.011}, DOI={10.1016/j.ab.2011.05.011}, abstractNote={Bone-specific compounds have been used effectively for the detection of bone mineralization, growth, and morphological changes. These agents typically contain iminodiacetic acid groups that can form complexes with apatite and fluoresce in the visible spectrum. We exploited a subset of these chemical chelators to produce a near-infrared (NIR) optical bone marker for preclinical animal imaging. By conjugating target compounds to IRDye 800CW, we extended the effective fluorescence signal detection to the NIR region without affecting the compound’s ability to function as a marker of the mineralization process. Calcein and a tetracycline derivative (BoneTag agent [BT]) bound specifically to differentiated mineralized osteoblast cultures, with the latter exhibiting 6-fold higher signal intensities. Subsequent in vivo testing demonstrated effective skeletal labeling with IRDye 800CW BT. We were able to identify a changing mineralization front in bone sections from (i) normal growing mice injected with IRDye 800CW BT 6 weeks prior to the administration of IRDye 680 BT and (ii) an osteoporosis mouse model comparing cortical bone in sham-treated and ovariectomized mice. These results provide evidence that the NIR-labeled BT is effective as a general marker of skeletal features and an indicator of the bone mineralization and remodeling processes.}, number={2}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Kovar, Joy L. and Xu, Xinshe and Draney, Dan and Cupp, Andrea and Simpson, Melanie A. and Olive, D. Michael}, year={2011}, month={Sep}, pages={167–173} }
 @article{huang_casale_tian_lele_pisarev_simpson_hemstreet_2010, title={Udp-glucose dehydrogenase as a novel field-specific candidate biomarker of prostate cancer}, volume={126}, ISSN={0020-7136 1097-0215}, url={http://dx.doi.org/10.1002/ijc.24820}, DOI={10.1002/ijc.24820}, abstractNote={Abstract}, number={2}, journal={International Journal of Cancer}, publisher={Wiley}, author={Huang, Dali and Casale, George P. and Tian, Jun and Lele, Subodh M. and Pisarev, Vladimir M. and Simpson, Melanie A. and Hemstreet, George P., III}, year={2010}, month={Jan}, pages={315–327} }
 @article{kovar_volcheck_sevick-muraca_simpson_olive_2009, title={Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models}, volume={384}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/j.ab.2008.09.050}, DOI={10.1016/j.ab.2008.09.050}, abstractNote={Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.}, number={2}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Kovar, Joy L. and Volcheck, William and Sevick-Muraca, Eva and Simpson, Melanie A. and Olive, D. Michael}, year={2009}, month={Jan}, pages={254–262} }
 @article{zhang_bharadwaj_casper_barkley_barycki_simpson_2009, title={Hyaluronidase Activity of Human Hyal1 Requires Active Site Acidic and Tyrosine Residues}, volume={284}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M900210200}, DOI={10.1074/jbc.M900210200}, abstractNote={Hyaluronidases are a family of endolytic glycoside hydrolases that cleave the β1-4 linkage between N-acetylglucosamine and glucuronic acid in hyaluronan polymers via a substrate-assisted mechanism. In humans, turnover of hyaluronan by this enzyme family is critical for normal extracellular matrix remodeling. However, elevated expression of the Hyal1 isozyme accelerates tumor growth and metastatic progression. In this study, we used structural information, site-directed mutagenesis, and steady state enzyme kinetics to probe molecular determinants of human Hyal1 function. Mutagenesis of active site residues Glu131 and Tyr247 to Gln and Phe, respectively, eliminated activity at all hyaluronan concentrations (to 125 μm or 2.5 mg/ml). Conservative mutagenesis of Asp129 and Tyr202 significantly impaired catalysis by increases of 5- and 10-fold in apparent Km and reductions in Vmax of 95 and 50%, respectively. Tyr247 and Asp129 are required for stabilization of the catalytic nucleophile, which arises as a resonance intermediate of N-acetylglucosamine on the substrate. Glu131 is a likely proton donor for the hydroxyl leaving group. Tyr202 is a substrate binding determinant. General disulfide reduction had no effect on activity in solution, but enzymatic deglycosylation reduced Hyal1 activity in a time-dependent fashion. Mutagenesis identified Asn350 glycosylation as the requisite modification. Deletion of the C-terminal epidermal growth factor-like domain, in which Asn350 is located, also eliminated activity, irrespective of glycosylation. Collectively, these studies define key components of Hyal1 active site catalysis, and structural factors critical for stability. Such detailed understanding will allow rational design of enzyme modulators.}, number={14}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Zhang, Ling and Bharadwaj, Alamelu G. and Casper, Andrew and Barkley, Joel and Barycki, Joseph J. and Simpson, Melanie A.}, year={2009}, month={Feb}, pages={9433–9442} }
 @article{kovar_simpson_schutz-geschwender_olive_2007, title={A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models}, volume={367}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/j.ab.2007.04.011}, DOI={10.1016/j.ab.2007.04.011}, abstractNote={Reduction of benzo[c] [1,7]naphthyridin-4(3H)-ones with sodium borohydride in acetic acid led to a series of 5-ethyl-5,6-dihydrobenzo[c] [1,7]naphthyridin-4(3H)-ones. The latter are photostable fluorophores emitting in the green region of the visible spectrum with a fluorescence quantum yield of up to 0.43 and a Stokes shift of up to 133 nm, containing an “embedded” amino acid motif. The localization and photobleaching of the obtained compounds in cryosections of rat skin was studied.}, number={1}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Kovar, Joy L. and Simpson, Melanie A. and Schutz-Geschwender, Amy and Olive, D. Michael}, year={2007}, month={Aug}, pages={1–12} }
 @article{easley_sommer_boanca_barycki_simpson_2007, title={Characterization of Human UDP-Glucose Dehydrogenase Reveals Critical Catalytic Roles for Lysine 220 and Aspartate 280}, volume={46}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi061537d}, DOI={10.1021/bi061537d}, abstractNote={Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.}, number={2}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Easley, Katherine E. and Sommer, Brandi J. and Boanca, Gina and Barycki, Joseph J. and Simpson, Melanie A.}, year={2007}, month={Jan}, pages={369–378} }
 @article{bharadwaj_rector_simpson_2007, title={Inducible Hyaluronan Production Reveals Differential Effects on Prostate Tumor Cell Growth and Tumor Angiogenesis}, volume={282}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M702964200}, DOI={10.1074/jbc.M702964200}, abstractNote={Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.}, number={28}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Bharadwaj, Alamelu G. and Rector, Katherine and Simpson, Melanie A.}, year={2007}, month={May}, pages={20561–20572} }
 @article{kovar_volcheck_chen_simpson_2007, title={Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice}, volume={361}, ISSN={0003-2697}, url={http://dx.doi.org/10.1016/j.ab.2006.11.021}, DOI={10.1016/j.ab.2006.11.021}, abstractNote={Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.}, number={1}, journal={Analytical Biochemistry}, publisher={Elsevier BV}, author={Kovar, Joy L. and Volcheck, William M. and Chen, Jiyan and Simpson, Melanie A.}, year={2007}, month={Feb}, pages={47–54} }
 @article{bharadwaj_kovar_simpson_2006, title={Hyaluronan synthesis and turnover induces metastatic growth}, volume={25}, ISSN={0945-053X}, url={http://dx.doi.org/10.1016/j.matbio.2006.08.110}, DOI={10.1016/j.matbio.2006.08.110}, journal={Matrix Biology}, publisher={Elsevier BV}, author={Bharadwaj, A and Kovar, J and Simpson, M}, year={2006}, month={Nov}, pages={S40–S40} }
 @article{sommer_barycki_simpson_2004, title={Characterization of Human UDP-glucose Dehydrogenase: CYS-276 is required for the second of two successive oxidations}, volume={279}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M401928200}, DOI={10.1074/jbc.M401928200}, abstractNote={UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.}, number={22}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Sommer, Brandi J. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2004}, month={Mar}, pages={23590–23596} }
 @article{yang_price_neudauer_wilson_ferrone_xia_iida_simpson_mccarthy_2004, title={Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms}, volume={165}, ISSN={1540-8140 0021-9525}, url={http://dx.doi.org/10.1083/jcb.200403174}, DOI={10.1083/jcb.200403174}, abstractNote={Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with α4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.}, number={6}, journal={The Journal of Cell Biology}, publisher={Rockefeller University Press}, author={Yang, Jianbo and Price, Matthew A. and Neudauer, Cheryl L. and Wilson, Christopher and Ferrone, Soldano and Xia, Hong and Iida, Joji and Simpson, Melanie A. and McCarthy, James B.}, year={2004}, month={Jun}, pages={881–891} }