bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effects of Nucleotides on the Conformational Flexibility and Stability of Glutamate Dehydrogenase
Authors: Tran Sirena , Bell Jessica K. , Bell Ellis 
Published: November 2023

Glutamate Dehydrogenase (GDH) catalyzes the reversible oxidative deamination of glutamate using NAD(P)+(H), and is involved in various metabolic pathways and energy cycles. GDH can be allosterically regulated by various nucleotides (ADP, GTP) as well as showing cofactor cooperativity. Previous work shows NADH binds to both the active site and a second regulatory (inhibitory) site while NADPH binds only to the active site. We hypothesize that changes in stability/flexibility correlate with inhibition and activation. To explore the allosteric mode for GDH, we have used Fluorescence‐based Thermal Shift Assays (FTSA), Circular Dichroism (CD), Thermal melts (Tm) and Limited Proteolysis using immobilized trypsin and tandem mass spectrometry (LP) to explore the stability (global and localized) of the protein in the presence or absence of various combinations of nucleotides and substrates (glutamate or 2‐oxo‐glutarate). CD and Tm studies indicate that while the overall structure of the protein is not changed by pH, the stability changes dramatically over the pH range 6–8.5 with a pKa of 7.4. ADP stabilizes the protein while GTP destabilizes the protein, with coligands having little effect. NADPH shows a small destabilizing effect, while NADH effects are complex depending upon concentration. Below the pKa, FbTSA experiments show that ADP and NADH binding to its regulatory site stabilize the protein significantly, while NAD(P)H binding to the active site, or GTP binding destabilize the protein, consistent with the conclusions reached from the CD thermal melt studies. With ADP, binding curves established by FTSA indicate biphasic binding in the presence or absence of cofactor bound at the active site suggesting negative cooperativity in ADP binding. The presence of glutamate has little effect. At pH 8, ADP and GTP have similar effects, while with NADH and NADPH destabilizes the protein. To determine localized regions of the protein responsible for these effects, we are using LP experiments and mapping regions of flexibility changes on the overall 3 dimensional structure of the protein.Support or Funding InformationWork was funded by the USD Knapp Chair and the USD McNair program.
Unmasking the High Affinity of Escherichia coli Glycogen Synthase Towards its Polyglucan Substrate
Authors: Iglesias Alberto Alvaro , Aleanzi Mabel , Diez Matias Damian Asencion , Machtey Matias , Yep Alejandra , Ballicora Miguel 
Published: November 2023

In bacteria, glycogen synthesis occurs by a 3‐step pathway where the glucosyl donor ADP‐glucose synthesized by ADP‐glucose pyrophosphorylase (EC 2.7.7.27) is used to elongate an α‐1,4‐glucan chain by glycogen‐synthase (EC 2.4.1.21; GlgSase), following by the action of polyglucan branching enzyme (EC 2.4.1.18). The pathway was extensively studied in Escherichia coli where the three enzymes were structurally and functionally characterized. The E. coli GlgSase is an enzyme with a relatively high specific activity (~500 U/mg) but an apparent low affinity towards glycogen, as judged by Km values in the order of 0.65 mg/ml. Interestingly, site‐directed mutants of the E. coli GlgSase with highly reduced Vmax decreased the Km. To have a Km equal to Ks requires the assumption that the enzyme is under rapid equilibrium, where kcat is negligible compared to the dissociation of substrate koff. This scenario sustains the hypothesis that a real (high) affinity towards glycogen by E. coli GlgSase could be masked in the kinetic measurement of Km by a relatively high kcat. It is important to note that the accurate determination of the actual affinity for glycogen is relevant to understand the enzyme action mechanism. For that reason, we conducted saturation curves of glycogen at different temperatures in order to analyze changes in Km under conditions where kcat is significantly reduced. We found that a decrease in one order of magnitude in the Vmax correlated with a ~200‐fold reduction in Km. This behavior was also observed for GlgSase mutants (H161A and K305A) and when the wild type enzyme used alternative (to ADP‐Glc) substrates. In addition, the high affinity of the enzyme for glycogen was confirmed by measurements of binding in the absence of catalysis, where Ks was determined to be in the order of μg/ml. This suggests that the kinetics of this enzyme could be better described by steady state models rather than rapid equilibrium.Support or Funding InformationSupported by: ANPCyT [PICT 2014 3362 and PICT 2015 0634 to MDAD, PICT 2014 3256 and PICT 2015 1767 to AAI], NSF MCB‐1616851 to MAB
The Firmicutes Case on the Regulation of Bacterial ADP‐Glucose Pyrophosphorylase
Authors: Diez Matias Damian Asencion , Cereijo Antonela Estefania , Demonte Ana , Ballicora Miguel , Iglesias Alberto Alvaro 
Published: November 2023

ADP‐glucose pyrophosphorylase (EC 2.7.7.27) catalyzes the key step in the prokaryotic pathway for glycogen synthesis. The enzyme from Firmicutes is encoded by two genes, glgC and glgD, leading to a heterotetrameric protein structure unlike other bacterial ADP‐glucose pyrophosphorylases. The enzyme from different orders (Bacillales, Clostridiales and Lactobacillales) in the Firmicutes phylum presents dissimilar kinetic and regulatory properties, as shown in this work. Thus, we comparatively studied the ADP‐glucose pyrophosphorylases from Ruminococcus albus (Clostridiales), Streptococcus mutans (Lactobacillales) and Geobacillus stearothemophilus (Bacillales) to shed light on the structure‐to‐function relationships of the firmicute enzyme. In each case, we produced separate GlgC or GlgD subunits, as well as the GlgC/GlgD co‐expressed forms. There were two active quaternary structures, homotetrameric GlgC and heterotetrameric GlgC/GlgD, whereas GlgD by itself was inactive for all species tested. Nevertheless, the presence of the GlgD in the co‐expressed enzymes depicted different kinetic and regulatory effects depending on the source, which suggests distinct evolutionary adaptations for the non‐catalytic subunit in each of the Firmicutes orders. For example, in the S. mutans, GlgC/GlgD was one order of magnitude more active than GlgC alone. In the others, the activity of GlgC/GlgD was 2‐fold higher than the respective GlgC. Two effectors (fructose‐1,6‐bisphosphate and phosphoenolpyruvate), opposing to the insensitive enzyme from G. stearotherophilus, modulated the S. mutans and R. albus ADP‐glucose pyrophosphorylases. Interestingly, phosphoenolpyruvate is a highly specific inhibitor of GlgC/GlgD ADP‐glucose pyrophosphorylase from S. mutans but an activator of both GlgC and GlgC/GlgD R. albus enzymes. The latter ones were also inhibited by fructose‐1,6‐bisphosphate, whereas this metabolite activated the S. mutans GlgC conformation. Thus, results suggest that allosteric control might be a common feature rather than an exception in the ADP‐glucose pyrophosphorylase from Firmicutes. Consequently, Glycogen synthesis may be regulated by metabolites from the main carbon utilization pathway, as reported in other bacteria.Support or Funding InformationSupported by: ANPCyT [PICT 2014 3362 and PICT 2015 0634 to MDAD, PICT 2014 3256 and PICT 2015 1767 to AAI], NSF MCB‐1616851 to MAB
Microarray and Pathway Analysis of Prostate Cancer Tumors Treated with Andrographolide
Authors: Forestier‐Roman Ingrid Samari , Sánchez María , Rohena Krizia , Ortiz‐Zuazaga Humberto , Martínez‐Ferrer Magaly 
Published: November 2023

Prostate cancer is the most frequently diagnosed non‐cutaneous cancer and the second cause of cancer‐related deaths in American men. Andrographolide, a labdane diterpenoid that is a component of the medicinal plant Andrographis paniculata, has been reported to have a wide range of biological activities including anticarcinogenic properties. In previous studies, we found that Andrographolide inhibits cell growth, cell migration and tumor growth in prostate cancer. The objective of this study is to examine the gene expression profile of prostate cancer tumors treated with Andrographolide. For this, we conducted microarray analysis of tumors treated with Andrographolide 10 mg/kg and their vehicle. Tumors were developed using a xenograft model in which the prostates of SCID mice were injected with 22RV1, and mice were treated three times per week with Andrographolide 10 mg/kg. Tumor tissues were collected and snap frozen. Gene expression was identified and analyzed using the Affymetrix GeneChip® Human Gene 2.0 array. Microarray studies showed that a total of 537 genes were upregulated and 138 were downregulated in tumors treated with Andrographolide 10 mg/kg when compared to their vehicle. By using Ingenuity Pathway Analysis (IPA) we found that Andrographolide 10 mg/kg altered molecular and cellular functions including “DNA Replication, Recombination and Repair”, “Cell Cycle”, “Cellular Compromise”, “Cellular Assembly and Organization” and “Post‐Translational Modification”. Understanding these biological pathways and networks is essential to determine possible targets of Andrographolide in prostate cancer.Support or Funding InformationNIH K01 CA140711 NIH RCMI #8G12MD007600 Seed Funds from University of Puerto Rico Comprehensive Cancer Center U54CA096297
A chemical biology approach to the study of Coenzyme Q biosynthesis and metabolism
Authors: Nag Anish , Fernandez Lucía , Villalba José M , Shepherd Jennifer N. , Kwon Ohyun , Clarke Catherine F. 
Published: November 2023

Coenzyme Q (ubiquinone or Q) is a redox active lipid molecule, with a fully substituted benzoquinone ring and a polyisoprenoid tail with variable number of isoprene units. Humans synthesize Q10 with ten isoprene units, while Saccharomyces cerevisiae synthesize Q6. In eukaryotic cells Q is an essential component of the mitochondrial respiratory electron transport chain and an essential antioxidant. Q deficiency has been linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The pathway of Q biosynthesis has still not been completely characterized. Therefore, it is vital to have a deeper understanding of the biosynthesis and metabolism of Q, to properly address its effects on health. In this study, farnesylated analogs (containing 3 isoprene units) of several Q‐intermediates have been chemically synthesized. The synthesis of 2‐farnesyl‐1,4‐hydroquinone serves as an analog of 3‐hexaprenyl‐4‐hydroxyphenol, which accumulates in yeast coq6 and coq9 mutants that over‐express the Coq8 polypeptide. In addition farnesylated analogs of 5‐demethoxy‐Q3 (DMQ3), 2‐demethyl‐5‐demethoxy‐Q3 (DDMQ3), and 2‐demethyl‐5‐demethoxy‐4‐amino‐Q3 (IDDMQ3H2) were chemically synthesized. The farnesylated analogs are added to yeast cell cultures to determine whether they serve as precursors and/or intermediates in the biosynthesis of Q3. Also, other selected farnesylated substrates such as 3‐farnesyl‐4‐hydroxybenzoic acid, 3‐farnesyl‐4,5‐dihydroxybenzoic acid, and other derivatives, are being synthesized to study Q metabolism. These substrates can also serve as ligands in protein binding assays and as substrates with isolated enzymes. Polyphenolic substrates, such as resveratrol, and p‐coumarate act as ring precursors for Q production in both yeast and mammalian cells. Here we investigate a number of other selected polyphenolic compounds that include kaempferol, ferulic acid, vanillin, and curcumin, to analyze their role as possible ring precursors for Q biosynthesis, in yeast and mammalian cells. Thereby we hope to understand and further characterize the biosynthetic and metabolic pathways responsible for Q biosynthesis, and function in different living systems.Support or Funding InformationThis research was supported by NSF MCB‐1330803.
Second Generation PS‐ASO Internalization and Endosomal Escape
Authors: Miller Colton , Egger Andrew , Kellar Bri , Hass Blake , Seth Punit P. , Harris Edward N. 
Published: November 2023

Phosphorothioate (PS)‐modified antisense oligonucleotides (ASOs) have been extensively investigated over the past few decades as therapeutic agents. Clearance of ASOs by liver and kidney greatly affect their efficacy for gene knockdown or modifications on the RNA level. The liver sinusoidal endothelial cells (LSECs) are highly endocytically active cells involved with the clearance of many ECM‐derived molecules as well as synthetic macromolecules such as heparan sulfates, hyaluronan, and ASOs. LSECS express the class H receptors known as the Stabilin‐1 and Stabilin‐2 receptors. Stabilin‐2 is also known as the Hyaluronic Acid Receptor for Endocytosis (HARE) which is found as two isoforms; 315‐kDa (315‐HARE) and 190‐kDa (190‐HARE). The Stabilin receptors are also found in the endothelium of many other tissues such as the bone marrow, spleen, and lymph node. Our previous work identified the Stabilin receptors as a primary mechanism for the internalization of ASOs in LSECs. ASO internalization occurs by clathrin‐mediated endocytosis, and are trafficked to the lysosome. Here we analyze how ASOs are able to escape the endosomal pathway to inhibit RN A expression in the cell.MethodsWe use recombinant cell lines stably expressing Stabilin‐1, 315‐HARE and 190‐HARE to analyze ASO vesicular escape. Using siRNAs for proteins involved in the early and late endosomal pathway, we will determine the levels of the non‐coding RNA, malat‐1, which is targeted by the ASO. Malat‐1 expression is directly correlated with escape efficiency of the ASO which targets it. The siRNA targeted gene that prevents the least amount of ASO functionality is likely the best candidate involved with ASO escape in the cytoplasm. We also analyzed vesicular transport by encapsulating ASOs in liposomes, and analyze ASO escape based on modifications resembling events of endosomal trafficking such as changes in pH, and lipid reshuffling, which will model vesicular maturation.Future AimsBy determining the precise component of the vesicular endosomal pathway by which the ASO escape mechanism occurs, we will be able to gain a better understanding of how these therapeutics function. This will help identify how these molecules, once internalized within the cell are able to functionally bind to mRNA in the cytosol and nucleus for RNase H recruitment and degradation. Understanding this mechanism will greatly assist in rational design of therapeutic ASOs.
Stereoelectronically Stabilized Flurogenic Probes for Esterase–Activated Biomolecule Imaging
Authors: Kilgore Henry R , Chyan Wen , Gold Brian , Raines Ronald T 
Published: November 2023

Esterase‐labile fluorogenic probes provide a means of interrogating the spatiotemporal distribution of molecules of interest across biological membranes. In designing such a probe, the primary consideration is maintaining orthogonality of activation to all intracellular reactions except those of interest. We are developing a strategy to curtail ambient hydrolysis of acyl masking groups on fluorescein dyes by carefully balancing n‐to‐pi* and steric interactions to enable the probes to maintain stability in the absence of esterases. The electronic properties of these probes is considered with respect to model compounds that maintain the n‐to‐pi* and steric interactions. These probes were used to visualize and quantify the unusual cytosolic and nuclear transport of large (≥70‐kDa) dextrans. Imaging results with the conjugated probes provide new insight on the permissivity of the nuclear envelope and demonstrate the efficacy of the esterase labile probe strategy.Support or Funding InformationThis work is supported by grant R01 CA073808.
Development of Red Fluorescent Protein pH Sensors
Authors: Rajendran Megha , Haynes Emily , Claywell Ben , Scales Umi , Henning Chace , Tantama Mathew 
Published: November 2023

The regulation of pH plays a crucial role in protein function, metabolic reactions, autophagy, and a wide range of other physiological processes. Cellular activities and diseases are known to perturb the pH in cells. For example, in cancer cells, increased reliance on aerobic glycolysis is thought to contribute to extracellular acidosis in the tumor microenvironment. Therefore, studying changes in pH in different compartments would allow us to better understand the role of pH in signaling and metabolism in both normal and disease states. Live‐cell imaging with fluorescent protein‐based sensors could allow us to monitor pH changes in situ; however, there are currently limited options for these sensors that are able to measure pH dynamics with subcellular resolution in live specimens. Furthermore, most of these genetically‐encoded pH sensors are generated from green fluorescent protein, limiting our ability to perform multiplex imaging to monitor pH simultaneously in different compartments. Therefore, it is the goal of this project to develop a range of genetically‐encoded fluorescent pH sensors. Here, we present the in vitro and live‐cell characterization of a new red fluorescent protein pH sensor. In proof‐of‐concept studies, we measure both intracellular and extracellular pH simultaneously as well as the pH of different subcellular compartments simultaneously. Our results demonstrate our progress towards the measurement of pH in the tumor microenvironment and understanding how pH is affected by aerobic glycolysis.Support or Funding InformationThis work is supported by National Institute of Health R21 NS092010 and R21 EY026425.
Integrated metabolomic studies on health effects of oxidized tyrosine administration in female mice
Authors: Yang Yuhui , Yan Biao , Zhang Tianyu , Gao Ying , Shi Yonghui , Le Guowei 
Published: November 2023

Oxidized tyrosine (O‐Tyr) which is associated with some pathological conditions and metabolic disorders (especially various age‐related disorders) in biological system, can be easily detected in protein‐rich food systems. However, the systemic effects of O‐Tyr in the animal body is unexplored. This study aims to investigate the effects of O‐Tyr on the overall metabolic status of mice. Twenty female Kunming mice were intragastrically administered with saline or O‐Tyr (320 μg/kg body weight) every day for six weeks. Urine and serum were sampled and analyzed by 1H nuclear magnetic resonance‐based metabolomics and coupled with multivariate data analysis methods. Body weight, serum biochemical profiles, organs index and histopathological data were obtained as complementary information. Compared with control group, O‐Tyr administration had a tendency to increase the body weight of mice (P>0.05), enhanced the activities of serum alanine aminotransferase and aspartate aminotransferase and the level of serum creatinine, and decreased the level of serum total protein (P<0.05). O‐Tyr administration changed systemic metabolic processes, including inhibited energy metabolism and the biosynthesis of protein, increased oxidative stress, elevated metabolism of vitamin‐B3, altered gut microbiota functions, decreased choline bioavailability. In addition, O‐Tyr administration also impaired liver and kidney function, lead to fat accumulation in liver. We concluded that metabolisms of vitamin‐B3 and choline together with gut microbiota may played crucially important roles in the deleterious effects of O‐Tyr. These findings provided a comprehensive and holistic perspective on the safety of O‐Tyr, and revealed the need for reduce the generation of oxidation protein products in food production process by improving the processing methods.Support or Funding InformationThis research was supported by program of “Collaborative innovation center of food safety and quality control in Jiangsu Province”, the National Natural Science Foundation of China (No. 31571841), State Key Laboratory of Food Science and Technology of Jiangnan University in China (SKLF‐ZZB‐201609), and China Postdoctoral Science Foundation (2015M571669).
Purification and Crystallization of the C‐terminal Interaction Domains of TRPP Ion Channel Proteins
Authors: Martin Hannah , Agbaje Zainab , Yu Yong 
Published: November 2023

Members of the transient receptor potential (TRP) protein family of ion channels are essential for various cell signaling pathways throughout the body. Proteins in the polycystin subfamily of TRP channels (TRPP) form receptor‐ion channel complexes with members of the polycystic kidney disease (PKD) protein family in 3:1 stoichiometry (3TRPP:1PKD). TRPP2 forms a flow‐sensing complex in primary cilia with its PKD partners that determines left‐right asymmetry in the embryonic node and proper morphology of kidney tubules. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD), which affects over 400,000 Americans and often leads to end stage renal failure. TRPP3, with its binding partner PKD1L3, is a sour taste receptor candidate found in taste cells in the tongue. The C‐terminus of the TRPP channel has been shown to be essential for channel assembly and function. In both TRPP2 and TRPP3, the C‐terminal coiled‐coil domains form a homotrimer and play a key role in the assembly of the TRPP/PKD complexes. Another C1 domain was found in TRPP3 that helps direct homomeric assembly. TRPP2 also has a calcium binding EF‐hand motif involved in regulation of channel activity, and an ER‐retention motif that is important for its trafficking from the ER to the cell membrane. Although the structures of the coiled‐coil domains of both proteins and the EF‐hand motif of TRPP2 have been solved separately, we still don't have an overall picture of how the different domains and motifs interact and assemble with each other. Recently, a cryo‐EM structure of TRPP2 has been reported. However, it lacks the C‐terminus. Clearly, the assembly of these proteins is multi‐faceted and significant for their function, and resolving the structure of that assembly is a necessary step towards understanding the function of the protein in vivo. Our preliminary data show oligomer formation of these proteins. In this structural study, we selected several C‐terminal fragments of all three TRPP proteins based on secondary structure prediction and functional importance, and expressed and purified them using E. coli. Our goal is to crystallize select members of the TRPP family and determine their structures through X‐ray diffraction. This study will elucidate the interaction and assembly of the C‐terminal domains of TRPP proteins and the relevance of key amino acid residues to channel function.Support or Funding InformationThis work was supported by NIH Grant DK102092.