bluuebunny/crossref_metadata_2025 huggingface dataset:
Metagenomic Analysis of Bacterial Communities in the Rhizosphere of Leguminous Crops and Trees.
Authors: Ahrenhoerster Carissa , Prasad Gyaneshwar , Martinez‐Vaz Betsy
Published: November 2023

Legumes account for 27% of the world's primary crop production and contribute 33% to the dietary protein needs of humans. Important legume crops include clover, soybeans, alfalfa and peas. These plants are notable for their ability to form nitrogen‐fixing symbioses with bacteria of the genera rhizobia. These organisms live in the rhizosphere (the region of soil in the vicinity of plant roots) of legumes. Symbiotic nitrogen fixation in legume crops takes place in plant structures known as root nodules. Recent research suggests that certain tree species of the Leguminosae family do not form nodules but still possess nitrogen fixation capabilities (Bryan, 1996). The main objective of this study was to investigate the microbial communities associated with the rhizosphere of nodulating legume crops (alfalfa, clover and soybeans) and non‐nodulating leguminous trees (Honeylocust and Redbud). We hypothesized that examining the microbial communities in the rhizosphere of these plants will provide insights into the diversity of N2‐fixing microbes and the mechanisms of nitrogen fixation in non‐nodulating legumes. Total DNA was extracted from the rhizosphere and bacterial community analyzes were performed by sequencing the V4 region of the 16S rRNA gene. QIIME and UPARSE were used to analyze the sequencing reads and select operational taxonomic units (OTUs). A total of 441,587 qualified reads were obtained from the eight rhizosphere samples. Rarefaction curves obtained with the normalized OTUs number nearly reached saturation level for all the samples, indicating that bacterial communities from the rhizospheres were well covered by the sequence analysis. Phylogenetic analysis showed that Proteobacteria was the most abundant phylum in the rhizosphere of legumes covering 45–70% of the amplicons detected in the samples analyzed. Bacteriodes was the second major phylum followed by Acidobacteria and Actinobacteria. Regarding bacterial classes, the alphaproteobacteria and betaproteobacteria were abundant in crops while gammaproteobacteria and alphaproteobacteria were prevalent in leguminous trees. Principal component analysis indicated that bacterial communities could be separated into two groups according to their association with crops or trees. Rhizobium species were present in the rhizosphere of all the legumes analyzed. The presence of rhizobial species in the rhizosphere of non‐nodulating leguminous trees suggests that these microbes might have a role in the nitrogen fixation mechanisms utilized by these plants. This study provided valuable insights about the distribution of bacteria in the rhizosphere of agriculturally relevant leguminous crops and trees.
Chemical and Genetic Analysis of Hopped Meads
Authors: Halmo Jason R. , Wolyniak Michael J. , Mueller Paul H.
Published: November 2023

Hops (Humulus lupulus) have been used in alcoholic beverages for centuries to enhance the aroma and provide a bitter flavor. The rise of homebrewing and craft beers has provided a resurgence of interest in understanding ideal hop usage. While hopped beer is well known and understood, the medieval drink Mead is gaining in popularity and is relatively not as well known. The aroma characteristics of commonly used hops are widely known, but much less is understood about the aroma effects in meads. This study aims to provide information on Volatile Organic Compounds (VOCs), which make up the aroma of the drink, found in the Head Space (HS) of six different homebrewed meads: S. cerevisiae, S. cerevisiae with Citra hops, S. cerevisiae with Williamette hops, S. bayanus, S. bayanus with Citra hops, S. bayanus with Williamette Hops.

Additionally, common strains of Baker's (S. cerevisiae) and Wine (S. bayanus) yeast have been used to produce commercially lucrative food and drink products. Despite their heavy usage, a great deal remains to be understood how the fermentation pathway can be maximized. The emergence of RNA‐seq allows for rapid detection of changes in RNA during fermentation. This study aims to explore changes in gene expression of the aforementioned yeast during fermentation conditions mimicking that of mead.
Response of the Energetic Metabolism of Saccharomyces cerevisiae to Different Nutritional Conditions of Carbon and Nitrogen
Authors: Olivares‐Marin Ivanna Karina , Madrigal‐Pérez Luis Alberto , Canizal‐Garcia Melina , González‐Hernández Juan Carlos , Regalado‐González Carlos
Published: November 2023

The energetic metabolism of Saccharomyces cerevisiae is oxidative/fermentative, and its balance can be altered by different factors such as oxygen supply and nutrient homeostasis. Carbon (C) is known as the main nutrient affecting alcoholic fermentation in S. cerevisiae, allowing fermentation as the principal pathway for energy production even in the presence of oxygen when high levels of fermentable sources are available (Crabtree effect). In addition, nitrogen (N) starvation can lead to sluggish or stuck fermentation, and the presence of certain N sources may enhance sugars consumption, associating in this way nutritional status with alcoholic fermentation. However, little is known about nutrients interactions and their effect on fermentation. The objective of this work was to investigate the impact of different C and N sources and their concentration, on the fermentative growth of S. cerevisiae. Fermentative and respiratory phenotypes of S. cerevisiae BY4742 was studied using the doubling time (Dt) to discriminate among phenotypes from 81 different conditions of N and C in duplicate, using five independent experiments. We used synthetic‐complete (SC) medium supplemented with glucose, sucrose or galactose used as C source, and proline, glutamate and ammonium as N source. Our results showed that the energetic metabolism of S. cerevisiae is directed toward fermentation or respiration depending on the type of carbon and nitrogen source and their concentrations. As expected, glucose supplementation resulted in a large number of conditions with a fermentative phenotype (19%), followed by sucrose utilization (17%). Cultures supplemented with galactose showed a fermentative phenotype only at 2% (w/v) when proline or glutamate was used as N donor (4%). The best N source was proline, irrespective of its concentration and the C source used in the culture media. Ammonium showed cellular toxicity when a concentration of 5% (w/v) was used in the cultures, and galactose supplementation showed more severe toxicity. Glutamate showed an intermediate number of fermentative phenotypes relative to proline and ammonium, despite of its quality as N donor. Our findings suggested a possible energetic role for proline that could be affecting fermentation induction.

Support or Funding Information

This worked was supported by a grant of the Consejo Nacional de Ciencia y Tecnología [grant number 427081] to IKOM.
Decreased Insulin Signaling Causes Loss of PFK‐2 and Impaired Glycolysis in the Heart
Authors: Humphries Kenneth , Bockus Lee , Eyster Craig
Published: November 2023

The heart has a dynamic capacity to orchestrate energy production with contractile demand. Normally, beta‐adrenergic signaling works through the cAMP‐dependent protein kinase (PKA) pathway to coordinate moment‐to‐moment changes in both contraction and metabolism. The main metabolic target of PKA is phosphofructose kinase‐2 (PFK‐2), which generates fructose‐2,6‐bisphosphate in response to PKA mediated phosphorylation. This metabolite is a potent allosteric activator of PFK‐1 and serves to increase glycolysis. We have previously found that PFK‐2 is unresponsive to PKA activation in the diabetic heart and we hypothesize that this may contribute to metabolic inflexibility. Despite the central role of PFK‐2 in cardiac metabolism it is unknown how its content and activity changes in either the healthy or diabetic heart. We have recently found that in the absence of insulin signaling there is a decrease in cardiac PFK‐2 content. This is true in types 1 and 2 diabetic models and with fasted animals, suggesting turnover of PFK‐2 is a normal glucose‐sparing response. Functionally, metabolomics studies have revealed that the loss of PFK‐2 is accompanied by an increase in glycolytic products upstream of PFK‐1, and a decrease of products downstream of PFK‐1. Ongoing mechanistic studies in primary adult mouse cardiomyocytes have identified that PFK‐2 levels are regulated by multiple means. In the absence of insulin PFK‐2 is rapidly degraded by both proteasomal and chaperone mediated autophagy mechanisms. Thus, the loss of PFK‐2 content as a result of reduced insulin signaling impairs its ability to dynamically regulate glycolysis. Although this may be beneficial in the fasted state to conserve systemic glucose, it represents a potential pathological impairment in diabetes.

Support or Funding Information

This work is supported by the National Institutes of Health grant R01HL125625.
Protein Modifying Enzyme Ate1 Controls Cellular Warburg Effects
Authors: Jiang Chunhua , Birnbaum Michael , Patel Devang , Kumar Akhilesh , Morgan William , Lampidis Theodore , Fontanesi Flavia , Barrientos Antonio , Zhang Fangliang
Published: November 2023

Protein arginylation by arginyl‐transfer RNA protein transferase (Ate1) is a post‐translational modification that targets a number of metabolic enzymes. However, the mechanisms and downstream effects of this modification in cellular metabolism are unknown.

In this study, wild type mouse embryonic fibroblast cells (MEF‐WT) and Ate1 knockout mouse embryonic fibroblast cells (MEF‐KO) were used to measure several key metabolic parameters including glucose uptake, glucose starvation sensitivity, lactate production, cellular oxygen consumption rate (OCR). Ate1 is also down‐regulated by shRNA to repeat some of the above experiments. The results showed that Ate1‐KO MEF is more sensitive to glucose starvation than MEF‐WT, and that KO cells have significant higher glucose uptake and lactate production than MEF‐WT. Conversely, Ate1‐KO cells have lower cellular OCR than MEF‐WT. While the WT and Ate1‐KO cells have similar levels of cellular ATP, a significantly larger proportion of ATP in KO cells are derived from the glycolysis pathway. We further showed that this phenomenon can be repeated when Ate1 is down‐regulated by shRNA in WT cells, and that this phenotype can also be reversed when a recombinant Ate1 protein is reintroduced in the KO cells.

CONCLUSION

Our data suggest that knockout of Ate1 in MEF shift cellular metabolic pathways from mitochondrial oxidative phosphorylation to aerobic glycolysis, which is the sign of the Warburg effect.

Support or Funding Information

NIGMS (NIH), R01, # GM107333.

DoD (CDMRP), Idea Award, PC140622

DoD (CDMRP), Exploratory Grant, PC141013
UCP1 is essential for mitochondrial structural integrity and function in brown adipose tissue
Authors: Riley Christopher L , Boutz Daniel , Bean Camilla , Kohno Shohei , Tioli Gaia , Genova Maria Luisa , Scorrano Luca , Mills Edward L
Published: November 2023

With the recent discovery of functional brown adipose tissue (BAT) in humans, there is a renewed interest in harnessing this thermogenic organ for the therapeutic treatment of diabetes and obesity. BAT activation increases energy expenditure and BAT mitochondria, through the actions of uncoupling protein 1 (UCP1), are integral to this energy wasting process. In response to adrenergic stimulation, UCP1 uncouples the proton pumping actions of the electron transport chain from ATP synthesis releasing the stored chemical energy as heat. While this phenomenon has been intensely investigated, surprisingly little is known about how BAT mitochondria distinctively adapt to the expression and activation of UCP1, which makes up a large percentage of BAT mitochondrial proteome. By taking advantage of UCP1‐null animals in combination with bioenergetic profiling and transmission electron microscopy, we provide clear evidence that UCP1 is necessary to maintain mitochondrial structure and function in brown fat. Furthermore, using novel ‘complexomic’ and proteomic profiling we demonstrate that mitochondrial supercomplex formation and stability in BAT requires UCP1. Finally, we show that the loss of UCP1 induces cellular stress, mitochondrial structural alterations and mitochondrial autophagy in an activation dependent manner. Taken together these data suggest that UCP1 is necessary for optimal mitochondrial function and health in brown adipose tissue and call into question any direct mitochondrial mechanism for heat generation in brown fat lacking UCP1.
Genetic and Functional Characterization of the Enzymes of Nicotinic Acid Degradation in Bacillus niacini
Authors: To Tsun Ki Jerrick , Snider Mark J.
Published: November 2023

N‐Heteroaromatic compounds (NHACs) are byproducts of fuel processing and are common components of herbicides, pesticides and pharmaceuticals that have been identified as environmental contaminants. A current approach to removing NHAC contaminants is bioremediation, which involves the use of bacteria and their degradation systems. Optimizing bioremediation efforts requires the understanding of different degradation strategies that have evolved in bacteria. The microbial degradation of nicotinic acid serves as a model system, the details of which can be translated to improving bioremediation of NHACs. Analysis of the recently sequenced genome of Bacillus niacini has putatively identified two gene clusters that collectively code for a distinct degradation pathway of nicotinic acid. This study describes the genetic characterization of key enzymes involved in the B. niacini pathway following the initial hydroxylation steps of the pyridine ring through gene knockouts and complementation studies. Further, reaction assays of the enzymes catalyzing the decarboxylation‐hydroxylation and the tripyridol ring‐opening steps will be established to confirm reaction substrates and products and to provide the platform for future mechanistic studies of these novel reactions.

Support or Funding Information

This work was supported by the Department of Chemistry and the Henry J. Copeland Fund for Independent Study at The College of Wooster.
Administration of NaoXinTong to db/db mice inhibits the development of diabetic nephropathy
Authors: Yang Shu , Liu Mengyang , Su Peng , Han Jihong
Published: November 2023

Buchang NaoXinTong capsule (NXT), a Chinese Materia Medica standardized product extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinennsis, Astragali Radix, is clinically used for treatment of patients with cerebrovascular and cardiovascular diseases. Clinically, it has been observed that NXT can demonstrate anti‐diabetic properties with unclear mechanisms. In this study, we used a pro‐diabetic animal model, db/db mice, to investigate if NXT can inhibit the development of diabetic nephropathy. The type 2 diabetic mice (~6‐week‐old) were randomly divided into two groups (10 mice/group), and started feeding normal chow and normal chow containing NXT, respectively, for 14 weeks. We collected blood and urine samples during the treatment, and kidney, adipose tissue and liver samples at the end of the experiment, respectively, for determination of serum glucose or lipid levels, renal functions as well as the involved mechanisms. We found that NXT reduced diabetes‐increased glucose, total and LDL‐cholesterol and triglyceride levels in the serum. NXT also reduced urinary protein levels. In the kidney, we determined that NXT inhibited mesangial matrix expansion, expression of vascular endothelial growth factor A (VEGFA), fibronectin and advanced glycation end (AGE) products while it repaired the diabetes‐induced podocyte injury and dysfunction by increasing expression of WT‐1 and nephrin. In addition, NXT inhibited renal accumulation of extracellular matrix (ECM) proteins by increasing matrix metalloproteinases (MMPs) expression. Mechanistically, we determined that NXT inhibited TGFβ/Smad signaling pathway and CTGF expression in kidney. Furthermore, we observed that NXT improved glucose metabolism in the liver and increase FGF21 expression in both liver and white adipose tissue, which is accompanied by increased expression of genes responsible for energy metabolism in the liver. Taken together, our study demonstrates that NXT inhibits the development of diabetic nephropathy by ameliorating glucose metabolism, maintaining kidney tissue structure and blocking the diabetes‐induced renal dysfunction. Our study further suggests the potential application of NXT for diabetes treatment in clinic.

Support or Funding Information

The National Science Foundation of China Grants 81473204 to J Han and 81573427 to Y Duan
Identifying a Source of Beta‐Alanine and its Broader Implications in Arabidopsis thaliana by GC/MS
Authors: Perrett Morgan , Gothard Megan , Ludwig Andrea , Rouhier Kerry A
Published: November 2023

β‐alanine, a naturally occurring non‐protein amino acid, serves as a precursor for the formation of vitamin B5 and, ultimately, coenzyme A in plants. The synthesis of β‐alanine has not been explored extensively in plants despite interest in the development of alternative methods for producing natural compounds. Previous work in plants showed that β‐alanine could be synthesized from propionyl‐CoA. Our lab studies the degradation of branched‐chain amino acids (BCAAs) ‐ valine, leucine, and isoleucine ‐ in Arabidopsis thaliana and the degradation of valine and isoleucine are direct sources of propionyl‐CoA. Therefore, we proposed that valine and isoleucine could serve as a source of β‐alanine via the production of propionyl‐CoA. Using metabolic tracing with radiolabeled precursors measured by Gas Chromatography/Mass Spectrometry (GC/MS), we were able to measure β‐alanine via isoleucine but not valine. However, valine treatment of young seedlings did result in increased levels of β‐alanine, which suggested an indirect link through amino acid homeostasis. Additionally, we used a mutant seedline that lacks expression of an enzyme necessary for valine and propionyl‐CoA degradation (mmsd‐1) to further emphasize the link between β‐alanine and propionyl‐CoA by shifting the equilibrium towards β‐alanine production. Here, we found that seeds had significantly high levels of β‐alanine compared to wild‐type seeds.

Support or Funding Information

This project was funded by the Jean Dreyfus Boissevain Lectureship for Undergraduate Institutions and Kenyon College.
Time‐resolved analysis of amino acid deprivation responses reveals dynamic relationship between GCN2 and mTORC1
Authors: Nikonorova Inna A , Goudie Michael P , Mirek Emily T , Wang Yongping , Anthony Tracy G
Published: November 2023

In higher eukaryotes, sensing of amino acids by liver is critical for fine tuning of whole body metabolism according to nutrient availability. Amino acid (AA) depletion activates General Control Nonderepressible 2 (GCN2, or eIF2AK4) kinase and consequential phosphorylation of eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 allows for preferential translation of specific mRNAs, such as activating transcription factor 4 (ATF4). AA depletion also reduces the activity of another AA sensor, mammalian target of rapamycin complex 1 (mTORC1), a major regulator of cell growth. The GCN2 pathway inhibits mTORC1 activity, but how remains unclear. Our study aimed to define the role of GCN2 in the regulation of mTORC1 activity to AA depletion in vivo. We used the anti‐cancer drug asparaginase (ASNase) as a molecular tool to induce AA depletion in wild type (WT) and Gcn2−/− mice. Following a single injection of ASNase, hepatic signaling via GCN2 and mTORC1 was analyzed. In this experimental setup we addressed three types of possible regulation: 1) immediate protein‐protein interactions (15–30 min), 2) early translational changes (1–3 h) and 3) long term transcriptional alterations (6–18 h). We found that genetic disruption of GCN2 lead to hyperactivation of hepatic mTORC1 at 15–30 min following ASNase. This indicates that GCN2 modulates hepatic mTORC1 activity via protein‐protein interaction(s). Examination of translational changes via polyribosome profiling revealed enhanced translation of ATF4 in WT, but not Gcn2−/− livers, at 1h. This suggests that: 1) translational induction of ATF4 by ASNase requires GCN2 and 2) hyperactivation of mTORC1 per se is not enough to induce ATF4 synthesis. Analysis of transcriptional changes induced by ATF4 revealed that Sestrin2, an inhibitor of mTORC1, is induced at a much later time point,12–18 h following ASNase, and is not involved in immediate or early control. Taken together, our data provide a mechanistic framework for dynamic interaction of the GCN2 pathway with mTORC1 during amino acid stress.

Support or Funding Information

This work was supported by NIH grant RO1HD070487 to T.G.A. and NIH K12 grant IRACDA New Jersey‐New York for Science Partnerships in Research and Education to I.A.N.
Hormonal Regulation of Glycine Metabolism And Its Potential Role in Diabetes Susceptibility
Authors: Jog Ruta , Wang Jian , Leff Todd
Published: November 2023

Type II diabetes (T2D) is characterized by insulin resistance, β cell dysfunction and impaired glucose tolerance. Multiple large‐scale metabolomics studies have demonstrated that diabetes is accompanied by alterations in amino acid metabolism. Interestingly, in both humans and animal models, circulating glycine levels are strongly reduced in obesity, glucose intolerance and T2D. These observations identify glycine as an important biomarker of T2D, and raise the possibility that altered glycine metabolism may play an active role in the development of the disease. The goals of our study are to determine if altered glycine metabolism plays a role in the etiology of diabetes, and to characterize the regulatory pathways that control plasma glycine levels. Glycine cleavage system (GCS), a multimeric enzyme complex, represents the only biochemical route that degrades glycine in human body. Human genetic evidences indicate that loss‐of‐function mutations of GCS cause hyperglycinemia and neurodevelopmental disorders resulting in early death. Given the key role of GCS in control of circulating level of glycine, we hypothesize that during progression of T2D, GCS‐mediated glycine catabolism may be altered in main metabolic organs including liver. Here, we show that in hepatoma cell lines both insulin and glucocorticoids strongly stimulate transcription of glycine decarboxylase (GLDC) gene that encodes the rate‐limiting component of GCS. We also show that the insulin responsive transcription factor sterol‐regulatory element binding protein‐1c (SREBP‐1c) regulates GLDC gene transcription and likely mediates the stimulatory effect of insulin. SREBP‐1c is known to mediate insulin's stimulatory effect on lipogenesis genes even in the presence of hepatic insulin resistance. These observations strengthen a working model that insulin‐mediated stimulation of GLDC, even in insulin resistant states, increases the activity of the glycine cleavage system and contributes to the low levels of glycine observed in T2D.

Support or Funding Information

Michigan Diabetes Research and Training Center Pilot/Feasibility Grant
Cloning and Characterization of Putative Histidinol‐Phosphatase not called by Machine Annotation
Authors: Ellis Samuel
Published: November 2023

The generation of genomic information has increased exponentially in the past decade, and while this has aided biological research immensely, it has also been shown to be problematic. The information being collected is compiled at a rate much faster than it can be interpreted, and this has led to potential annotation errors in genomic databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a widely used genomic database that records compilations of biochemical pathways. KEGG uses the genome of the bacterium Escherichia coli as a reference genome for some of these pathways. The biosynthesis of the amino acid histidine is a complex, multistep process that requires the use of a multitude of enzymes to catalyze a sequence of biochemical reactions. E. coli utilize a fused, bifunctional imidazoleglycerol‐phosphate dehydratase/histidinol‐phosphatase encoded by the HisB gene to catalyze the sixth and eighth steps in the pathway. However, other bacteria may be able to catalyze these steps using two separate proteins instead of a single fused protein similar to the one utilized by E. coli. The deep‐sea autotrophic bacterium Thiomicrospira crunogena has been proposed to have evolved to catalyze these two steps in this manner by using a separated pair of proteins, encoded by the Tcr_0356 and Tcr_1966 genes, instead of a fused protein. As a consequence, when the genome of T. crunogena was compared against the reference genome, KEGG determined that this organism cannot perform the phosphatase activity in the eighth step and therefore machine calling a histidine auxotroph. By expressing and isolating the proteins and performing biochemical assays, evidence has been collected revealing a protein hypothesized to be a histidinol‐phosphatase. A malachite green phosphatase assay was used to assess the phosphatase activity of the protein encoded by Tcr_0356, while an IGP‐Dehydratase assay was used to assess activity of the protein encoded by the Tcr_1966 gene. The implications of this study further solidify the need for accurate annotations in databases in order to maintain the integrity of genomic data.

Support or Funding Information

This work is based upon ideas generated and supported by the National Science Foundation under Grant Number 0954829, funding the Microbial Genome Annotation network.
Identifying the Metal That Activates the Prenyltransferase That Catalyzes Formation of Geranyl Diphosphate in the Diatom Pseudo‐nitzschia multiseries
Authors: Tran Hai , Savage Thomas
Published: November 2023

Domoic acid (DA) is a neurotoxin synthesized by marine diatoms of the genus Pseudo‐nitzschia which causes amnesiac shellfish poisoning in humans and death of marine wildlife. Geranyl diphosphate (GPP), a key intermediate in domoic acid biosynthesis, is formed from the “head‐to‐tail” condensation of isopentenyl diphosphate and dimethylallyl diphosphate in a reaction catalyzed by a member of the prenyltransferase family of enzymes. These prenyltransferases can generate different chain‐length products, including geranyl diphosphate (C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20), all of which are precursors to terpenoid natural products. Prenyltransferases require a divalent metal cation for activity. Previous work has shown that a single prenyltransferase can generate multiple products (GPP, FPP, and GGPP), and that the product distribution will vary depending on the species of divalent cation used to support the reaction (Mg2+, Mn2+, or Co2+). Here, we measured the levels of Mg2+, Mn2+, and Co2+ in Pseudo‐nitzschia cells to determine which ion is most likely to support GPP formation. Three samples of Pseudo‐nitzschia multiseries cultured in sea water were harvested by centrifugation. Samples were digested with HNO3 before analysis using atomic absorption spectroscopy. The average concentration of Mg2+ present, per cell and per μm3, is over 100‐fold higher than the concentrations of Mn2+ and Co2+. This is consistent with results from studies in other systems, suggesting that Mg2+ is the main cofactor that activates the prenyltransferase that generates GPP for domoic acid biosynthesis in Pseudo‐nitzschia.

Support or Funding Information

This project is supported by the SURE (Summer Undergraduate Research Experience) Award Program from the College of Natural Science and Mathematics, California State University Sacramento.
Molecular Basis of TyrA Substrate Specificity Underlying the Evolution of Alternative Tyrosine Biosynthetic Pathways
Authors: Schenck Craig , Holland Cynthia , Schneider Matthew , Jez Joseph , Maeda Hiroshi
Published: November 2023

An aromatic amino acid, tyrosine, is essential for protein synthesis in all orders of life and also serves as a precursor to numerous specialized metabolites crucial for plant and human health (e.g., vitamin E, isoquinoline alkaloids, and plastoquinone). Most plants synthesize tyrosine via arogenate dehydrogenase (ADH/TyrAa) that is strongly feedback inhibited by tyrosine, whereas we recently found that soybean and Medicago truncatula have, besides ADHs, prephenate dehydrogenase (PDH/TyrAp) enzymes, which are completely insensitive to tyrosine. However, molecular and evolutionary mechanisms underlying TyrA substrate specificity and the two alternative tyrosine pathways are currently unknown. In this study, we identified non‐canonical ADHs (ncADHs), which are phylogenetically very similar to PDHs, but prefer arogenate substrate and are partially inhibited by tyrosine. By combining the x‐ray crystal structure of soybean PDH (GmPDH) and the phylogenetic distribution of PDH enzymes, we identified putative substrate specificity determining residues in the recently diverged PDH and ncADH homologs. Site‐directed mutagenesis of Asn222 on GmPDH, which corresponds to an Asp in soybean ncADH (GmncADH) resulted in loss of PDH and gain of ADH activity. Consistently, the reciprocal mutation on GmncADH, D218N, introduced PDH at the expense of ADH activity. The introduction of an Asp residue on GmPDH also resulted in gain of tyrosine inhibition, which allowed co‐crystallization of the GmPDH N222D mutant with tyrosine in the active site and demonstrated that Asp222 directly interacts with the side chain amine of tyrosine and likely arogenate. Furthermore, mutating the corresponding Asp on divergent plant ADHs resulted in a gain of PDH activity and relaxation of tyrosine inhibition. Thus, the corresponding Asn of PDHs and Asp of ADHs simultaneously control substrate specificity and tyrosine regulation, which can be altered in various plants to enhance the production of tyrosine and its derived plant natural products.
Does sensitivity to FACs result in higher resistance against herbivory?
Authors: Conner Allie , Grisset Laquita , Stratmann Johannes
Published: November 2023

Nicotiana sylvestris, N. knightiana, N. tabacum and N. benthamiana are members of the tobacco family, two of which have displayed sensitivity to fatty acid‐amino acid conjugates (FAC's) present in the salivary secretions of herbivorous insects, including the tobacco hornworm, Manduca sexta. We have determined through mitogen‐activated protein kinase (MAPK) assays that N. benthamiana and N. tabacum, but not N. sylvestris and N. knightiana, have MAPK activity when exposed to FAC's and we will test the hypothesis that FAC‐sensitive species are better defended against herbivory. M. sexta larvae on N. benthamiana and N. tabacum are expected to be smaller with a higher rate of mortality due to the plants' ability to detect FAC's as danger signals and then produce anti‐digestive proteins that inhibit growth. Inversely, we expect M. sexta larvae on N. sylvestris and N. knightiana to grow faster and have a lower rate of mortality as they lack sensitivity to FACs and thus a means to induce a defense response.
cDNA cloning and characterization of UDP‐glucosyltransferase from Indigofera tinctoria
Authors: Inoue Shintaro , Morita Rhito , Thul Sang T , Sarangi Bijaya K , Minami Yoshiko
Published: November 2023

Indigofera tinctoria is a cultivated plant that is utilized as a source of the natural blue dye, indigo. The plant produces a colorless secondary metabolite known as indican (indoxyl β‐D‐glucoside). Following its released from the cells, indican is enzymatically degraded into indoxyl and glucose. Indoxyl is an unstable compound that is easily oxidized and dimerizes to produce the stable indigo. Unlike the degradation reaction of indican, there is limited knowledge about indican biosynthesis pathway in the cells. We have previously reported that Polygonum tinctorium, an alternative indigo‐producing plant, has UDP‐glucosyltransferase (UGT) that catalyzes the synthesis reaction of indican from indoxyl and UDP‐glucose. However, there are several subjects that need to be studied including the cDNA cloning, the subcellular localization, the transport pathway of substrate and product during indican metabolism, etc. We hypothesize that the indican metabolic pathway in Indigofera is similar to that in Polygonum. Here, we report cDNA cloning and characterization of UGT from Indigofera tinctoria. The sample containing UGT that was partially purified from Indigofera leaves was separated on SDS‐PAGE gel. After staining the gel with CBB, several bands were cut off and analyzed by peptide mass fingerprinting. We identified two fragments homologous to UGT from the transcriptomic data that was generated from Indigofera leaf tissue. Based on the sequence of the fragments, RACE method was performed, leading to amplification of two types of full‐length Ugt cDNAs, itUgt1 and itUgt2. The polypeptide sequence of 477 amino acids encoded by itUgt1 shows high homology (max identity is 89%) with the 475 amino acids encoded by itUgt2. Their primary structures contain the motif of UDP‐glucoronosyl and ‐glucosyl transferase and show the high homology to some UGTs from other plants. To analyze the properties and functions of itUGT1 and itUGT2, the recombinant proteins were expressed in E. coli and purified. The antibody was raised against recombinant itUGT2. The recombinant itUGT1 and itUGT2 show high activity of indican synthesis as reported for the Polygonum enzyme. Although there was no difference between values of optimum pH, itUGT2 was more stable with temperature variations than itUGT1. Further, the expression of itUGT proteins and mRNA levels in various tissues from Indigofera were examined by Western blot and semi‐quantitative RT‐PCR, respectively. Consequently, it was observed that UGT1 synthesizes indican in Indigofera tissues and not UGT2. Further work on cell fractionation, for determining the subcellular localization of itUGTs, is in progress
Tyrosine Biosynthesis Revisited: Characterization of novel arogenate dehydrogenases from legumes
Authors: Schneider Matthew R. , Schenck Craig A. , Holland Cynthia K. , Jez Joseph M. , Maeda Hiroshi A.
Published: November 2023

The aromatic amino acid tyrosine (Tyr) serves the central role as both a building block for proteins and a precursor to a wide variety of derived secondary metabolites. An increasing interest in Tyr‐derived plant natural products for their application to industry and human health has cited the need to revisit and gain a better understanding of the Tyr biosynthetic pathways in plants. It has previously been shown that Tyr synthesis in plants occurs through tightly feedback regulated arogenate dehydrogenase (ADH/TyrAa) enzymes. However, recent research has discovered that some legumes, including Medicago truncatula, have prephenate dehydrogenase (PDH/TyrAp) enzymes, which deviate from this model by acting on a different substrate, prephenate, and do not show sensitivity to Tyr feedback inhibition. A phylogenetic analysis of PDH enzymes discovered a new group of ADH enzymes which show significantly relaxed sensitivity to Tyr inhibition. Phylogeny/structure‐guided site‐directed mutagenesis identified a single active site residue in these ADHs, which could be mutated to confer PDH activity and reduce Tyr sensitivity. The reciprocal mutation of the corresponding residue in M. truncatula PDH enzymes was found to reduce and increase affinity for prephenate and arogenate, respectively, and introduce Tyr sensitivity. These results suggest a biochemical link between PDH activity and sensitivity to Tyr feedback inhibition and represent a potential target for metabolic engineering to optimize Tyr biosynthesis and increase the production of Tyr‐derived metabolites in plants.
Characterization of Mutant Sunflower Acetoacetyl CoA Thiolase
Authors: Dyer James
Published: November 2023

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.
Lipoprotein Lipase regulates the expression of genes responsible for cellular cholesterol uptake and efflux in human and mouse macrophages.
Authors: Moctar Khadija , Madhwani Kimberly Rose R. , Kim Aidan J.M , Medh Jheem D.
Published: November 2023

Background

Cellular cholesterol accumulation is the net result of receptor‐mediated cellular uptake and transporter‐mediated efflux. Scavenger receptors (CD36, SR‐AI, SR‐BI) mediate the cellular uptake of cholesterol whereas the ATP‐binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. SR‐BI also mediates bidirectional cholesterol transport. Lipoprotein lipase (LPL) is secreted by macrophages and catalyzes the hydrolysis of circulating triglycerides. Macrophage LPL expression was shown to be inversely correlated with ABCA1 levels. We hypothesized that LPL may regulate the expression of macrophage scavenger receptors as well.

Objective

To examine if the expression of macrophage scavenger receptors and cholesterol transporter are regulated by LPL. Regulation of gene expression was studied in LPL‐knock‐down THP‐1 macrophages and LPL‐over‐expressing mouse macrophages. Additionally we are preparing recombinant wild type and mutant LPL to determine which structural components of LPL modulate cholesterol efflux.

Methods

THP‐1 monocytes were cultured and were induced to differentiate into macrophages with phorbol 12‐myristate 13‐acetate. Thioglycolate‐stimulated macrophages were isolated from the peritoneal cavity of transgenic mice with macrophage‐specific LPL overexpression. The expression of CD36, SR‐AI, SR‐BI and ABCA1 was compared in normal and LPL‐(THP‐1) or LPL+ (mice) macrophages by RT‐PCR analysis. b‐actin served as a control for RNA mass. Gel images were quantified by ImageJ analysis.

Results

Silencing of the LPL gene in THP‐1 macrophages correlated with a significant increase in ABCA1 expression (161% of WT). This was corroborated in mouse macrophages since overexpression of LPL resulted in a 50% decrease in ABCA1 expression. The expression of scavenger receptors SR‐BI and CD36 was significantly lower in LPL‐THP‐1 cells compared to WT macrophages (55% and 60%, respectively, compared to WT). Contrary to results in THP‐1 cells, LPL‐overexpressing mouse macrophages showed a decrease in SR‐BI levels (20% of WT), but no change in CD36 expression. The expression of SR‐AI was not influenced by LPL silencing or overexpression.

Conclusion

Manipulation of LPL expression modulates the transcription of genes relevant to cholesterol accumulation in isolated THP‐1 cells. In LPL+ transgenic mice, a decrease in SR‐BI may be due to the influence of other organs in cholesterol homeostasis and SR‐BI's role in cholesterol efflux.

Support or Funding Information

Acknowledgements: This work was supported by National Institutes of Health Awards R15HL083946, SC3GM095413, GM008395 and GM063787.
Cardiolipin Content has a Stronger Influence than Acyl Chain Composition on Select Membrane Properties of Biomimetic Mitochondrial Membranes
Authors: Pennington Edward R
Published: November 2023

Cardiolipin (CL), the hallmark phospholipid of the mitochondria, plays a fundamental role in maintaining mitochondrial inner membrane structure and function. In several ailments including cardiovascular diseases and Barth Syndrome, in which mitochondrial dysfunction contributes towards disease pathology, there is loss of CL content and remodeling of CL's acyl chains. However, it remains unclear how CL content and acyl chain composition impact the biophysical lipid organization of the inner mitochondrial membrane and thereby protein activity. Therefore, we discriminated how CL content and acyl chain composition influence distinct membrane properties of biomimetic mitochondrial membranes. In monolayer studies, the loss of tetralinoleoyl [(18:2)4] CL promoted lipid‐lipid miscibility, while replacement of (18:2)4CL acyl chains with tetraoleoyl [(18:1)4] CL or tetradocosahexaenoyl [(22:6)4] CL had no influence on lipid‐lipid miscibility. Replacement of (18:2)4CL acyl chains with tetramyristoyl [(14:0)4] CL, which has limited biological relevance for humans, promoted lipid‐lipid immiscibility. Supporting studies in bilayers revealed that substitution of (18:2)4CL with (18:1)4CL or (22:6)4CL lowered the phase transition temperature of phosphatidylcholine vesicles, whereas (14:0)4CL had no effect. Finally, quantitative confocal imaging of giant unilamellar vesicles (GUVs) showed that loss of CL content had a stronger influence than remodeling of CL acyl chains. Taken together, we propose that loss of (18:2)4CL content may have a stronger influence on select biophysical properties of membranes than CL acyl chain composition, which has implications for mitochondrial inner membrane structure/function and the design of CL‐specific therapeutics.

Support or Funding Information

Research was supported by NIH R01HL123647 (D.B. and S.R.S), an East‐West Collaboration Award (S.R.S. and A.K.), NIH R15HL122922 (D.B. and S.R.S.), Brody Brothers Foundation Award 213147 (S.R.S.), NIH K25GM100480 (S.R.S. and P. Wang), and NIH R01AT008375 (S.R.S.).
Impact of altering the headgroup‐acylated glycerophospholipid levels on Escherichia coli cells deficient in phosphatidylserine and phosphatidylethanolamine
Authors: Oke Oluwadamilola , Garrett Teresa A
Published: November 2023

Escherichia coli contains a variety of glycerophospholipids (GPLs). Phosphatidylethanolamine (PE) is particularly important as it is the only zwitterionic phospholipid and makes up 70–80% of total GPLs. It is formed from CDP‐diacylglycerol by the action of phosphatidylserine synthase (Pss) and phosphatidylserine decarboxylase (Psd). Pss condenses CDP‐diacylglycerol and serine to form phosphatidylserine (PS) and releases cytidine monophosphate. PS is a short‐lived intermediate that is rapidly decarboxylated to PE by the Psd enzyme. This study investigates the impact of altering headgroup‐acylated GPLs (HAGPLs) has on E. coli that are deficient in Pss and Psd. Overexpression of the E. coli genes pldB and pagP or the Arabidopsis thaliana gene At1g78690, elevates the levels of the HAGPL acyl phosphatidylglycerol (acyl PG). The overexpression of the human calcium independent N‐acyl transferase (iNAT) increases the levels of the HAGPL N‐acyl phosphatidylethanolamine (N‐acyl PE) in the cells. We transformed E. coli with temperature sensitive mutations in pss and psd with plasmids that contain pldB, pagP, At1g78690 or iNAT behind an arabinose inducible promotor. For each strain, a variety of cellular phenotypes, such as temperature sensitivity, cell morphology, antibiotic resistance, membrane permeability, membrane fluidity and lipid composition, were determined. Expression of At1g78690 promotes reversion of the temperature sensitive phenotype of the pss mutant by, we hypothesize, creating conditions under which compensatory mutations are more likely to support high temperature growth. Expression of pagP results in a longer cell phenotype that will be further investigated using scanning electron microscopy. Additional experiments will use fluorescently‐tagged proteins involved in cell division to determine if the increased cell length is the result of cell division defects promoted by accumulation of HAGPLs.

Support or Funding Information

National Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659
Modulation of Endothelial Membrane Biomechanics by Oxidized Phospholipids: A Combined Experimental and Computational Approach
Authors: Ayee Manuela A.A. , LeMaster Elizabeth , Shentu Tzu Pin , Barbera Nicolas , Akpa Belinda S , Levitan Irena
Published: November 2023

Elevated concentrations of biologically active oxidized phospholipids (oxPCs) are known to exert various pathophysiological effects, including disruption of the endothelial barrier and induction of monocyte adhesion to endothelial cells. These effects are further implicated in a range of disorders, including certain cardiovascular, metabolic, and immunological diseases. The specific mechanisms by which oxPCs modulate membrane barrier function are poorly understood.

We investigated the differential influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, fluidity, elastic properties, and endothelial cell biomechanics both experimentally, using Laurdan multi‐photon imaging and atomic force microscopy (AFM) microindentation experiments, and computationally, using coarse‐grained molecular dynamics simulations. Specifically, we focused on two major oxidation products of 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine (PAPC), which are known to exert multiple pathophysiological effects. The oxidized phospholipids 1‐palmitoyl‐2‐(5‐oxovaleroyl)‐sn‐glycero‐3‐phosphocholine (POVPC ‐ aldehyde‐terminated) and 1‐palmitoyl‐2‐glutaroyl‐sn‐glycero‐3‐phosphocholine (PGPC ‐ carboxyl‐terminated) have been found to induce inflammation, cell proliferation, and cell death.

Our molecular simulations of multicomponent phospholipid bilayers predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails. We showed that for the analogous oxPCs incorporated in a phosphatidylcholine bilayer containing sphingomyelin, the carboxylic analog, PGPC, experienced more significant reorientation of the truncated tail and displacement of the oxidized lipid from the plane of the bilayer. These phenomena induced greater perturbation of the structure of the lipid bilayer than the aldehyde analog, POVPC. Decreased bilayer packing and increased water permeation was observed, which was consistent with Laurdan imaging results. Spectral analysis of bilayer undulations further indicated increased elasticity of bilayer systems containing both oxPCs. Computational predictions of larger membrane perturbation by PGPC corresponded with greater stiffness observed for PGPC‐treated endothelial cells measured by cellular elastic moduli using AFM. These analyses confirmed the paradoxical relationship observed between membrane softening by oxPCs and increased overall cell stiffness in the presence of POVPC and PGPC.

Support or Funding Information

NIH grants: T32 HL‐82547, HL‐073965, HL‐083298; AHA MWA Pre‐doctoral Fellowship: 14PRE20490156