bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and Functional Characterization of an F17‐Like CUP Adhesin from Uropathogenic E. coli Isolate UTI89
Authors: Klein Roger Davies , Spaulding Caitlin , Hultgren Scott
Published: November 2023

Adhesion to biotic and abiotic surfaces is an essential step in the infectious cycle of many human pathogens. To this end, both Gram‐positive and Gram‐negative bacteria have developed a variety of strategies to overcome innate clearance mechanisms and persist within the host. The expression and biogenesis of Chaperone‐Usher Pathway (CUP) pili represent one such strategy. In addition to mediating the formation of bacterial biofilms, these proteinaceous extracellular appendages contain, at their tip, a two‐domain adhesin that is composed of an N‐terminal lectin domain and a C‐terminal pilin domain. The structure of the binding pocket within the lectin domain determines the specificity of that pilus for a given epitope. Individual Escherichia coli species encode and express up to 16 distinct CUP operons, each likely mediating a tropism for a specific habitat or environment within the host. While the importance of many of these pili, such as Type 1, P, and others, in human infection has been extensively elucidated, there still exist hundreds of pili across a broad range of bacterial species whose function has yet to be determined. Using a murine model of gastrointestinal infection, we have demonstrated that the F17‐Like (Ucl) pilus is an important virulence factor in the colonization of the gastrointestinal tract by E. coli species. Analysis of the distribution of this pilus type across E. coli clinical isolates reveals an enrichment in uropathogenic E. coli (UPEC) strains that cause recurrent urinary tract infection (rUTI). These findings suggest that the F17‐like pilus allows UPEC to persist in the gastrointestinal habitat before seeding the urinary tract. To probe the molecular mechanism of this attachment, we have obtained an X‐ray crystal structure of the adhesive domain of the F17‐Like pilus tip. Structural and biochemical comparison of this tip to other known bacterial adhesins involved in intestinal colonization indicates that the F17‐like pilus binds an epitope in the gastrointestinal tract that is distinct from both the Fim and F17 molecular receptor.

Support or Funding Information

This work was supported by Washington University MSTP Training Grant #5T32GM007200‐39, NIH/NIDDK R01 #AI048689, and the Department of Molecular Microbiology Sondra Schlesinger Graduate Student Fellowship
Structural and functional insights into σ1 receptor ligand binding
Authors: Schmidt Hayden Riley , Kruse Andrew C
Published: November 2023

Originally misclassified as an opioid receptor, the σ1 receptor is a single‐pass transmembrane protein with a diverse pharmacological profile. Though its precise biological function has proven elusive, the σ1 receptor is thought to play a role in neurodegenerative diseases such as Parkinson's disease and ALS, as well as neurological pathologies, with the most notable being addiction and depression. A rich repertoire of ligands have emerged for the receptor, including both compounds traditionally associated with other targets, including haloperidol or cocaine, as well as (+) benzomorphans such as (+)‐pentazocine or unique ligands like PD 144418 or ditolylguanidine. Many of these compounds have been classified as agonists and antagonists, but the molecular distinction between the two classifications is not entirely clear. Here, we employ a combination of biochemical, pharmacological, and structural techniques to examine the basis for σ1 receptor ligand binding and attempt to determine the molecular basis of agonism and antagonism for this enigmatic receptor.

Support or Funding Information

This work is supported by the National Science Foundation Graduate Research Fellowship awarded to HRS.
Stress‐triggered self‐association of an enzyme reverses its catalytic activity
Authors: Yoo Haneul , Goyal Rohan , Drummond D. Allan
Published: November 2023

A recent explosion of studies centered around phase‐separating proteins is revolutionizing our understanding of cell biology. These proteins can reorganize themselves into massive assemblies, which lack the defined stoichiometry of quaternary structures and therefore go beyond our concept of protein quaternary structure, and form membraneless compartments such as stress granules in response to changes in cellular environment. Most studies in this new field have been focused on probing physical properties of membraneless organelles and their constituent proteins. However, equally important questions arise regarding whether and how the biochemical activities of these proteins change upon reorganization and, if so, what regulatory roles they play.

We recently identified dozens of proteins that form quinary assemblies in response to heat shock in budding yeast. Consistent with the general idea of quinary assemblies and stress granules as a transient protein inactivation and/or storage mechanism, most of the identified proteins also experience transcriptional repression during heat shock. However, our attention was drawn to one enzyme that forms quinary assemblies but is translationally upregulated during heat shock, hinting that the self‐assembly of this enzyme unlocks its normally concealed enzymatic activity needed during stress. We have successfully reconstituted the in vitro self‐association of this enzyme, diadenosine tetraphosphate (Ap4A) phosphorylase. Using this system, we explore the relationship between the enzymatic activity and the physical transition of the enzyme from soluble monomers to quinary assemblies.

Support or Funding Information

Molecular and Cellular Biology Training Grant (T32 GM007183)
NMR Studies of Ubc9 Mutant Identify Structural Basis for SUMO Target Selection
Authors: Placzek William J , Bjornsti Mary‐Ann , Whitaker Robert H , Wright Christine , Onuiri Joshua
Published: November 2023

Background

The regulated biosynthesis, trafficking, interaction, and degradation of proteins are essential for normal cell growth. SUMO is a conserved ubiquitin‐like protein that is conjugated to a multitude of cellular proteins and mediates these functions. The transient SUMO modification (SUMOylation) of proteins affects protein function, allowing cells to respond to environmental stress without the need for de novo protein synthesis. Not surprisingly, dysregulation of SUMO conjugation has been directly implicated in the pathogenesis of human cancer and in modulating cellular responses to chemotherapeutics. Yet, the underlying determinants of SUMO target specificity.

Materials and Methods

SUMO conjugation is accomplished by an ATP‐dependent cascade of enzymes, which includes a single activating enzyme (SAE1/2 heterodimer), SUMO‐1 or SUMO‐2/3, and a single conjugating enzyme (Ubc9). We used yeast genetics, biochemical SUMO conjugation assays, and NMR spectroscopy to define the basis for alterations in substrate selection and SUMO chain formation.

Results

Our studies suggest that the altered substrate specificity induced by the Ubc9P123L mutant enhances cell sensitivity to a wide range of DNA damaging agents. NMR analyses of wild‐type and mutant Ubc9 enzymes have identified critical flexibility in Ubc9 necessary for SUMO target selection. In parallel, biochemical and genetic studies demonstrate the impact this dysregulation of Ubc9 function has on SUMO conjugation and cell viability.

Conclusions

Our results provide new insights into the structural features of Ubc9 that underlie substrate selection and the impact of SUMO chain linkages on cell viability. These findings constitute a paradigm shift in our understanding of SUMOylation and the potential contribution of SUMO chain linkages to tumorigenesis and chemotherapeutic drug response.
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 Information

Work was funded by the USD Knapp Chair and the USD McNair program.
Investigation of cellular signaling and epigenetic dynamics via optogenetic control of nuclear cytoplasmic distribution
Authors: Yumerefendi Hayretin , Kuhlman Brian
Published: November 2023

The ability to control nuclear cytoplasmic distribution of proteins in vivo with spatial and temporal precision would allow us to probe the functions of a broad range of proteins and uncouple their roles in cellular signaling. Here we report achieving reversible, blue light‐dependent control of nuclear import and nuclear export by engineering a nuclear localization and export signals into the Light Oxygen Voltage (LOV2) domain of phototropin 1 from Avena sativa. We validated predictions first in mammalian cells and in yeast. The Light Activated Nuclear Shuttle (LANS) and the Light Induced Nuclear eXporter (LINX) interact with the endogenous nucleocytoplasmic transport machinery for rapid nuclear import and export in mammalian cells, yeast and C. elegans embryos. In yeast, LANS allowed robust control of transcriptional activation via light‐induced nuclear accumulation of a transcription factor demonstrating its potential to activate nuclear proteins. Similarly, we demonstrated the use of LINX for transcriptional repression in yeast by ejecting a transcription factor out of the nucleus. Moreover, fusing the chromatin modifier Bre1 to LINX, we achieved light‐dependent control of histon H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. This inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.
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 Information

Supported 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 Information

Supported 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 Information

NIH K01 CA140711 NIH RCMI #8G12MD007600 Seed Funds from University of Puerto Rico Comprehensive Cancer Center U54CA096297
Effects of Aspirin on Expression of Proteins Implicated in Airway Remodeling in Human Lung Fibroblasts
Authors: Geary Kyla , Hasenmeyer Don , Daghigh Farzaneh
Published: November 2023

Airway remodeling (AR) in asthma is characterized by increased airway smooth muscle mass, subepithelial fibrosis, epithelial changes, and increased numbers of fibroblasts and myofibroblasts. Established AR is resistant to current asthma therapies and can lead to irreversible airflow limitation, thereby increasing the severity of asthma. We hypothesized that L‐arginine metabolism is altered in AR, ultimately affecting collagen synthesis which may be evidenced by upregulation of expression of genes involved in arginine metabolism. We investigated expression levels of key regulatory enzymes in the arginine pathway: arginase 1, arginase 2, ornithine decarboxylase (ODC), and ornithine aminotransferase (OAT). mRNA levels were detected by reverse‐transcriptase quantitative polymerase chain reaction (RT‐qPCR). Cell immunochemistry and/ or enzyme‐linked immunosorbent assay (ELISA) were utilized to identify protein levels in human lung fibroblast (HLF) from normal (NHLF) and diseased (DHLF) asthmatic donors. ELISA was performed with nuclear, mitochondrial, and cytosolic protein fractions. Cells were treated with acetylsalicylic acid (ASA), a known trigger of symptoms for aspirin‐induced asthma, for 24 hours. Arginase 1 mRNA was not detected. ASA upregulated arginase 2 mRNA 5‐fold and 7‐fold in NHLF and DHLF, respectively after 24 hours. ODC and OAT mRNA were not significantly altered by treatment. Cell immunochemistry experiments indicated arginase 1, arginase 2, ODC, and OAT proteins were present in NHLF and DHLF with or without ASA treatment. Arginase 2 levels were further analyzed via ELISA. In NHLF and DHLF mitochondrial fractions, increased levels of arginase 2 were observed after 24 hours of ASA treatment compared to untreated cells. In the NHLF and DHLF cytosolic fractions, there were decreased levels of arginase 2 compared to untreated cells. In the nuclear NHLF and DHLF fractions, we observed increased arginase 2 after 24 hours of treatment compared to untreated cells. Both mRNA and protein levels of arginase 2 increase in human lung fibroblasts after 24 hours of aspirin treatment. As arginase 2 may be involved in airway remodeling during the development of asthma, limiting exposure to aspirin may have an effect on lowering the rate of airway remodeling, and thus have an effect on asthma development. Further investigation into the role of the arginase pathway in aspirin‐intolerant asthma needs to be pursued.
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 Information

This 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.

Methods

We 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 Aims

By 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.
Abscisic Acid/Dormin, a Plant Hormone that Inhibits Angiogenesis in vitro and Neovascular Growth in vivo
Authors: Chaqour Julienne Y , Lee Sangmi , Chaqour Brahim
Published: November 2023

Vessel development and regeneration by angiogenesis is critical for adequate oxygen and nutrient delivery to every organ in the body. However, the concept of anti‐angiogenesis therapy has previously been proposed due to the important role of blood vessels in solid tumor growth and dissemination. Non‐neoplastic disorders associated with numerous proliferative diseases (e.g. diabetes or chronic inflammation) also are promoted by pathological angiogenesis that leads to blinding diseases. Current anti‐angiogenic treatments are based on the use of synthetic drugs, many of which, not only cause serious side effects without correcting the underlying pathology, but also have an unfavorable cost to benefit ratio. The objective of this study is to explore the anti‐angiogenic potential of abscisic acid (ABA), a natural plant hormone that inhibits germination from occurring when the environmental conditions are not favorable and contributes to the acquisition of stress tolerance. Using a fibrin beads‐based sprouting assay, we show that ABA effectively inhibits in a dose‐dependent manner, migration, growth and expansion of endothelial tubes without affecting cell viability. In the in vivo model of postnatal retinal angiogenesis, administration of ABA affects neither the differentiation and normal development of the retinal vasculature nor the growth of the animals although it incidentally increases fetal hyaloid vessel persistence. In the mouse model of oxygen‐induced retinopathy (OIR) characterized by a hyperoxia‐induced vaso‐obliteration phase and a subsequent ischemia‐induced neovascular growth, administration of ABA reduces aberrant growth of retinal vessels during the ischemic phase of OIR without altering the normal vascularization of the retina. Thus, as a natural vascular stabilization signal restricting neovascular growth, ABA is an anti‐angiogenic molecule with potential translational implications in vascular diseases of the eye.
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 Information

This 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 Information

This 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 Information

This 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).
Understanding the Effects of Arginine Kinase Gene Deletion on Mitochondrial Volume in Caenorhabditis elegans
Authors: Beal Megan Anne , Fraga Dean , Timar Kathryn
Published: November 2023

The Arginine Kinase‐Phosphoarginine (AK‐PAg) system serves an analogous function to invertebrate systems as the Creatine Kinase‐Phosphocreatine (CK‐PCr) system does to vertebrates. Comparative studies found CK and AK enzymes exist in similar cellular regions within the organism, in “burst” muscles that depend on glycolysis for energy production. Structurally, the AK and CK enzymes resemble each other as well. Gene knockout experiments in mice highlighted the significance of the CK‐PCr system to a vertebrate's response to burst activity as the knockout mice had a decrease in peak twitch force, but were still able to react. It was seen that these knockout mice had an increase in mitochondrial volume, which suggested the cell tries to compensate for the CK loss by increasing mitochondrial volume and content. I plan to test this in an invertebrate system by imaging mutant C. elegans in which an AK gene has been knocked out to see if they compensate by increasing their mitochondrial volume. This will be accomplished using fluorescence microscopy, and complemented by a Real‐time PCR analysis as a secondary measure to determine if there is an increased mitochondrial volume and content. This comparative analysis will help us better understand how invertebrate and vertebrate systems have evolved to manage energy homeostasis by investigating how they compensate for a similar perturbation in the phosphagen kinase systems.
Subcellular Localization and Functional Characterization of cAMP‐dependent Protein Kinase A Isoforms: Painting Specificity by Mosaic Brain Mapping
Authors: Ilouz Ronit , Ram Varda Lev , Ellisman Mark , Taylor Susan S.
Published: November 2023

cAMP dependent Protein kinase (PKA) plays a critical role in numerous neuronal functions. Specificity in PKA signaling is achieved in part by the four functionally non‐redundant regulatory (R) subunits. The inactive holoenzyme has a dimeric R‐subunit bound to two Catalytic (C) subunits. The full‐length holoenzyme crystal structure allowed me to understand how isoform‐specific assembly can create distinct holoenzyme structures that each defines its allosteric regulation. High‐resolution large‐scale mosaic images provide global views of brain sections and allow identification of subcellular features. Analysis of multiple regions demonstrates that the R‐isoforms are concentrated within discrete regions and express unique patterns of subcellular localization. Using correlated light and electron microscopy I confirmed mitochondrial and nuclear localization of RIb isoform, modifying the existing dogma of cAMP‐PKA in the nucleus. To show the functional significance of nuclear localization, I demonstrated that RIb down‐regulation, but not RIIb, decreased CREB phosphorylation in primary neuronal cultures, consistent with deficits observed in RIb mutant mice. Furthermore, I show that a point mutation in the RIb gene, found in patients diagnosed with a neurodegenerative disease, abolishes dimerization while retaining robust interaction with its catalytic subunit. As a consequence, the interaction with A Kinase Anchoring Proteins (AKAPs) is also abolished. Quantitative mass spectroscopy analysis of patient samples identified a set of proteins that were up or down regulated in this disease. This point mutation further emphasizes the importance of precisely controlled PKA isoform subcellular localization as defined by my mosaic maps.
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 Information

This work was supported by NIH Grant DK102092.
The Nucleotide Exchange Factor Sil1 Modulates Redox Signaling through the Molecular Chaperone BiP
Authors: Siegenthaler Kevin , Pareja Kristeen , Wang Jie , Sevier Carolyn
Published: November 2023

The endoplasmic reticulum (ER) is responsible for the secretion of approximately one third of the eukaryotic proteome. The oxidizing environment within the ER lumen allows for the formation of disulfide bonds present in many secreted proteins. However, oxidative protein folding generates hydrogen peroxide, which can over oxidize the ER and disrupt protein folding. Since protein flux through the ER can vary widely in a cell depending on developmental phase and environmental factors, the oxidative status of the organelle can fluctuate. It is critical that cells maintain the fidelity of protein folding despite the fluctuating redox environment. One way cells do this is by regulating the activity of the lumenal Hsp70 chaperone, BiP. When hydrogen peroxide accumulates in the ER, a conserved cysteine residue in BiP is oxidized to cysteine sulfenic acid. This decouples the normally ATPase driven chaperones nucleotide hydrolysis cycle from its peptide binding, resulting in constitutively higher peptide binding by the chaperone. BiP therefore tightly binds translocating proteins and maintains them in a folding‐competent state until oxidative stress levels subside.

While BiP oxidation is protective during periods of oxidative stress, constitutive modification decreases cell fitness, raising the question of how BiP is reduced post stress. Unexpectedly, we have found that Sil1, one of BiP's nucleotide exchange factors, is able to reduce BiP and restore progression through the chaperones ATPase cycle. Sil1's reductase activity is tractable to a catalytic cysteine pair near the proteins N‐terminus and is distinct from its activity as a NEF. We are currently studying the role Sil1's reducing activity plays in the cells oxidative stress response and how this connects to human health. Interestingly, mutations in the Sil1 gene are associated with the cerebellar ataxia Marinesco Sjögren syndrome (MSS), suggesting loss of Sil1's reducing activity may cause ER dysfunction that manifests itself in a multi‐system disorder.

Support or Funding Information

R01 GM105958 T32 GM007273
A Novel Mediator of Heart Failure Development and Progression
Authors: Miriyala Sumitra , Panchatcharam Manikandan , Chandra Mini , Maxey Benajamin , Orr wayne , Harrison Lynn , McCarthy Kevin , Bhuiyan Shenuarin , Kevil Christopher
Published: November 2023

Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese Superoxide Dismutase (MnSOD), an antioxidant enzyme that catalyzes the conversion of superoxide radicals (O2•−) in mitochondria. Absence of SOD2 (gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, MnTnBuOE‐2‐PyP5+ distributes 3‐fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE‐2‐PyP5+ to dismute O2•− parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Myh6‐SOD2Δ). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O2 consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito‐Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Myh6‐SOD2Δ to the wild type. We then examined OXPHOS levels in Myh6‐SOD2Δ v.s. Wild type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was significantly lower in Myh6‐SOD2Δ cardiomyocytes than that in wild type. The most noticeable difference in the O2 consumption was found in the presence of FCCP (H+ ionophore/uncoupler). FCCP is an inner membrane pore opener which resets the proton gradient between the mitochondrial matrix and the interspace, resulting in continuous transport of protons and consuming O2 at the maximum potential. Remarkably, while the FCCP treatment increased O2 consumption in wild type, the treatment showed no effect on the O2 consumption in the Myh6‐SOD2Δ cardiomyocytes. The result indicated that the low basal OXPHOS activity in Myh6‐SOD2Δ was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidification (ECAR) and the pattern exactly opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Myh6‐SOD2Δ and Wild type.
Synthesis of a Novel Endoperoxide and Evaluation of the Drug's Ability to Induce Apoptosis in Colon Cancer Cells
Authors: Devlin Jason R , Gornick Emily S , Vavrek Tyler R , Rubush David M , Sarathy Jayashree
Published: November 2023

Artemisinin is currently the frontline treatment for malaria and it has recently demonstrated potential as an anticancer drug. The key pharmacophore in artemisinin is an endoperoxide which generates ROS in the presence of reducing agents. It has been shown to work by decreasing cell proliferations, inducing apoptosis and cell death, and might involve ROS generation in intracellular compartments. In this study, in an effort to probe this reactivity and develop more potent endoperoxides, we developed a simple and economical synthesis of novel endoperoxide containing small molecules. Our unique methodology combines imines and peroxyquinols to form novel 1,2,4‐dioxazinanes in a one‐pot cascade reaction. 1,2,4‐dioxazinanes are a largely unexplored class of molecules with potential anticancer activity. One therapy to control cancer with minimal side effects would be to use non‐toxic natural products or their derivatives that could cause cell death by inducing apoptosis. Therefore, the efficacy of one of the synthetic derivatives, namely DMR‐1, to induce apoptosis in cancer cells was studied using a human colon carcinoma cell line, T84. Cells were grown to confluency in 12 well plates in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle medium supplemented with 2.5 mM L‐‐glutamine and fetal bovine serum, 5%. Cells treated with increasing doses (5 – 100μM) of DMR‐1 in serum‐free media for 18 hrs were labeled with Annexin V and propidium iodide to assess apoptosis and cell death using an Accuri Flow cytometer. We report that DMR‐1 increased apoptosis at concentration as low as 5μM; however, there was no dose‐dependent increase in apoptosis [AnnexinV positive cells, in %; Control: 9±1; 5 μM: 20±0.4; 10 μM: 15±2; 50 μM: 12±1; 100 μM: 11±1]. Other artemisinin derivatives have been shown to be more effective in rapidly growing cell lines and at higher concentrations (>200 μM). T84 cells are slow growing cells and also have a tendency to secrete mucin, thereby requiring higher doses. Therefore, our future studies will include using higher doses of DMR‐1 and also studying the effect of DMR‐1 (and other derivatives synthesized in our laboratory) on rapidly growing cancer cells such as A549 lung cancer cells. If DMR‐1 is found to be effective in inducing apoptosis in these cells, the mechanism of action will be determined.

Support or Funding Information

This research is supported by NSF ‐ MRI: DBI‐1427937 and Institutional Funds, Ben. U. to JS and to DMR