bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the HECT Domain Ubiquitin Ligase Nedd4‐2
Authors: Todaro Dustin , Augustus‐Wallace Allison , Klein Jenny , Haas Arthur 
Published: November 2023

Nedd4‐2 is a WW domain‐containing HECT (Homologous to E6AP C‐Terminus) ligase best known for its role in targeting cell surface receptors, including ENaC in the distal kidney, for Lys63‐linked polyubiquitin chain‐mediated endocytic uptake and lysosomal degradation. The Nedd4‐like ligases, including Nedd4‐2, also function in the pathway for viral budding of Ebola and Marburg viruses, among others. By employing rates of 125I‐polyubiquitin chain assembly as a functional readout of ligase activity, we quantitatively explore the mechanism of Nedd4‐2 in biochemically‐defined assays. We demonstrate that Nedd4‐2 exhibits cooperative allosteric kinetics (n=1.8±0.7) with varying Ubc5B~125I‐ubiquitin thioester substrate concentrations ([S]1/2=103±29 nM; kcat=0.07±0.01 s−1), requiring a priori that the enzyme exist as an oligomer. Gel filtration analysis reveals that the active form of Nedd4‐2 migrates as a trimer of 250 kDa, larger than the predicted 110 kDa monomer, confirmed by dynamic light scattering. Successive addition of a dominant negative Nedd4‐2C922A active site mutant inhibits wild type Nedd4‐2 to limiting 30% activity, consistent with polyubiquitin chain assembly requiring intersubunit interactions within an intact trimer. Based on sequence and structural homology of the predicted Nedd4‐2 trimer to E6AP, we identify Glu646 and Arg604 as conserved putative catalytic residues by point mutagenesis. Our studies demonstrate Ca2+‐independent polyubiquitin chain assembly, contrary to previous reports of Ca2+‐dependent C2 domain inhibition of Nedd4‐2 activity. These findings led us to further investigate the role of the N‐terminal C2 domain on Nedd4‐2‐catalyzed chain assembly. Truncation of the C2 domain of Nedd4‐2 to yield Nedd4‐2ΔC2 leads to a 2‐4‐fold increase in kcat and loss of cooperativity. Exogenous C2 domain non‐competitively inhibits Nedd4‐2ΔC2 (Ki=68±0.93 μM) without restoring cooperativity, consistent with a modest Ca2+‐independent allosteric autoinhibition by the C2 domain via intersubunit interactions. Using the proteasomal subunit S5a as a model substrate in vitro, we demonstrate that Nedd4‐2 catalyzes S5a conjugation with apparent hyperbolic kinetics (KM=254±0.078 nM; kcat= 0.020±0.002 s−1), requiring the polyubiquitin chain assembly and substrate conjugation half reactions to be conformationally independent. The good agreement between kcat for S5a conjugation (0.020±0.002 s−1) and that for free polyubiquitin chain assembly (0.07±0.01 s−1) suggests that Nedd4‐2 does not discriminate between lysine donors and that both conjugation half reactions share similar transition states. Parallels to our recent observations with E6AP suggest mechanistic conservation with subtle differences based on distinct sequence and structural variations for Nedd4‐2. These findings support mechanistic conservation within the HECT ligase superfamily.Support or Funding Information[Supported by GM034009 to A.L.H]
Uptake and Lysosomal Delivery of Recombinant Human Alpha‐N‐Acetylglucosamine‐6‐Sulfatase to Mucopolysaccharidosis IIID Fibroblasts
Authors: Moen Derek R , Wang Daniel M , Zhang Xiaoyi , Kan Shih‐hsin , Wood Jill , Ekins Sean , Chou Tsui‐Fen , Dickson Patricia I 
Published: November 2023

Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha‐N‐acetylglucosamine‐6‐sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity >10,000 nmol/hr/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37°C and showed it was stable for over one month at 4°C in artificial cerebrospinal fluid storage buffer. Additionally we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33–65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale‐up is feasible in preparation for proof of concept studies in the MPS IIID knock‐out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families.Support or Funding InformationThis work was funded by Grant R41 NS089061‐01 from NIH NINDS
Examining FOXO1 and DNA Interactions through Computational and Direct Methods
Authors: McDaniel Brianyell , Cherlin Tess , Haynes Rashad , Mottamal Madhusoodanan , Anderson David , Johanson Kelly 
Published: November 2023

FOXO transcription factors are a subgroup of the FOX (forkhead) family and are important for regulation of cellular processes, gene expression, apoptosis, cell‐ cycle progression, and oxidative stress resistance. Several FOXO translocations have also been implicated in cancer development, with the most common being the PAX3‐FOXO1 translocation linked to the development of Alveolar Rhabdomyosarcoma (ARMS). All FOXO proteins share sequence similarity and a highly conserved DNA‐ binding domain (DBD). This DNA‐binding domain consists of three alpha helices accompanied by two winged domains; this domain binds the Daf‐16 binding element (DBE) and the insulin response element (IRE), with the crystal structure indicating that the majority of the DNA contacts are located within Helix 3. There have been reports in the literature of sequences flanking the IRE causing an increase in binding affinity; however, no mechanism has been reported to explain this effect. Using Molecular Dynamics Simulation (MDS) we observed that the number of direct interactions between protein and DNA increases over time. Interestingly, MDS showed that wing 1 has no contact with DNA at 0 ns but eventually makes contact with a region flanking the IRE consensus sequence at 10 ns. In order to verify the new interactions observed in the 10 ns model, we mutated several bases within the IRE and observed the effect of these mutations on the interaction between protein and DNA through Electrophoretic Mobility Shift Assays (EMSA) and intrinsic fluorescence. Mutation of several bases in the IRE consensus sequence as well as in the immediate 5′ and 3′ flanking regions decreased the interaction between FOXO1 and DNA. In support of our model, mutation of the new base contact identified with Wing 1 also decreased FOXO1 DNA‐binding. The truncated FOXO1 DNA‐binding domain found in the PAX3‐FOXO1 fusion was also examined by MDS, and was predicted to retain several contacts within Helix 3 as well as the newly identified contact in Wing 1. Using this information, we can further examine the DNA‐binding of PAX3‐FOXO1 to determine if the presence of flanking sequences surrounding the IRE consensus sequence also affect the affinity of the fusion protein for DNA. Knowledge of the contribution of the FOXO1 domain to PAX3‐FOXO1 DNA targeting is important for understanding the activity of this fusion protein and its role in ARMS development.Support or Funding InformationNIH SC2GM095430RCMI‐Molecular Structure and Modeling core facility (NIH‐RCMI 2G12MD007595‐06 )NIH 5RL5GM118966‐03RISE (R25GM060926)
Gene Expression Profiles of Various Rat Brain Regions Following Exposure to Soman
Authors: Gautam Aarti , Sowe Bintu , Donohue Duncan , Miller Stacy‐Ann , Hammamieh Rasha , Meyerhoff James , Pham Michael , Lumley Lucille , Jett Marti 
Published: November 2023

Soman (GD) is a highly toxic man‐made substance and a potential chemical weapon. It belongs to a class of chemicals known as organophosphorus (OP) nerve agents, which may cause seizures, respiratory failure, and death. Soman undergoes aging and is one of the more difficult nerve agent exposures to treat since field available oxime therapy is not effective against soman. Understanding the effects of soman is vital to developing a method of detection and treatment for exposure. Exposure to GD can consequently result in neuropathological abnormalities in various regions of the brain, including the hippocampus, piriform cortex, amygdala, thalamus, and hypothalamus. In this study, rats were exposed to either 0.8 LD50 GD or 1.0 LD50 GD versus a saline control group and evaluated for seizure activity. Rats were sacrificed and brain regions were harvested at 72 hours post‐exposure. Brain tissues were homogenized and RNA was isolated using TRIzol reagent. In order to explore the pathways and biological processes associated with GD exposure, transcripts in the aforementioned brain regions were assayed using Agilent's rat gene expression arrays. Detailed analysis is currently underway to compare the expression profile generated from different regions of the brain and to understand the differences in response to the variation in the concentration of GD exposure.DISCLAIMER “Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.”
Analysis of the expression of the Basigin subset of the immunoglobulin superfamily in mouse brain
Authors: Hall Tavia N , Ochrietor Judith D 
Published: November 2023

Basigin gene products are members of the immunoglobulin (Ig) superfamily and are structurally related to immunoglobulin molecules. Basigin protein possesses two extracellular Ig domains, a single transmembrane domain, and a cytoplasmic tail, whereas the splice variant Basigin‐2 protein possesses three extracellular Ig domains, a single transmembrane domain, and a cytoplasmic tail. There are two other members of the Basigin subset of the Ig superfamily – the Neuroplastins and Embigin. Neuroplastin gp55 and Embigin are similar in structure to Basigin and Neuroplastin gp65 is similar in structure to Basigin‐2. All five of these proteins are cell adhesion molecules within neural tissues and play various roles in development, structure, and function of those tissues. The purpose of the present study was to analyze the expression of the Basigins, the Neuroplastins, and Embigin during the development of the mouse brain. Mouse brains were obtained using an accepted protocol and RNA was isolated for use in quantitative reverse transcription polymerase chain reaction (q‐RT‐PCR) using primer sets specific for Basigin gene products, Neuroplastin gene products, and Embigin. The values obtained were normalized to 18s rRNA. It was determined that Neuroplastin gene product expression is ten times greater than that of Basigin or Embigin. Their relative expression levels remain the same throughout the life of the mouse. These data suggest that Neuroplastin gene products have a more prominent role in the structure and function of the mouse brain than the other two members of the subset of the Ig superfamily.Support or Funding InformationUniversity of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
Identification and purification of a serine protease involved in cell‐mediated immunity in fish
Authors: Matsuura Yuta , Yabu Takeshi , Shiba Hajime , Nakanishi Teruyuki 
Published: November 2023

Cell‐mediated immunity is crucial immune response against non‐self antigen such as allograft, tumor and virus infected cells. Non‐self antigens could be eliminated by effector cells (e.g. cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) with secretory and/or non‐secretory cell‐death pathways in mammal. Also in fish, cytotoxic effector cells have been characterized in lymphocyte subsets level; however, biochemical mechanisms of the killing remain unknown. Here we tried to identify the protease involved in the killing adopting ginbuna crucian carp which has been used as a nice model for the study on cell‐mediated immunity in fish.In order to identify the protease involved in the killing of allogeneic target cells, we performed cytotoxicity assays in the presence of serine or cysteine protease inhibitors using leukocytes of ginbuna crucian carp. Cytotoxicity was significantly inhibited by serine protease inhibitor, 3,4‐Dichloroisocoumarin: “DCI”. By contrast the cytotoxicity was partially inhibited by cysteine protease inhibitor. Then we tried to explore a substrate of the serine protease involved in killing activity, and we found that a protease activity against a peptide substrate “z‐Gly‐Pro‐Arg‐4‐Methylcoumaryl‐7‐amide (MCA) was inhibited by “DCI”. In addition, z‐Gly‐Pro‐Arg‐MCA cleaving activity was significantly enhanced by allo‐antigen stimulation. These results suggest that the serine protease which cleaved z‐Gly‐Pro‐Arg‐MCA substrate was involved in killing against allogeneic cells.In order to analyze the enzymatic function of the serine protease, we tried to purify the protease. Purification was sequentially performed on two steps of benzamidine‐sepharose and SP‐sepharose chromatography. The purified protease was visualized as a single protein band and a molecular mass was estimated to be approximately 27,000 Da by using SDS‐PAGE. The protease was certainly serine protease because it was not inhibited by cysteine, metallo‐ and aspartic protease inhibitors but serine protease inhibitor.In conclusion, we succeeded to purify the serine protease involved in the killing against allogeneic cells. Now we initiate proceeding to examine a function and to find endogenous substrate of the protease with the purified protease.Support or Funding InformationSasakawa Scientific Research Grant from the Japan Science Society
Investigating the GCN5 Histone Acetyltransferase Chemical Mechanism
Authors: Young Byron H. , Berndsen Christopher E. 
Published: November 2023

Enzyme catalyzed protein acetylation involves the transfer of an acetyl group to a lysine side chain within the substrate. This process serves many vital cellular purposes including the regulation of gene expression and controlling of the circadian rhythm. Defects in enzymatic acetylation have been linked to many common diseases such as cancer, insomnia, and anemia. Despite decades of research into the biological function of protein acetylation, the enzymatic mechanism of acetyl‐transfer is unknown. We have worked to characterize the mechanism of acetyl‐transfer utilized by the yeast protein acetyltransferase General Control Nonderepressible 5 (GCN5) using deuterium solvent isotope effects, Brønsted‐beta analysis, and competitive inhibition studies. These analyses have allowed us to characterize the occurrence of proton transfer during catalysis as well as gain a general knowledge of the transition state structure. These results will be coupled with QM/MM computational modeling of the GCN5 acetyl‐transfer reaction in order to gain an explicit understanding of the mechanism of catalysis. The collection of these results set the groundwork for further investigation into the catalysis mechanisms of other acetyltransferases and may aid in development of treatment for acetyl‐transfer associated illness.Support or Funding Information‐Jeffress Trust Awards Program in Interdisciplinary Research‐NSF REU: CHE‐1461175
Synthesis of Effective Water‐Soluble Fluorescein Dibenzyl Ether Cytochrome P450 Substrates
Authors: Stepler Cameron M , Gallivan Maxwell A , Wysocki Laura M 
Published: November 2023

Cytochrome P450 is a key family of enzymes in the breakdown of drugs and toxins in the body. We are interested in probing the activity of these enzymes with different organic ethers through a library of fluorescein diether derivatives. These compounds are hydrolytically stable and exhibit very low background fluorescence before unmasking with high contrast, making them ideal targets for sensitive assays. The first‐generation library revealed challenges related to solubility of the fluorogenic compounds in the desired assay. Through the design and synthesis of a variety of substrates, progress was made toward water‐soluble probes that display reasonable kinetics for evaluation and investigation of enzymatic activity.Support or Funding InformationThis work was supported by the Henry and Nellie Pence Trust.
Experimentally‐Induced Melanoma has an Heterogeneous Oxidative Metabolism and Induces Peri‐Tumoral Innervation Remodeling
Authors: Noriega‐Cisneros Ruth , Rosas‐Plaza F. Ximena , Ramírez‐Santos Jesús , Gutierrez‐Ospina Gabriel 
Published: November 2023

Conventional wisdom assumes that carcinogenesis is caused by mutations in genes involved in the control of cell proliferation and survival, replicative senescence, oxidative metabolism, cell adhesion, immune recognition and angiogenesis. Over the last two decades, however, mounting evidence supports that “healthy” leukocytes, endothelial and fibroblasts “invade” the extracellular matrix of tumors and provide soluble factors that promote malignancy. Somatic and autonomic innervation, however, are factors that have remained out of the equation in spite of the evidence that shows that malignant tumors may attract or repel them. In addition, various neuro‐messengers are now known to modulate cancer progression. This is why in this work we begin studying the pattern of autonomic innervation in melanoma, putting emphasis on the cytological relationship between innervation and tumor metabolic compartmentalization; it is known that innervation regulates oxidative metabolism in normal tissues.MethodologyNon‐metastatic, B16F0 melanoma cell line were inoculated subcutaneously (5×105 cells) in the back of C57BL/6 mice (n=5). Twenty days after inoculation, the mice were euthanized (pentobarbital 45mg/Kg of body weight) and perfused with buffered 4% paraformaldehyde. The excised tumors and the surrounding skin were then dissected and processed through histological procedures that allowed the tumor mapping of cytochrome oxidase and of cholinergic and catecholaminergic innervation.Results1) Oxidative metabolism was heterogeneous throughout the tumor. A trend for the tumor's core to display greater activity of cytochrome oxidase was observed. 2) Under normal conditions, cholinergic innervation was observed surrounding hair follicles. This innervation was absent in hair follicles adjacent to the tumor in melanoma‐inoculated mice. 3) Non‐inoculated mice showed numerous catecholaminergic fibers running throughout the dermis in association or not with blood vessels. This innervation qualitatively increased in melanoma‐inoculated mice. 4) No autonomic innervation was observed within the tumor. Only catecholaminergic fibers surrounded it.ConclusionsAutonomic innervation remodels significantly around the tumor. Melanoma likely prevents the autonomic innervation to “invade” it. No obvious associations between tumor metabolic activity and autonomic innervation distribution were observed.Support or Funding InformationThe work was supported by PAPIIT Project, UNAM IN203912‐3. NCR is a postdoctoral fellow supported by CONACyT (No. 247328).
Expression of Folate Transport Proteins are Decreased in Head and Neck Squamous Cell Carcinoma
Authors: Green Jacalyn M. , Cuadros Kayla , Goral Joanna , Jham Bruno , Guimaraes Eduardo Pereira , Hanneman João Adolfo Costa 
Published: November 2023

IntroductionHead and neck squamous cell carcinoma (HNSCC) is currently ranked tenth and eleventh in incidence and mortality, respectively, in the US. These cancers are often diagnosed at an advanced stage, making the prognosis poor for these patients. Cancers often have an increased need for folate, an essential vitamin that is involved in cell division and growth. Folate is transported into cells through three proteins: folate receptors (FR), reduced folate carrier (RFC), and proton coupled folate transporter (PCFT). Several other cancers have been found to have increased expression of at least one of these transporters. Changes in expression of these transporters have not been studied systematically in HNSCC.HypothesisCompared to normal cells, HNSCC cells will show different patterns of expression in at least one of the three folate transporters.MethodsPathological samples with head and neck cancer were obtained from fifteen patients; eleven (80%) were male and 4 (20%) were female. The average age was 54 years; the range was from 38 to 76 years old. Cadaveric samples of tongue were used as controls. Expression of FR, RFC, and PCFT in formalin‐fixed tissues was detected by immunohistochemistry. Patient records on age, sex, lesion location, symptoms, treatment and prognosis were collected. Statistical analysis was performed to determine the correlation of expression of the protein of interest and clinical data.ResultsNormal tongue samples demonstrated maximal expression of all three folate transport proteins in the oral epithelium with wide distribution of expression in all cell layers of the stratified squamous epithelium. In contrast, HNSCC samples generally demonstrated low intensity of expression of FR, RFC, and PCFT, with wide distribution of expression in the invading cancer cells. There were no observed differences related to gender or age in patterns of FR, RFC, and PCFT expression in HNSCC and control tissues.ConclusionThe majority of HNSCC showed decreased expression in folate transport proteins compared to normal tongue epithelial cells. This would be consistent with the idea that decreased folate availability might influence carcinogenesis through alterations in DNA methylation, and concomitant alterations in gene expression.Support or Funding InformationAcknowledgements: Kayla Cuadros was supported by the Midwestern University Biomedical Sciences Program and Joanna Goral, Jacalyn Green and Bruno Jham were supported by Midwestern University. We thank Alice Meyer and Sabiha Raziuddin for their technical assistance.