bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tau and α‐synuclein protein expression, purification, and their amyloid inhibitor discovery
Authors: Marcus Madeleine , Henderson Frances , Kelly Katherine , Umana Ariana , Velander Paul , Wu Ling , Xu Bin 
Published: November 2023

Alzheimer's and Parkinson's diseases (AD/PD) are two major neurodegenerative diseases. AD is characterized by the accumulation in brain of two types of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Prevalent evidence now indicates that the respective building blocks of plaques and tangles – oligomers of Aβ peptide and the neuron‐specific, axon‐enriched microtubule‐associated protein, tau, represent the most toxic molecular species contributing to AD pathogenesis. Similarly, alpha‐synuclein protein amyloid is a major constitute of Lewy body, protein aggregates that are the pathological hallmark of Parkinson's disease.Currently there are no effective drugs to prevent or cure AD and PD. Because of protein aggregates, including oligomers, mediate neuronal toxicity in AD/PD, small molecule aggregation inhibitors are attractive drug candidates. Many drug trials aimed at preventing or clearing Aβ pathology have failed in recent years. Investigations on tau yielded innovative targets backed by recent findings about the central role of tau in AD pathogenesis (1,2). Preliminary data from a phase 2 trial with the small molecule, methylene blue chloride, have shown some promise and thereby validate the potential of tau aggregation inhibitors (TAIs), but superior TAIs are needed.In this poster we will present our work on expression and purification of human tau and α‐synuclein proteins and their amyloid formation and inhibitor discovery. Tau and α‐synuclein are expressed as recombinant proteins in E. coli and were purified to homogeneity using multiple chromatographic steps. They are further validated by western blot analysis and mass spectrometry. We have developed multiple biochemical, biophysical, and cell‐based assays to monitor oligomer/amyloid formation and amyloid‐induced cytotoxicity assays in characterizing amyloidogenic proteins (3). We use ThT fluorescence‐based assay for semi‐high throughput library screens. Upon identification of “hit” compounds, we validate them with multiple secondary assays including transmission electron microscopy (TEM), photo‐induced crosslinking of unmodified proteins (PICUP), IC50 measurements, and cell‐based cytotoxicity assays. We will further perform mechanistic studies for these validated inhibitors.Support or Funding InformationThis work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, and Diabetes Action Research and Education Foundation (DAREF) to B.X.
Metal Induced Conformational Changes of Alpha‐Synuclein and the Role of Ambient Oxygen
Authors: Lucas Heather R 
Published: November 2023

Hypotheses surrounding the native conformational states of α‐synuclein (αS), which is known to be a key player in Parkinson's Disease (PD) pathology, have evolved in recent years to adopt both a monomeric and tetrameric form. Traditionally, the conformation of αS was described as an intrinsically disordered monomer that aggregates to form β‐sheet fibrils, potentially through oligomeric intermediates. Upon interaction with membranes, this monomeric conformation of αS is known to shift from its dynamic unfolded state to α‐helical. Tetrameric αS has been characterized as both α‐helical and aggregation resistant, likely a passive storage form of the protein. The pathological conformation of αS remains controversial but each functional fold and dysfunctional misfold must originate from the monomeric protein. Membrane‐protein interactions are thought to facilitate the transition of monomeric αS to the α‐helical tetramer due to the similarity in folding patterns.Physiological levels of cerebral biometals, such as copper and iron, change in various regions of the brain with aging and with advanced disease states. Metal dyshomeostasis has long been linked to PD but most of the work predated the discovery of the tetramer and the acceptance of αS as being post‐translationally acetylated at the N‐terminus. The latter has shown biophysical variation in regards to both αS conformational dynamics and metal coordination. The presented research will highlight metal‐induced changes in the folding pattern of αS and the distinguishable differences observed between various metal oxidation states. Unique changes are observed in the protein secondary structure and the hydrodynamic radius upon the introduction of stoichiometric quantities of individual metal ions based on circular dichroism and dynamic light scattering analyses respectively. Under oxidatively stressed conditions, metal‐bound αS can promote the generation of reactive oxygen species. Immunoblotting techniques have revealed that metal‐bound αS can follow distinct pathways towards toxic oligomers or β‐sheet fibrils depending on the metal. Furthermore, a clear involvement of ambient oxygen in the production of post‐translational modifications has been observed. Such metal‐induced conformational changes result in altered membrane interactions, which could have physiological implications. Taken together, these results contribute to the ongoing conversation within the field about the role of metals and αS in PD progression.Support or Funding InformationSupported by Virginia Commonwealth University, College of Humanities & Sciences
Optimization of the Paired Enzyme Assay Synthesizing UDP‐Xylose from UDP‐Glucose
Authors: Cook Matthew D , Culbertson Alan , Zabotina Olga 
Published: November 2023

UDP‐xylose (UDPX) is the substrate used for xylosylation in most organisms. Xylosyl residues are components of vital molecules in the cell walls of plants and bacteria and in the extracellular matrices of animals. Despite the proliferation of xylosylated compounds, UDPX is a markedly expensive chemical which limits research potential into these areas. It is known that the pair of enzymes UDP‐glucose dehydrogenase (UGD) and UDPX synthase (UXS) can catalyze the set of reactions leading from UDP‐glucose, a much cheaper chemical, to UDPX. This project optimized the enzyme assay of both UGD and UXS to improve the efficiency and rate of UDPX synthesis in order to develop an economical means of producing UDPX. Additionally, the kinetics of the previously uncharacterized UGD and UXS isoforms used in the assay were investigated.Enzyme assays were performed, systematically comparing the rates of reaction and final UDPX‐production efficiency under different assay compositions and component concentrations for both enzymes individually and then together. The UGD assay was optimized first. As a result of reacting UDPG to the product UDP‐glucuronic acid, two equivalents of NAD+ were reduced to NADH. The generation of NADH could be analyzed spectrophotometrically at a 340 nm wavelength. Results from optimization have assay efficiency approaching 100% and the rate of reaction has improved appreciably. The earliest assays reacted with a maximum rate of 1.4 × 10−5 umol UDPG/min/ug UGD. Optimized assays have improved to 1.0 × 10−3 umol UDPG/min/ug UGD, a nearly 100‐fold improvement. Work on UXS proceeds.Support or Funding InformationNSF‐MCB Grant # 1121163
Reactivity of Neuroglobin with H2S
Authors: Ruetz Markus , Kumutima Jacques , Filipovic Milos , Lehnert Nicolai , Banerjee Ruma 
Published: November 2023

H2S is toxic at high concentrations due to the inhibition of cytochrome c oxidase. However, it is also an important signaling molecule. Inside the cell, H2S is mainly produced by three enzymes, cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE) and 3‐mercaptopyruvate sulfur transferase (MST). The low steady‐state levels of H2S in tissues are maintained by the efficient oxidation of H2S by the mitochondrial sulfide oxidation pathway that converts H2S to thiosulfate and sulfate.[1]Recently, we reported that H2S can be cleared by a noncanonical sulfide oxidation reaction catalyzed by hemoglobin, the oxygen carrier in blood.[2] In this study, we report that neurogloin, a relatively recently discovered member of the globin family also exhibits the capacity for sulfide oxidation. In contrast to hemoglobin and myoglobin, the heme cofactor in neuroglobin is coordinated by two histidine residues. The function of neuroglobin, which is primarily expressed in neuronal tissue, is still unclear. It has been suggested that neuroglobin plays a role in protecting neuronal cell from cell death.In this study, we have elucidated the reactivity of human neuroglobin with H2S. Under aerobic conditions, H2S is oxidized by neuroglobin to hydropolysulfides and thiosulfate. EPR and resonance Raman spectroscopic analyses reveal that the heme cofactor is reduced during the reaction, following the initial formation of a ferric sulfide intermediate. To determine the effect of the bis‐histidine coordination on sulfide oxidation, the distal histidine was substituted by alanine by site directed mutagenesis to create a myoglobin‐like mutant. The H64A mutant shows a 4000‐fold increase in the bimolecular rate constant for sulfide binding as well as in the rate of formation of the oxidation products, thiosulfate and hydropolysulfides.
Spectroscopic insight into the mechanism of nickel‐substituted rubredoxin, a bioinspired hydrogenase mimic
Authors: Stevenson Michael James , Slater Jeffrey Worthington , Marguet Sean Conrad , Shafaat Hannah S 
Published: November 2023

Nickel‐substituted rubredoxin (NiRd) has been shown to serve as a functional mimic of nickel‐containing hydrogenases, able to produce hydrogen gas with high catalytic rates. In the native enzyme, the nickel center is coordinated by four cysteine thiolates. A similar environment is seen in NiRd, which represents the first functional model to reproduce this coordination motif. This 52‐residue protein is stable under a wide range of conditions, making it well‐suited for catalytic hydrogen production or in future fuel cells. Although much is known about the structure and function of native rubredoxin and its variants, the catalytic mechanism for hydrogen generation remains unknown. It has been postulated that NiRd may follow a similar mechanism to the well‐studied [NiFe] hydrogenase, with a terminal, rather than a bridging, hydride on the nickel(III) center. Using multiple spectroscopic methods, structures of key catalytic intermediates have been elucidated and compared to those reported for the native [NiFe] hydrogenase. This study of NiRd not only resolves the mechanistic details of an efficient, robust, bioinspired catalyst, but also provides insight into proposed intermediates of the [NiFe] hydrogenase.Support or Funding InformationThe Ohio State University, The Institute for Materials Research, The National Science Foundation
Investigation of the Substrate Specificity of L‐Idonate Dehydrogenase By Site‐directed Mutagenesis
Authors: Steiner Steve , Terpening Allison , McCurdy Chelsea 
Published: November 2023

The enzyme L‐idonate dehydrogenase (L‐IdDH) is an important enzyme in the pathway for the biosynthesis of tartaric acid from ascorbic acid in grapes. L‐IdDH is an NAD+/NADH‐dependent enzyme which catalyzes the conversion of L‐idonate to 5‐keto‐D‐gluconate. Analysis of the amino acid sequence indicates that L‐IdDH from Vitis vinifera is a member of the family of medium chain dehydrogenases/reductases and is 79% identical with the putative sorbitol dehydrogenase (SDH) from Arabidopsis thaliana. Despite the high degree of homology, each enzyme is specific for its substrate with neither enzyme showing activity toward the substrate of the other. One of the notable differences in the amino acid sequences of L‐IdDH compared to SDH is at position 42 where His is located in L‐IdDH in contrast to Tyr in SDH. It is possible that the cationic His in L‐IdDH is essential to the binding of the anionic substrates L‐idonate and 5‐keto‐D‐gluconate. For this study, site‐directed mutagenesis was performed to convert 42H to 42R and to 42Y in L‐IdDH. Assay results indicate that mutation of His (positive charge) to Arg (positive charge) causes a significant reduction in activity toward 5‐keto‐D‐gluconate and a mutation to Tyr (uncharged) at this position completely eliminates activity. These finding illustrate the critical role of 42H in the activity and substrate specificity of L‐IdDH.Support or Funding InformationThis work is supported by a grant from the Indiana Academy of Science.
Substrate specificity of the novel serine hydrolase, LipN, implicated in the virulence of Mycobacterium tuberculosis
Authors: Schemenauer Daniel , Johnson R. Jeremy 
Published: November 2023

Tuberculosis (TB) remains one of the most prevalent diseases in the world, infecting one third of the world's population. The causative agent, Mycobacterium tuberculosis, relies on a plethora of lipases to maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, especially as drug targets for the dormant state of M. tuberculosis. This research aimed to determine the substrate specificity, biochemical properties, and potential structural information of LipN, one proposed mycobacterial lipase. Initially, wild type LipN was expressed in E. coli, purified to homogeneity, and its substrate specificity characterized against a library of 35 latent fluorophore substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon chains with a kcat/Km over 105 M−1s−1 for its best single acetyl ester substrate and maintained high activity (kcat/Km > 104) with small polar groups, including esters and oxazole rings. LipN's preference for short nonpolar substrates (three carbons or less) and polar substituents suggests its physiological role as an esterase rather than a lipase. This specificity also suggests hydrogen‐bonding capabilities within the active site of LipN. To categorize the structural factors controlling this substrate specificity, nine variants of LipN were made with substitutions in predicted binding pocket and active site residues and their relative kinetics against the top three ester substrates were characterized. Based on this analysis, the serine and histidine residues of the catalytic triad were positively identified, with 100‐fold decreases in catalytic activity upon their substitution. The aspartate residue in the catalytic triad was tentatively assigned, as multiple aspartate substituted variants showed similar reductions in catalytic efficiency. Future work will be aimed at definitely assigning the catalytic triad as well obtaining a three‐dimensional structure of LipN to confirm the structural features controlling its strict substrate specificity.Support or Funding InformationFunded by NIH 1R15GM110641‐01A1
Elucidating the Mechanism of Thiol Oxidase Activity of a B12‐trafficking Protein
Authors: Li Zhu , Shanmuganathan Aranganathan , Ruetz Markus , Lesniak Nick , Koutmos Markos , Banerjee Ruma 
Published: November 2023

CblC is involved in an early step in cobalamin (or B12) processing following entry of the cofactor into the cytoplasm. CblC catalyzes glutathione‐dependent dealkylation of alkylcobalamin and reductive decyanation of cyanocobalamin (CNCbl). Recently, we found that Caenorhabdiitis elegans CblC (ceCblC) exhibits a robust thiol oxidase activity, converting GSH to GSSG accompanied by O2 scrubbing. This futile oxidative cycle is suppressed in wild‐type human CblC (hCblC) but significant in two of the pathogenic mutants, R161G and R161Q. In this study, we sought to understand the catalytic mechanism of the thiol oxidase activity of ceCblC using biochemical and structural analyses. We propose the following mechanism: i) the binding pocket of ceCblC is tighter than hCblC, which facilitates formation of a thiol‐ligated glutathionyl‐cob(II)alamin, (GS‐Co(II); ii) GS‐Co(II) is oxidized by superoxide ion to form glutathionyl‐cob(III)alamin which undergoes GSH‐dependent dethiolation catalyzed by ceCblC, iii) the product of dethiolation, cob(I)alamin, is subsequently oxidized in air to cob(II)alamin, and thereby, the redox cycling continues.Support or Funding InformationThis work was supported in part by the National Institutes of Health (DK45776) to RB and the American Heart Association (13SDG14560056) to MK
The Regulatory Subunit Type Iα of Protein Kinase A: A Study of Carney Complex and Acrodysostosis Mutations
Authors: Del Rio Jason C , Nielsen Cole R. , Taylor Susan S. 
Published: November 2023

cAMP‐dependent Protein Kinase A (PKA), a tetrameric holoenzyme comprised of two Catalytic (C) and one dimer of Regulatory (R) subunits, is the prototypical kinase in response to intracellular cAMP. Of the four functionally non‐redundant R subunits (RIa, RIb, RIIa, RIIb), RIa is the dominant, ubiquitously expressed isoform and is the only R subunit which is embryonic lethal in homozygous mouse knockout models due to defective cardiogenesis at E8.5. Recently, two rare phenotypically dichotomous diseases have been linked to mutations in the same gene encoding RIa. Hindering the capacity of RIα to bind and respond to cAMP results in overregulation (inhibition) of the C subunit, a rare condition called Acrodysostosis type I (ACRDYS1), where patients display severe skeletal defects, mental retardation, and hormonal resistance. In contrast, patients with gain of function mutations result in Carney Complex (CNC) disease, caused by under‐regulation of C subunit activity, and present with lentigenosis, recurrent cutaneous and cardiac myxomas, and a Cushings‐like endocrinopathy. While the majority of CNC mutations result in happloinsufficiency via nonsense mediated decay (NMD), the mutations which survive NMD implore multiple mechanisms for disease manifestation. Using these disease mutations with functional effects of opposite magnitude will provide insight into the allosteric communication networks present within the RIa subunit necessary to tightly regulate the critical and finely tuned PKA signaling node for biological processes. Furthermore, structural studies of these two rare diseases will provide a pharmacological platform for rational drug design in their treatment.Support or Funding InformationJCDR was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752
Development of Algorithmic Techniques for Designing Electrochemical DNA Biosensors
Authors: Bonham Andrew J , Bulow Aviva J 
Published: November 2023

Oligonucleotide‐based, electrochemical (E‐DNA) biosensors have been shown to be effective tools for detecting heavy metals, small molecule drugs, and protein targets. There is thus considerable interest in moving these biosensors from the lab bench to applied fields, such as medical, environmental, and food safety diagnostics. A significant hurdle to this transition is the development of effective methods to rapidly and effectively adapt E‐DNA biosensors to the detection of new targets of interest. While great strides have been made in computationally predicting the folded structures of DNA and RNA‐based biosensors, interpreting whether such predicted structures would be effective biosensors and using that knowledge to design functional E‐DNA biosensors ab initio remains challenging. To overcome this we have designed a tool, Fealden, which models potential E‐DNA biosensors using the framework of graph theory and automates the process of structure interpretation and sorting. Using this tool, a researcher can provide a core sequence of interest (such as a DNA recognition element or aptamer) and the software will generate optimized, functional E‐DNA biosensor designs for the sensitive detection of that target. For example, our approach has allowed order‐of‐magnitude improvement in the affinity of biosensors directed towards the antibiotic tobramycin, without human intervention in the design process. This software incorporates literature knowledge of oligonucleotide biosensor design constraints, such as the proximity of appended chemical moieties and predicted folding free energies, and can traverse a search space of thousands to millions of possible sequences to determine the “best candidate” biosensor. This work provides insights into applying an algorithmic approach to utilize the wide pool of existing aptamers as E‐DNA biosensors in practical applications spanning such diverse fields as food safety, environmental monitoring, and clinical diagnostics.Support or Funding InformationSupport for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
An Inexpensive and Effective Aptamer‐Based Evanescent Wave Biosensor
Authors: Mazin Ilia , Bonham Andrew J 
Published: November 2023

We have developed an inexpensive and effective evanescent wave biosensor utilizing 3D printed scaffolding and readily available electronics. This biosensor consists of a 3D printed brace, an inexpensive LED light source and cylindrical lens to direct the light in an elliptical pattern, and an acrylic slide on which DNA is immobilized. This scaffold allows light to enter the slide at above the critical angle for total internal reflectance, ensuring that the immobilized DNA experiences excitation by an evanescent wave. If the immobilized DNA is an aptamer‐based biosensor with a fluorescent reporter tag, binding of target that is washed across the slide surface will result in characteristic and measurable fluorescence changes. The utility of this inexpensive DIY setup was evaluated with image‐analysis software to create dose‐response curves for the response of both a botulinum toxin serotype A‐binding aptamer biosensor and a uranium ion‐binding aptamer biosensor, and demonstrates the potential utility of these convenient evanescent wave biosensors.Support or Funding InformationSupport for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.