bluuebunny/crossref_metadata_2025_split huggingface dataset:
Are heme proteins major targets for homocysteine‐induced modifications?
Authors: Sharma Gurumayum Suraj , Singh Laishram Rajendrakumar 
Published: June 2021

The prime cause of homocysteine (Hcy) toxicity towards proteins has been attributed to the modification of protein by homocysteine thiolactone (HTL). But the effects of Hcy (the parent molecule) on protein structure and function are still scarce. Hcy also has the ability to modify proteins, which could in turn affect the functionality and proper folding of the modified protein. Hcy is a highly pro‐oxidizing agent, thus Hcy‐induced oxidative stress has been commonly used to explain the toxicity of Hcy on endothelial cells and other tissues. Moreover, Hcy is known to modify cysteine residues in proteins in a process termed as S‐homocysteinylation. However, the structural and functional consequences of such modification are not yet clearly understood. The present study aims at investigating possible effects of Hcy on proteins structure and function. Using RNase‐A, Lyz and horseradish peroxidise (HRP) as models, we studied the effects of Hcy on the enzyme function and conformational status of these proteins. Enzyme activity measurements after overnight incubation with Hcy led to gradual decrease in enzyme function of HRP in a concentration dependent manner but not in Lyz and RNase‐A. Conformational analyses revealed no gross structural alterations in all proteins studied. However, certain alteration in the heme‐Trp distance was observed in case of HRP as evident from Trp fluorescence measurements. The loss of function in HRP was due to disruption of heme redox state in the presence of Hcy. Similar findings were obtained for three other common heme metalloprotein (Cytochrome c, Cyt c; Hemoglobin, Hb; and Myoglobin, Myo), wherein Hcy induced certain alterations in the heme environment rendering it reduced with further disruption of the heme‐Trp distance. In addition, Hcy was found to alter the heme redox states without direct interaction with the proteins (in case of Cyt c and Myo). The findings suggest that heme and heme containing proteins could be major targets for Hcy‐induced modifications and involved in Hcy‐induced cytotoxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing the Three Dimensional Structure of Cortactin Protein
Authors: Gurumani Margaret , Kruchten Anne 
Published: June 2021

Cortactin is a cytoskeletal protein which increases the metastasis of cancer cells. Cortactin is commonly located and over‐expressed in cancer cells. Because of its role in cancer, we sought to better understand the structure of cortactin. Our analysis involved expression of cortactin from E. coli, purification using His‐tag and ion exchange column purification, dialysis, and analysis by SDS‐PAGE. Previous work in the laboratory suggested that ion exchange purification resulted in the isolation of folding variants of cortactin. To understand the structures of these folding variants, we used the apoptotic enzymes calpain and caspase to probe solvent exposed regions of the protein and analyze the resulting protein fragments by SDS‐PAGE. There are three putative caspase sites on cortactin (at the N‐terminus, middle, and C‐terminus) and several putative calpain sites distributed throughout the structure. In the future, we plan on further analyzing cortactin by digging deeper into its conformation through other chemical analysis.Support or Funding InformationThis work is supported by the College of Saint Scholastica SSC Summer Research Fellowships.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Study of Arc and Its Interactions with Endocytic Binding Partners
Authors: Boldridge Melissa , Wang Lei 
Published: June 2021

Alzheimer's disease is a devastating neurodegenerative disease that causes great hardship to afflicted individuals as well as their caregivers. Currently available treatments seek to slow the disease's progression, but there is no known cure. Recent studies have shown that the activity‐regulated cytoskeleton‐associated protein Arc could act as a potential new drug target for the treatment of Alzheimer's disease. The Arc gene is an immediate early gene whose expression is induced by neuronal activation. Its expression is essential for memory consolidation. Behavioral studies of Arc−/− mice in implicit and explicit learning tasks demonstrate deficits in long‐term memory formation, though short‐term memory is unaffected. Still, the mechanism of Arc's role in memory formation is unknown. Previous studies have identified the endocytic proteins dynamin and endophilin as binding partners of the Arc protein. Dynamin is a large GTPase that plays a role in pinching off and fissioning the membrane during endocytosis as well as recruitment other proteins involved in endosome formation. Endophilin is implicated in vesicle formation and function in the brain. Arc interacts with both dynamin and endophilin to enhance AMPA receptor endocytosis and decrease surface expression. In this study, we have produced fragment proteins of dynamin, endophilin, and Arc using the E. coli expression system, and we have collected structural data on these proteins and aim to identify their structures and interactions.Support or Funding InformationThis project is sponsored by the grants from the National Institute of Health (NIGMS‐INBRE III, award number: P20GM103466; NIGMS‐AREA, award number: 1R15GM126499‐01) and the Hawaii Community Foundation (Alan M. Krassner Fund, award number: 16ADVC‐78888). The content is solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the Hawaii Community Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical characterization of Suppressor of IKKepsilon structure, stability, and metal binding properties
Authors: Shikwana Flowreen , Bell J. Ellis , Bell Jessica K. 
Published: June 2021

Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and purification of human Brain and Muscle Arnt‐like 1 protein (hBMAL1)
Authors: Moreno Brenda , Li Diyang , Sarabia‐Gonzalez Alejandro , Rodriguez Jose , Choi Jennie , Ray Supriyo , Xiao Chuan 
Published: June 2021

The circadian rhythm is an intrinsic biological clock in living organisms that oscillates every 24‐hours. It is influenced by external environmental cues to regulate many behavioral and physiological processes. Prolonged disruptions to circadian rhythms in humans have been associated to sleep disorders, cardiovascular and metabolic diseases, as well as high risks of cancer. Within every individual cell at a molecular level, the circadian rhythm is maintained by a negative feedback loop made of several core proteins' accumulation and degradation in a daily cycle. One of the core proteins involved in this cycle is Brain and Muscle ARNT‐Like 1 (BMAL1). Crystal structures of the N‐terminal DNA binding part of BMAL1 has provided some understanding of its DNA‐binding function, however, the lack of C‐terminal structural information of BMAL1 hinders our understanding of its regulatory role in the circadian rhythm. With the purpose of obtaining the atomic structure of full length circadian protein BMAL1, the complete human BMAL1 (hBMAL1) gene was cloned into bacterial systems for protein expression. However, the recombinant hBMAL1 is in its mis‐folded and aggregated form. Various methods in expression and purification of hBMAL1 are being developed to obtain high homogenous soluble hBMAL1. After achieving that objective, the full length hBMAL1 protein will be used in in vitro studies using various structural and functional analysis tools. The results will deepen our understanding of the regulatory mechanism of the circadian rhythm and build the foundation to develop therapeutic agents for circadian rhythm related diseases. In addition, the research project has been chosen for the Research‐Teaching‐Integration program. A teaching module will be developed and delivered to senior biochemistry students to raise their interests of scientific research and apply the biochemistry principles that they learn during the semester to analyze real experimental data.Support or Funding InformationResearch reported has used instruments sponsored by the BUILDing SCHOLARS program funded by National Institute of General Medical Sciences of NIH under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970.Research reported has used the core facility at the BBRC supported by National Institute of Minority Health and Health Disparities under award number 5G12MD007592.Research reported was also supported by the Campus Office of Undergraduate Research Initiatives (COURI), Summer Undergraduate Research Mentoring Experience (SURME), BUILD Research Teaching Integration (RTI), and Research Initiative for Scientific Enhancement (RISE) program awards.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using TTR50‐127 fragments as probes to detect amyloidogenic seeding
Authors: Nelson Luke Thomas , Schonhoft J.D. , Kelly Jeffrey , Duerr Jeffrey 
Published: June 2021

The aggregation and amyloid deposition of soluble transthyretin (TTR) has been linked to the development sporadic and familial forms of cardiac amyloidosis. TTR associated cardiomyopathies are often diagnosed when the disease state has reached advanced stages (if at all), precluding effective clinical intervention. This diagnostic problem can largely be attributed to a lack of fundamental understanding of the mechanism of disease pathogenesis and the clinical similarities of TTR cardiomyopathies to other heart failure related conditions. In the human prion diseases and tau protein related neurodegenerative diseases, the amyloidogenic protein conformations have the ability to “seed” and transform non‐amyloidogenic states into pathogenic amyloid‐like states. Therefore, we hypothesized that TTR related cardiomyopathies may undergo similar seed based aggregation mechanisms. To test this hypothesis, we synthesized C‐terminal fragments of Transthyretin that are fluorescently labeled and upon assembly, the fluorescent signal is quenched and provides a convenient method to measure aggregation. We specifically chose the C‐terminal peptide fragment (TTR50–127) because at autopsy it has been shown that the C‐terminal region is preferentially deposited in heart tissue compared to the full length sequence. First we used these fluorescent probes to analyze the kinetics involved in the transition from monomeric TTR to the stable polymeric form of TTR and thus demonstrated proof‐of‐principle. As C‐terminally cleaved TTR is primarily found in biopsies of cardiomyopathy patients, introduction of rhodamine labeled TTR50–127 to cardiomyopathy and polyneuropathy patient plasma revealed distinct aggregation profiles in Native PAGE gels. While preliminary in nature, these results suggest that plasma of symptomatic patients may accelerate the aggregation of Transthyretin.Support or Funding InformationSpecial thanks to staff of the Kelly Lab at TSRI as well as mentors James Smart Ph.D. and Jeff Duerr Ph.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Evaluation of Quorum Sensing Signal Synthase Inhibitors
Authors: Higgins Erin Leigh , Payne Sterling , Ulrich Scott 
Published: June 2021

Quorum sensing is a system through which bacteria sense and respond to their cell density by altering their gene expression. This behavior is executed through the synthesis, secretion, and detection of a class of small molecules called autoinducers. Some bacteria that are clinically relevant human pathogens contain a quorum sensing system that controls genes related to virulence and biofilm formation. As such, quorum sensing represents a reasonable antibacterial drug target for the control of pathogenic behavior without targeting bacterial growth or survival. Acyl‐homoserine lactones, the autoinducer for gram‐negative bacteria, is catalyzed by the LuxI family of autoinducer synthases. This project involves the expression, purification, and kinetic characterization of the LuxI family of autoinducer synthases and design of synthase inhibitors to block quorum‐sensing induced gene expression.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screemomg, expression, and characterization of Baeyer‐Villiger Monooxygenase for the biotransformation of ricinoleic acid
Authors: Yun Joohyun , Choi Kwon‐Young 
Published: June 2021

Here, the biotransformation of ricinoleic acid into of 9‐(nonanoyloxy)nonanoic acidusing Baeyer‐Villiger monooxygenases (BVMOs) was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrogenase), and BVMO Enzymes consecutively. BVMOs are known to catalyze regiospecific Baeyer‐Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9‐(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co‐expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N‐terminal fusion tag. The whole cell biotransformation with this fusion enzyme resulted in 3 to 5‐fold enhancement in product formation compared with the non‐fusion counterpart. Final productivity up to 105.3 mgL−1 was achieved. Besides Di_BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357, were screened for production of 9‐(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.Support or Funding InformationThis work was supported by the Industrial Strategic Technology Development program (No.10044604), funded by the Ministry of Trade, Industry & Energy (MI, Korea)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Snapshots of the catalytic cycle of an O2, pyridoxal phosphate‐dependent hydroxylase
Authors: Hedges Jason B. , Kuatsjah Eugene , Du Yi‐Ling , Eltis Lindsay D. , Ryan Katherine S. 
Published: June 2021

The organic cofactor pyridoxal phosphate (PLP) is used by enzymes to catalyze numerous modifications of amino acid substrates. Recently, the diversity of PLP‐dependent enzymes has been expanded with the discovery of several enzymes that use O2 as a co‐substrate to perform chemically challenging oxidative reactions. The interesting chemistry catalyzed by such O2‐, PLP‐dependent oxidases prompted us to undertake a bioinformatic search for new O2‐, PLP‐dependent enzymes. We identified one widely distributed enzyme, which we named RohP. Through in vitro biochemical studies we characterized the activity of RohP and found that it uses PLP and O2 to catalyze the hydroxylation of an sp3‐hybridized carbon in l‐arginine, giving 4‐hydroxy‐2‐ketoarginine, along with stoichiometric conversion of O2 to H2O2. Surprisingly, we found that the hydroxyl group in the 4‐hydroxy‐2‐ketoarginine product is derived from water, raising questions about the mechanism of RohP. To provide insight into the mechanism behind this hydroxylation, we used X‐ray crystallography to obtain four ~1.5 Å resolution snapshots interpreted to represent RohP at different stages of its catalytic cycle: the holo enzyme, two different PLP‐bound intermediates, and the enzyme in complex with (S)‐4‐hydroxy‐2‐ketoarginine. These structures reveal that the dynamic N‐terminus of RohP, which is required for catalysis, becomes ordered upon substrate binding, sealing off the active site from the bulk solvent. Concurrently, several conformational changes of residues in the active site help to properly orient the substrate for catalysis. Through our structural work we also identify a conserved histidine, which we demonstrate is essential for the later steps in catalysis, and we suggest that this residue may be key to catalyzing a stereospecific alkene hydration. Collectively, our biochemical and X‐ray crystallographic investigation provides insights into how O2‐, PLP‐dependent oxidases can catalyze the challenging hydroxylation of an unactivated sp3‐hybridized carbon in l‐arginine and sets the stage for further mechanistic studies on this exciting group of enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Monoamine Oxidases (MAOs) by Green Tea Extracts
Authors: Topaz Gemma Rose , Rodriguez Joyce , Mahmood Farah , Depeiza Damien , Lorenzo Loren , Destine Rodely , Stieglitz Kimberly 
Published: June 2021

Monoamine oxidase (MAO) performs deamination of amines and is found bound to the outer mitochondrial membrane at high‐concentration in neuronal cells. There are two isoforms of MAO: MAO_A which oxidizes serotonin, noradrenaline and adrenaline, and MAO_B which oxidizes dopamine, b‐phenylethylamine (PEA), and benzylamine. Alterations in MAO activity can occur in some central and peripheral nervous system diseases. More specifically, heightened MAO_B activity in the brain occurs in Alzheimer's disease, Huntington's disease, Parkinson's disease and normal aging. Abnormal MAO_A activity has found to be associated with depression, anxiety and psychiatric disorders. Drugs have been developed and continue to be developed for both MAO_A and MAO_B as targets. MAO_A is inhibited by clorgyline and MAO_B is potently inhibited by both deprenyl and pargyline. Using these inhibitors as controls, a fluorescent activity assay was performed with commercially available catechins (green tea extracts), serotonin, and benzylamine substrates for MAO_A and MAO_B respectively, to investigate and confirm recent studies suggesting that green tea catechins (polyphenols) may be preventative for certain degenerative diseases and emotional illnesses utilizing MAOs as a target. Using a fluorescent assay, the Km value of serotonin to MAO_A was 1.75 μM and the Km value of benzylamine to MAO_B was 0.75 μM. The IC50 of clorgyline to MAO_A was 3.25 nM and that of deprenyl to MAO_B was 7.25 nM, in close agreement with the literature. The commercial catechins tested were found to have IC50s in the low‐to‐mid μM range (~50–750 μM). Efforts to purify catechins are underway to repeat these studies. Molecular docking of specific catechins into the MAO_A and MAO_B active sites resulted in binding constants in the low μM range (in agreement with experimentally determined Km values for natural substrates). Crystallization studies of MAO/catechin complexes are in progress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.