bluuebunny/crossref_metadata_2025 huggingface dataset:
Expanding Electrochemical DNA Biosensors to Detect Ricin
Authors: Fetter Lisa , Bonham Andrew
Published: June 2021

Ricin toxin chain A (RTA), a byproduct of the production of castor oil from castor bean plants, can cause serious health problems if ingested, inhaled, or injected. In this project, we have designed and tested an electrochemical biosensor that is capable of ‐‐ and sensitive enough ‐‐ to detect small, biomedically relevant concentrations of RTA. To create this biosensor, we drew from an existing aptamer, previously shown to bind to RTA, and rationally designed a custom biosensor scaffold that coupled RTA binding to a conformational change. Ultimately, this oligonucleotide biosensor design allows voltammetric interrogation to detect RTA concentration in complex media (such as coffee, blood, and river water). This biosensor is convenient and collects real‐time data, offering beneficial applications in monitoring of areas of castor oil production, and furthermore offers potential diagnostic applications in assessing Ricin exposure.
Electrochemical Biosensor Development for Detection of Botulinum Toxin A (BoNT/A)
Authors: Richards Jonathan , Bonham Andrew
Published: June 2021

Effective methods for the detection of biological toxins are vital for food safety, human medicine, environmental maintenance, and safeguarding against biological warfare. In this project, we are developing DNA‐based electrochemical biosensors to sensitively detect botulinum toxin A (BoNT/A), the target involved in the life‐threatening paralytic disease botulism. We are utilizing rationally designed and synthesized oligonucleotide biosensors to bind to this target, generating measurable electrochemical changes upon binding the toxin in a potentiometric analysis. These biosensors are based on BoNT/A binding aptamers discovered through modified SELEX procedures. They are uniquely able to capture these data in real‐time in complex fluids, making them advantageous over current detection techniques. This approach should enable the detection of minute levels of BoNT/A in media such as foodstuffs, blood, or water, improving both prevention and diagnosis of botulism disease. Together, development of a BoNT/A biosensor will allow for detection in safety, research, commercial, and/or at‐home applications.
Amyloid Formation by Human Apolipoproteins: Sequence Analyses and Structural Insights
Authors: Gursky Olga , Das Madhurima
Published: June 2021

Apolipoproteins are protein components of lipoproteins that transport lipids in circulation and are central to cardiovascular health and disease. Apolipoprotein dissociation from the lipid renders them labile to misfolding that is implicated in atherosclerosis, systemic amyloidoses and Alzheimer's disease. We used 12 bioinformatics approaches to assess amyloid‐forming potential of 10 members of this protein family and identify segments that initiate aggregation. Mapping such segments on the available atomic structures helps explain why some apolipoproteins readily form amyloid while others do not. We show that nearly all amyloidogenic segments are hydrophobic, are located in the native lipid‐binding amphipathic a‐helices or b‐sheets, and are likely to form parallel in‐register beta‐sheet in amyloid. These predictions have been verified experimentally for several proteins. Surprisingly, the rank order of the sequence propensity to form amyloid (apoB > apoA‐II > apoC‐II 蠅 apoA‐I, apoC‐III, SAA, apoC‐I> apoA‐IV, apoA‐V, apoE) does not correlate with the proteins' involvement in amyloidosis. Rather, it correlates directly with the strength of the protein‐lipid association, which increases with increasing protein hydrophobicity. Therefore, the lipid surface‐binding function and the amyloid‐forming propensity are both rooted in apolipoproteins' hydrophobicity, suggesting that functional constraints make it difficult to completely eliminate pathogenic apolipoprotein misfolding. We propose that apolipoproteins have evolved protective mechanisms against misfolding, such as the sequestration of the amyloidogenic segments via the native protein‐lipid and protein‐protein interactions involving amphipathic a‐helices or b‐sheets. Funded by NIH GM067260.
Cysteine Cathepsins Are Essential in Lysosomal Degradation of α‐Synuclein
Authors: McGlinchey Ryan , Lee Jennifer
Published: June 2021

A cellular feature of Parkinson's disease is cytosolic accumulation and amyloid formation of α‐synuclein (α‐syn), implicating a misregulation or impairment of protein degradation pathways that involve the proteasome and lysosome. Within lysosomes, Cathepsin (Cat) D, an aspartyl protease is suggested to be responsible for α‐syn clearance; however, the protease alone only generates amyloidogenic C‐terminal truncated species (e.g. 1–94 and 5–94), arguing that other proteases and/or environmental factors within the lysosome are needed to facilitate degradation and to avoid α‐syn aggregation. Here, we show that in addition to aspartyl cathepsin activity, purified mouse brain and liver lysosomes harbor cysteine cathepsin activity identified as Cat B and L by using liquid chromatography mass spectrometry and peptide mapping. In contrast to Cat D, both Cat B and L cleave α‐syn within its amyloidogenic region (residues 20–100) generating short peptides that are not aggregation prone. Surprisingly, we discovered that Cat L is efficient in degrading α‐syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. Based on these results, stimulating Cat L activity or increasing its level is a potential strategy for clearance of aggregated α‐syn in lysosomes.
Role of ATP synthase as a molecular drug target against bacterial infections
Authors: Ahmad Zulfiqar
Published: June 2021

Background

F1Fo ATP synthase is the main source of cellular energy in almost all organisms from bacteria to man through oxidative phosphorylation. Binding of ADP and Pi in the catalytic sites of ATP synthase is essential for the formation of ATP. Inhibition of ATP synthase deprives cells of required energy in the form of ATP resulting in cell death. Here, we propose that ATP synthase can be used as a molecular drug target against microbial infections in general and bacterial infections in particular. This can be achieved by targeting the bacterial ATP synthase. A wide variety of phytochemicals and peptides are known to potently in inhibit the ATP synthase. Thus, identification of natural phytochemicals or antibacterial peptides is an important goal towards using ATP synthase as a molecular drug target.

Methods

Phytochemicals or peptide induced inhibition of wild type purified F1 or membrane bound F1Fo ATP synthase were performed. Many polyphenolic compounds were structurally modified to increase the extent of inhibition and potency on molar scale. Mutagenic analysis of polyphenol binding and peptide binding site was done to understand the mechanism of inhibition. Growth yield in limiting glucose and non‐fermentable carbon source succinate in presence or absence of inhibitors was done to prove the ATP synthase related cell death.

Results

Lately, we found that structural medication of polyphenolic compound such as resveratrol resulted in ~60% additional inhibition of ATP synthase and caused complete abrogation of wild type E. coli cells while mutant cells along with null cells were not affected. Currently, we are studying the role of βDELSEED‐motif in peptide binding to elucidate the mechanism of inhibition of ATP synthase.
rLosac, a Hemolin, Prevents Human Chondrocyte Aging and Degeneration of Articular Cartilage
Authors: Chudzinski‐tavassi Ana , Alvarez‐Flores Miryan , Bosch Rose , Antonoili Eliana , Pereira‐Junior Edgard , Ferreti Mario , Cohen Moises
Published: June 2021

Chrondrocyte aging is associated with the degeneration of articular cartilage such as rheumatoid arthritis and osteoarthritis (OA). One of the most prominent changes in cartilage affected by these diseases is loss of extracellular matrix due to its elevated degradation and lowered synthesis. Moreover, pro‐inflammatory cytokines such as TNF‐a and IL‐1b play roles in the progression of these degenerative changes.

The aim was to investigate of rLosac as an anti‐inflammatory/chondroprotective agent and to analyze its capacity to stimulate the production of extracellular matrix molecules. Primary cultures of human chondrocytes from cartilage of osteoarthritic knee joints was cultured in the absence or in the presence of IL‐1b and treated with rLosac. Specific senescence‐associated β‐galactosidase activity, cell viability, production of extracellular matrix molecules, cell cycle, expression of collagen I receptor and IL‐1 β were analysed. rLosac inhibited cellular death and reduced the IL‐1β‐induced expression of β‐galactosidade and stimulated the production of collagen type I, laminin and fibronectin. Increased expression of Col‐I and decreased expression of IL‐1β, proposing a down‐regulation of the inflammatory condition, suggesting that rLosac may have the potential to prevent inflammation, aging and cellular death induced in pro‐inflammatory chondrocytes.

Supported by FAPESP (CETICs), Capes, INCTTox‐CNPq.
Intracellular Zinc Dyshomeostasis Caused by a Disrupted TRPML1‐TMEM163 Protein Interaction
Authors: Silva Joshua , Cuajungco Math
Published: June 2021

Dysfunctional Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder Mucolipidosis type IV (MLIV). Cells affected by MLIV display higher intracellular zinc levels compared to normal cells; however, the mechanism behind this zinc accumulation is unknown. We recently identified a putative zinc transporter called transmembrane‐163 protein (TMEM163) as an interacting partner for TRPML1. We hypothesize that TMEM163′s interaction with TRPML1 influences its subcellular localization. We also propose that the interaction between the two proteins regulates intracellular zinc homeostasis. To examine their functional relationship, we created N‐terminus deletion mutants of TMEM163. Co‐immunoprecipitation shows disruption of TRPML1 binding to the deletion mutants compared against wild‐type (WT) TMEM163. Cell surface biotinylation reveals that plasma membrane (PM) localization of TMEM163 mutants increased upon co‐expression with TRPML1 in human embryonic kidney (HEK)‐293 cells, while WT TMEM163 PM localization decreases with co‐expressed TRPML1, suggesting that TRPML1 influences TMEM163′s internalization. Spectrofluorometric analysis of HEK‐293 cells co‐expressing TMEM163 mutants and TRPML1 shows an increase in intracellular zinc levels relative to control when the cells are transiently exposed to zinc. Overall, these data suggest that the loss of TMEM163 protein interaction with TRPML1 results in its abnormal subcellular localization, which consequently affects intracellular zinc homeostasis. Future studies characterizing the role of TMEM163 on MLIV pathology could further explain the contribution of zinc in progressive cell death and open new research avenues to develop potential therapies for the disease.
Investigating the Interaction between the Dopamine Transporter and the Sigma 1 Receptor
Authors: Rodriquez Meghan , Perley Danielle , Henry Keith , Khoshbouei Habibeh , North Ashley , Sambo Danielle
Published: June 2021

Addictive psychostimulants such as cocaine and amphetamines bind to the dopamine transporter (DAT) and alter its ability to recycle dopamine (DA), resulting in adverse psychological and physiological effects. Several of these stimulants also bind the sigma 1 receptor (S1R), which modulates signal amplification of neurotransmitters, calcium and potassium through protein‐protein interactions at the plasma membrane. The S1R is expressed in dopaminergic neurons however; a physiological relationship between DAT and S1R has not been identified. Here we investigate physical interaction between DAT and S1R through co‐immunoprecipitation techniques in COS7 cells expressing the two proteins. Our data support that DAT and S1R are at least involved in a single protein complex as antibodies against DAT also purify S1R and vice versa.

Grant Support: This work is supported by an NIH Grants DA026947 and NS071122 awarded to HK.
Label‐Free Quantitative Proteomics Identify Global Protein Interactors for the Androgen Receptor Associated Cochaperone SGTA
Authors: Prieto Luis , Garcia Yenni , Cox Marc
Published: June 2021

Molecular chaperones facilitate proper folding and regulation of steroid hormone receptors (SHRs). Upon proper folding, SHRs bind ligand with high affinity, translocate to the nucleus and initiate gene expression accordingly. A novel cochaperone, human small glutamine rich TPR (tetratricopeptide repeat) containing protein alpha (SGTA), is a down‐regulator of androgen (AR), glucocorticoid (GR), and progesterone (PR) receptors. It binds to heat shock protein (Hsp) 70 and 90 kDa. Additionally, SGTA plays a role in cellular processes such as cell cycle progression and apoptosis. Therefore, label‐free quantitative proteomics was utilized to determine novel interactors in LNCaP human prostate cancer cells. Unknown protein interactions were purified using a Nickel resin and further analyzed with liquid chromatography mass spectrometry (LC‐MS/MS). To determine if interactions were transient or strong, a comparison of a 3‐hour versus an overnight incubation was used. Preliminary studies using rigorous statistical analysis have identified a broad range of proteins involved in different pathways other than the chaperoning pathway. Thus, future studies aim to validate these interactions with SGTA using nickel purification and assess their functional relevance, which will contribute to the understanding of the role SGTA plays within the chaperoning pathway and any cross‐talk with other pathways.
Discovering Relationships Between Lysine Ubiquitylation and Acetylation Sites from Proteomic Datasets
Authors: Elwood Jake , Maki Jennifer , Freund Dana
Published: June 2021

Ubiquitylation (Ub) and acetylation (Ac) are post translational modifications that both occur on lysine residues. Ac of non‐nuclear proteins is a new area of research, especially in the plant biology field. Ac of proteins can alter enzyme activities and protein functions, while Ub is well known for marking proteins for degradation by the proteasome as well as many other cellular functions. Recently, it has been suggested that competition may exist between Ub and Ac of specific lysine residues. It may be that the neutralization of the lysine side chain caused by Ac prevents Ub from taking place. If proven, this Ac‐Ub competition has the potential to affect processes such as the regulation of the cell cycle and protein degradation. The purpose of this study was to manually data mine previously published Ac and Ub sites in Arabidopsis thaliana to determine the amount of overlap. Sites identified in three publications were compared: Finkemeier et al (2011) Plant Physiol, 155:1779‐90; Kim et al (2013) The Plant Cell, 25: 1523‐40; and Wu et al (2011) Plant Physiol, 155:1769‐78. Thus far, seven of the modified proteins are subjected to both of these post‐translational modifications. Further analysis has been initiated to locate the specific Ub lysine residues and compare sites to known Ac lysine sites of the same protein. Currently, more extensive mass spectrometry based proteomics studies are underway to expand the number of identified Ac sites. Future work will investigate other plant species and other types of acylations. This project is part of a collaboration between The College of St. Scholastica and The University of Minnesota and provides research opportunities for undergraduates.
Investigation of Evolutionary Events in Corynebacteriaceae Involving Prenyltransferases in Corynebacterium glutamicum
Authors: Allen Michael , Nelson Kacie , Mann Francis
Published: June 2021

Terpenoids, a class of compounds with 30,000 unique structures, are known to include antitumor compounds, pigments, vitamins, antibiotics, and flavor molecules. Prenyltransferases are the primary enzyme responsible for assembling the carbon backbone of these compounds from five carbon isoprene units. Bioinformatic analysis has indicated that an evolutionary event has resulted in the acquisition of an additional prenyltransferase in Corynebacterium glutamicum (C. glutamicum) compared to the majority of the Corynebacteriaceae. This result suggests that C. glutamicum may produce a non‐essential terpenoid compound, or secondary metabolite. Sequencing has confirmed the successful cloning of the aforementioned genes. Present work includes expressing these genes in Escherichia coli, isolating the protein product, and assessing the function in vitro. This data will be used to expand our understanding of the evolutionary path of C. glutamicum compared to other members of the genus. Continuing work will shed light on the function of the third gene for C. glutamicum, and understanding the role of terpenoids in the family Corynebacteriaceae and greater Actinobacteria.
Lipid Metabolism Regulation by Hyperosmolarity in Renal Cells
Authors: Casali Cecilia , Weber Karen , Morel Gómez Emanuel , Fernández María del Carmen
Published: June 2021

Physiologically, renal medullary cells are immersed in an unusual extracellular matrix that shows a high osmolarity and plays a major role in the urine concentrating system. To survive and work in such environment, cells have developed adaptive and protective mechanisms. Various works from our laboratory showed that the preservation of membrane structure helps to maintain cell architecture and viability. We showed that changes in environmental osmolarity regulate membrane phospholipid (PL) synthesis and turnover. PL de novo synthesis requires fatty acids which usually come from cellular storage in triglycerides (TG) molecules.

The aim of the present work was to evaluate whether hyperosmolarity modulates lipid metabolism through the activation of the transcription factor sterol response element binding protein (SREBP). For this purpose, MDCK cells, derived from renal structures, were incubated in isosmolar and hyperosmolar media. Then, SREBP participation in TG synthesis and storage, and in the expression of the main TG biosynthetic enzymes, was evaluated. Results showed that hyperosmolarity increased TG storage and synthesis, and also the expression of fatty acid synthase (FAS), acetyl‐CoA carboxylase and lipin‐2 mRNA levels. Fatostatin (20 mM), a SREBP inhibitor, blocked hyperosmolar‐increased TG content and synthesis, and the expression of FAS enzyme. Fatostatin treatment also decreased the number of cells that survive to the treatment. These results suggest that hyperosmolarity activates SREBP which in turn modulates lipid synthesis. Thus, SREBP would play a key role in cell adaptation to osmotic changes by ensuring the substrates for PL synthesis that leads to membrane homeostasis

Supported by CONICET 0327 and PICT 2013‐1132.
Yeast Pah1 Phosphatidate Phosphatase Regulates the Expression of the CHO1‐encoded Phosphatidylserine Synthase for Membrane Phospholipid Synthesis
Authors: Han Gil‐Soo , Carman George
Published: June 2021

Pah1 phosphatidate (PA) phosphatase catalyzes the committed step for the synthesis of triacylglycerol, whereas Cho1 phosphatidylserine (PS) synthase catalyzes the committed step for the synthesis of membrane phospholipids. In the exponential phase, the expression of Cho1 PS synthase is largely governed by the cellular concentration of PA and the Ino2/Ino4/Opi1 regulatory circuit acting on the UASINO cis‐acting element in the CHO1 promoter. In this work, we examined the effect of the PA‐controlling Pah1 PA phosphatase on the expression of Cho1 PS synthase. Compared with the wild type control, the pah1Δ mutant in the exponential phase exhibited elevated levels of Cho1 PS synthase. In the stationary phase when Cho1 PS synthase expression was repressed in wild type, the enzyme expression in the pah1Δ mutant was further induced over that observed in the exponential phase. Analysis of the CHO1 promoter by its deletion and site‐directed mutation indicated that the expression of Cho1 PS synthase in the pah1Δ mutant was elevated by transcriptional regulation through the UASINO element. Lipid analysis showed that the increased phospholipid synthesis in the pah1Δ mutant was significantly reduced by the expression of a UASINO‐deficient CHO1 allele. These results indicate that Pah1 PA phosphatase activity plays a major role in the regulation of Cho1 PS synthase expression through the UASINO‐mediated transcriptional control. Supported by NIH grant GM028140.
Interaction of Ceramide Synthase with Long Chain Fatty Acyl‐CoA Synthase 5 Channels de novo Ceramide to Acylceramide Generation by Diacylglycerol Acyltransferase 2 on Lipid Droplets
Authors: Senkal Can , Hannun Yusuf , Obeid Lina
Published: June 2021

The central molecule of the sphingolipid pathway, ceramide, has been shown to regulate multiple cellular responses including growth arrest, senescence, apoptosis and autophagy. Fatty acyl‐CoA (FA‐CoA) dependent ceramide generation is catalyzed by ceramide synthases in the de novo pathway. Importantly, six CerS isoforms displaying different preferences to generate ceramides with distinct fatty acyl chains have been identified. Currently, there is little insight into the regulation of the distinct CerS enzymes. In an approach aimed at defining interacting partners with CerSs, we found that that fatty acyl CoA synthase ACSL5 interacts with CerS. Contrary to our initial hypothesis, ACSL5 generated FA‐CoA was not a direct substrate utilized by CerS to generate ceramide as measured by LC/MS. Rather, ACSL5 generated FA‐CoA was utilized with de novo ceramide for the generation of acylceramides, poorly studied ceramide metabolites. Moreover, inhibition of ceramide channeling to acylceramide enhanced accumulation of de novo ceramide and resulted in augmentation of ceramide‐mediated apoptosis. Mechanistically, we show that acylceramide generation is catalyzed by DGAT2 and involves the formation of an ACSL5‐CerS‐DGAT2 complex on lipid droplets. In summary, this study identifies the molecular mechanism of a novel metabolic pathway of acylceramide generation and implicates the importance of this novel pathway in ceramide‐mediated apoptosis.

This study is supported by NIGMS, NIH 9R01GM097741‐13A1
Kinetic Mechanism of Lipidprotein Nanodisc Dissociation in Bicelles
Authors: Muñoz‐Forti Kevin , Renthal Robert
Published: June 2021

Nanodisc soluble lipid bilayer systems are vital in rendering membrane proteins soluble in aqueous solutions where they remain monodisperse and active in a membrane‐like environment. Nanodiscs or nanolipidprotein particles (NLPs), are bordered by an amphipathic helical protein belt or membrane scaffolding protein (MSP) mimicking a cell membrane. Past works have shown that fragments of bacteriorhodopsin when placed in separate nanodisc do not interact to form the functional protein. The introduction of bicelles enabled the fragments to form oligomers inside the bicelles. An analysis of the kinetics of the aforementioned NLP‐bicelle interaction showed a sigmoid curve suggesting the possibility of an autocatalyzed reaction. We hypothesized that the kinetics where due to MSP1E3D1′s dissociation from the nanodisc which catalyzed the reaction. Using fluorescently labeled lipids 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(lissamine rhodamine B sulfonyl), 1,2‐diphytanoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl) LR‐PE and NBD‐PE, respectively with 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) and MSP1E3D1 we constructed NLPs. Bicelles were prepared using 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) and DMPC. Kinetics of NLP‐bicelle interactions and fluorescently labeled lipid dispersion were observed measuring fluorescence resonance energy transfer (FRET) using stopped‐flow spectroscopy. The NLP‐bicelle interaction followed previously observed kinetics and continued to do so after the addition of excess MSP1E3D1. Our results show that MSP1E3D1 is not responsible for the observed kinetics of the transfer of lipids between NLPs and bicelles.
Regulation of the Yeast DAG Kinase by Phosphorylation
Authors: Qiu Yixuan , Carman George
Published: June 2021

In the yeast Saccharomyces cerevisiae, DGK1‐encoded diacylglycerol (DAG) kinase is a membrane‐associated enzyme that catalyzes the CTP‐dependent phosphorylation of DAG to form phosphatidic acid (PA). This enzyme, in conjunction with PAH1‐encoded PA phosphatase, controls the levels of PA and DAG for phospholipid synthesis, membrane growth, and lipid droplet formation. When cells resume vegetative growth from stasis in the absence of de novo fatty acid synthesis, DAG kinase is required. In addition, the activity of DAG kinase is induced when cells resume growth and it is not due to the increased protein expression. Therefore, we speculated that the increased activity was due to a biochemical mechanism such as phosphorylation. Several phosphorylation sites are located in the hydrophilic N‐terminal region of Dgk1p. We expressed and purified the N‐terminal His6‐Dgk1p1‐77 (residues 1‐77) for in vitro phosphorylation studies. His6‐Dgk1p1‐77 was phosphorylated on serine residues by casein kinase II (CKII) and cAMP‐dependent protein kinase A (PKA). Site‐directed mutagenesis coupled with two‐dimensional phosphopeptide mapping analyses identified Ser‐44, Ser‐45, and Ser‐46 as target sites of CKII and Ser‐25 and Ser‐26 as target sites of PKA. Phosphorylation‐deficient and phosphorylation‐mimic mutations will be introduced into full‐length Dgk1p to examine the effects of phosphorylation by CKII and PKA on DAG kinase function in vivo. (Supported by NIH grant GM028140).
Development of Resistance to Intrinsic Apoptotic Stimuli in Lymphocytes without Alterations in Bcl‐2 Family Member Gene Expression
Authors: Bortner Carl , Scoltock Alyson , Cidlowski John
Published: June 2021

Apoptosis is a physiological mode of cell death that removes unwanted cells at a specific time or in response to a given cell death signal. Apoptotic resistance is a common feature in many disease states that impedes the treatment of these pathologic conditions. We have developed a model system to investigate apoptotic resistance in T‐cells using osmotic stress to drive selection of death resistant cells. Exposure of S49 (Neo) cells to multiple rounds of osmotic stress which induces apoptosis, followed by recovery of surviving cells resulted in the production of a stable population of T‐cells (S49 (OS 4‐25)), resistant to acute osmotic stress and failed to die. These cells are also resistant to multiple intrinsic apoptotic stimuli. In contrast, S49 (OS 4‐25) cells remain sensitive to extrinsic apoptotic stimuli, suggesting the cellular apoptotic machinery remains intact. The S49 (OS 4‐25) cells undergo aerobic glycolysis, characteristic of the Warburg effect, and have a total dependence on glucose; whereupon its withdrawal results in apoptosis. Microarray analysis of S49 (OS 4‐25) compared to the parent S49 (Neo) cells revealed over 8,500 differentially regulated genes with almost 90% of the genes having repressed expression. Pathway analysis indicates apoptosis and mitochondrial dysfunction having major changes in gene expression. Interestingly, gene expression of pro‐ and/or anti‐apoptotic Bcl‐2 family members were either repressed or unchanged. Our findings implicate stimulus‐specific recognition defects and altered signaling mechanisms, but not specific pro/anti‐apoptotic Bcl‐2 proteins are critical determinants of apoptotic resistance in lymphoid cells.
O‐GlcNAcylation Stabilizes Nod2, an Innate Immune Receptor Involved in Crohn's Disease
Authors: Hou Ching‐Wen , Zachara Natasha , Grimes Catherine
Published: June 2021

Nucleotide‐binding oligomerization domain2 (Nod2) is an intracellular receptor that can sense bacterial components, such as, Muramyl dipeptide (MDP). MDP is a peptidoglycan fragment from bacterial cell wall that can activate NF‐kB, a transcriptional factor that can induce the production of various inflammatory molecules. Nod2 mutations have been linked to Crohn's disease with a decreased ability to activate NF‐kB. Recently, using the classic cycloheximide stabilization assay, we demonstrated that Nod2 variants have a lower half‐life compared to wild type Nod2 in cells. To further characterize this protein, the objective of this study is to determine if Nod2 is post‐translationally modified. O‐GlcNAcylation is a post translational modification in which the O‐GlcNAc transferase (OGT) transfers N‐acetylglucosamine (GlcNAc) from UDP‐GlcNAc to selected serine and threonine residues of a target protein. As GlcNAc is a major component of peptidoglycan of bacterial cell wall and a large amount of GlcNAc is released from bacterial cell wall during cell wall remodeling, we hypothesized that Nod2 could be O‐GlcNAcylated. Preliminary data show that wild type Nod2 and Nod2 variants are O‐GlcNAcylated. In addition, increasing the O‐GlcNAcylation level can increase the half‐life of wild type Nod2 or Nod2 variants. In future experiments, we will determine which residue of Nod2 is O‐GlcNAcylated and investigate if this modification affects Nod2 mediated NF‐kB pathway in wild type Nod2 or Nod2 variants.

Funding of this project is provided by University of Delaware.
Chemistry‐Driven Approaches to Deconstruct Redox‐Linked Signal Transduction Networks
Authors: Haegele Joseph , Lin Hongyu , Long Marcus , Parvez Saba , Aye Yimon
Published: June 2021

Oxidative and electrophilic stress that results from loss of redox homeostasis has become increasingly appreciated to underlie numerous disease states including inflammation, cardiovascular disease, cancer, neurodegeneration, and psychiatric disorders. Utilizing our novel T‐REX (Targeted Reactive Electrophiles and oXidants) methodology, we aim to better resolve electrophile‐ and oxidant‐mediated signaling effects on any protein of interest (POI). In contrast to widely used methodologies, in which live specimens are treated globally with inherently reactive electrophiles and oxidants (RES/ROS) in excess quantities, our technique allows us to selectively deliver these reactive species to specific POIs with spatiotemporal control. Herein, T‐REX is exploited to delineate the functional consequences of H2O2 signaling along individual disease relevant redox‐dependent pathways in live cells and animals.
Histone Post‐translational Modifications Differentially Regulated in a Mouse Mode of Heart Disease
Authors: Miller Mickey , Makaju Aman , Wang Li , Franklin Sarah
Published: June 2021

While global changes in gene expression are a hallmark of heart disease, much less is known regarding the epigenetic factors driving these changes. Local chromatin packing and gene accessibility, which governs transcriptional status, has been correlated with specific post‐translational modifications (PTMs) on the histone tails of nucleosomes occupying these regions. However, the specific alterations in histone PTMs driving the conserved gene expression changes observed in heart disease and how these ultimately affect cardiac physiology are largely unknown. To identify changes in histone PTMs during disease progression, we performed label‐free quantitation of acid‐extracted proteins from mouse heart (under basal, hypertrophic, and failing conditions) and from isolated ventricular myocytes exposed to hypertrophic agonists, a cell model of cardiac hypertrophy. Protein samples were propionylated, enzymatically digested and analyzed on an Orbitrap Velos mass spectrometer and differential expression analysis was performed using MaxQuant and Perseus software. Collectively we identified 55 and 30 PTMs spanning the five histone families, from cardiac tissue and isolated myocytes, respectively, 16 of which were differentially regulated in the setting of disease. Additionally our analysis of large peptides containing multiple PTMs enabled us to determine how these modifications are regulated with respect to one another providing insights into the broader histone code. This study presents the first global characterization of histone post‐translational modifications in the heart and highlights basic mechanisms of genomic reprogramming operative in disease.
Production and characterization of a monoclonal antibody against eel glycoprotein hormone
Authors: Min Kwan‐Sik , Park Chae‐Won , Seo Eon‐Bi , Kim Dae‐Jung , Kang Myung‐Hwa
Published: June 2021

The glycoprotein hormone family consists of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and thyroid‐stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin (CG) secreted by placental trophoblast cells in primates and equids. These hormones consists of non‐covalently associated α and β subunits. Within a species, the amino acid sequence of the α subunit is identical across all glycoprotein hormones and is encoded by a single gene. The α/β dimer is the active form of the hormone, and biological specificity is conferred by the β subunit. Also in fish, the duality of gonadotropin hormone (GTH), GTH‐1 (FSH‐like GTH), and GTH‐II (LH‐like GTH) is found in certain teleost. In this study, to understand the fundamental mechanisms involved in teleost reproduction and to prove a broader basis for comparative study of teleost GTHs, we produced tethered rec‐eelFSH protein, the cDNA encoding the full‐length eel FSH β‐subunit (signal sequence of 22 amino acid residues and the mature protein of 105 amino acid residues) was fused with the mature protein (93 amino acids) of eel α‐subunit, immunized and A positive hybridoma was selected by ELISA using eel FSH. Anti‐eel glycoprotein mAbs was purified from culture supernatants by affinity chromatography. Six monoclonal antibodies for eel FSH were produced and 2 clones (#11, #5) of these antibodies were bound with α‐subunit of eel glycoprotein. And the other one (#14) was bound with FSHβ. Recombinant FSHβ/α also produced into the CHO cells. Its molecular weight was about 34 kDa. The deglycosylated rec‐FSHβ/α was about 25 kDa. This mAb can specially recognize eel glycoprotein and may serve as a component of eel glycoprotein ELISA kit. (This work was supported by NFRDI. Eun Bi Seo was supported by a scholarship from the BK21 Plus Program (31Z20130012928). the Ministry of Education, Science and Technology, korea)
Impact of Environmental Conditions on the Pathogenesis of the Common Cold
Authors: huang Chao
Published: June 2021

The common cold is a worldwide health issue, and is associated with severe complications in patients with chronic illnesses and disability, which can even be life‐threatening. More than 200 types of viruses are associated with the common cold. However, until now, there is still no effective protection against the cold. The fact that a cold cannot be prevented is reflective of a poor understanding of the etiology of the disease. The present study identified a relationship between the incidence of the common cold and environmental conditions, namely low temperature and air humidity. Three basic etiological factors of the common cold were identified: (1) Dry air, defined as relative humidity of <40%, challenges the lungs to maintain exhaled air humidity of approximately 100%. (2) Low temperature (<5°C) in combination with excessive relative humidity (>90%), challenges the lungs to maintain exhaled air temperature of approximately 37°C. (3) Common cold induced by lung chilling during sleep.

Conclusion

Extreme weather reduces airway defenses against viral infections. Therefore, adequate regulation of indoor air humidity (55%–70%) is expected to prevent most colds.
An Intricate Link between MicroRNAs and Regulation of Autophagy in Cystic Fibrosis
Authors: Tazi Mia , Dakhlallah Duaa , Caution Kyle , Marsh Clay , Amer Amal
Published: June 2021

Each year 1,000 individuals are diagnosed with Cystic Fibrosis (CF), a lethal, inherited disease characterized by severe infection and inflammation. Burkholderia cenocepacia (B.c), the most‐feared CF pathogen, harbors an innate antibiotic resistance to the majority of treatments and further exacerbates inflammation. Normally, invading microbes can be cleared through a highly‐regulated biological process called autophagy, however, this process is defective in CF. Furthermore, we demonstrated that B.c interrupts autophagic flux as it indirectly downregulates autophagy genes, causing increased bacterial burden and inflammation in CF macrophages. MicroRNAs (miRs) post‐transcriptionally regulate gene expression essential to numerous biological processes. The underlying mechanism of dysfunctional autophagy in CF macrophages remains unknown. As autophagy is a cellular innate immune response to pathogen clearance, we hypothesize that CF macrophages exhibit elevated miRNA expression that downregulate autophagy‐targeted genes thus contributing to autophagy dysfunction and facilitating infection and inflammation. qRT‐PCR results reveal a specific cluster of miRNAs, predicted to target autophagy genes, is elevated in CF macrophages. Macrophages lacking these miRs exhibit increased autophagy protein expression. Luciferase assays validate the autophagy targets of these miRs. Increased levels of active transcription factor, c‐myc, propose a mechanism for the elevated cluster level, as inhibiting c‐myc increases autophagy protein expression in CF macrophages. Together, these data demonstrate miRNA regulation of autophagy modulates the pathophysiology of CF.
Triphosphate Re‐orientation of the Incoming Nucleotide as A Fidelity Checkpoint in Viral RNA‐dependent RNA Polymerases
Authors: Liu Xinran , Yang Xiaorong , Musser Derek , Boehr David
Published: June 2021

The fidelity of the virus‐encoded RNA‐dependent RNA polymerase (RdRp) plays an essential role in generating sufficient genetic diversity to escape the bottlenecks of the host's anti‐viral response. For many nucleic acid polymerases, a pre‐chemistry conformational change is at least partially rate‐limiting and represents a key fidelity checkpoint. There has been disagreement in the polymerase field about the structural rearrangements important in this step. One suggestion has been that this step represents a rearrangement of the triphosphate of the incoming nucleotide that will facilitate nucleophilic attack by the primer 3′‐hydroxyl group. We have now gained insight into the triphosphate re‐orientation in the poliovirus (PV) RdRp via 31P NMR. Modeling studies suggest that Lys167 and Arg174 on structural motif F make important interactions with the triphosphate. 31P NMR results demonstrate that WT RdRp interacts with “correct” nucleotide in a way different from “incorrect” nucleotide; R174K RdRp is unable to rearrange correct nucleotide like WT enzyme and instead interacts with correct nucleotide in a similar fashion as incorrect nucleotide; The R174K substitution also substantially reduces enzyme efficiency and leads to very high fidelity. The K167R variant has similar catalytic efficiency as WT when correct nucleotide is incorporated, however, incorrect nucleotide is more readily incorporated. Altogether these results suggest that triphosphate re‐orientation is an essential fidelity checkpoint and interactions with motif F residues are important for this alignment.

The research is supported from Penn State start‐up funds and NIH R01 AI104878 to DDB.
MTHFD1 and Nuclear Thymidylate Biosynthesis
Authors: Liebenthal Rebecca , Stover Patrick , Kamynina Elena , Field Martha
Published: June 2021

Thymidylate (dTMP) is the only DNA nucleotide formed at the sites of DNA replication. dTMP is synthesized by a multi‐enzyme complex, which includes serine hydroxymethylase 1 (SHMT1) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). When dTMP synthesis is impaired, dU is incorporated into DNA, which can lead to DNA strand breaks. SHMT1 has been shown to act as a scaffold to anchor the thymidylate biosynthesis complex to the nuclear lamina and MTHFD1 supplies folate‐activated single carbons for dTMP synthesis. Both proteins have been shown to be SUMOylated in vitro and in vivo. Protein SUMOylation serves many functions including trafficking proteins to the nucleus, protecting proteins from ubiquitin dependent proteasome degradation, and assembling proteins into complexes. It is hypothesized that the non‐covalent interaction between SUMOylated proteins and SUMO interacting motifs (SIMs) on other proteins facilitate multiprotein complex formation and stabilization. In this study, MTHFD1 is shown to localize to the nucleus during S‐phase in HeLa cells and when MTHFD1 is mutated at its SUMOylation site, it's nuclear localization is impaired. Furthermore, we have identified a SIM on SHMT1 that may function in complex formation and may be regulated by CK2 phosphorylation. We also demonstrate that arsenic exposure affects SHMT1 and MTHFD1 SUMOylation, causes genome instability, and that the effects of folate‐depletion and arsenic on genome stability are additive. Collectively, these studies reveal the factors that regulate the metabolic complex responsible for dTMP biosynthesis to maintain genome stability.
Potential New Chemical Roles for Diheme Enzymes in Burkholderia
Authors: Rizzolo Kimberly , Elliott Sean
Published: June 2021

Recent bioinformatics analysis led to the discovery of unreported diheme enzymes found in all strains of Burkholderia, which share sequence similarity to known bacterial cytochrome c peroxidases (bCCPs). bCCPs are periplasmic enzymes that reduce hydrogen peroxide (H2O2) to water. Traditional bCCPs contain a low potential, peroxidatic heme (LP) and a high‐potential heme (HP), which acts as a conduit for the delivery of reducing equivalents to the active site. Alternatively, within the bacterial diheme family there is MauG from Paracoccus denitrificans, an enzyme that utilizes peroxide to generate an unusual high valent bis‐Fe(IV) species required for the post translational modification of two tryptophan residues present in protein precursor, preMADH, to yield tryptophyltryptophan quinone (TTQ), the catalytic cofactor required for methylamine dehydrogenase (MADH). To explore how sequence divergence within the peroxidase superfamily reflects differences in functionality, we cloned, expressed and purified various diheme enzymes from Burkholderia. Of the two classes identified, BmvA, from B. multivorans and BpmB, from B. pseudomallei, show novel reactivity compared to bCCPs and MauG. BmvA has peroxidase activity comparable to bCCPs, yet nearly identical electron paramagnetic resonance (EPR) spectrum to MauG as well as a predicted bis‐Fe(IV) species, as identified by near‐infrared (NIR) spectroscopy. Unlike BmvA, BpmB has been observed to react in the presence of oxygen, suggesting novel O2 chemistry. Additionally, orthologs to these proteins in the non‐pathogenic strain B. thailandensis (BthA and BthB), behave similarly. We hypothesize that the sequence divergence of the Burkholderia enzymes correlates to the observed reactivity, ultimately introducing a new class of enzymes to the peroxidase superfamily.
Peroxidation and redox reactions catalyzed by truncated hemoglobins
Authors: Tang Joseph , Couture Manon
Published: June 2021

Roseobacter denitrificans is one member of widespread marine Rosoebacters and performs photosynthesis and denitrification aerobically. We report a native heme‐bound R. denitrificans truncated hemoglobin (Rd. trHb) from heterologous expression in E. coli. Rd. trHb exhibits absorbance at 412, 538 and 572 nm bands with predominately alpha‐helical secondary structure. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares important heme pocket residues with both groups I and II trHbs. The resonance Raman spectra indicate that ferric Rd. trHb is mostly 6‐coordinate low‐spin and the ferrous form is a mixture of 5‐ and 6‐coordinated states. Two Fe‐His stretching modes were detected: one at 228 cm‐1, which represents an unconstrained neutral histidine as in other trHbs, and the other at 248 cm‐1, which has been reported in peroxidases and some flavohemoglobins that contain Fe‐His‐Asp (or Glu) catalytic triad, but not in trHbs. Further, Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than bovine Hb and only one order lower than horseradish peroxidase (HRP). The pH‐rate profiles yield a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme‐bound ligands in group II trHbs from M. tuberculosis and B. subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinct features and appears equipped to help Roseobacters to cope with reactive oxygen/nitrogen species and/or operate redox biochemistry.
Small molecule‐induced catalytic ubiquitination of non‐natural substrates
Authors: Bondeson Daniel , Pancevac Christina , Kruidenier Laurens , Carter Paul , Churcher Ian , Crews Craig
Published: June 2021

To achieve more potent and selective inhibition of disease‐causing proteins, we have generated PROTACs, or PROteolysis TArgetting Chimera. PROTACs are small, hetero‐bifunctional small molecules which simultaneously bind target proteins and E3 ubiquitin Ligases. Upon formation of a ternary complex, the Cullin‐based RING E3 ligase catalyzes the ubiquitination of the target protein, leading to its degradation. PROTACs have shown efficacy against a variety of targets in tissue culture and animal models, but mechanistic questions remain. Specifically, a prediction of the event‐based nature of PROTAC function is an ability to catalytically inhibit the target protein (i.e. the induction of supra‐stoichiometric ubiquitination of that target protein with respect to PROTAC). To rigorously define this catalysis, we have reconstituted the E1/E2/Cullin‐RING E3 ubiquitination reaction, and have shown that PROTACs are indeed capable of modifying supra‐stoichiometric amounts of target protein. Furthermore, as the ubiquitination system is presented with a non‐natural substrate, we hope to use this system to address questions surrounding lysine‐selection and ubiquitin chain‐type formation.
Mutational Analysis of a Putative Drug Resistance Transporter in the Human Parasite T. gondii
Authors: Rivera‐Lugo Rafael , Dou Zhicheng , Carruthers Vernon
Published: June 2021

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in animals and humans. At least 30% of the human population is infected. The infection can lead to lethal disease in immunocompromised individuals including AIDS and organ‐transplant patients. No existing drugs are capable of eradicating the parasites from infected people. Opening new options, we recently reported that Toxoplasma parasites intercept host cytosolic proteins by funneling them into a lysosome‐equivalent organelle termed vacuolar compartment (VAC) where they are digested. Additional work revealed that the VAC harbors a homolog of the malaria chloroquine resistance transporter (CRT). A single point mutation in CRT confers resistance to chloroquine treatment for malaria, profoundly impacting fatalities. Knocking out the CRT gene in Toxoplasma results in an enlarged VAC and reduced fitness. Topology analysis of the CRT primary sequence showed that four cysteine residues localize to a loop oriented in the lumen of the VAC. We hypothesized that these conserved cysteine residues function to regulate redox potential of the VAC and/or bind to metal ions to optimize the activity of VAC proteases. We performed site‐directed mutagenesis to mutate these four cysteine amino acids to serine residues individually and introduce them into CRT‐deficient parasites. We found that ΔcrtCRTC585S and ΔcrtCRTC596S did not restore the size of the VAC, and that ΔcrtCRTC609S did restored it but about 50% of the parasites showed mistrafficking of CRT. By identifying important conserved residues in a therapeutically relevant transporter this work forms a basis for future mechanistic dissection of CRT function