bluuebunny/crossref_metadata_2025 huggingface dataset:
The Impact of mRNA Structures Regulating Transcription Attenuation on Bacterial Fitness
Authors: Meyer Michelle , Babina Arianne
Published: June 2021

In bacteria regulation of transcription attenuation through modulations in mRNA structure is a common mechanism for coordinating gene expression. The formation of rho‐independent transcription terminators may be controlled by variety of factors including direct interactions with small molecules, tRNAs, and proteins. However, generally such mRNA structures are one part of several regulatory layers that include regulation of transcription initiation and in many cases appear to act as fine‐tuning mechanisms. The role such mRNA structures play in organism fitness and the forces that maintain their presence in microbial genomes remain largely undetermined. In this work we examine this question by assessing the impact of disrupting the protein‐binding site within an mRNA structure that regulates ribosomal proteins L20 and L35 in Bacillus subtilis. The mutation prevents formation of a rho‐independent terminator that allows autoregulation in response to ribosomal protein L20, but otherwise has minimal effects on gene expression. B. subtilis carrying the altered RNA display a cold‐sensitive increase in lag‐time, but the mutation has little effect on logarithmic phase growth rate. Our findings suggest that such RNA regulators may be most important within a rapidly changing environment and at the transitions between growth phases.
Changes in Water Availability or Molecular Crowding Induce Folding in Instrinsically Disordered Stress Proteins from Plants
Authors: Covarrubias Alejandra , Cuevas‐Velazquez César , Rivera‐Najera Lucero , Rendon‐Luna David , Saab‐Rincón Gloria , Martinez‐Yamout Maria , Dyson H Jane , Amero Carlos
Published: June 2021

Late Embryogenesis Abundant (LEA) proteins are a broadly distributed group involved in the plant response to water deficit. Most of them are predicted to be intrinsically disordered. By partial dehydration and freeze‐thaw in vitro assays, it was shown that some LEA proteins protect other proteins from the effects of water deficit. Given the presumed unstructured character of LEA proteins, we hypothesized that their flexible nature plays a critical role in their interaction with diverse partners. This structural flexibility might be modulated by the cell water status, promoting conformations needed to interact with specific targets. In this work, we report some of the structural and functional properties of LEA proteins from groups 4 and 6. We showed that proteins in both groups are intrinsically unstructured in solution; however, their intrinsic potential to form secondary structure was exhibited by treatment with a structure inducer, which promoted a significant α‐helix formation. A decrease in water availability and an increase in molecular crowding induced folding in both LEA protein groups but in different level. We also determined the protection efficiency of group 4 LEA proteins and proposed a mechanism for this function.

This work was partially supported by CONACyT (132258) and DGAPA‐UNAM (IN208212).
An Intrinsically Disordered Region of MBD2 Both Recruits the Histone Deacetylase Core Complex of NuRD and Modifies Kinetics of DNA Binding
Authors: Williams David , Desai Megha , Ginder Gordon
Published: June 2021

The MBD2 protein recruits and assembles the Nucleosome Remodeling and Deacetylase (NuRD) complex and thereby uniquely combines binding specificity for methylated DNA with histone deacetylation and chromatin remodeling. This MBD2‐NuRD complex has been implicated in methylation dependent silencing of genes during development and aberrant silencing of tumor suppressor genes during carcinogenesis. We have previously characterized and determined the structures of a coiled‐coil and methylcytosine binding (MBD) domains of MBD2. More recently we have characterized an intrinsically disordered region (IDR) of ~120 amino acids linking the MBD and coiled‐coil. NMR, CD, and AUC analyses show that this region, MBD2IDR, does not adopt a regular structure in isolation or in the context of full‐length protein. Yet the MBD2IDR stably binds three proteins that form the histone deacetylase core of NuRD. The first two‐thirds of the MBD2IDR are necessary and sufficient to bind the histone deacetylase core; while mutating two consecutive residues within this region abrogates binding and disrupts the function of MBD2 in cells. At the same time, adding the MBD2IDR to the MBD2MBD in vitro reduces the off rate and increases DNA binding affinity by ~100 fold. The MBD2IDR does not adopt a regular fold in the presence of DNA thereby functioning as a fuzzy DNA binding region. Hence the MBD2IDR plays a dual role both augmenting DNA binding affinity and recruiting a large portion of the NuRD complex.
Total Mutagenesis and the Effects on the MDM2‐p53 Interaction
Authors: Beyer George , Thayer Kelly
Published: June 2021

The aim of this study is to assess the impacts of mutation on the p53‐MDMD2 interaction.

While the available literature explores alanine‐scanning mutagenesis of the six necessary amino acids of the p53 binding subdomain in order to obtain precise ΔG values for the wild‐type amino acids in the p53‐MDM2 binding domain, the effects of non‐alanine point mutations have not been explored. Many mutations of MDM2 that could affect the binding to p53 have largely gone unstudied, but several SNPs in MDM2 have been directly linked to tumor formation. The aim of this study is to assess the impacts of mutation on the p53‐MDMD2 interaction utilizing both MUMBO and MM‐PBSA. MUMBO was used in order to perform total mutagenesis of the p53‐MDM2 domain in order to determine key mutations that may influence MDM2 binding activity. Data achieved from exhaustive point‐by‐point mutagenesis suggests great variation in the mutant structures. Mutants of interest will then be further explored utilizing the MM‐PBSA approach, and structural models will be constructed. Many mutations not shown through alanine‐scanning mutagenesis may greatly impact the p53‐MDM2 interaction.
Intrinsic Structural Disorder Observed in the Cell Cycle Regulatory GTPase Nucleostemin
Authors: Daman Tyler , Robinson Victoria
Published: June 2021

To maintain tissue homeostasis, it is necessary to control cell proliferation based on nutrient availability and environmental factors. Numerous lines of evidence suggest that the balance of the cellular concentrations of nucleostemin (NS), a non‐canonical circularly permuted GTPase, is critical for cell cycle progression. It does so by interacting with different partners involved in a wide range of cellular processes such as ribosome biogenesis, tumor suppression, and DNA maintenance. The cellular function of NS is regulated by guanine nucleotide driven localization to the nucleolus. Currently, little is known about the structure of NS and how it is modulated during this event.

Using an ensemble of biophysical techniques, we present the first structural characterization of Drosophila melanogaster Nucleostemin (dNS1), which is 33% identical and 67% similar to the human homolog. This represents the first structural data collected for a eukaryotic cpGTPase. Bioinformatic analysis, limited proteolysis, and circular dichroism were used in combination to determine that dNS1 contains large intrinsically disordered regions, primarily at the N‐ and C‐ termini. Small‐angle X‐ray scattering (SAXS) experiments, supported by analytical ultracentrifugation, provided a solution description of dNS1. Data obtained from these techniques establish that dNS1 exists as a highly elongated monomer, and confirms the lack of secondary structure. In summary, our studies show that NS contains large regions of structural disorder, which likely facilitates its broad partner recognition.
A Novel Mechanism to Control Cell/Tissue Specific Activities of Glucocorticoid Receptor
Authors: Kumar Raj , Khan Shagufta
Published: June 2021

Cell/tissue‐specific differential actions of glucocorticoids are mediated by the glucocorticoid receptor (GR) splice variants that possess truncated N‐terminal domain (NTD), which exists in an intrinsically disordered (ID) conformation and consists of GR's major activation function, AF1. Human GR variants lacking AF1 sequences are virtually inactive whereas those lacking amino acids on the NT flanking sequences of AF1 are most active in regulating GR target genes. However, the underlying mechanism of these GR isoform specific effects is largely unknown. We hypothesize that the GR isoform specific activities are allosterically regulated through order‐disorder transition of ID NTD sequences upon interaction with TATA‐box binding protein (TBP). First, using surface plasmon resonance method, we determined the binding kinetics of TBP with different fragments of GR containing AF1 sequences. We found that placing AF1 immediately upstream from the DNA binding domain leads to strongest AF1‐TBP interaction (KD = 0.07 µM) whereas AF1C (a core sub‐domain of AF1, important for AF1 activity) showed the weakest binding (KD = 3.0 µM) when compared to AF1 (KD = 0.5 µM). Interestingly, the GR fragment consisting of entire NTD showed a biphasic binding response to TBP. Our secondary/tertiary structural analyses using biophysical methods showed that upon TBP binding, each GR fragment adopts a compact/stable structure, and the stability is proportional to the TBP binding affinity. Consistent with GR isoform specific activity, TBP‐bound GR fragment lacking NT flanking sequences showed highest AF1‐mediated GR activity. Our studies for the first time have shown a mechanism for differential activities of GR isoforms that could open unique opportunities for cell/tissue‐specific endocrine‐based therapies.
The Intrinsically Disordered C‐terminal Domain Triggers Nucleolus Localization and Protein Interactions of Poly(A)‐specific Ribonuclease
Authors: Yan Yong‐Bin , He Guang‐Jun
Published: June 2021

Poly(A)‐specific ribonuclease (PARN) is a highly regulated deadenylase with diverse physiological functions via modulating poly(A)‐tail length of eukaryotic mRNAs. PARN is a multi‐domain protein with three well‐structured RNA‐binding domains and one C‐terminal domain (CTD) with unknown functions. Herein we investigated the structural features and potential functions of CTD. Spectroscopic studies indicated that CTD was intrinsically disordered with some local structures. The full‐length PARN had an intensive distribution in the nucleoli, while truncations of CTD impaired the nucleolus location of PARN. The nuclear‐localization and nucleolus‐localization sequences were identified by both deletion studies and fusion of the sequence with GFP. Furthermore, CTD was found to be the site triggering protein‐protein interactions and could selectively bind with its partners via the status of phosphorylation. Thus the intrinsically unstructured CTD is indispensible in the nucleolus localization and binding with PARN partners, which in turn regulate PARN activity. We proposed a model in which PARN is resistant in the nucleoli and can recruit dissimilar binding partners via the intrinsically disordered CTD to respond to various cellular stresses.

This study was supported by grant 31370797 from the National Natural Science Foundation of China.
Specificity of the Yeast Auxilin Homolog Swa2 in [URE3] Prion Propagation is Solely Determined by the TPR Domain
Authors: Hines Justin , Troisi Elizabeth , Rockman Michael , Oliver Emily
Published: June 2021

Previous results from our laboratory assert that the yeast Hsp40 chaperone protein Swa2 is essential for the propagation of the prion [URE3] in a yeast population. Swa2 is the sole yeast homolog of mammalian protein auxilin, which functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. The objective of this study was to find the determinants of the specific genetic interaction between Swa2 and [URE3]. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast Saccharomyces cerevisiae. Summary of Results: Our investigation revealed that two regions of the Swa2 protein, the J‐domain and the tetratricopeptide repeat region (TPR) are both necessary and sufficient to maintain [URE3], but that the TPR domain is solely responsible for prion/chaperone specificity. While the function of the TPR domain in Swa2 is undetermined, TPR domains typically act as docking platforms for a variety of protein complexes. As such we propose that the Swa2 TPR domain either participates in a bipartite interaction with Hsp70, analogous to the C‐terminal domains of other yeast Hsp40s, or that the TPR domain mediates the formation of a ternary complex with another undetermined protein in [URE3] propagation. We expect that understanding the function of the Swa2 TPR domain will provide new insight into both J‐protein function and the complex mechanisms of prion propagation.

This work was supported by a Single‐Investigator Cottrell College Science Award (Award #21010) from the Research Corporation for Science Advancement and by by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606.
Characterization of a Dihydropyridine‐insensitive Cav1.3 Channel
Authors: Wang Yuchen , Salyer Amy , Li Tingjiao , Hockerman Greg
Published: June 2021

The L‐type voltage‐gated calcium channels (L‐VGCCs), Cav1.2 and Cav1.3, play an important role in different physiological processes such as action potential firing, exocytosis and muscle contraction. Due to a lack of selectivity for the dihydropyridine (DHP) class of L‐VGCC blockers, a Cav1.3 mutant that is insensitive to DHPs (Cav1.3/DHPi) has been used to dissect the role of Cav1.3 in glucose‐stimulated insulin secretion and other cellular activities. However, the Cav1.3/DHPi mutant channel has not been biophysically characterized. Our lab created the Cav1.3/DHPi by mutating Thr1033 and Gln1037 that are located in segment 5 of domain III (IIIS5) of the rat neuronal Cav1.3 clone (accession no. AF30010.1) to Tyr and Met, respectively. Ba2+ current (IBa) elicited by different voltage steps from tsA cells expressing Cav channels with and without the presence of drugs was measured. The voltage‐dependent activation and inactivation of Cav1.3/DHPi (V1/2 inact.= ‐26.6±1.2mV, V1/2 act.= ‐22.3±1.1mV) resembles that of wild type Cav1.3 (Cav1.3/WT) (V1/2 inact.= ‐36.0±1.3mV, V1/2 act.= ‐29.8±1.5mV). Cav1.3/DHPi is over 100 fold more insensitive to nifedipine compared to Cav1.3/WT (IC50 for Cav1.3/WT= ~0.55μM; IC50 for Cav1.3/DHPi=~70μM). However, the non‐DHP blockers, diltiazem (500μM) and the recently debated Cav1.3 selective blocker, compound 8 (50μM) achieved comparable inhibition of Cav1.3/WT and Cav1.3/DHPi‐mediated current. Our findings showed that Thr1033 and Gln1037 are not only critical, but also specific for DHP block of Cav1.3. The Cav1.3/DHPi mutant is a useful tool to screen for Cav1.3 specific blockers by removing the DHP binding site which is common to both Cav1.2 and Cav1.3.
Regulation of the Human Ether‐a‐go‐go Related Gene (hERG) Potassium Channel by Nedd4 Family Interacting Proteins (Ndfips)
Authors: KANG YUDI , Guo Jun , Yang Tonghua , Li Wentao , Zhang Shetuan
Published: June 2021

The cardiac electrical disorder long QT syndrome (LQTS) predisposes affected individuals to high risks of ventricular arrhythmias and sudden death. Dysfunction of the human ether‐a‐go‐go related gene (hERG)‐encoded rapidly activating delayed rectifier K+ channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by the anterograde trafficking of newly synthesized channels to and the retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin ligase Nedd4‐2 (neuronal precursor cell expressed, developmentally down‐regulated protein 4 type 2) targets the PY motif of hERG channels to initiate channel degradation. Although both premature and mature hERG channels contain the PY motif, Nedd4‐2 selectively mediates the degradation of mature hERG channels. Here, using whole‐cell patch clamp, Western blot and immunocytochemical analyses, we demonstrate that Nedd4‐2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Ndfip1 and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4‐2 to target mature hERG proteins for degradation during channel trafficking to the plasma membrane. Although Ndfip2 also recruits Nedd4‐2 to the Golgi apparatus to some extent, it mainly recruits Nedd4‐2 to the multivesicular bodies (MVBs), which impedes the degradation of internalized hERG proteins. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.

(Supported by the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of Ontario)
Regulation of Lipid Droplet Formation by Group IVA Phospholipase A2
Authors: Balsinde Jesús , Guijas Carlos , Rodríguez Juan , Rubio Julio , Balboa María
Published: June 2021

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. LD are formed by two different routes, namely (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling leading to increased neutral lipid formation utilizing fatty acids coming from the de novo biosynthetic route. The latter predominates, accounting for 60‐70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2a, pointing out this enzyme as a key regulator of AA‐induced signaling. LD formation in AA‐treated monocytes can also be blocked by the combined inhibition of the mitogen‐activated protein kinase family member p38 and JNK, which correlates with inhibition of cPLA2a activation by phosphorylation. Since cPLA2a has no effect on neutral lipid formation there must be a step in the mechanism of LD biogenesis that is critically dependent on the enzyme, e.g. generation of a positive curvature at the organelle baseline.

Supported by Grant SAF2013‐48201‐R from the Spanish Ministry of Economy and Competitivenes
Reverse Effect of Coconut Water on Lipidomics of Low Level Cadmium‐Induced Rat
Authors: Ogunrinola Olabisi , Wusu Adedoja , Adu Benedict , Elemo Babajide , Ademuyiwa Oladipo
Published: June 2021

Coconut water is one of the world natural products with increasing scientific evidence that support the role of coconut water in health and medicinal application. This study was undertaken to investigate the reverse effect of coconut water on the lipid metabolism of cadmium‐induced Wister male albino rats that might result in vascular diseases. Sixty‐four animals were exposed to 100, 200, and 300ppm cadmium doses for 6 weeks in their drinking water, then administered with coconut water for a week. Control animals received distilled water and coconut water for the same period. At the end of 6 weeks, dyslipidemia induced by the cadmium doses exhibited different patterns. Dose‐dependent hypocholesterolemia, hypotriacylglyceridemia and hypophospholipidosis characterized the effect of cadmium exposure at all doses while coconut water ameliorate these effects. Hepatic and brain cholesterol and phospholipid were dose‐dependently decreased by cadmium exposure respectively. While brain triacylglyceride decreased by cadmium exposure, hepatic triacylglyceride was increased dose‐dependently. On the other hand, administration of coconut significantly (p<0.05) enhanced cholesterol, triacylglyceride content and resulted in a fluctuation in phospholipid concentration. Our findings indicate that in contrast to strengthening a dose‐dependent effect phenomenon as observed with many other compounds, cadmium up‐ or down‐regulate different pathways in the lipid metabolism spectrum at “low” or “high” doses and this might be responsible for the insidious vascular effects and the administration of coconut water was able to reverse the damage caused by cadmium intoxication.
Single Molecule Dynamics of Lipid Bilayer Associated PTEN
Authors: Gericke Arne , Harishchandra Rakesh , Bryant Anne , Ross Alonzo , Neumann Brittany
Published: June 2021

Objective

Study the dynamics of PTEN associated with laterally heterogeneous lipid bilayers using single molecule microscopy.

Background

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), is one of the most frequent genes deleted/mutated in a wide variety of tumors. PTEN acts as an antagonist to PI 3‐kinase signaling, thereby affecting cellular processes such as cell proliferation and survival. PTEN activity is regulated by dynamic shuttling between the cytoplasm and the plasma membrane.

Methods

We use single‐molecule TIRF microscopy and stopped‐flow fluorescence measurements to observe PTEN molecules as they dynamically associate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN lipid binding is investigated using supported lipid bilayers of binary and ternary lipid mixtures of phosphatidylcholine (PC) with physiological relevant levels of phosphatidylserine (PS), phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) and/or phosphatidylinositol (PI).

Results

Using single molecule trajectories of PTEN molecules we find significant differences in PTEN dynamic behavior when bound to different membrane environments. Interestingly, we determine the existence of three membrane bound states with different diffusion coefficients. To gain insight into the molecular mechanisms of PTEN membrane association, we compared the lipid binding of wt‐PTEN, PTEN‐(Δ1‐15aa) and the recently discovered, PTEN‐L with a 173 aa N‐terminal extension. We find profound differences in the dynamic behavior of these PTEN derivatives at the membrane. We also found that PI may replace PS as the second lipid component to which PTEN binds.
Ionization Properties of the enigmatic plant lipid DGPP
Authors: Bazilevich Gloria , Budge Kevin , Rapovy Shannon , Khalifa Hady , Koukounas Yannis , Graber Zachary , Kooijman Edgar
Published: June 2021

Diacyglycerol pyrophosphate (DGPP) an uncommon membrane lipid found in plants, and yeast, but never in mammals is formed from phosphatidic acid. The function of DGPP during stress signaling is unclear. Therefore, the physiochemical properties, specifically the ionization of the head group, were analyzed to elucidate the function of DGPP. The presence of the trans‐membrane peptide KALP23, to mimic the interaction with DGPP binding proteins, caused an increase in the ionization of DGPP. This increase in negative charge might be due to the decrease in interfacial pH by the cationic lysine residues of KALP23, or might be due to direct hydrogen bond interaction between KALP23 and DGPP. We investigated the effect of positive charge on the negative charge of DGPP to delineate these separate effects. Next, we investigated the interaction of PA with DGPP and the resultant effect on the charge of both of these signaling lipids. The results are discussed in terms of the electrostatic‐hydrogen bond switch model previously described for the ionization and protein interaction of PA.
The PHO4 Transcription Factor Regulates Triacylglycerol Metabolism under Low Phosphate Conditions in Saccharomyces cerevisiae
Authors: Yadav Kamlesh , Singh Neelima , Rajasekharan Ram
Published: June 2021

Objective

To study the transcriptional regulation of lipid metabolism in Saccharomyces cerevisiae under low phosphate (Pi) conditions.

Methodology

[14C]Acetate labeling, confocal microscopy, gas chromatography, semi quantitative and quantitative PCR. Electromobility shift assay (EMSA) and β‐galactosidase assay. To validate the binding site‐directed mutagenesis was performed. Enzyme kinetics and characterization.

Results

The expression of PHM8 is high under low phosphate conditions, which encodes a LPA phosphatase and nucleotidase. In an electrophoretic mobility shift assay, Pho4p was shown to bind specifically in PHM8 promoter. Analyses of PHM8 mRNA, LPA phosphatase activity and triacylglycerol levels under low phosphate conditions in phm8Δ showed that the plasmid harbouring mutated promoter along with the PHM8 gene had much lower PHM8 expression than the plasmid harbouring the native promoter with the gene. Enzyme kinetics with the differentially phosphorylated Phm8p using LPA and CMP as substrates showed that LPA is the preferred over CMP under low phosphate conditions. The results indicate that the transcriptional activator Pho4p binds to the PHM8 promoter under low phosphate conditions, activating PHM8 expression and leading to the formation of monoacylglycerol, which is successively acylated by Dga1p to form diacylglycerol and triacylglycerol.

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Conclusion

The phosphate‐mediated transcriptional activation of PHM8 by Pho4p affects triacylglycerol biosynthesis by controlling lysophosphatidic acid and MAG flux.
Ubiquitin ligase Idol Inhibitors for Treating Hypercholesterolemia
Authors: Marblestone Jeffrey , Wu Jian , LaRocque James , Agarwal Saket , Kodrasov Matthew , Weinstock Joseph , Kumar Suresh , Mattern Michael , Sterner David
Published: June 2021

Despite widespread use of statins and other therapeutics, hypercholesterolemia remains a significant medical issue for a many patients unable to effectively regulate cholesterol levels. Cholesterol uptake is primarily mediated by the hepatic low density lipoprotein receptor (LDLR), which binds and internalizes plasma LDL. Elevated LDLR is associated with reduced LDL levels. Recently, the E3 ligase Idol (Inducible Degrader of LDLR) was shown to be a key regulator of LDLR levels. IDOL is a unique E3 ligase that utilizes its FERM domain to specifically target LDLR for polyubiquitylation and subsequent lysosomal degradation. Genetic ablation of IDOL raises levels of LDLR. Thus, inhibition of IDOL may be beneficial for the treatment of hypercholesterolemia. The ubiquitin‐proteasome system is a rich landscape for drug discovery. E3 ligases in particular are attractive therapeutic targets in various disorders, including metabolic disorders. Nevertheless, it has been challenging to discover and develop E3 ligase inhibitors as first in class clinical candidates. Here, we report the identification of novel IDOL inhibitors that modulate cellular cholesterol homeostasis. These compounds increased LDLR levels and increased LDL association in various cellular models. Biophysical characterization revealed direct binding of the compounds to IDOL and perturbation of IDOL:LDLR interactions. The most promising compounds were used as starting points to develop novel drug like molecules and the lead compounds are being evaluated in translational models of hypercholesterolemia. Data will be presented summarizing our progress to date targeting IDOL for the treatment of hypercholesterolemia. Work supported in part by NIH grant HL127893.
CD36 Deficiency Reduces Obesity‐Associated Inflammation and Oxidative Stress in Heart
Authors: Hajri Tahar , Gharib Mohamed , Tao Huan , Fungwe Thomas
Published: June 2021

Mounting evidence shows that obesity and diabetes are associated with structural and functional changes in the heart. CD36 protein is highly expressed in heart and regulates lipid utilization in cardiac myocytes. In this study, we investigate the impact of CD36 expression in obesity‐associated inflammation in heart.
Studies were conducted in control lean, obese leptin deficient (LepOb/Ob) and leptin deficient‐CD36 null (LepOb/Ob CD36‐/‐) mice. Histology and immunohistochemisty were conducted to examine heart structure and presence of macrophages. Uptake and oxidation glucose and fatty acids was tested in primary cultures of ventricular myocytes. Finally, the effects of fatty acid treatment on oxidative stress and activity of pro‐inflammatory kinases were examined in isolated cardiac myocytes. In LepOb/Ob, glucose uptake and oxidation in heart was lower than normal weight mice, while cardiac FA oxidation was strongly higher than normal. Silencing CD36 markedly improved insulin sensitivity and glucose uptake in heart, but marked reduced FA oxidation. Moreover, CD36 deficiency reduced macrophage content, oxidative stress markers and expression of pro‐inflammatory cytokines in heart. Chronic treatment with palmitate strongly induced oxidative stress and inflammatory markers in LepOb/Ob cardiac myocytes, but was clearly less effective in LepOb/Ob‐CD36‐/‐ cardiacmyocytes.
These results show that CD36 deficiency induces a significant reduction of obesity‐associated oxidative stress and inflammation in heart, in parallel to a drop of lipid infiltration and improved insulin sensitivity
The effects of Kolaviron, a Natural Flavonoid from the Seeds of Garcinia Kola on LPS‐induced Inflammation in Macrophages
Authors: Abarikwu sunny
Published: June 2021

Kolaviron (Kol‐v), an important component of Garcinia kola seed has a variety of biologic activities, including anti‐inflammatory properties. In this study, we investigated the possible anti‐inflammatory effect of Kol‐v against pro‐inflammatory responses induced by LPS and the associated signalling pathways in vitro. The protective effects of Kol‐v were determined by measuring gene expressions and activation of inflammatory molecules in LPS‐activated RAW macrophages. We found that Kol‐v inhibited LPS‐induced phosphorylation of p‐c‐JUN and p38 but not IκBα, NF‐κB (p65), ERK1/2, JNK, and IκBα degradation. Kol‐v also suppressed LPS‐induced IL‐18 and LPS‐down‐regulated IP‐10 gene expressions but had no effect on the LPS‐induced gene expression levels of IL‐1α, IL‐33, IL‐1β, IFNβ1‐1, and TNF‐α. Kol‐v concentrations (50 and 100 μM), blocked the phosphorylation of Akt, ERK1/2, IκBα, p38, and NF‐κB but not CREB. The protective effect on JNK phosphorylation was observed at 100 μM Kol‐v. At all concentrations of Kol‐v (10‐100 μM) tested, Kol‐v suppressed the production of IL‐6 in a concentration dependent manner but had no effect on LPS‐induced secretion of TNF‐α. Collectively, these results suggest that Kol‐v interferes with LPS signalling by reducing the activation of several inflammatory transcription factors and that its inhibitory action on IL‐6 secretion correlates with inhibition of ERK1/2, p38, Akt, p‐c‐JUN and JNK signalling pathways.
Discovery of Isolndolo[2,1‐a]quinazoline derivatives as anti‐Hepatitis C virus agents by suppressing HCV associated inflammation
Authors: Manvar Dinesh , Kaushik‐Basu Neerja , Kulkarni Shridhar , Talele Tanaji
Published: June 2021

Chronic Hepatitis C Virus (HCV) infection leads to serious liver disease with sequelae including cirrhosis, steatosis, and hepatocellular carcinoma. HCV infection triggers vigorous immune response which in the long term leads to upregulation of COX‐2 and other pro‐inflammatory cytokines. Together, this condition likely contributes to persistent viral infection and subsequent liver damage. We have identified a series of small molecules that exhibited potent anti‐HCV activity and inhibited the replication of HCV replicon of both 1b and 2a genotype origin at low micromolar range EC50 values. Active compounds had minimal effect on cell viability at concentrations in far excess of their EC50 values and showed excellent selectivity indexes. We will present the molecular mechanisms of anti‐HCV activity of Isolndolo[2,1‐a]quinazoline derivatives. Active compounds suppressed HCV‐induced COX‐2 mRNA and protein expression. Conversely, the anti‐HCV activities of these compounds were abrogated upon transient over‐expression of COX‐2. Further, treatment with the active compounds mitigated both Nuclear Factor‐κB activation as well as the effects of tumor necrosis factor‐α in HCV replicon cells. Our data suggests that these compounds function as anti‐HCV agents via targeting COX‐2 at both the transcription and translation levels and thus have the potential to be developed as novel anti‐HCV therapeutic agents with the ability to dampen inflammation during chronic HCV infection.
Oxidation of Salicylic Acid and Aspirin by Human Cytochromes P450
Authors: Sedgeman Carl , Bojic Mirza , Guengerich F.
Published: June 2021

Aspirin (acetylsalicylic acid) is a commonly used analgesic. It is rapidly deacetylated both enzymatically and non‐enzymatically to salicylic acid. Salicylic acid can then be further metabolized by conjugation with glycine or glucuronides or by hydroxylation to form 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and 2,5‐dihydroxybenzoic acid (2,5‐DHBA). This oxidation has been suspected to occur from oxygen radicals via Fenton reactions as well as by cytochrome P450 enzymes. We analyzed the formation of 2,3‐DHBA and 2,5‐DHBA in the presence of human liver microsomes by HPLC with fluorescence detection. We were able to determine that microsomal oxidation was much faster for salicylic acid than aspirin. 2,3‐DHBA was found to be the major oxidative product formed from salicylic acid, whereas aspirin could detect only 2,5‐DHBA. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5‐hydroxylation of salicylic acid. Inhibitor studies were able to confirm that all six of these enzymes played a role in the formation of 2,3‐ and 2,5‐DHBA, as well as found that 2A6 and 2B6 played a role in the hydroxylation to 2,5‐DHBA. It was also shown in the inhibitor studies that P450 2E1 was the major enzyme contributing to the in both 3‐ and 5‐hydroxylations.

This work was supported in part by National Institutes of Health grant R37 CA090426.
MD‐2 as the Target of a Novel Small Molecule, L6H21, in the Attenuation of LPS‐induced Inflammatory Response and Sepsis
Authors: Liang Guang , Wang Yi , Chen Gaozhi , Fang Qilu
Published: June 2021

Background

Myeloid differentiation 2 (MD‐2) recognizes LPS, which is required for TLR4 activation, and represents an attractive therapeutic target for severe inflammatory disorders. We previously found that a chalcone derivative, L6H21, could inhibit LPS‐induced overexpression of TNF‐α and IL‐6 in macrophages.

Methods

The binding affinity of L6H21 to MD‐2 protein was analyzed using computer docking, surface plasmon resonance analysis, ELISA, fluorescence measurements, and flow cytometric analysis. The effects of L6H21 on MAPK and NF‐κB signaling were determined using EMSA, fluorescence staining, western blotting, and immunoprecipitation. The anti‐inflammatory effects of L6H21 were confirmed using ELISA and RT‐qPCR in vitro. Finally, the anti‐inflammatory effects of L6H21 in septic C57BL/6 mice were evaluated.

Results

Compound L6H21 was inserted into the hydrophobic region of MD‐2 pocket, forming hydrogen bonds with Arg90 and Tyr102 in the MD‐2 pocket. In vitro, L6H21 subsequently suppressed MAPK phosphorylation, NF‐κB activation, and cytokine expression in macrophages stimulated by LPS. In vivo, L6H21 pretreatment improved survival, protected lung injury, decreased serum and hepatic cytokine level in mice subjected to LPS. In addition, the MD‐2 gene knockout led to universal protection from LPS‐induced septic shock in mice.

Conclusions

This work identified a new chalcone derivative, L6H21, as a potential candidate in the treatment of sepsis; more importantly, the data confirmed that MD‐2 is an important therapeutic target against inflammatory disorders.
PADlock Cryptanalysis: a Bayesian Approach to Identifying Trends in the Post‐Translational Modification Deimination
Authors: Olson Justin , Church James , Grant Jennifer
Published: June 2021

Cellular proteins are subject to the post‐translational modification known as deimination in which the amino acid arginine is converted into citrulline. Deiminated proteins are widespread in disease states and thought to play a role in Rheumatoid Arthritis, Multiple Sclerosis, Alzheimer's disease, Parkinson's disease, and cancer. Unlike phosphorylation, substrate specificity of the peptidyl‐arginine deiminase (PAD) family of enzymes, which are responsible for deimination, remains elusive and ill defined. Our objective was to identify patterns in the sequence primary and secondary structures, which may underpin this modification. We found that deiminated sequences were most likely to be categorized as having a disorganized secondary structure, and we identify trends in how the sequence primary structure influences the likelihood of deimination, based on amino acid property.
Deciphering the Functional O‐GlcNAc Sub‐Proteome
Authors: Martinez Marissa , Pandey Akilesh , Zachara Natasha
Published: June 2021

O‐linked N‐acetyl‐β‐D‐glucosamine (O‐GlcNAc) is a post‐translational modification of serine and threonine residues that regulates protein function and signaling. O‐GlcNAc has emerged as a novel regulator of the cellular response to stress, including oxidative stress and ischemia reperfusion (IR) injury of the heart. A recent study has identified over 200 proteins whose state of O‐GlcNAcylation changes in response to oxidative stress. However, the specific pathways that regulate this response remain ill‐defined. This is in‐part due to the promiscuous nature of proteins to associate in complexes in order to mediate cellular pathways. Thus, the goal of this study is to characterize the O‐GlcNAc modified protein complexes that change in response to oxidative stress. Proteins were isolated from mouse embryonic fibroblasts, followed by affinity enrichment for O‐GlcNAcylated protein complexes. Complexes were then separated by size exclusion chromatography (SEC). Preliminary data demonstrates differential elution of O‐GlcNAc modified proteins in distinct SEC fractions. In addition, known O‐GlcNAc modified proteins such as CARM1 elute in the same SEC fractions as known binding proteins, FUS and HNRNPU. Together these data support the use of affinity enrichment coupled to SEC for the characterization of O‐GlcNAc modified protein complexes. Current studies are now focused on the identification of proteins within complexes by mass spectrometry with the ultimate goal of quantification by stable isotopic labeling of amino acids in cell culture. Together, these data will provide a more detailed understanding of the role of O‐GlcNAc in response to oxidative stress and IR injury.
Effect of Pathway‐Interconnectors in SEB‐Induced Apoptosis Related Events in Human PBMCs
Authors: Mills Whitney , Wampfler Ashlynne , Rothschadl Kendra , Gilman Kim , Mendis Chanaka
Published: June 2021

Staphylococcus enterotoxin B (SEB) is produced by the bacterium Staphylococcus aureus. SEB is the most common cause of food poisoning often accompanied with nausea, diarrhea, and intestinal cramping. The purpose of this study is to use human peripheral blood mononuclear cells (PBMCs) in order to better understand the apoptosis related events induced by SEB. This study is focused on inhibiting signaling pathways with the use of the inhibitor 4‐[5‐(4‐Fluorophenyl)‐2‐[4‐(methylsulfonyl)phenyl]‐1‐H‐imidazol‐4‐yl]pyridine (SB203580), followed by analyzing alterations to known expression patterns of genes associated with apoptosis. Here we attempt to better understand the type of pathway utilized by SEB by evaluating gene expression patterns using RT‐PCR. We will also investigate the Protein expression patterns of some genes through ELISA. We believe that our work will allow us to better understand the complex interactions of multiple signal transduction pathways as well as see the effects of inhibiting pathway interconnectors when induced by SEB.
CXCL12/SDF‐1 Stimulates Megakaryocyte β‐1,4‐Galactosyltransferase 1 Activity to Enhance Platelet Production in Vivo
Authors: Giannini Silvia , Adelman Max , Jurak Begonja Antonija , Hoffmeister Karin
Published: June 2021

Platelet recovery following bone marrow (BM) transplant and chemotherapy is crucial to prevent bleeding complications. The chemokine SDF‐1 promotes megakaryocyte (MK) migration towards BM sinusoids increasing platelet production. Here we show that SDF‐1 upregulates expression of the glycan structure Type‐2‐Lactosaminoglycan (LacNAc), synthesized by β4Galactosyltransferase1 (β4galt1). We hypothesized that SDF‐1 increases LacNAc synthesis promoting platelet production. Supporting this hypothesis, β4galt1–/– mice had severe macrothrombocytopenia (80% reduction) with increased BMMK numbers (35%), which localized poorly with BM sinusoids (50%), compared to WT MKs (72%). β4galt1–/– hematopoietic stem cells (HSCs) transplanted into lethally irradiated WT mice restored BMMKs, which failed to migrate to sinusoids and produce circulating platelets. We next treated WT and β4galt1–/– mice with the myeloablative chemotherapeutic agent 5‐Fluorouracil (5‐FU), which decreased platelet counts by 50% in both phenotypes. SDF‐1 injections increased platelet counts by 70% in 5‐FU treated WT mice but not in β4galt1–/– mice. Importantly, WT but not β4galt1–/– platelets had increased LacNAc expression after SDF‐1 injections.β4galt1–/– BMMKs had increased β1 integrin expression. Expression of laminin, β1 integrin ligand, was upregulated in β4galt1–/– MKs and sinusoids. Our results suggest that SDF‐1 upregulates β4GalT1‐dependentLacNAc expression promoting MK interaction with BM sinusoids, likely regulating β1 integrin expression and interaction with components of the extracellular matrix, specifically laminin.
Loranthus micranthus Leaves Extract Attenuates Risk Factors of Cardiovascular Disease in Fructose Fed Rats
Authors: Ajani Emmanuel , Ogunlabi Olugbenga , Adegbesan Bukunola
Published: June 2021

There are concerns about adverse effects of dietary sugars. This study compared dietary fructose induced metabolic dysfunction in male rats with the female and the modulatory effect of Loranthus micranthus. Fifty six rats assigned to four groups of 7 male and 7 females per group were used in the study. The water of group B, C and D rats were supplemented with 10% fructose for the first two 2 weeks and was later increased to 20%, 30% and 40% after every 2 other weeks respectively. Nifedipine was administered to group C while L. micranthus (600mg/Kg) was orally administered to group D. All administrations were carried out daily as a single dose.The rats were sacrificed and the serum analyzed for the lipid components. Serum glucose level was measured every 2 weeks interval. Fructose administration increased serum total cholesterol, triglyceride, LDL‐C, VLDL‐C, atherogenic and coronary risk indexes but decreased serum HDL‐C significantly. Serum glucose was not altered during the first 6 weeks of study but was significantly increased above the initial value after 8 weeks of study. Both L. micranthus and nifedipine prevented this metabolic dysfunction but the effect was more pronounced with L. micranthus extract. The study concludes that male subjects are more prone to metabolic dysfunction of fructose than the female group and that L. micranthus is efficacious in preventing this defect.
Targeted Inhibitors of P‐glycoprotein Overcome Multidrug Resistance in Human Cancer Cell Lines
Authors: Follit Courtney , Brewer Frances , Wise John , Vogel Pia
Published: June 2021

Multidrug resistance (MDR), the intrinsic or acquired simultaneous resistance to structurally and chemically unrelated drugs, is arguably the largest barrier to the successful chemotherapeutic treatment of cancer. The best studied mechanism of MDR is due to the overexpression of ABC‐transporter member P‐glycoprotein (P‐gp). All ABC‐transporters utilize ATP hydrolysis to actively export substrates out of the cell, but P‐gp is exceptional in that its substrates are diverse enough to include the majority of clinically relevant chemotherapeutics. Researchers have searched for drugs to inhibit P‐gp for decades and largely failed because the majority interacted with the drug‐binding domain of P‐gp and were themselves transported. Using targeted molecular dynamic simulations and massively parallel drug docking studies, we identified novel compounds that inhibit P‐gp by specifically targeting the ATP binding domain of P‐gp. Biochemical and biophysical studies have confirmed ATP hydrolysis is inhibited by these novel compounds. In the studies reported here, we have evaluated these compounds for their ability to restore drug sensitivity to MDR human cancer cell lines that overexpress P‐gp. Effects on other ABC‐transporters capable of conferring the MDR phenotype are being evaluated and lead compounds are undergoing chemical optimization to develop co‐therapeutics capable of reversing MDR in human cancers.

This work is supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, SMU Research Council, SMU Dean's Research Council, the SMU Center for Drug Discovery, Design and Delivery, and the Communities Foundation of Texas, Dallas.
A QM/MM Investigation of the Antitumor Drug Neocarzinostatin
Authors: McKoy Quincy , Miller Bill , Parish Carol
Published: June 2021

Under the influence of heat or light, enediynes convert to para‐benzenes via the Bergman cyclization reaction. The resulting para‐benzyne moiety can abstract hydrogen atoms from DNA leading to DNA unraveling and subsequent cell death. Neocarzinostatin is a natural product drug molecule that contains an enediyne. In order to evaluate the effectiveness of neocarzinostatin as a cancer drug, we utilized computational chemistry to evaluate the mechanism whereby neocarzinostatin undergoes the Bergman cyclization and abstracts H atoms in the presence of DNA. We used docking methods available in GLIDE to generate initial poses which were subsequently subjected to long time scale molecular dynamics to explore the conformational flexibility and binding affinity of the DNA:neocarzinostatin complex. We then used the QM/MM (Quantum Mechanics/Molecular Mechanics) ONIOM method to study the barriers for neocarzinostatin Bergman cyclization in the presence of DNA as well as the mechanism for H abstraction. This study will allow us to investigate both the interaction of a cancer drug with DNA as well as the energy necessary to influence the Bergman cyclization reaction.
Identifying activators of the mobile element L1 endonuclease
Authors: Meyers Jade , Darthard Loryn , Servant Geraldine , Streva Vincent , DeFreece Cecily
Published: June 2021

Long interspersed element‐1 (L1) is the only active autonomous retroelement in the human genome. Studies characterizing L1 function show that the L1 endonuclease activity is associated with the formation of DNA double strand breaks (DSBs). While L1 mutagenic insertion events have been associated with diseases such as breast and colon cancer as well as muscular dystrophy, the full effect of L1 endonuclease upon stability of the genome is undetermined. Data suggests that the damage from L1 endonuclease activity may be greater that previously considered seeing as though only a small fraction of L1‐induced DSBs results in a retrotransposition event. We hypothesis that an activator of L1 endonuclease would be useful for determining the effects of L1 endonuclease DNA damage to genetic instability and disease. We have tested small molecule activators of L1 endonuclease in vitro using an endonuclease cleavage assay. This assay uses an annealed oligonucleotide substrate that is labeled with a fluorophore and a quencher on the opposite strand. Once the cleavage occurs, the dissociation of the fluorophore allows us to observe fluorescence over time. We also tested the ability of the small molecule to increase the activity of a related endonuclease, Ape1, to determine if the molecule is specific. The molecules increase L1 endonuclease activity in vitro and appear to be toxic to cells. This toxicity may be due to increase in L1‐associated DNA DSB formation in cells following treatment with the small molecule.