bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic Digestion Reveals Novel Insights into the Kv1.5 Structure‐Function Relationship
Authors: Hogan‐Cann Andrew , Zhang Shetuan
Published: June 2021

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Kv1.5 channel mediated ultra‐rapidly activating delayed rectifier potassium current (IKur) is important for atrial repolarization. Since cardiac IKur is expressed solely in the atria, therapies that target Kv1.5 represent promising strategies for treating AF. Pathologies linked to AF, such as ischemia and hypoxia, have been reported to activate various proteases. It has been shown that cell‐surface Kv1.5 channels are sensitive to cleavage by extracellular proteases, such as proteinase K (PK). In this study, we investigated the consequences of proteolytic digestion on Kv1.5 channels. Our data demonstrate that extracellularly applied PK cleaves mature (75 kDa) Kv1.5 channels at a single locus in the S1‐S2 linker, yielding two digestion products of 42 and 33 kDa, corresponding to the N‐ and C‐terminal fragments, respectively. Surprisingly, whole‐cell patch clamp analysis showed that IKv1.5 was unaltered by the PK cut. Plasma membrane protein isolation via biotinylation indicated that both the N‐ and C‐terminal fragments following PK digestion retain cell‐surface expression over several hours. Co‐immunoprecipitation studies after PK cleavage indicate that the two fragments of the channel do not associate. The time‐dependent decline in the Kv1.5 fragment lacking the N‐terminus and the S1 domain paralleled the amplitude of recorded Kv1.5 currents. Our results suggest that Kv1.5 remains functional without the NH2‐S1 portion of the channel. The resistance of IKv1.5 to proteolytic cleavage may represent an inherent protective mechanism for Kv1.5 function. These findings extend our understanding of the Kv1.5 channel structure‐function relationship.

Supported by CIHR & the Heart and Stroke Foundation
Monitoring the Dynamic Change of Inositol Lipid Pools upon EGFR and M3R Activation in Live Cells
Authors: Toth Jozsef , Gulyas Gergo , Toth Daniel , Hunyady Laszlo , Balla Tamas , Varnai Peter
Published: June 2021

Phosphoinositides (PI) in the plasma membrane (PM) can dynamically change upon hormonal stimulation which can influence several cellular processes thus measuring the level of PIs can help us to better understand their distinct functions. We developed a highly sensitive method which enables us to follow the dynamic change of these lipids in living cells. We performed BRET measurements between various luciferase‐labeled PI‐binding domains and a PM‐targeted Venus in HEK 293T cells. To monitor the inositol lipid pools the followings were used: the 2xP4M domain of SidM for PI(4)P, the PH domain of PLCδ1 for PI(4,5)P2, the PH domain of BTK for PI(3,4,5)P3 and an intramolecular IP3 BRET sensor based on the ligand binding domain of the type‐1 IP3 receptor. Stimulation of cells with 100 ng/ml EGF resulted in a sustained increase of PI(3,4,5)P3, a slowly developing IP3 signal a transiently decreased PI(4,5)P2 level and an unexpected increase of PI(4)P. We also tested the lipid changes in case of M3R activation and we found that 100 µM carbachol causes more robust increase in the IP3, and decrease in the PI(4,5)P2 and PI4P levels however decreasing the carbachol concentration to 100 nM resulted in a very similar lipid changes as were seen in case of the EGF stimulation. Using different PI4K inhibitors we found that the increase in the PI(4)P level was caused by the activation of PI4KIIIα, and could be prevented by PKC inhibitors. With the help of our highly sensitive approach we were able to follow the dynamic inositol lipid changes upon stimulation of the cells with various compounds, and we could identify a possible signaling route how the cells can resynthesize PIs.
Walnuts Rearrange the Lipid Mediator Composition of Lipoproteins Independent of Changes in Fatty Acid Precursors
Authors: Shearer Gregory , Newman John , Holt Roberta , Keen Carl , Hackman Robert
Published: June 2021

Lipoproteins transport lipid mediators through the body, making them an important component of lipid mediator biology. Walnuts are an abundant dietary source of linoleic (LA) and alpha‐linolenic (aLA) acids. They also improve plasma lipoprotein profiles. Both are likely to impact the quality and quantity of lipoprotein lipid mediators. We measured mid‐chain alcohols as HODEs from LA, HOTEs from aLA, HETEs from arachidonate (AA) and HEPEs from eicosapentaenoate(EPA) and fatty acid epoxides as EpOMEs from LA, EpODEs from aLA, EpETrEs (EETs) from AA, and EpETEs from EPA in High‐, Low‐, and Very Low‐Density Lipoproteins (HDL, LDL and VLDL respectively) in 20 subjects consuming Low (LWD; 5g/d) or High (HWD; 40g/d) walnut diets. HWD increased aLA content of all lipoproteins by 53% [12, 109]. In contrast to nominal fatty acid changes, HWD decreased HODEs by 41% [‐15, ‐59], but reduced HDL‐ and LDL‐HETEs (‐56% [‐25, ‐75] & ‐63% [‐37, ‐79]) and HDL‐ and LDL‐HEPEs (‐53% [‐12, ‐75] & ‐71% [‐45, ‐84]). HWD had no effect on HOTEs. EpOMEs, EpODEs and EpETrEs, were increased by HWD specifically in HDL by 63% [10, 141], 102% [28,220], and 58% [9, 130] respectively. EpETEs were reduced by HWD in LDL ‐54% [‐24, ‐72]. The rearrangements are consistent with reduced inflammatory burden of lipid mediators but are not explained simply by changes in precursor fatty acid abundance. This indicates some component of walnuts beyond their fatty acid composition influences lipid mediator distribution within lipoproteins.
Cordycepin Inhibits LPS‐Induced Inflammatory Responses by Modulating NOD‐Like Receptor Protein 3 Inflammasome Activation
Authors: Yang Jing , Li Yunzhou , Zhang Luyong , Zhou Huiping
Published: June 2021

The inflammasome is a multi‐protein complexthat induces maturation of inflammatory cytokines interleukin (IL)‐1β and IL‐18 through activation of caspase‐1. Activation of inflammasome contributes to host defense during diverse bacterial infections. But prolonged activation of inflammasome can be deleterious due to tissue damage associated with chronic inflammatory responses. Cordycepin, a natural compound and adenosine analogue derived from Cordyceps militaris, has been shown to have a great potential for therapeutic use against a number of inflammatory diseases. However, the molecular mechanisms by which cordycepin regulates inflammatory responses have not been clearly elucidated. The aim of this study is to examine the effects of cordycepin on LPS‐induced inflammatory response in macrophages and further investigate the underlying molecular mechanisms.

Methods

Cultured mouse RAW263.4 macrophages and human THP‐1‐derived macrophages were used in this study. The mRNA and protein levels of caspase‐1, IL‐1β, IL‐6, TNF‐α, MCP‐1, and COX‐2 were detected by real time RT‐PCR, ELISA and Western blot, respectively. The activation of NLRP3 inflammasome and ERK was determined by Western blot analysis. Results: Cordycepin significantly inhibited LPS‐induced expression of proinflammatory cytokines (IL‐1β, IL‐6, TNF‐α, MCP‐1) and COX‐2. LPS‐mediated NLRP3 and ERK activation was inhibited by cordycepin.

Conclusion

Cordycepin prevents LPS‐induced inflammatory response through influencing NLRP3 inflammasome activation and inhibiting the ERK pathway.
InflammatoryCaspase‐1 Activation and Vegfr‐2 Signaling Regulate Each Other in Lipid Stimulated Endothelial Cells
Authors: Lopez‐Pastrana Jahaira , Ferrer Lucas , Cueto Ramon , Choi Eric , Wang Hong , Yang Xiao‐Feng
Published: June 2021

Background

Vascular inflammation is reported to be an antiangiogenic state, however, the underlying mechanism remains unknown. More specifically, the interplays between danger‐associated molecular pattern (DAMP) sensing in endothelial cell (EC), EC activation and EC growth are poorly defined. We tested a novel hypothesis that the caspase‐1 activation and VEGFR‐2 pathway regulate each other in inflammatory lipid stimulated EC.

Methods and Results

We assayed caspase‐1 activity in human aortic endothelial cells (HAECs) stimulated with lysophosphatidylcholine (LPC). Using flow cytometry we analyzed the EC size, death, caspase‐1 activation and VEGFR‐2 pathway in LPC stimulated EC. We made the following findings: LPC increases caspase‐1 activity in larger size EC first that is associated with EC activation; and then in smaller size EC, which is associated with inflammatory cell death as judged by 7‐AAD staining. LPC decreases VEGFR‐2 expression in EC, whereas the inhibition of caspase‐1 increases the expression of VEGFR‐2 after LPC treatment in EC, which leads to an improvement in tube formation. The caspase‐1 deficient mice have an increase in the blood flow after hind limb ischemia presumably via enhancing angiogenesis.

Conclusion

Our results indicate for the first time that caspase‐1 inhibition increases VEGFR‐2 signaling pathway after lipid stimuli in EC. The interplays of inflammatory caspase‐1activation and VEGFR‐2 signaling pathway could be a novel therapeutic target for vascular inflammation and angiogenesis.
Decreasing Phosphatidylcholine on the Surface of the Lipid Droplet Alters Protein Binding
Authors: Townsend Elizabeth , Hains Anastasia , Brown Elizabeth , Rickertsen Cassandra , Kharbanda Kusum , Listenberger Laura
Published: June 2021

Excess lipid is stored in an intracellular organelle known as the lipid droplet. While the core of the lipid droplet is rich in neutral lipids, the surface is primarily comprised of two phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The lipid droplet surface is further decorated with proteins, such as perilipin 2, which facilitate lipid storage and utilization. Here, we show that changes to the phospholipid composition of the lipid droplet surface alters protein binding to the lipid droplet. We utilize studies with synthetic lipid droplets to demonstrate that reduced ratios of PC:PE increase the binding of perilipin 2 in vitro. Moreover, altering the availability of intracellular PC by growing NIH 3T3 cells in choline deficient media decreases the PC:PE ratio on lipid droplets and increases intracellular perilipin 2 association with the lipid droplet. While total levels of lipid droplet‐associated proteins are not changed, choline deficiency alters the ability of other unidentified proteins to associate with the lipid droplet. Future experiments will seek to identify these currently unknown proteins, and continue to explore the connections between the lipid droplet phospholipid composition, protein binding, and the ability of the lipid droplet to store and utilize fat.
Amino Acid Deprivation Increases Lipid Accumulation in HepG2 Hepatoma Cells through Repression of Histone Deacetylase 3 (HDAC3)
Authors: Wang Huan , Hess Kathryn , Guetterman Heather , Chen Hong , Pan Yuan‐Xiang
Published: June 2021

Hepatic lipid overload is considered a predisposition for the development of fatty liver disease. Amino acid deprivation has been linked to lipogenesis in mouse liver. The present study investigated mechanisms of amino acid deprivation on hepatic lipid accumulation. Knockout of histone deacetylase HDAC3 in mice leads to genome‐wide histone hyperacetylation and increased expressions of lipogenic genes in mouse liver. Amino acid deprivation was induced by incubating HepG2 human hepatoma cells with amino acid‐free media. mRNA expression of Hdac3 was decreased significantly by 4 h amino acid deprivation. This is correlated with a significant increase in lipid accumulation in the cells using Oil red O and immunofluorescence staining of BODIPY. The lipid accumulation was accompanied with an increased mRNA expression of lipogenic genes including Srebf1 and Fasn in HepG2 cells. To confirm the involvement of HDAC3 in the amino acid deprivation‐induced lipid accumulation, HDAC3 was overexpressed in HepG2 cells and the overexpression abolished lipid accumulation by amino acid deprivation. Knockdown of HDAC3 induced mRNA expressions of Srebf1 and Fasn in HepG2 cells, leading to lipid accumulation. Therefore amino acid deprivation caused reduction of HDAC3, leading to the increased expression of lipogenic genes and lipid accumulation in HepG2 cells.
Lysophosphatidylcholine Counteracts LPS‐Induced Nitric Oxide Production through the Activation of PKC Zeta and Induction of the M2 Macrophage Phenotype
Authors: Silva‐Neto Mario , Moreira Isabel , Carneiro Alan , De Melo Luiz Dione , Miguel Nadia , Seabra Sergio , DaMatta Roberto , Lopes Angela , Atella Georgia
Published: June 2021

TLR4‐mediated pathways are central to several human diseases, such as sepsis and pathogen infection. Pharmacological down‐regulation of bacterially‐activated TLR4 signaling provides a potential way to treat sepsis. Also, TLR4 activation by invading pathogens triggers innate immunity. We have previously shown that lysophosphatidylcholine (LPC) is a potent modulator of innate immunity through suppression of LPS‐induced nitric oxide (NO) production. LPC is a true ligand of Toll‐like receptors (TLR) 2/1 and 4 induces a pro‐inflammatory phenotype in the absence of LPS. Macrophages are central to mount the immune response and display two different phenotypes, M1 and M2, which represent different ends of functional states of cell polarization. Here, we show that LPC counteracts the proinflammatory phenotype M1. Instead it induces the morphology of the M2 phenotype as demonstrated by both electron and optical microscopy. Cells simultaneously exposed to LPS and LPC display a downregulation of inducible nitric oxide synthase and express high levels of arginase. Such effects are largely associated to the modulation of actin cytoskeleton and are induced upon LPC‐mediated activation of protein kinase C zeta. Thus, in the presence of LPC and LPS macrophages display an anti‐inflammatory phenotype that is critical for the resolution of inflammation. In conclusion, detailing of signaling pathways triggered by lysophospholipids in immune cells may ultimately lead to novel modulators of innate immunity and disease therapies.
12/15‐Lipoxygenase‐Dependent ROS Production Is Required For Diet‐Induced Endothelial Barrier Dysfunction
Authors: Chattopadhyay Rima , Tinnikov Alexander , Dyukova Elena , Singh Nikhlesh , Kotla Sivareddy , Mobley James , Rao Gadiparthi
Published: June 2021

To understand the mechanisms of 15(S)‐hydroxyeicosatetraenoic acid (15(S)‐HETE)‐induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)‐HETE induced JamA phosphorylation on Y164, Y218 and Y280 involving XO‐mediated ROS production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction disruption, increased vascular permeability and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appear to be critical for its role in 15(S)‐HETE‐induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high‐fat diet (HFD) feeding, WT but not 12/15‐LO‐/‐ mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by Allopurinol. These observations provide novel insights on the role of XO in 12/15‐LO‐induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.
SCH 66712 and EMTPP are the First Potent Mechanism‐Based Inactivators of Both Human CYP2D6 and CYP3A4
Authors: Fujiwara Rina , Bolles Amanda , Briggs Erran , Nomeir Amin , Furge Laura
Published: June 2021

Cytochrome P450s (CYPs) are heme‐containing enzymes that metabolize small organic molecules including drugs. CYP3A4 and CYP2D6 are responsible for more than 70% of pharmaceutical drug metabolism and inactivation of these enzymes can lead to drug‐drug interactions. The substituted imidazole compounds, 5‐fluoro‐2‐[4‐[(2‐phenyl‐1H‐imidazol‐5‐yl)methyl]‐1‐piperazinyl]pyrimidine (SCH 66712) and 1‐[(2‐ethyl‐4‐methyl‐1H‐imidazol‐5‐yl)methyl]‐4‐[4‐(trifluoromethyl)‐2‐pyridinyl]piperazine (EMTPP), have been previously identified as mechanism‐based inactivators (MBIs) of CYP2D6. The current study shows SCH 66712 and EMTPP are also MBIs of CYP3A4. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. The rates of inactivation, kinact, and inhibition constant, Ki,were 0.21 min‐1 and 1.6 µM for SCH 66712, and 0.046 min‐1 and 11.7 µM for EMTPP, respectively. Whole protein MS analysis was consistent with a binding stoichiometry of 1:1 for both MBIs on CYP3A4 apoprotein. To determine the site of modification of the enzymes, MS analysis of digested peptides of inactivated enzymes was performed. Additionally, inactivation mechanisms of enzymes by possible electrophiles of SCH 66712 and EMTPP are proposed based in part on metabolite studies (Support: NIH 1R15‐GM086767‐02).
The Determination of the Biological Activity of Three South Texas Fabaceae Leaf Extracts
Authors: Palacios Patrick , Ynalvez Ruby
Published: June 2021

Plant extracts are known to possess biologically active components. Many of these plant extracts have been reported to have anti‐bacterial, anti‐fungal and anti‐HIV activities. The objective of this study is to determine any potential biological activities present in plant extracts of three native South Texas legumes. These are Vachellia rigidula (Blackbrush), Senegalia berlandieri (Guajillo) and Senegalia gregii (Catsclaw). V. rigidula has been noted to possess anti‐bacterial activity when tested against Staphylococcus aureus. As for anti‐fungal activity, S. berlandieri has been shown to induce damage to the hyphal structures of Aspergillus niger when observed via scanning electron microscopy. All three plant extracts possess anti‐HIV RT activities. In addition, of particular interest in this study are lectins and tannins known to be present in the three plant extracts under study. Thus, this study will also attempt to isolate the biomolecule responsible for the anti‐HIV RT property observed in Blackbrush, Guajillo and Catsclaw. The determination of biological properties as well as isolation of the biomolecule responsible for anti‐HIV capabilities will provide useful information about the plants' biochemistry as well as potentially exploitable properties in the medical realm.
SUMO‐binding Sites in Mouse Nrf2: Impact on the Transcriptional Activity of Nrf2
Authors: Walters Treniqka , McIntosh Deneshia , Arinze Ifeanyi
Published: June 2021

The transcription factor Nrf2 induces transcription by binding to the antioxidant response element(s) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. Recently, we showed that Nrf2 undergoes posttranslational modification by sumoylation after it is activated [Malloy et al. (2013) JBC 288, 14569‐14583], but the SUMO‐binding sites were not identified. Here, we have used three SUMO site‐predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to locate putative SUMO‐binding sites in mouse Nrf2. Three of the fifteen predicted sumoylation sites identified by these algorithms have high probability scores on all three prediction algorithms. Of these sites, only one (524LKDE527) matched the classical definition (ΨKXD/E) of a canonical SUMO‐binding site. Using site‐directed mutagenesis and in vitro sumoylation assays we show that this site is a bona fide SUMO‐binding site. Three other lysine residues (K53, K110, and K535) were also identified as SUMO‐acceptor residues. To test whether sumoylation of Nrf2 influenced transcription Nrf2‐dependent gene transcription, we mutated (K→R) some of the SUMO‐acceptor lysines. We found that mutations at K525, K535, and K591 resulted in impaired Nrf2‐dependent gene transcription, as measured by the luciferase reporter gene assay. To understand how sumoylation influenced transcription we assessed, using the Re‐ChIP (Active Motif, Carlsbad,CA) assay, whether sumoylation impacted the ability of Nrf2 to interact with its binding partners, specifically MafG, at the ARE.

Supported by NIH grants SC1CA143985 and 5T32 GM07628‐34 and by CTSA award No. UL1TR000445.
Exploring the Importance of Dimerization for DJ‐1 Function through Engineered Domain Fusions
Authors: Hansen Sierra , Lin Jiusheng , Wilson Mark
Published: June 2021

Parkinson's Disease is a progressive neurodegenerative disease that affects approximately 6.3 million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. DJ‐1 (PARK7) is one of several genes that are mutated in rare forms of familial parkinsonism. DJ‐1 is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. Dimerization of DJ‐1 is thought to be essential for this function, as some disease‐associated mutations cause poor folding and disrupt the DJ‐1 dimer. However, recent reports suggest that DJ‐1 may be functional as a monomer. To test this, we have engineered a non‐dissociable DJ‐1 dimer that is a fusion of two human DJ‐1 domains. This construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric DJ‐1. Our engineered construct is modeled on plant DJ‐1 homologs, which feature naturally occurring duplicate DJ‐1 domains separated by a small (19 amino acid) linker region. Using X‐ray crystallography, we confirmed that this engineered non‐dissociable human DJ‐1 dimer has identical structure to the naturally occurring dimeric protein. We have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian L166P and L10P mutations. CD spectroscopic analysis reveals that single and double L166P mutations in the non‐dissociable DJ‐1 dimer maintain a higher degree of structure than L166P mutations in the native protein. Additional characterization of the protective capacity and subcellular trafficking of this non‐dissociable DJ‐1 dimer in human cells is underway.
Effects of Landscape Fragmentation, Habitat, and Vegetation on Abundance of Ixodesscapularis as a Lyme Disease Carrier in Essex County, Massachusetts
Authors: Kruguer Samantha , Mason Samuel , Keller Gregory , Topp Justin
Published: June 2021

The blacklegged tick (Ixodes scapularis) is the main vector for the Lyme disease‐causing spirochete Borrelia burgdorferi, which is transmitted to humans through the tick's bite. One explanation for the increased incidences in Lyme disease and tick abundance in the Northeast is fragmentation, in which continuous forest is transformed into small isolated patches. This study sought to connect landscape, habitat, and vegetation variables with Lyme disease prevalence. We monitored tick abundance among larva, nymph, and adult stages at 16 study sites representing 4 different habitat types (natural edge, pasture edge, residential edge, interior forest) in Essex County, MA. In order to detect the etiologic agent, B. burgdorferi, we isolated DNA and performed nested PCR to the 16S‐23S rRNA intergenic spacer region. Results showed that larvae correlated positively with % vegetation cover, total basal area (TBA) deciduous, and tree richness, but negatively to coniferous density. Nymphs demonstrated a positive relationship with % vegetation cover, TBA deciduous, and tree richness, but a negative relationship with coniferous density, deciduous density, and leaf litter. At a landscape scale, adults showed a positive relationship to human edge (forest edge created by humans) within a 500 m radius of the sampling point. Based on preliminary results, nested PCR has yielded adult ticks positive for B. burgdorferi, but additional data is needed in order to determine the relationship between Lyme disease prevalence and landscape, habitat, and vegetation variables. This work is supported by internal funding from Gordon College.
The Surfactant Protein A Receptor SP‐R210L Variant Medicates Infection and Resolution of Influenza A Virus in the Lung
Authors: Yang Linlin , Hu Sanmei , Halstead E Scott , Chroneos Zissis
Published: June 2021

Alveolar macrophages (AM) are critical for controlling influenza virus (IAV) infection by limiting virus release and initiating immunity. Surfactant protein A (SP‐A) and D (SP‐D) are a primary line of lung host defense against IAV. Previous studies showed that the SP‐A receptor SP‐R210 mediates phagocytosis of SP‐A‐opsonized pathogens. Macrophages express SP‐R210L and SP‐R210S variants, though SP‐R210L is predominant on AM. Here we studied the role of SP‐R210 in IAV infection. The SP‐R210L deficient cell was constructed in RAW264.7 macrophage. The expression and localization of virus nuclear protein (NP) and hemagglutinin (HA) were detected by flow cytometry and immunofluorescence staining. Results show that disruption of SP‐R210L in vitro blocked IAV infection with H3N2 and H1N1 strains. SP‐R210L is required for endocytic trafficking of IAV to the nucleus. Furthermore, SP‐R210L‐mediated infection of macrophages was coupled to the production of TNFa. To address these results in vivo, we studied transgenic mice with conditional disruption of SP‐R210L in macrophages. SP‐R210L‐deficient mice were significantly more susceptible to IAV infection as indicated by increased morbidity and mortality. The increased mortality was associated with reduced clearance of apoptotic cells. These findings support the model that IAV co‐opts SP‐R210L to initiate infection, but the lack of SP‐R210L impairs clearance of apoptotic cells resulting in excessive inflammation and poor resolution of viral pneumonia.
Mapping the DNA and RNA Binding Domains of the HIV Restriction Factor APOBEC3G
Authors: Smith Harold , McDougall William , Polevoda Bogdan , Friedman Alan , Bennett Ryan , Stewart Ryan
Published: June 2021

APOBEC3G (A3G) is an ssDNA binding protein and HIV restriction factor. A3G mutates the viral genome during reverse transcription through deamination of dC to dU. The C‐terminal catalytic domain only supported ssDNA binding and deaminase activity within the full length protein. Cellular RNAs inhibit A3G binding and deamination of ssDNA, and form ribonucleoprotein complexes with A3G. Point mutations mapped RNA binding to the N‐terminus but again only full‐length A3G bound RNA. We evaluated quaternary complexes of A3G bound to ssDNA and RNA using near equilibrium binding, gel shift analyses and fluorescence anisotropy. The domains of A3G in contact with ssDNA or RNA were found by UV crosslinking and mass spectral sequencing. The data showed a lax ssDNA and RNA sequence requirement for A3G binding, but oligonucleotides below 15 nt showed impaired binding. A3G bound ssDNA as a homodimer that became catalytically active upon the formation of A3G homotetramers as a highly ordered process. RNA formed differently sized complexes with A3G. RNA dissociated ssDNA from A3G and simultaneously disrupted dimers and tetramers of A3G. Mass spectrometry demonstrated that ssDNA and RNA bound primarily unique A3G peptides dispersed along the entire protein. The data suggested a mechanism for the assembly of A3G catalytic complexes that explains the processivity of mutagenesis through dimer and tetramer formation. We predict the stochastic dissociation of A3G:ssDNA complexes by RNA is due to both conformational changes from RNA binding to allosteric sites not occupied with ssDNA as well as competitive binding. The work was supported by grants awarded to HCS from NIAID 095007 and NIGM 110568.
Bioethanol Production: Optimization of Pretreatment Condition of Sugarcane Bagasse
Authors: Igwo‐Ezikpe Miriam , Odumade Olusegun , Ayanshina Oluwamuyiwa , Babalola Musa
Published: June 2021

Interest in the bioconversion of renewable lignocellulosic biomass into biofuel as an alternative to petroleum is rising globally. This study investigated the optimum chemical pretreatment concentration for bioethanol production using sugarcane bagasse. The bagasse was ground and subjected to various concentration of chemical treatment which include Hydrothermal (Hot‐H2O), Hydrogen peroxide (H2O2), Tetraoxosulphate VI acid (H2SO4), Ethanol‐Tetraoxosulphate VI acid (C2H5OH and H2SO4), Sodium hydroxide (NaOH), and Combined Ethanol‐Sodium hydroxide (C2H5OH and NaOH). This was followed by enzymatic hydrolysis using crude enzyme extract of Tricoderma viride and Aspergillus niger prior to anaerobic production of ethanol by fermentation using Saccharomyces cerevisiae. Results showed that pre‐treatment significantly (p<0.05) altered the percentage weight loss and cellulose content of the bagasse in a concentration dependent manner compared to the untreated control. Cellulose content and reducing sugars were maximal with NaOH pre‐treatment concentration. Crude enzyme of T. viride and A. niger co‐culture released significant (p<0.05) reducing sugar. The various pre‐treatment resulted in varying amount of ethanol comparable to control with NaOH and C2H5OH‐NaOH‐H2O treated showing improved yield. In conclusion, the effectiveness of any chemical pretreatment method is concentration dependent and essential for the release of cellulose for hydrolysis to fermentable sugar. A mutual synergy of co‐culture of A. niger and T. viride which resulted in increased cellulase, endo‐glucanase and β‐glucosidase activities could further be explored in bioethanol production.
Biological Evaluation of Structurally Simplified Staurosporine Analogs on Protein Kinase C Inhibition
Authors: Mowery Patricia , Pelkey Erin
Published: June 2021

Protein kinase C (PKC) is involved in a number of diseases including certain cancers. The most studied inhibitor of PKC is staurosporine, a small molecule produced by Streptomyces spp. We have generated new analogs of staurosporine with simplified heterocyclic backbones using coupling reactions of tetramic acid derivatives as a key synthetic step. We have tested our analogs for their impacts in cell cytotoxicty MTT assays in order to examine their potency. Initial results indicate that size and planar characteristics of the analogs impact their effectiveness at inducing cell toxicity. From these studies we hope to determine structural lessons for PKC inhibition.
Selective and Bioavailable Inhibitors of Collagen Prolyl 4‐Hydroxylase
Authors: Vasta James , Andersen Kristen , Raines Ronald
Published: June 2021

Collagen is the most abundant protein in animals. A variety of diseases are associated with the overproduction of collagen including cancer metastasis. The stability of collagen relies on post‐translational modifications that occur throughout the secretory pathway. One of the most important modifications is the hydroxylation of collagen strands by collagen prolyl 4‐hydroxylases (CP4Hs). Catalysis by CP4Hs converts proline residues to hydroxyproline residues, which are essential for the conformational stability of mature collagen strands. Importantly, CP4Hs are validated targets for the treatment of metastatic breast cancer, a significant unmet clinical need. Nonetheless, a therapy based upon this strategy has yet to emerge, primarily due to the difficulty in designing selective inhibitors for members of this enzyme family. Herein, we use a chemical approach based upon the classic 2,2′‐bipyridine (bipy) scaffold to probe the active site of human CP4H1 and understand the determinants of selectivity for this enzyme. Then, we redesign the bipy scaffold to enhance its pharmacological properties and generate potent biheteroaryl inhibitors that are selective for human CP4H1. Evaluations of these inhibitors in cell culture suggest that they are bioavailable and inhibit collagen synthesis at concentrations that do not disrupt iron homeostasis or cause general cytotoxicity. These results suggest the potential use of these inhibitors as leads for the development of antimetastatic therapeutics.

This work was supported by grant R01 AR044276 (NIH).
Kinetic studies examining the inhibitory potential of compounds selected using a computationally derived active site model of glutamate racemase from S. Pneumoniae
Authors: Dinsmore Olivia , Wells Emily , Krall Danny , Stockert Amy , Mahfouz Tarek
Published: June 2021

Antibiotic resistance is continually developing and diminishing the efficacy of current antibiotics and exploring novel targets is essential. Glutamate racemase, the enzyme responsible for the inter‐conversion between D‐ and L‐glutamate in bacteria, is a potential target. Glutamate racemase is essential for viability of several pathogens, making it an ideal target for developing a new class of antibiotics. Comparative modeling was employed to develop a three‐dimensional structure for the S. Pneumoniae glutamate racemase using Prime®. Several inhibitors were docked into the model's active site to investigate their binding modes. Our results indicate that inhibitor binding induces active site structural changles and that two active site water molecules are important for proper orientation. Our lab has synthesized and expressed the gene MurI, which encodes glutamate racemase. We have explored the inhibition potential of several compounds and present here the kinetic analysis. Inhibitor and substrate was reacted with glutamate racemase, followed by heat inactivation and subsequent reaction of L‐glutamate product with glutamate dehydrogenase. We were able to determine the inhibitory potential of the most promising compounds and compare them to the results obtained in whole cell assays.
Exposure to Phosphodiesterase Inhibitors Impairs Physiological Processes in Juvenile Nematodes
Authors: Schuster Kevin , Danquah Wiseborn , Williamson Valerie , Cote Rick
Published: June 2021

Meloidogyne spp. (root‐knot nematodes) are the most damaging plant‐parasitic nematodes to commercial agriculture. Currently used nematicides are of limited effectiveness as well as toxic to other organisms. The present study examines whether disruption of cyclic nucleotide signaling pathways using phosphodiesterase (PDE) inhibitors could serve as the basis for novel nematicides with greater efficacy and reduced environmental impact. To explore this, we carried out an evolutionary analysis of nematode phosphodiesterases (PDEs) and found that nematodes contain only 6 of the 11 members of the vertebrate PDE superfamily. Evolutionary trace analysis reveals nematode‐specific amino acid residues in the drug binding pocket of several nematode PDEs, suggesting the feasibility of developing compounds that selectively inhibit individual nematode PDEs. We also examined the physiological effects of PDE inhibitors on both C. elegans and M. hapla. We found that IBMX (non‐selective), cilostazol and milrinone (PDE3‐selective), rolipram (PDE4‐selective), and papaverine (PDE10 inhibitor) can reduce C. elegans dauer locomotion velocity by 30‐50%. IBMX, cilostazol, and rolipram also reduce by 50‐70% the chemotactic response of C. elegans to salt. Treatment of juvenile M. hapla with cilostazol or rolipram reduced by 蠅50% their ability to invade plant roots. We conclude that inhibitors that target individual PDE families present in nematodes can disrupt locomotion, chemosensation, and/or processes required to invade plant roots, and thus may have nematicidal applications.

This work was supported by the New Hampshire Agricultural Experiment Station (USDA).
AraL from Bacillus subtilis, a Phosphatase Member of the Nitrophenyl Phosphatase Family of the HAD Superfamily
Authors: Armeli Jordan , Madaio Michael , Hill Jacqueline , O'Handley Suzanne
Published: June 2021

The HAD (Haloacid Dehalogenase) superfamily is a diverse superfamily with a majority of the enzymes being phosphatases. One family of the HAD superfamily is the nitrophenyl phosphatase family, so named because the first family members were yeast enzymes found only to hydrolyze p‐nitrophenyl phosphate. Since that time, a number of enzymes with a variety of activities have been identified and characterized. There are pyridoxal phosphatases, a UMPase, phosphoglycolate phosphatases, and most recently, AraL from B. subtilis. We have cloned the araL gene, expressed and purified the AraL protein, and partially determined its activity. Like the other family members, AraL is a phosphatase, but unlike any other member, it was proposed that AraL is a sugar phosphatase. Currently, we have found activity on ribose 5‐phosphate, ribulose 5‐phosphate, and arabinose 5‐phosphate, and more recently glyceraldehyde 3‐phosphate (G3P), phosphoenolpyruvate (PEP), and dihydroxyacetone phosphate (DHAP). The kinetic parameters that we determined for the latter three substrates were better than for the sugar phosphates, indicating that a sugar phosphate may not be the optimal substrate for AraL. If AraL G3P, PEP, and DHAP are the best substrates, then AraL's cellular role may be to help regulate glycolysis. Homologs in pathogenic bacteria could be novel antibiotic targets since glycolysis is such an important metabolic pathway for most if not all organisms. This research was supported by an NIH AREA grant, an ASM SURF, and an ASBMB undergraduate research grant.
Gemcitabine is a Competitive Inhibitor of Cytidine‐5′‐triphosphate Synthase (CTPS) that Induces Enzyme Filament Polymerization
Authors: McCluskey Gregory , Bearne Stephen
Published: June 2021

CTP plays a central role in nucleotide and lipid biosynthesis and is synthesized de novo by the catalytic action of CTP synthase (CTPS). CTPS is an important modulator of lymphocyte proliferation and has been identified as a potential therapeutic target for the treatment of acute lymphoblastic leukemia. The cytidine analogue gemcitabine is used to treat various cancers by inhibiting DNA polymerase, and also depleting intracellular CTP levels. While depletion of CTP levels suggested that gemcitabine inhibits CTPS activity in vivo, in vitro characterization of the inhibition is lacking. We demonstrate that gemcitabine is a potent competitive inhibitor of CTPS, binding with an affinity that exceeds that exhibited for UTP by 100‐fold. Site‐directed mutagenesis (E149D) studies reveal that a conserved glutamate (E149) is necessary for gemcitabine recognition, and this residue is also required for catalyzing the conversion of UTP to CTP. CTPS polymerization is an additional mode of inhibition where filament formation apparently prevents conformational changes conducive to enzymatic activity. Using electron microscopy, we detected CTPS filaments in gemcitabine‐treated samples of wild‐type enzyme, but not in E149D CTPS which cannot bind gemcitabine. These studies highlight the critical role that E149 plays in catalysis and the conformational changes of the enzyme.

Supported by the Canadian Institutes for Health Research
Synthesis and Evaluation of Novel Photoisomerizable Small Molecules as Chemical Tools
Authors: Engdahl Ashton , Streu Craig
Published: June 2021

Tubulin, a central component of the cellular cytoskeleton is critical for the formation of the mitotic spindle in mitosis. Without this dynamic protein, a cell is unable to properly divide. As a result, a number of cancer drugs target tubulin, and prevent the cell from properly undergoing mitosis. A class of drugs known as mitotic inhibitors disrupt tubulin polymerization by acting to stabilize or destabilize tubulin polymer formation. While these drugs are often used to treat disease, they may also be used as biochemical tools to study tubulin polymerization. However, these molecules often lack precise spatial and temporal control, rendering them inadequate to fully study a dynamic process such as tubulin polymerization. Recently, photoisomerizable compounds have gained attention due to their increased spatial and temporal control. Azo‐stilbene compounds are well known to be photoisomerizable between the cis and the trans form at 400‐600nm. This photoisomerization gives azo‐stilbene compounds great power as biochemical tools. Certain mitotic inhibitors have a stilbene backbone, which make azo‐stilbene compounds the ideal starting point for the discovery of photoisomerizeable tubulin polymerization inhibitors. We have developed a library of azo‐stilbenes, which we have supplemented with rationally designed compounds, for the purpose of inhibiting tubulin polymerization. We will discuss the synthesis of these compounds and their activity as novel azo‐stilbene tubulin binders, as well as the implications of our findings for the synthesis of next‐generation compounds to inhibit tubulin polymerization.
Isoform specificity and phosphorylation site specificity for 14‐3‐3 protein binding to tyrosine hydroxylase studied by fluorescence anisotropy and chemical cross‐linking
Authors: Daubner Susan Colette , Vogt Ellysa , Cruz Eduardo , Rodriguez Augustin
Published: June 2021

14‐3‐3 proteins are chaperone proteins that bind phosphorylated proteins and affect their activity or stability. Tyrosine hydroxylase (TyrH) is reported to be activated by 14‐3‐3. TyrH is phosphorylated at serine19 by calmodulin‐dependent protein kinase and by cAMP‐dependent protein kinase (PKA) in vivo. A variant of TyrH was constructed to enable phosphorylation of both serines in vitro by PKA, plus a TyrH variant to limit phosphorylation by PKA to serine19. Therefore TyrH phosphorylated at only serine19, only serine40, at both, or neither, has been studied. Binding between 14‐3‐3 and TyrH using chemical cross‐linking and fluorescence anisotropy will be reported. The TyrH forms variants were phosphorylated and cross‐linked to two of the 14‐3‐3 isoforms, eta and zeta via dimethylpimelimidate protein chemistry. Further, fluorescence polarization anisotropy of dansylated 14‐3‐3zeta has been measured in the presence and absence of these TyrH phosphorylated forms.

Anisotropy of dansylated 14‐3‐3zeta is increased upon the addition of TyrH even if unphosphorylated, but it increases more if the enzyme is phosphorylated at serine40, and even more if phosphorylated at serine19. The greatest increase in anisotropy is seen if TyrH is phosphorylated at both positions.

The chemical cross‐linking of the same forms of TyrH with 14‐3‐3 suggests that double phosphorylation results in a tighter complex than single phosphorylation at serine19. Cross‐linking between doubly‐phosphorylated TyrH and 14‐3‐3eta is tighter than with 14‐3‐3zeta. These results clearly suggest a 14‐3‐3 isoform specificity for TyrH binding.
Exploring the Structural Basis for Negative Cooperativity in the Phosphagen Kinase Superfamily
Authors: Kerwood Gentry , Aryal Manish , Van Houten Brittany , Fraga Dean , Snider Mark
Published: June 2021

Phosphagen kinases (PKs) are a family of enzymes that catalyze a reversible phosphoryl transfer from ATP to various guanidino compounds. While PK substrate specificity is currently well understood, far less is known about negative cooperativity in this family. Structural and comparative analyses have led us to a hypotheses regarding the structural basis for negative cooperativity in PKs. Previous assessment of negative cooperativity in P. sojae hypotaurocyamine kinase (HTK), O. cuniculus creatine kinase (CK), H. diversicolor glycocyamine kinase (GK), and M. brevicollis arginine kinase (AK) via isothermal titration calorimetry (ITC) provided some initial support for this hypothesis. Not only are we extending this comparative analysis, but we are also analyzing specific O. cuniculus CK mutants in an attempt to further validate this hypothesis. This research has been made possible by the Henry J. Copeland Fund.
PP2A phosphatase regulates autophagy in budding yeast
Authors: Talukdar Muhammad , Akter Mst , Kondo Akihiro , Ushimaru Takashi
Published: June 2021

Autophagy is a highly conserved, degradative process in eukaryotic cells. It maintains homeostasis by degradation of damaged or unnecessary proteins and intracellular organelles, and supplied nutrients as degraded products. Dysfunction of autophagy leads to cellular disorder, such as cancer and neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases. In budding yeast Saccharomyces cerevisiae, TORC1 phosphorylates Atg13 to prevent complex formation between Atg13 and other ATG proteins including the protein kinase Atg1. When TORC1 is inactivated by nutrient depletion, Atg13 is rapidly dephosphorylated and forms the Atg1‐13‐17‐29‐31 complex that triggers autophagy. In addition, protein kinase A (PKA) is involved in autophagy. Thus, various protein kinases involve in autophagy regulation are discovered, whereas protein phosphatases (PPs) involved autophagy are largely unknown. To find out protein phosphatases involved autophagy regulation, we assessed various autophagy profiles in 36 budding yeast PP‐knockout mutants and found that cells lacking PP2A are defective in autophagy and assembly of the pre‐autophagosomal structure (PAS). This study reveals that PP2A promotes autophagy after TORC1 inactivation.
Self‐phosphorylation Primes JNK2 For Activation by MKK4 in Glioblastoma and Non‐small Cell Lung Carcinoma.
Authors: Kaoud Tamer , Dalby Kevin
Published: June 2021

JNKs (c‐Jun N‐terminal kinases) are members of the mitogen‐activated protein kinase (MAPK) family, known primarily for their response to cell stress. JNK activation is mediated cooperatively by both MKK4 and MKK7. Uniquely, c‐Jun N‐terminal kinase 2 (JNK2) isoform has been reported to be constitutively activated (without MKK4/7). Recent studies in glioblastoma and lung carcinoma have identified important roles for JNK2 constitutive activation (but not JNK1). Using phospho‐specific antibodies, recombinant activated (MKK4 & MKK7), inactivated (JNK1 & JNK2) and phos‐tag PAGE, we have identified that JNK2 self‐phosphorylation results in the production of active JNK2 (phosphorylated on both Thr‐183 and Tyr‐185) and a form of mono‐phosphorylated JNK2 (phosphorylated only on Thr‐183) whose activation is MKK4‐dependent (phosphorylates Tyr‐185), but MKK7‐independent. The effect of MKK4 on JNK2 constitutive activation has been further investigated in mouse embryonic stem cells that have MKK4 and/or MKK7 knocked out. MKK4 but not MKK7 existence in these cell lines enhanced JNK2 autoactivation. Interestingly, knockdown of MKK4 or JNK2 in U78MG glioblastoma cell line, A549 non‐small cell lung carcinoma cell line and HEK293 cells dramatically decreased JNK2 constitutive activation and inhibited A549 cellular growth and anchorage‐independent growth that have been reported to be regulated by JNK2 constitutive activity. As a conclusion, our data demonstrates for the first time that JNK2 autophosphorylation primes its activation by MKK4. This phenomenon can allow JNK2 activity to be further regulated by discrete cellular signals.
Pin1‐mediated Sp1 Phosphorylation by CDK1 Increases Sp1 Stability and Decreases its DNA Binding Activity during Mitosis
Authors: Hung Jan‐Jong , Yang Hang‐Che
Published: June 2021

We previously showed that Sp1 phosphorylation at Thr739 decreases its DNA binding activity. In this study, we found that phosphorylation of Sp1 at Thr739 alone is necessary, but not essential for the inhibition of its DNA binding activity during mitosis. Further study showed that Pin1 could be recruited to the Thr739(p)‐Pro motif of Sp1 to modulate the interaction between phospho‐Sp1 and CDK1, thereby CDK1‐mediated phosphorylation of Sp1 at Ser720, Thr723 and Thr737 during mitosis. Loss of the C‐terminal end of Sp1 (741‐785 aa) significantly increased Sp1 phosphorylation, implying that the C‐terminus inhibits CDK1‐mediated Sp1 phosphorylation. Binding analysis of Sp1 peptides to Pin1 by isothermal titration calorimetry indicated that Pin1 interacted with Thr739(p)‐Sp1 peptide but not with Thr739‐Sp1 peptide. X‐ray crystallography data showed that the Thr739(p)‐Sp1 peptide occupies the active site of Pin1. Increased Sp1 phosphorylation by CDK1 during mitosis not only stabilized the Sp1 levels via decreasing interaction with RNF4 but also caused Sp1 to move out of the chromosomes completely via decreasing its DNA binding activity, facilitating cell cycle progression. Thus, Pin1‐mediated conformational changes in the C‐terminal region of Sp1 are critical for increased CDK1‐mediated Sp1 phosphorylation to facilitate cell cycle progression during mitosis.