bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling
Authors: Barman Priyanka , Ferdoush Jannatul , Sen Rwik , Kaja Amala , Bhaumik Sukesh R.
Published: June 2021

SAGA (Spt‐Ada‐Gcn5‐Acetyltransferase) and TFIID (transcription factor IID) promote the formation of the PIC (pre‐initiation complex) at the promoters of two distinct sets of genes for transcriptional initiation. However, it is unknown whether SAGA and TFIID are involved in the PIC formation at the same gene. Interestingly, we find that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high‐affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. Thus, a gene can be regulated by both TFIID and SAGA. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID‐dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4‐TFIID to SAGA dependency. Further, we find that both NuA4‐TFIID‐ and SAGA‐dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID‐independent and ‐dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling. Further, we find that proteolytic function of the proteasome is also involved in transcriptional regulation of PHO84.

Support or Funding Information

NIH 2R15GM088798‐01, NIH 2R15GM088798‐02 and NIH 2R15GM088798‐03

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Conditional Mutations in MTR4: an RNA Helicase Associated with the Nuclear Exosome
Authors: Isenhour Anthony , Fulop Hannah , Pingali Hema , Hilliker Angie
Published: June 2021

The nuclear exosome is a complex of proteins in the nucleus that processes and degrades various RNAs. In doing so, it associates with various other proteins, including Mtr4, an RNA helicase that processes and degrades RNA. MTR4 is an essential gene conserved from yeast to humans. Mtr4 associates with various complexes, such as TRAMP and the nuclear exosome, to degrade aberrant RNAs and process RNAs such as the 5.8s rRNA and the U4 snRNA (1, 2). The ATPase region of Mtr4 has been shown to be essential, but the N‐ and C‐termini are not fully characterized (3). Current questions about MTR4 focus on how Mtr4 chooses which RNAs to process and degrade, as well as what regions of the protein affect said functions.

Through random mutagenesis of the N‐ and C‐ termini of Mtr4, we selected for conditional mutants that are phenotypically defective only at high temperature. Because Mtr4 is essential, conditional mutants serve as in vivo tools to investigate how minor alterations to MTR4's protein sequence affect the cell. We have generated five C‐terminal conditional mutants, three N‐terminal conditional mutants, and a large library of lethal mutants. Through chemiluminescent northern blotting we have been able to confirm that our C‐terminal mutants are not defective in 5.8s rRNA processing. N‐terminal mutants testing is still underway, as well as U4 processing tests. Understanding the integral residues of Mtr4 will allow us to greater understand how it processes some RNAs and degrades others.

Support or Funding Information

NIH R15 GM128068‐01; Arts & Sciences (UR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Elucidation of Novel Viral Factors that Hijack Host Ubiquitination Pathways
Authors: Lippa Geoffrey Michael , Muehl Madison , Lapham Joshua
Published: June 2021

Viruses, like human immunodeficiency virus (HIV), have the ability to hijack host pathways to promote disease pathogenesis. The process of ubiquitination is used by the cell for regulating signal transduction, DNA replication, cellular development, transcription, and the cell cycle. Ubiquitination and the proteasome work together to degrade a variety of cellular target proteins involved in the processes described. The covalent attachment of a ubiquitin protein to a cellular substrate requires a multi‐protein complex consisting of a scaffold protein, adaptor module, substrate receptor, RING protein, and E2 protein. In HIV, the Cullin‐RING E3 ligase complex utilizing Cullin5, Rbx2, E2, Elongin B, Elongin C, and Vif (viral infectivity factor), amongst other accessory proteins results in the targeted degradation of antiretroviral factor APOBEC3G. Other viruses have followed suit countering these natural processes by expressing viral proteins capable of sequestering or enhancing host ubiquitination conducted by CRL complexes. Research efforts have been made in discovering both host proteins targeted in this fashion and those viral proteins responsible for this aberrant activity. Through bioinformatics and structural analysis new potential viral proteins involved in the above CRL complex have been discovered. These targets are predicted to play a role in viral fitness for Hepatitis C virus (NS5A), West Nile Virus (NS1), and Lassa virus (Z‐protein) due to their similarities with the host substrate receptor SOCS2. We are currently in the process of co‐expressing parts of the CRL complex with these viral targets in E. coli with the goal of evaluating their interactions. Utilizing in vitro pull‐down assays and thermodynamic techniques we hope to verify the viral proteins involved in the CRL complex and their mechanisms of interaction laying the groundwork for new avenues of anti‐viral therapeutics.

Support or Funding Information

Funding was achieved through Alfred Research Grants for Undergraduate Research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Protein Palmitoylation Via Click Chemistry and Flow Cytometry
Authors: Sime Drake , Wilander Benjamin , Hamann Michael J
Published: June 2021

Palmitoylation is the process of attaching palmitate groups to protein cysteine residues. Many proteins have dynamic palmitoylation modifications that are added and removed to regulate protein function. Techniques to study protein palmitoylation, however, are slow and difficult to perform. We are in the process of developing a rapid technique for measuring protein palmitoylation using the palmitate analogue17‐ODYA and click chemistry followed by flow cytometry. To work out assay conditions, the Rho family GTPase TC10 is being used as a positive palmitoylation control, as it is known to be palmitoylated on its C‐terminal tail. To perform the analysis, 17‐ODYA is added to cells transfected with YFP‐tagged versions of TC10, and lysates from the cells are treated with click chemistry reagents to selectively conjugate the terminal alkyne group of 17‐ODYA with Alexa‐647 azide. The protein is isolated with magnetic anti‐FLAG beads which then are analyzed for YFP and Alexa‐647 fluorescence using flow cytometry. The current results indicate the method may be effectively detecting palmitoylation of TC10; however, the results will need to be validated using Western blotting as well as TC10 mutants that are incapable of being palmitoylated. Overall, the method will significantly enhance the time required to detect protein palmitoylation compared to traditional blot‐based techniques.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insight into the recognition mechanism of EcoRV
Authors: Zahran Mai , Imhof Petra
Published: June 2021

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV–cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base‐pair step (TA). In a previous research, we have examined the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme, and have understood the mechanism of action of the enzyme and the intrinsically behavior of the cognate sequence to undergo a kink allowing the proper placement of the atoms for cleavage. We have also demonstrated that the enzyme requires 2 magnesium ions at the catalytic site to perform the cleavage.

In the present study, we perform multiple independent molecular dynamics simulations to investigate the behavior of a mutant EcoRV (E45A), in the presence of one or two magnesium ions and complexed to three DNA sequences: the cognate sequence, GATATC (TA); the non‐cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GACH3TATC (TA‐CH3). The results give further insight into the recognition and cleavage mechanism of EcoRV.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amyloid‐Beta Mediated Pro‐Oxidative Effects in Human Retinal Cells Involve the Small GTPase Rac‐1: Potential Therapeutic Role of FTI277 for Age‐Related Macular Degeneration
Authors: Owens‐Walton Jeunice , Thounaojam Menaka , Gutsaeva Diana , Bartoli Manuela
Published: June 2021

There is a need for new therapies for age‐related macular degeneration (AMD), the leading cause of blindness in elderly. Fibrils of amyloid beta (Aβ), a causative factor of Alzheimer's disease (AD), have been identified among the constituents of “drusen”, yellowish deposits located in the retinal pigment epithelium (RPE) of AMD patients and considered diagnostic hallmarks of the disease. Increased production of reactive oxygen species in RPE is in important pathogenic step for AMD and involves NAD(P)H oxidases such as NOX2 and NOX4. Here we have conducted a study analyzing the effects of oligomers of Aβ (1–42) in inducing oxidative stress in human retinal pigment epithelial cells (HRPE) and evaluated the role of the small GTPase Rac‐1, required for NOX2 and 4 activation, in this process. Using a Cell‐ROX assay we found that Aβ (1–42) (5mM for 12hours) stimulated the formation of reactive oxygen species (ROS) in HRPE. Aβ (1–42)‐promoted ROS production and contemporaneous activation of Rac‐1, measured by translocation of the protein to the plasma membrane (cell fractionation and Western blotting) and by increased ratio of the GTP bound versus the GDP bound form of the protein (assessed by immunoprecipitation). Same doses of the inverse peptide Aβ (42‐1), used as negative control, had no effects. Activation of Rac‐1 is preceded and dependent on farnesylation. Treatment of the cells with the farnesyl transferase inhibitor FTI‐277 (25mM) blocked Aβ (1–42)‐induced activation of Rac‐1 and consequent ROS formation in HRPE. In summary, our results suggest a role for Aβ in promoting oxidative stress and dysfunction of the RPE, two effects seen during AMD. In addition, we have identified FTI‐277 as a potential new therapy for AMD because on its inhibitory effects towards Rac‐1.

Support or Funding Information

Supported by the Medical Scholars Program, Medical College of Georgia, Augusta, GA.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the nuclease resistance of intramolecular DNA four‐way junction composed of phosphorothioate bonds
Authors: Klein Mitchell , Parker Elliott , Troisi Maria , Smith Samara , Wade Elizabeth , Moorehead Gaston , Bell Anthony Jerome
Published: June 2021

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also target proteins to control gene expression. One strategy uses double stranded DNA as “decoy” sequences that are directed against transcription factors such as NF‐kB and specificity protein 1. More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, cruciform and bent DNA are directed against the damage‐associated molecular pattern molecule, High Mobility Group B1 (HMGB1). We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of immobilized 4WJs for ex vivo studies, the helical termini are modified to generate the intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the 4WJ lattice to enhance nuclease resistance. In this study, the nuclease resistance of five intramolecular 4WJs and the DNA control, J1 are evaluated. J1 is a classic immobilized 4WJ that is composed of four asymmetric strands of DNA. Three nucleases are used: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I is an endonuclease that cleaves dsDNA and ssDNA in a largely non‐specific manner. Exo III has expanded functionally. Here, we focus on the 3′®5′ exonuclease activity of the enzyme. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction. The nuclease protection data shows that each intramolecular 4WJ has significantly higher nuclease resistance values than J1 against DNase I and Exo III. Circular dichroism studies are being developed to investigate the minor groove binding properties of J1 vs. the intramolecular 4WJs. We hypothesize that perturbations within the minor groove of intramolecular 4WJs may enhance the nuclease resistance of these constructs vs. J1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Activity Relationships of Chalcone Derivatives as Inhibitors of Breast Cancer Cell Growth
Authors: Neal Hadley E. , Stantliff Trevor M. , Harrod Tighe , Hill Amber , Barrios Francis , Krzysiak Amanda
Published: June 2021

Chalcones are polyphenols composed of two aromatic rings linked by an aliphatic three carbon chain. Chalcones and their derivatives have been shown to have numerous types of biological activity including anti‐inflammatory, antioxidant, anti‐infective, antiviral, anticancer and antitumor. Previous work in our lab discovered that anti‐cancer activity benefitted from electron donating substitutions to the B ring. Here, we are exploring the flexibility of the bridge structure using tetralone and indanone to generate a more rigid link between the aromatic rings. Using tetralone and indanone combined with benzaldehydes bearing carious electron donating groups a diverse library of chalcones was generated. These different compounds were first tested in noncancerous HEK 293 cells and have continued being tested in breast cancer cells (MCF7). An initial screening assay is performed at 50 μM. Compounds showing fifty percent or greater inhibition are taken on for more detailed analysis to determine an IC50. In both cases, cells are counted and seeded at specific densities, followed by drug treatment forty‐eight hours later, then cell viability is measured using an MTS assay on a visible spectrophotometer forty‐eight hours after drug treatment. Our results indicate the flexibility is an important feature for biological activity.

Support or Funding Information

Bellarmine University Chemistry and Physics Department

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In silico Prediction of Immunogenic Sites on PMN Antigen and Design of Epitope Blocking Caps
Authors: Welsh Colin L , Hayes Candace A , May Xavier A , Stoddard Shana V
Published: June 2021

Autoimmune disorders (AD) are a type of disease in which an individual's immune system attacks the cells of its own body. Currently, AD are the 2nd leading cause of chronic long term illness. The primary method of treating AD is the use of non‐specific immunosuppressant drugs, suppressing the entire immune system and leaving the patient vulnerable to infections. Antigen specific therapies have been proposed in order to replace treatment with immunosuppressant drugs for AD. The work here focuses on developing a new approach to AD therapy which targets the epitope site (ES) on the antigen, with an emphasis on the kidney specific AD primary membranous nephropathy, which affects 10–12 million people worldwide. Using in silico mutagenesis the ES on the phospholipase A2 receptor (PLA2R) were predicted and epitope binding proteins (EBP) were developed to target these regions. Using Epitopia and EPCES two regions on the CTLD1 domain of PLA2R were identified as potential ES. These ES are characterized in terms of the number of residues, their electrostatic potential, and their hydrophobicity. In silico prediction of immunogenicity showed the single nucleotide polymorphisms M292V, H300D, and G1106S do not directly contribute to immunogenic sites on PLA2R. In silico mutagenesis of two protein templates (5IMK and 4JE4) was performed to design EBP structures. The Rosetta protein‐protein docking server was used to evaluate the interface score of designed EBP to the PLA2R antigen, and disruption of binding to their original binding partner via molecular mechanics calculations. In silico mutations to the 5IMK monobody increased the binding interface score from −4.259 rosetta energy units (REU) to −8.903 REU. Similarly, in silico mutations to the 4JE4 monobdy increased the interface score from −3.964 REU to −5.867 REU. Data shows the introduction of charged residues to the monobody contributes to the most significant gains in interface score. Further in silico mutations were introduced to disrupt the binding preference of 5IMK and 4JE4 to their original targets. Binding potency was reduced from −7.851 REU to −3.87 REU for 5IMK, and from −12.263 REU to −3.693 REU for 4JE4. This work could be used to develop a new antigen specific therapy approach for PMN and AD in general.

Support or Funding Information

William Randolph Hearst Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative transcriptome profiling of genes and pathways related to acquired and intrinsic resistance to BRAF inhibitor
Authors: Lee Michael , Hwang Sung‐Hee
Published: June 2021

Melanoma cells have been shown to respond to BRAF inhibitors; however, intrinsic and acquired resistance limits their clinical application. In this study, we performed RNA‐Seq analysis with BRAF inhibitor‐sensitive (A375P) and ‐resistant (A375P/Mdr with acquired resistance and SK‐MEL‐2 with intrinsic resistance) melanoma cell lines, to reveal the genes and pathways potentially involved in intrinsic and acquired resistance to BRAF inhibitors. A total of 546 differentially expressed genes (DEGs), including 239 up‐regulated and 307 down‐regulated genes, were identified in both intrinsic and acquired resistant cells. Gene ontology (GO) analysis revealed that the top 10 biological processes associated with these genes included angiogenesis, immune response, cell adhesion, antigen processing and presentation, extracellular matrix organization, osteoblast differentiation, collagen catabolic process, viral entry into host cell, cell migration, and positive regulation of protein kinase B signaling. In addition, using the PANTHER GO classification system, we showed that the highest enriched GOs targeted by the 546 DEGs were responses to cellular processes (ontology: biological process), binding (ontology: molecular function), and cell subcellular localization (ontology: cellular component). Ingenuity pathway analysis (IPA) network analysis showed a network that was common to two BRAF inhibitor‐resistant cells. Taken together, the present study may provide a useful platform to further reveal biological processes associated with BRAF inhibitor resistance, and present areas for therapeutic tool development to overcome BRAF inhibitor resistance.

Support or Funding Information

Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016R1D1A1B03930193)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Black Crappie Sarcoma: Searching for a Piece of the Puzzle
Authors: BOYD KAYLA
Published: June 2021

Black Crappie Sarcoma (BCS) is a disease primarily affecting fresh water lake systems in western Wisconsin. It manifests in the form of a tumor in front of the pelvic fin on black crappies, Pomoxis nigromaculatus. A defining characteristic are lesions on the body that are also found on the pectoral, anal, and caudal fins. The cause of the sarcoma and its complete effects on the body of the infected fish is unknown. Past efforts in revealing any information about BCS have yielded little to no results, in part because of the complexity of the pathogen. Lack of information is causing a rise in concern for lake residents as the disease appears to be spreading, although the route of transmission is unclear. The primary goal of the study is to investigate differential protein expression in sick fish tissue including muscle, heart, and spleen. These tissues were selected because damage had been reported in those areas on infected fish in past investigations. This study also explores techniques for protein extraction from fish organ tissue. The findings in this study will provide evidence of a molecular change in infected black crappies and will provide future ichthyologists the means to perform proteome studies more efficiently and effectively when examining disease.

Support or Funding Information

Supported by donations from Bone Lake Management District, Polk County Association of Lakes and Rivers, Lake Wapogasset/Bear Trap Improvement Association, Pipe & North Pipe Lake Rehabilitation and Protection District, and private donations

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and characterization of a lipid peroxidase from Nitrosomonas europeae structurally related to prostaglandin H2 synthase
Authors: Selinsky Barry Steven , Cunniff Alecia M , Skaf Rebecca S , Butchy Margaret
Published: June 2021

Prostaglandin‐endoperoxide synthase (PGHS; EC 1.14.99.1) catalyzes the two step synthesis of prostaglandin H2, an important precursor in the synthesis of prostaglandins and eicosanoids. The first step of the reaction involves the addition of two molecules of oxygen to arachidonic acid to form prostaglandin G2. In the second step, the C15 hydroperoxide in prostaglandin G2 is reduced to an alcohol, generating prostaglandin H2. PGHS has been isolated and characterized from many different eukaryotes (both vertebrates and invertebrates) and has recently been expressed from an open reading frame in the red algae Gracilaria vermiculophylla. A number of putative PGHS orthologs have been identified in bacterial genomes. In this study, a putative PGHS protein from Nitrosomosas europeae has been cloned, expressed, and characterized. Soluble Nitrosomonas protein was isolated to greater than 90% purity exploiting an N‐terminal (his)6 tag. The expressed protein exists in solution as an oligomer, likely a tetramer, and binds heme at a ratio of approximately 0.40 heme molecules per protein monomer. The expressed protein is incapable of adding oxygen to unsaturated lipid substrates, but is a capable peroxidase using both hydrogen peroxide and 15(S)‐hydroperoxy eicosapentaenoic acid as substrates. This result contrasts experiments using a similar protein previously expressed from the genome of Nostoc punctiforme, which exhibits lipoxygenase, but not peroxidase, activity. Structural modeling of the Nostoc and Nitrosomonas proteins yields superimposable structures. Mutagenesis studies of the Nostoc protein are in progress to generate peroxidase activity in the protein product.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Technique for Sickle Cell Anemia Diagnosis
Authors: Braga Camila Pereira , Grove Ryan Albert , Adamec Jiri
Published: June 2021

Sickle cell anemia (SCA) is a global public health problem that occurs mostly in low‐ and middle‐income countries, and underserved communities. Diagnosis and early treatment are critical to prevent mortality from this disease; however, a large‐scale screening is difficult due to a need for blood collection by trained phlebotomist in designated health centers. The primary goal of this study was to test a new self‐blood collection platform to enable screening in rural and underserved communities. To address this need, a simple, miniaturized device ‐ Noviplex™ card from Novilytic was evaluated. The advantages of this device include the ability to collect 2.7 μL of biofluid as well as the fact that the collected samples are stable at ambient temperature for several weeks and can be mailed to a clinical laboratory for further testing. To obtain the whole blood samples, 1% sodium deoxycholate was added to the blood prior the application or directly to the collection disk of the device. Two mass spectrometry (MS) approaches including MALDI and Multiple Reaction Monitoring (MRM) techniques were developed and validated to 1) detect the normal peptide present in β‐globulin (HBB) from a healthy donor (VHLTPEEK, Mw= 952.50 Da); 2) detect the mutated peptide present in HBB from a SCA donor (VHLTPVEK, Mw= 922.53 Da); and 3) detect a control peptide present in HBB of both healthy and SCA donors (LLVVYPWTQR, Mw= 1,274.72 Da). For MALDI based technique, blood proteins were transferred from the dried collection disc to a PVDF membrane by a semi‐dry blot system, digested with trypsin on the membrane and matrix directly deposited on the top of the blotted sample. Presence or absence of the peptides was determined using a Bruker 7T SolariX Fourier Transform Ion Cyclotron Resonance (FTICR) MS. For MRM, the dried collection disc was transferred to a 96‐well plate, and trypsin was added to cover the collection disc completely. Following digestion, samples were directly analyzed by LC‐MRM for normal, mutated and control peptides. Both techniques were evaluated using the samples from a donor with SCA and from a healthy individual and showed good digestion efficiency and reproducible detection of the peptides of interest. In addition, both methods can be fully automatized for high‐throughput testing in clinical laboratory environment. Sample preparation times for MALDI and MRM techniques were 25 and 3 min, respectively.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of the inhibition of UCP2 on the activity and presence of Amylin in RIN‐5F cells
Authors: Manuel Gygeria , Clemons Tameka
Published: June 2021

Amylin is a fibrillogenic protein co‐secreted with insulin from pancreatic beta cells. Amylin has been shown to function in metabolism through its influence on glucose homeostasis, which includes sending satiety signals to the brain, inhibiting glucagon secretion and delaying gastric emptying. In addition to amylin's direct role on metabolism it has also been proposed to have an impact on amyloid beta, a major component of the amyloid plaques found in Alzheimer's disease. Alzheimer's disease brains have been shown to have amyloid plaque formation and decreased expression of Uncoupling Protein 2 (UCP2), which is a protein that also has a role in metabolism. Traditionally, uncoupling proteins have been known to function as mitochondrial transporter proteins that create proton leaks across the inner mitochondrial membrane resulting in the dissipation of energy in the form of heat. Knowing that UCP2 is expressed in pancreatic beta cells and has been shown to have diminished levels in Alzheimer's disease brains the focus of this study was to examine the influence of UCP2 on the presence and expression of amylin in RIN‐5F cells (a pancreatic beta cell line). siRNA UCP2 constructs were created and immunocytochemistry experiments and ELISA assays were conducted to examine the presence of amylin and the activity level of amylin, respectively. Initial studies suggest that inhibition of UCP2 may impact the activity level and presence of amylin.

Support or Funding Information

Tameka Clemons at Spelman College Department of Chemistry and Biochemistry

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optogenetic activation and inactivation of the neurotrophin pathway in live cells
Authors: Mondal Payel , Krishnamurthy Vishnu Vardhan , Sharum Savanna , Haack Neeka , Zhang Kai
Published: June 2021

Signaling pathways extensively crosstalk among each other and result in different cellular phenotypes depending on the dynamic profile of protein activity. Conventional genetic and pharmacological approaches such as gene overexpression, use growth factors or inhibitors have helped us understand different aspects of signaling pathways, however these techniques provide limited means to determine contribution of a target protein for specific cellular phenotype. Therefore, to find out the molecular mechanism for a cellular outcome, there is an urgent need for a tool that can specifically activate or inactivate a protein of interest and study it's role towards a specific cell fate. Optogenetic techniques utilize light to control protein functions with high spatial and temporal resolution. Here we introduce a generalizable method for optical control of protein stability that enables fine spatiotemporal control of specific signaling pathways. This optogenetic system has been used to re‐purpose the protein degradation machinery by combining a small peptide sequence called degron, an evolved LOV (eLOV) protein and a light‐inducible nuclear export system (LEXY). Our system has been used to activate the Raf/MEK/ERK signaling cascade by light induced stabilization of a constitutively active cytosolic MEK (CA MEK). Light induced activation of the ERK pathway results in elevated phosphor‐ERK levels in human embryonic kidney cells (HEK) and rat PC12 pheochromocytoma cells as well as PC12 neurite outgrowth in the absence of growth factors. Furthermore, current non‐neuronal optogenetic methods provide fewer tools for inactivation of a specific signaling pathway compared to optogenetic activation. To bridge this gap, we have demonstrated inhibition of the ERK pathway by light induced stabilization of mitogen activated protein kinase phosphatase 3 (MKP3), an inhibitor of the ERK pathway. Thus, our system allows light‐mediated rescue of protein stability by using a degradation‐rescue strategy and offers the potential for regulation of a plethora of biological processes.

Support or Funding Information

This work was supported by the University of Illinois at Urbana‐Champaign (UIUC) (K.Z.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Tributyltin Exposures on Translation Regulation in Human Immune Cells
Authors: Ruff Amanda Elizabeth , Hamza Nafisa M , Whalen Margaret M
Published: June 2021

Tributyltin (TBT) contaminates the environment due to its use as a biocide. It is found in human blood (ranging as high as 261 nM). TBT is able to increase the synthesis of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) in human immune cells. This TBT‐induced increase in pro‐inflammatory cytokines could contribute to chronic inflammation, which is a risk factor for a number of diseases including several cancers. TBT appears to utilize the ERK1/2 and/or p38 MAPK pathways to stimulate pro‐inflammatory cytokine synthesis in immune cells. MAPK pathways have the capacity to regulate translation including processes leading to the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), eIF4B, and the S6 ribosomal subunit. We hypothesize that TBT may influence the activation state of these translational regulators as part of its mechanism of increasing cytokine synthesis. Studies to examine the levels and phosphorylation state of eIF4E, eIF4B and S6K after varying lengths of exposure to TBT in monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs) were carried out. Initial results suggest that TBT caused increased phosphorylation (activation) of eIF4E and S6. These results may indicate that TBT is elevating the synthesis of key pro‐inflammatory cytokines in immune cells by its ability to activate translation.

Support or Funding Information

NIH Grant 5U54CA163066

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ang‐II‐Induced Phosphorylation and Nuclear Export of Histone Deacetylase 5 is Mediated by an AKT‐Dependent Pathway in Vascular Smooth Muscle Cells
Authors: Truong Vanessa , Jain Ashish , Srivastava Ashok K.
Published: June 2021

Augmented levels of angiotensin‐II (Ang‐II) are associated with the development of cardiovascular disorders such as atherosclerosis and hypertension. Exaggerated activation of Ang‐II‐induced signaling events and expression of genes linked to cell proliferation, hypertrophy and migration have been demonstrated to contribute to the development of these vascular pathologies. Ang‐II induces the phosphorylation of class II histone deacetylases (HDACs), particularly HDAC4 and HDAC5. HDACs regulate gene transcription by their ability to modify the acetylation status of the lysine residues in histone and non‐histone proteins. A heightened activation of HDACs, notably HDAC5, has been associated with vascular disorders such as atherosclerosis. We have shown earlier that Ang‐II induces the expression of early growth response factor‐1 (Egr‐1), a zinc finger transcription factor which is upregulated in atherosclerotic lesions and in animal models of vascular injury. We have also reported that the PI3Kinase/AKT pathway plays a key role in inducing the expression of Egr‐1 in vascular smooth muscle cells (VSMC). However, the role of the AKT pathway in HDAC5 phosphorylation and the contribution of HDAC5 in Egr‐1 expression in VSMC remains unexplored. Here, we show that Ang‐II induced the phosphorylation of HDAC5 in a time and dose‐dependent manner, and pharmacological blockade of the PI3K/AKT pathway by wortmannin or SC66 attenuated Ang‐II‐induced HDAC5 phosphorylation in A10 VSMC. In addition, Ang‐II treatment of VSMC resulted in the translocation of HDAC5 from the nucleus to the cytosol, and the inhibition of AKT by SC66 almost completely attenuated the nuclear export of HDAC5 induced by Ang‐II. In contrast, pharmacological blockade of the MAPK pathway by UO126 was ineffective in reducing Ang‐II‐induced HDAC5 phosphorylation. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or siRNA‐induced silencing of HDAC5 suppressed Ang‐II‐induced Egr‐1 expression. In summary, our results suggest that Ang‐II induces the phosphorylation and nuclear export of HDAC5 via the PI3K/AKT‐dependent signaling pathway and that HDAC5 is an upstream modulator of Egr‐1 expression in VSMC.

Support or Funding Information

Supported by Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Drosophila P5B ‐Type ATPase is Required for Polyamine Transport
Authors: von Kalm Laurence , Brown David
Published: June 2021

In eukaryotes P‐type ATPases are represented by a superfamily of membrane ion and lipid transporters divided into five subclasses. Substrates for subclasses 1–4 have been identified, however little is known about the substrate specificity of the P‐type 5A and 5B transporters. Here we show that the Drosophila P5B‐type ATPase is required for polyamine transport. The gene encoding the Drosophila P5B‐type ATPase produces multiple protein isoforms. Using a combination of genetic mutational analysis and CRISPR‐Cas9 gene editing we show that some of these isoforms are required for polyamine transport and others are not. In addition, we show that the Drosophila P5A‐type ATPase is not required for polyamine transport indicating a different substrate specificity for this transporter subfamily. In summary, we provide evidence for the first time of substrate specificity for the P5B‐type ATPase subfamily of transporters.

Support or Funding Information

University of Central Florida, College of Sciences Seed Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tribbles Homolog 2 (TRIB2) plays a potent role in ovarian granulosa cells proliferation and function
Authors: Warma Aly , Lussier Jacques G. , Ndiaye Kalidou
Published: June 2021

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells of bovine preovulatory follicles. This study aimed to further investigate TRIB2mRNA and protein regulation, to identify its binding partners, and study its function in granulosa cells of bovine dominant follicles. Granulosa cells (GC) were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the oestrous), ovulatory follicles 24 hours following injection of an ovulatory dose of hCG (OF), and corpus luteum (CL) at day 5 of the oestrous cycle. In addition to this in vivo model, an in vitro model of cultured GC was used for functional studies using the CRISPR‐Cas9 approach. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF and CL (P < 0.0001). Temporal expression of TRIB2mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 hours after hCG injection. There was a significant reduction of TRIB2 steady‐state mRNA levels in follicular walls, starting at 6 hours through 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression (P < 0.05) while inhibition of TRIB2 using CRISPR‐Cas9 resulted in significantly reduced GC proliferation (P < 0.05). These results provide strong evidence that TRIB2 is a potent regulator of GC proliferation.

Support or Funding Information

Research supported by NSERC of Canada grant #RGPIN‐2018‐04516 to KN.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of ATM Positively Regulates Rac1 Activity and Cellular Migration Through Oxidative Stress
Authors: Srougi Melissa C. , Tolbert Caitlin E. , Smith Aaron , Beck Matt V.
Published: June 2021

Ataxia‐telangiectasia mutated (ATM) is a serine‐threonine kinase that is integral in the response to DNA double‐stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM‐deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM‐associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor‐driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N‐acetyl cysteine restores wild‐type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro‐migratory cellular behavior.

Support or Funding Information

This work was funded by High Point University's Research and Sponsored Programs (MCS), the Summer Undergraduate Research Program in the Sciences (MVB, MCS), and Undergraduate Research and Creative Works (AS, MVB, MCS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAP Kinases Mediate Regulation of eNOS through Phosphorylation of Different Sites
Authors: Solone Xzaviar , Wells Brady , Chrestensen Carol
Published: June 2021

Mitogen activated protein kinases (MAPK) p38 and ERK, have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via hypothesized interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK. In the present study, eNOS phosphorylation by MAPKs (P38, ERK, JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at pS116 contrasts with ERK, which phosphorylated pS602, and p38, which phosphorylated both sites. Literature reports that JNK1/2 activation does not alter eNOS activity while prior work in our lab shows that ERK phosphorylation negativity impacts NO output. We will present in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy‐Hb) assay. In HMEC‐1 cells and in vitro we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (pS1179), creating antagonistically phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.

Support or Funding Information

R15GM110634‐01A1 supported this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interrogating signaling pathways activation and deactivation with homogeneous NanoBiT Cell based kinase immunoassays
Authors: Zegzouti Hicham , Hwang Brian (Byounghoon) , Engel Laurie , Goueli Said
Published: June 2021

Cellular responses such as gene expression, enzyme activities, protein synthesis and translocation are orchestrated through activation of diverse cell signaling pathways. In these pathways, phosphorylation of specific proteins by specific kinases constitute important nodes by which the signal is transduced from an upstream activation event to downstream cellular responses. As a corollary, alteration of these kinase activities can have a detrimental effect on these cellular processes, leading to serious pathologies such as cancer, inflammation and diabetes. Therefore, monitoring these signaling events through the analysis of specific node phosphorylation states can help better understand cell behavior in health and disease states. We developed the NanoBiT cell based kinase immunoassay platform which is a suite of simple homogeneous assays to detect phosphorylation of endogenous proteins of interest in lysed cells. NanoLuc Binary Technology (NanoBiT) is a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments. A cell lysate containing the protein target is incubated with two primary antibodies that recognize separate epitopes on the protein. These bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of the target protein phosphorylation. Unlike current assays such as Western blot or ELISA, that are tedious, require multiple washing steps, and not easily adaptable to HTS, the NanoBiT immunoassay method presented here takes less than two hours to complete in a homogeneous “Add and Read” format. Other advantages include no cell engineering as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. We tested this system by monitoring the activation of multiple signaling pathways including NF‐κB, mTOR/AKT and JAK/STAT, through specific nodes of phosphorylation such as pIκB, pAKT, and pSTAT3. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, mTOR, or JAKs) and obtained the expected pharmacology. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.