bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of Breast Cancer Resistance Protein (BCRP) Purification from PichiaPinkTM
Authors: Jensen Madeline K , Oliveira Maisa C , Lavigne Collette A , Okwuone Dakota D. D. , Farokhnia Roxana , Edgar Jennifer M , Wise John G , Vogel Pia D 
Published: June 2021

The breast cancer resistance protein (BCRP) is an ATP Binding Cassette (ABC) transporter responsible for the export of many toxins across cellular membranes. Overexpression of these ABC transporters is often correlated to multidrug resistances in cancer and decrease in the success of chemotherapy. We have performed high‐throughput in silico ligand docking studies to find drug‐like compounds that are predicted to inhibit the function of BCRP and the related transporter, P‐glycoprotein. A number of drug like molecules were identified that reversed multidrug resistance of a BCRP‐overexpressing breast cancer cell line generated in our lab. Biochemical and biophysical analyses of these inhibitors are needed to evaluate the inhibition mechanism of these drug‐like compounds. For that purpose, relatively large amounts of the purified protein are required. We previously generated a gene for human BCRP that was codon optimized for expression in the yeast, Pichia Pastoris, and cloned into the new expression system PichiaPinkTM. BCRP purification proved to be challenging as reported previously by others. Optimization of growth and BCRP expression in the PichiaPinkTM cells revealed highest cell density and satisfactory BCRP expression after methanol induction when the cells were fed with their carbon source, methanol, more frequently than originally described. Protein purification techniques including cell breakage and solubilization conditions were optimized to achieve a consistent 60% membrane solubilization. Here we report our attempts to purify BCRP into detergent mixed micelles using Ni‐NTA affinity and ion exchange chromatography. Our attempts to assemble BCRP into native‐like membrane nanodiscs to achieve maximum ATP‐hydrolysis activity are also reported. Obtaining BCRP in a near native like environment with high biological activity will be necessary for the assessment of efficiency as well as the mechanism of BCRP inhibition of our newly found drug‐like compounds.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors wish to thank Ms. Lynn McBee and New England Biolabs for their generous support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and Synthesis of Photoaffinity Probe for Identification of Protein Targets of Glycosaminoglycan Mimetics
Authors: Ongolu Ravi Kumar , Afosah Daniel , Sistla Jyothi , Hawkridge Adam , Desai Umesh R. 
Published: June 2021

Glycosaminoglycans (GAGs) are complex biomacromolecules ubiquitously distributed in the animal kingdom where they play major roles in the large number of physiological and pathological processes. Identification of protein targets of GAGs and GAG mimetics has been a major challenge because of the size of the library of GAG‐binding proteins. We reasoned that photoaffinity probes of bioactive GAG mimetics would serve as a useful method for identification of protein targets. This work presents synthesis and mass spectrometric characterization of novel photoactivatable analogs of a lead anti‐cancer agent for identification of its plausible protein targets. Photoactivatable analog of the lead GAG mimetic G2.2 containing diazirine and alkyne groups G2PA was synthesized through a series of 10 steps. Structural characterization was performed using 1H/13C NMR and HRMS analysis. A test case of photoactivation was performed by first incubating insulin‐like growth factor‐1 with mimetic G2PA at 4 °C and then photo‐irradiated for 5 min at 368 nm. The sample was reduced using DTT, alkylated with iodoacetamide in dark, digested with trypsin and cleaned up using C18 StageTip. Proteomics analysis of the photoactivated IGF‐1–G2PA complex was performed on nLC‐Q Exactive (Thermo) in a top 12 data dependent acquisition. Proteomic analysis of the IGF‐1–G2PA complex gave a mass peak at 2688 m/z corresponding to the peptide APQTGIVDEccFR covalently linked to the photoactivated G2PA. MS/MS analysis of this fragment showed peaks 1) at 2391 m/z corresponding to TGIVDEccFR+G2PA; 2) 1098 m/z corresponding IVDEccFR; and 3) 297 m/z corresponding APQ. Proteomic analysis indicated G2PA covalently modified the thyronine residue present in APQTGIVDEccFR sequence of IGF‐1. Thus, the diazirine‐based photo‐activation approach for the test case of IGF‐1 identified the site of covalent conjugation and validated the approach. This approach, in principle, could be more generally used to identify protein targets of GAGs and GAG mimetics.Support or Funding InformationThis work is supported by HL107152, HL090586 from National Heart, Lung and Blood Institute to URDThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Investigation of Melatonin Metabolites as Calmodulin Antagonists and potential Nitric Oxide Synthase Inhibitors
Authors: Pimlott Douglas , Guillemette Guy , Dieckmann Thorsten 
Published: June 2021

Nitric oxide synthase (NOS) is a flavo‐hemoprotein that catalyzes the conversion of L‐arginine to L‐citrulline in the production nitric oxide (NO). Mammalian NOS exists in three isoforms: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Each isoform requires calmodulin (CaM) for activation as CaM binding induces a conformational change, which facilitates electron transfer within the NOS peptide via a highly dynamic process. NO overproduction is implicated in several neurodegenerative and inflammatory conditions including septic shock, epilepsy, myocarditis and Alzheimer's, thus the development of a NOS isoform‐specific inhibitor is of clinical significance. Previous research has shown that N1‐acetyl‐5‐methoxykynuramine (AMK), a metabolite of melatonin (aMT), is a CaM‐dependant inhibitor of nNOS in rat striatum1,2. Despite attenuation of nNOS activity demonstrated both in vitro and in vivo, there has yet to be a comprehensive structural analysis of AMK‐CaM‐NOS complexes. To further validate possible therapeutic applications, we conducted an investigation of AMK interactions with CaM and the CaM‐NOS binding domain for all three NOS isoforms. Our results showed CaM‐AMK binding using gel shift assays, while circular dichroism (CD) results displayed the greatest spectral differences when AMK was added to the CaM‐nNOS complex. This indicates potential isoform‐dependent variation in NOS inhibition due to differences in the CaM‐NOS binding domain. These results were substantiated by subsequent multidimensional NMR experiments, in which the largest differences in chemical shifts occurred when inhibitors were added to the CaM‐nNOS sample, primarily amongst residues found in the N‐lobe of CaM. Our investigation provides significant insight into the potential of AMK as an isoform‐specific NOS inhibitor.Support or Funding InformationThis work was funded by the NSERC of Canada.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
pM to μM: Elucidating the Interdependence of Highly Resistant HIV‐1 Proteases
Authors: Lockbaum Gordon J , Henes Mina , Leidner Florian , Kosovrasti Klajdi , Nalivaika Ellen , Kurt‐Yilmaz Nese , Schiffer Celia A 
Published: June 2021

HIV‐1 protease is an ideal model system to investigate the molecular mechanisms of resistance against small molecule inhibitors. Darunavir (DRV) binds WT HIV‐1 protease with a potency of <5 pM. We have identified a protease variant that loses potency to DRV 150,000‐fold, with 11 mutations in and outside the active site. Traditionally, active site mutations were thought to confer resistance while other “secondary” mutations compensate for losses in enzymatic activity. To elucidate the molecular mechanisms and roles of these selected mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10 and 11 mutations showed that the primary active site mutations caused ~50‐fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 760 nM (11 mutations). Structural analysis indicates distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that many of the changes remote from the active site directly and dramatically impact the potency of the inhibitor. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets and these insights have implications for the design of more robust inhibitors.Support or Funding InformationThis work was supported in part by a grant from the National Institute of General Medical Sciences, National Institutes of Health (P01‐GM109767)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells
Authors: Phelan Shelley Ann , Fulco Natalie , Fagan Sophia 
Published: June 2021

Oleuropein is one of the two main phenolic compounds derived from olive leaves. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, and increase apoptosis in many types of cancer cells, suggesting its possible efficacy as an anti‐cancer treatments. The purpose of our study was to examine the effects of oleuropein extract on cell growth and viability of the K562 human leukemia cell line, and to investigate possible molecular mechanisms of action. Oleuropein of 98% purity was used for our studies. We first demonstrated that K562 cells treated with 200 and 400 μg/ml oleuropein exhibited a significant reduction in cell growth and viability after four days as compared to ethanol‐treated controls. Cells treated with 200μg/ml oleuropein showed decreased cell proliferation and viability after two days, and exhibited approximately 90% reduction in cell density and viability after four days of treatment. A concentration of 400 μg/ml oleuropein resulted in 100% toxicity by day four. We also examined the effects of oleuropein on cellular cytotoxicity using an LDH release assay and found a two‐fold elevation in LDH release within 24 hours of treatment with 200 μg/mL oleuropein. To begin to explore a mechanisms of action, we used western blotting to examine known antioxidant and signaling pathways for possible regulation by oleuropein in these cells. We examined expression of the Peroxiredoxin (Prdx) family of proteins, a group of thiol‐specific antioxidants found to be elevated in many types of cancer. We found that 200 ug/ml oleuropein reduced Prdx1 expression by about 50% after eight and twenty‐four hours, while having no significant effect on expression levels of Prdx‐2, ‐3, ‐4, and ‐6. We are currently investigating the mechanism of cell death induced by oleuropein, as well as the role of Prdx1 in oleuropein‐induced toxicity. Together with previously reported cancer‐cell specific effects, this research provides a promising new avenue for the use of natural products as anti‐cancer agents.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exosomes Promote a Metastatic Mircroenvironment in Triple Negative Breast Cancer
Authors: Roberts Paige A , Coward Jordon A , Johnson Jasmine P , Reeves Alexavier , Lemieux KiTani P 
Published: June 2021

Triple negative breast cancer (TNBC) is the most aggressive subtype and is characterized by its patterns of metastasis and abnormal morphology. Due to the molecular heterogeneity in TNBC's, discovering effective targeted therapies has proven to be difficult. Our research group has designed a series of experiments to gain insight on how role of the microenvironment plays a role in TNBC proliferation, migration and invasion. Studies indicate that cellular stress causes exosomes to be released from both tumorigenic and nontumorigenic cells. Given that exosomes are systemic cell‐to‐cell transporters, our research goal is to better understand the role of exosomes harvested from non‐tumorigenic breast epithelial cells have on TNBC cells by measuring the proliferation, migration, and invasion when TNBC cells are exposed to these exosomes. Exosomes were harvested and purified from MCF‐10A cells. Proliferation was measured by plating 5 × 105 MDA‐MB‐231 cells in a 96 well plate which were exposed to either exosomes from MCF‐10A cells or growth media. Absorbance was measured at 560nm after 48 hours. Migration was measured by plating 5 × 10 MDA‐MD‐231 cells with the exosomal media or growth media for 48 hours. Invasion was measured using the Matrigel assay at a density of 5 × 10 5 MDA‐MB‐23 cells were exposed to exosomes from MCF10A cells. The migration and invasion assays were measured by counting cells at 4x magnification. Our data indicate that MCF‐10A exosomes increased the growth of TNBC MDA‐MB231 cells by an average of approximately 30% compared to the control (p>0.05). The MDA‐MB‐231 cells demonstrated enhanced migration and invasion when exposed to exosomes from MCF‐10A cells at 10% and 4%, respectively (p> 0.05) when compared to the control. Exosomes from nontumorigenic breast epithelial cell line MCF‐10A cells caused increases in proliferation, migration, and invasion in TNBC cell line MDA‐MB‐231. Our data suggests that exosomes are key mediators in the progression of cancer.Support or Funding InformationD34HP00006‐31‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Fluorescently Tagged Proteins to Determine Potential Interactions in a Non‐native Cellular System
Authors: Soska Mallory A , Sheridan David C 
Published: June 2021

L‐type calcium channels couple membrane depolarization to muscle contraction and aid in signal conduction between cells. Cav1.3 and Cav1.2 are calcium channel isoforms that are expressed in neurons and endocrine cells. Nervous and endocrine tissues also express both Glucocorticoid (GR) and Mineralocorticoid receptors (MR) that are responsible for binding serum cortisol to induce immediate and long term changes within the cell. While neurons co‐express both GRs and MRs, GR activation occurs in response to high concentrations of cortisol. There is a direct correlation between GR activation and the amplitude of calcium currents (Champeau, 2007). To determine potential interactions between the GR and the calcium channel isoforms, we will co‐express a GFP‐tagged GR, purchased from Addgene, and a mRubyC1‐tagged L‐type calcium channel in a simplified, non‐native cellular system, Human Embryonic Kidney (HEK) cells. Specifically targeted primers were designed to clone the Cav1.3 sequence in preparation for its insertion into the mRubyC1 red fluorescent vector. The Cav1.2+mRubyC1 and Cav1.3+mRubyC1 constructs will then be separately transfected into HEK cells expressing GFP‐tagged GR. Injection of a high concentration of cortisol into the cellular environment will activate the GR receptor and fluorescence capture imaging will determine if there is an in vitro interaction between Cav1.2 and GR and / or Cav1.3 and GR.Support or Funding InformationOtterbein University Student Research Fund Bert and Jane Horn Endowed Research AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tract‐Tracing in Rhesus Macaques Reveals Anatomical Connectivity of the Posterior Cingulate Cortex and Medial Temporal Lobe
Authors: Drake Olivia Rose , Monko Megan , Casta Tanya , Heilbronner Sarah 
Published: June 2021

Brain connectivity often correlates to function, and the understanding of connectivity may also lend itself to bettering the diagnoses and treatments of psychological disorders. One brain network that is often disrupted in psychological disorders such as Alzheimer's and depression is the default mode network (DMN). The exact function of the DMN is disputed. It is well known that areas within the DMN are functionally correlated, but whether they have anatomical connections remains less clear. Using tract‐tracing in rhesus macaques, we investigated the connectivity between the posterior cingulate cortex (PCC), a primary node in the DMN, and the medial temporal lobe (MTL). The activity in these areas has previously been found to be abnormal in those with disorders, such as Alzheimer's, schizophrenia, depression, and autism. We found that these areas do have axonal connections to each other, primarily between the PCC and the entorhinal cortex of the MTL. Our findings offer information about the exact function of the DMN and could lead to a target for treatment of some disorders. These results can also be used to compare macaque to rat brain connectivity to determine whether rats are an appropriate DMN model for humans.Support or Funding InformationSponsored by NIH NINDS 2R25 NS083059‐06 University of Minnesota Summer Research in Neuroscience PIs: Dr. Robert Meisel & Dr. Timothy EbnerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glia Maturation Factor Dependent Mast Cell Activation and Calpain 1 Synergize Dopaminergic Neuronal Loss and Behavioral Deficits in an MPTP Mouse Model of Parkinson's Disease
Authors: Pushpavathi Selvakumar Govindhasamy , Duraisamy Kempuraj , Ahmed Mohammad Ejaz , Thangavel Ramasamy , Dubova Iuliia , Mentor Shireen , Saeed Daniyal , Zaheer Smita , Raikwar Sudhanshu P , Iyer Shankar S , Zaheer Asgar 
Published: June 2021

The molecular mechanism mediating nigrostriatal dopaminergic neurons degeneration in Parkinson's disease (PD) is not yet fully understood. We have shown that glia maturation factor (GMF), a proinflammatory protein mediated mast cells activation in the dopaminergic neurodegenerations. Here, we show that deficiency of GMF (GMF‐KO) and mast cell reduces the synergistic effects of mast cell proteases and calpain 1 dependent dopaminergic neuronal loss in the SN, and STR of the midbrain leads to improves motor behavioral impairments in an MPTP mouse model of PD. The MPTP induced nigrostriatal neurodegeneration and astro‐glial activations were determined by western blotting and immunocytochemistry. We found that MPTP administrated wild type (Wt) mice exhibits oxidative stress by significantly increased level of MDA and activity of SOD and reduced the level of GSH and the activity of GPx when compared with both mast cell deficient (MC‐KO) and GMF‐KO mice. The number of TH‐positive neurons in the ventral tegmental area (VTA), SN and the fibers in STR were significantly reduced and GFAP, IBA1, calpain 1 and ICAM 1 expressions were significantly increased in Wt mice. Similarly, we found that TH, DAT and VMAT2 proteins expression significantly reduced in the SN of Wt mice when compared with both the MC‐KO and GMF‐KO mice. The motor behavior in rotarod and hang test performance significantly reduced Wt mice as compared with both the MC‐KO and GMF‐KO mice. Our study shows that deficiency of GMF and mast cells protects nigrostriatal degeneration via inhibition of astro‐glial activation‐mediated oxidative stress and neuroinflammation in the MPTP mouse model of PD. These results suggest that GMF dependent mast cells activations enhances the detrimental effect of dopaminergic degeneration and its inhibition may be a beneficial therapeutic target of PD and other neurodegenerative disorders that are associated with neuroinflammation, mast cell‐astro‐glial activation‐derived cell death.Support or Funding InformationThis work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 and NS073670.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The E3 Ligase TRAF6 directs FOXP3 localization and facilitates Treg function through K63‐type ubiquitination
Authors: Barbi Joseph , Ni Xuhao , Kou Wei , Wei Ping , Sass Stephanie , Newman Stephanie M. , Li Bin , Lu Ling , Pan Fan 
Published: June 2021

For the immune system to function properly, the activation of leukocytes must be tightly regulated. Failures of immune control mechanisms underlie the aberrant targeting of self‐antigens (i.e. autoimmunity) and the collateral damage of healthy tissues during excessive responses to infectious threats or the unnecessary immune mobilization against innocuous commensal microbes. Foxp3‐expressing CD4+ Regulatory T cells (Tregs) are a major cellular force working to prevent these pathologies of immune dysregulation. These cells characteristically exhibit multiple suppressive function that is absolutely required for the maintenance of immune homeostasis. However, these cells can hamper desirable anti‐tumor immune responses in cancer patients. Accordingly, a relative abundance of Tregs in the tumor microenvironment (TME) is a negative prognostic factor in many cancers. Treg‐depleting or antagonizing strategies show promise as potential interventions capable of unleashing anti‐tumor immunity from suppression. The characteristic gene expression and suppressive function of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. While transcriptional regulation of the Foxp3gene has been studied in depth, both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. We have found that in the absence of the E3 ligase TRAF6 Tregs were dysfunctional in vivo. In these studies, mice with Treg‐restricted deletion of TRAF6 were highly resistant to implanted tumors and displayed enhanced anti‐tumor immunity. We further determined that FOXP3 undergoes lysine‐63 linked (K63) ubiquitination at lysine 262, and this posttranslational modification was mediated by TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63 ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene‐regulating activity in Tregs. These results implicate TRAF6 as a key, hitherto unappreciated posttranslational facilitator of FOXP3 stabilization and Treg suppressive function that may be targeted by novel tolerance‐breaking immunotherapies to improve anti‐tumor immunity and combat disease progression in cancer patients.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.