bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Hydrogen Bond Donors on Reactivity of Sulfite Oxidase
Authors: Bali Sofia
Published: June 2021

Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate as the last step in the degradation of cysteine and methionine [1]. Severe neonatal neurological problems are caused by SO deficiency in humans [2]. The overall mechanism of SO suggested that the reductive half reaction of SO is the rate limiting step for enzyme activity [3]. Using a computational approach, first I will study the elementary steps for the half reaction to determine the rate limiting step, then by introducing mutations in the active site of SO I will determine the effects of the hydrogen donors present in the active site.

SO is part of the Molybdenum cofactor family of enzymes as it contains a mononuclear Molybdenum (Mo) that is penta‐coordinated [4]. At the Mo active site, the reductive half reaction of sulfite to sulfate takes place in two steps. The first step is a nucleophilic attack by the sulfite ion to the equatorial oxygen group. The second step is the release of sulfate facilitated by the spectator oxygen group [4]. By determining the energy profiles for both steps in this reaction I will determine which step is rate limiting and determine the route, associative or dissociative interchange, of the reaction.

Model enzyme complexes have suggested that hydrogen donors in the active site play a key role in mediating this reaction. In previous studies performed we determined that the product release step is facilitated by pyridinium a hydrogen donor for the reaction. Pyridinium lowers the activation energy for the product release step (ΔH~ 30 kJ/mol) and promotes a dissociative interchange reaction route. In this study I will determine if hydrogen donors in the Mo active site behave as pyridinium does in the model enzyme complexes.

Using Gaussian ONIOM to perform QM/MM calculations for the reaction of sulfite to sulfate catalyzed by SO, I will determine the energy profile of the reaction to identify the rate limiting step and the role of possible hydrogen donors in the active site of SO by comparing the reaction mechanism of two variants of SO with wild type SO. The variants will demonstrate the role as possible hydrogen bond donors of Tyrosine and water that are located in the active site of MO.

The role of sulfite oxidase is crucial to the degradation of sulfite in the body and prevention of the accumulation of toxic sulfite in the body. Understanding how SO functions at a molecular level is crucial for the design and implementation of treatment for SO deficiency. The elementary steps of the enzyme activity can help determine specific roles of key residues in the enzyme. These steps cannot be seen in large scale enzyme kinetics studies, but computational studies allow the study of small movements and allow the study of these small‐scale reaction mechanics.

Support or Funding Information

Louis Stokes Alliance for Minority Participation (LSAMP) HRD # 1305011

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidant and Ameliorative Potential of Aqueous Seed extracts of Delonix regia on High Fat Diet and Streptozotocin Induced Diabetes in Female Wistar Rats
Authors: ONYEGEME‐OKERENTA BLESSING MINAOPUNYE , OGBOYE PHILIP OSEIKHUENMHEN , MONAGO‐IGHORODJE COMFORT CHINAZO , MONAGO IFEOMA NWAMAKA
Published: June 2021

Antioxidant and ameliorative potential of aqueous seed extracts of Delonix regia on high fat diet (HFD) and streptozotocin (STZ) induced diabetes in female wistar rats were investigated. Forty eight rats were grouped into eight, consisting of six per group. Diabetes was induced in all the groups except for group 1 which was used as normal control (NC) and was fed with normal diet. Diabetic state was achieved by feeding the rats with HFD which contained 20% sucrose, 20% lard and 60% grower mash for six weeks, followed by 40mg/kg body weight (BW) of a single dose intraperitoneal injection of STZ. Seven days after induction of diabetes, treatment of cooked Delonix regia seeds (A) and uncooked Delonix regia seeds (B) extracts commences and this lasted for six weeks. Rats in groups 3(MET) and 4(MET+VDG) received metformin 100 mg/kg and metformin/vildagliptin 50/25 mg/kg (BW) respectively as standard drugs. While the rats in groups, 5 (A1), 6(A2) and 7 (B1), 8(B2) were induced and treated with A and B extracts respectively by receiving 150 and 300 mg/kg (BW) orally and daily. Blood was obtained through cardiac puncture after the rats were anaesthetized and sacrificed. Proximate analysis showed that the seeds contained, moisture, ash, lipid, protein, carbohydrate and fiber contents of 3.83 ± 0.07, 3.50 ± 0.00, 7.82 ± 0.02, 17.50 ± 0.00, 21.79 ± 0.21 and 45.57 ± 0.26 respectively. The treatment with both extracts A and B significantly decreased (p<0.05) the fasting blood glucose concentration and pancreatic amylase activities in dose and time dependent manner, when compared to the DC. Similarly, treatments with A and B extracts significantly decreased (p<0.05) the pack cell volume (PCV), and platelets (PLT) count with, a significant decreased (p<0.05) in the granulocyte count in a dose dependent manner. The potassium and bicarbonate concentrations of the treated diabetic rats significantly decreased (p<0.05) when compared to the DC except for group 5(A1) extract treated group. Results obtained for superoxide dismutase (SOD) showed a significant increase (p<0.05) in activities of treated diabetic rats in a time dependent manner when compared to DC. Conclusively, treatments exhibited immune boosting and antioxidant properties, protective against disturbance of acid‐base balance, in treated diabetic rats. Therefore, the plant exhibited very impressive potency and promise in the management of diabetes and its complications and hence a potential source for the discovery of new orally active anti‐diabetic drugs and alternative source for food nutrients

Support or Funding Information

Self funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SuFEx based click chemistry for peptide stapling
Authors: Han Jennifer , Celaje Jeff , Thomas Joice , Fokin Valery
Published: June 2021

Click chemistry is a chemical synthesis strategy which relies on covalent assembly of functional building blocks using high‐yielding reactions that are broad in scope, chemoselective, and compatible with water and oxygen. The sulfur (VI) fluoride exchange (SuFEx) is the latest addition to the click family due to the unique stability of the SO2F moiety, which confers inertness against acid‐base reactions, resistance to oxidation and reduction, and generally high yields of sulfonylated products. Ethenesulfonyl fluoride (ESF) is a powerful Michael acceptor for installing the SO2F group. We are exploring the use of SuFEx chemistry as a powerful tool in peptide stapling, a technique that locks peptide conformation to improve its pharmacological properties. Specifically, we take advantage of a complementary reactivity of two neighboring nucleophilic side chains of a peptide which can undergo a conjugate addition to ESF, followed by fluoride exchange, thus forming a chemical “staple” and locking a specific conformation of the resulting peptide. Such selective modification can inform the development of chemical probes to improve efficacy of a peptide therapeutic, may improve drug delivery, and facilitate the discovery process. Preliminary studies described herein demonstrate feasibility of this approach.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1
Authors: Bagnell Kyle P. , Provost Joseph J. , Wallert Mark
Published: June 2021

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping Domain Interaction Networks in Protein Kinases with Optical Tweezers
Authors: Maillard Rodrigo
Published: June 2021

Signaling proteins are dynamic molecular switches that sample multiple conformational states. Such conformational plasticity allows these proteins to adapt and respond to different biological signals. Protein Kinase A (PKA) is a signaling protein complex composed of regulatory and catalytic subunits. The regulatory subunit harbors two cAMP binding domains (A and B) that oscillate between inactive and active conformations depending on cAMP concentration. In this study, we use optical tweezers to identify the networks of communication between cAMP binding domains that enable PKA to switch efficiently and cooperatively from inactive to active states. We show that in the inactive PKA conformation, domain B of the regulatory subunit serves as an energetic hub, controlling the magnitude of interaction between domain A and the catalytic subunit, and whose mechanical perturbation triggers the highly coordinated dissociation of the PKA complex. In the presence of cAMP, domains A and B establish an intricate network of communication that involves direct, interfacial domain contacts as well as long‐range interactions between non‐contiguous structural motifs. In contrast, without ligand the two domains behave as independent, non‐interacting structures. Lastly, we demonstrate the application of optical tweezers to dissect how loss‐of‐function mutations can disrupt very distinct communication networks in PKA. The experimental approach based on optical tweezers should be readily applicable to study disease mutations, the mechanism of action of small molecules, or other signaling proteins.

Support or Funding Information

NSF MCB 1715572

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MK5 and ERK3 play overlapping but distinct roles in regulating cardiac fibroblast function
Authors: Sahadevan Pramod , Nawaito Sherin A. , Calderone Angelo , Gaestel Matthias , Allen Bruce G.
Published: June 2021

Background and objectives

Immunoreactivity for MAP kinase‐activated protein kinase‐5 (MK5), a protein serine/threonine kinase, is detected in cardiac ventricular fibroblasts but not myocytes. p38 and ERK3/4 MAP kinases are activators of MK5. However, the regulation of MK5 remains controversial and its physiological functions are poorly understood. Following hemodynamic overload, the increase in collagen mRNA was attenuated in ERK3 and MK5 haploinsufficient mice. In addition, following myocardial infarction, scar size and collagen content were reduced in MK5+/− mice, compared with MK5+/+ mice. In addition, MK5−/− fibroblasts show reduced motility and proliferation. Thus, MK5 may play a role in fibroblast function. The present study was to examine the role of ERK3, a putative activator of MK5, in cardiac fibroblast function.

Methods

Cardiac ventricular fibroblasts were isolated from MK5+/+ mice and male Sprague‐Dawley rats. Subconfluent cultures of fibroblasts from passages 2 and 3 were used. Ventricular myocytes were prepared from adult MK5+/+ mice. Protein expression was determined by immunoblotting. The presence of ERK3‐MK5 complexes was determined by proximity ligation assay (PLA) and co‐immunoprecipitation assays. Cell motility and myofibroblast contraction were studied by scratch‐wound and collagen gel contraction assays, respectively.

Results

siRNA‐mediated knockdown of MK5 (MK5‐kd) resulted in reduced contraction of collagen gels, reduced cell spreading, and formation of fewer dendritic extensions compared to fibroblasts transfected with scrambled RNA. ERK3 immunoreactivity was detected in fibroblasts but negligible in cardiomyocytes. MK5 immunoprecipitates from fibroblast lysates contained ERK3 immunoreactivity. Proximity ligation assays revealed ERK3‐MK5 complexes in the cytoplasm, which were less abundant following knockdown of MK5. ERK3‐MK5 complexes were not observed in the nucleus. Cell migration, in response to serum and/or angiotensin II, was reduced upon siRNA‐mediated knockdown of ERK3. However, whereas the secretion of type 1 collagen and fibronectin was increased in MK5−/− or MK5‐kd cells relative to MK5+/+ fibroblasts, knocking down ERK3 did not affect collagen secretion.

Conclusion

MK5 and ERK3 immunoreactivity was detected in fibroblasts but not myocytes. Co‐immunoprecipitation and PLA suggest that ERK3 and MK5 form cytoplasmic complexes in cardiac fibroblasts. Suppressing ERK3 or MK5 expression decreased cell motility; however, type 1 collagen secretion was enhanced by reduced MK5 expression but unaffected by knockdown of ERK3. Hence, MK5 and ERK3 play overlapping but distinct roles in regulating cardiac fibroblast function.

Support or Funding Information

This study was supported by a grant from the Heart and Stroke Foundation of Canada.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Teaching Biochemistry based Research Driven Course through Active Learning to Accelerate Student's Adaptation to College Study and Promote Freshman Research
Authors: Ray Supriyo , Fresquez Joseph , Esper Ronda , Clark Jeni , Corral Guadalupe , Xiao Chuan
Published: June 2021

Freshman Year Research Intensive Sequence (FYRIS) program was implemented at The University of Texas at El Paso (UTEP) in 2015. The program promotes student enrollment in research driven course (RDCs) for two semesters to enhance student development and increase retention in STEM through early interventions by engaging them in research activities during the freshman and sophomore years. As the undergraduate population at UTEP is 85% Hispanic, programs such as FYRIS can play an important role in increasing the representation of minorities in STEM. UTEP has a near 100% acceptance for the undergraduate courses implies a class population represents students of diverse academic proficiencies. In our special freshman chemistry laboratory sections (CHEM 1105 and CHEM1106/4176), students conduct research on expression and purification of proteins. In the first semester, students gain understanding of basic chemistry and biochemistry concepts prior optimizing protein expression. In the second semester, they continue expressing proteins and also learn protein purification using Fast Protein Liquid Chromatography. To enhance student success in advanced research driven courses, it's important that all students have an accelerated understanding in the basic chemistry, biochemistry and molecular biology. Active learning methodologies such as personal response systems, case studies, problem based learning, flipped classroom, model building, game based quizzes, think pair share, peer led team learning were used to promote accelerated learning. Research‐Teaching Integration through peer led undergraduate students were implemented to boost student understanding and confidence. Overall, our evaluation showed that students' enjoyed the class sessions more and had a better clarity of complex research based concepts in our newly designed research driven courses

Support or Funding Information

RDCs; S.R.; J.F. & R.E. National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Three web tools to aid genetics instruction developed by students in a unique undergraduate bioinformatics projects course
Authors: Rutherford Robert , Garcia Nic , Charczenko Richard , Levy Samuel , Bjelajac Jeremy
Published: June 2021

At Seattle University, some of our undergraduate Cellular and Molecular biology (CMOL) majors enroll in 10 week bioinformatics course. In addition to learning to use a range of bioinformatics tools written by the research community, students learn to use Python programming language. Some of these students take a second course, where they work in teams to develop novel biological software to support the teaching and learning of threshold genetics concepts. Here we describe this method of teaching bioinformatics and programming to CMOL majors and also features three student projects that have extended beyond this class to become independent and ongoing research endeavors. These web ready, graphical software projects include: 1) EVO.py, a new pedagogical tool that simulates genetic drift and natural selection in snowshoe hare populations. Users control a multitude of variables, including climate, allele frequency, and population size, and run the simulation, observing how procedurally generated snowshoe hare populations evolve over time in response to those inputs. 2) Lactose Operon (LacOp), a new program that simulates the regulation and biochemistry of the E.coli Lac operon. LacOp allows students configure this pathway with a wide range of mutations and/or substrate concentrations and then observe the full set of molecular outputs through time in a graphical format. 3) An enhanced FaSE (FauxSequenceEvolver) tool, which subjects user supplied molecular sequence(s) to faux evolution, automating the generation of novel but biologically credible derivative sequences. These derivative sequences have been used, for example, as ‘unknown sequences' suitable to develop and test a classroom of students' abilities to use bioinformatics tools for characterization and annotation. Student software development on this project included integrating FaSE with BioPython to allow NCBI interactivity, to capture and import known characterization data, and to automate scanning for domains.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Apoptotic Stage of HCC827 Lung Adenocarcinoma Cell Line, Following Calcium Sulfide Nanoclusters Treatment
Authors: Rodriguez Irizarry Valerie J. , Torres Geraline Trossi , Forti Kevin Muñoz , Rivera Abigail Ruiz , Castro Miguel E. , Suarez Martínez Edu B.
Published: June 2021

Lung cancer is the number one cause of death independently of gender. By the time of the initial diagnosis, more than 50% of all lung cancer patients have already developed secondary tumors due to metastases. Despite current treatments such as surgery, chemotherapy, and radiotherapy, the prognosis for these patients still poor. Subsequently, there exists the need for more efficient treatments in order to increase the survival rate of patients suffering this disease. Previously, in‐vitro experiments in our lab demonstrated that Calcium Sulfide (CaS) nanoclusters induced apoptosis and necrosis in the non‐small lung cell adenocarcinoma (NSCLC) cell line HCC827. Using the Flow Cytometry technology, in this project we aimed to determine the effect of CaS in these cells evaluating apoptotic markers including, Caspase‐3/7 activity, Bcl‐2 phosphorylation, mitochondria potential, DNA‐damage, and oxidative stress. We hypothesized that CaS will show significant differences in these assays when compared to the Dimethyl sulfoxide (DMSO) control. We added a single dose of CaS [3.8μM] (experimental treatment), DMSO (control vehicle) or Etoposide [10 nM] (positive control) at time 0 and then collected the data after 24, 48, and 72 hours. As our results, we did not observe any statistically significant effect on apoptotic markers, Caspase‐3/7 and Bcl‐2. Analysis of the Mitopotential assay showed an statistical increase (p≤0.01) of dead cells population at 48 hours and of depolarized/lives cells at 72 hours compared to DMSO, in HCC827 cells treated with CaS. However, at 48 hours cells treated with CaS decrease the numbers of non‐affected cells (p≤0.05), increase DNA double‐strand breaks (p≤0.001), decrease the activation of Ataxia‐telangiectasia mutated kinase (ATM) and the phosphorylation of H2A.X significantly (p≤0.01). These results suggested that the detrimental effect of CaS on malignant HCC827 cell line is influenced by the accumulation of oxidative stress damage also affecting the cell cycle as our lab previously reported. Additional studies are ongoing to validate these results. Within the next month we plan to study the lung non‐malignant cells MRC‐5 using the same assays.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploration of the mechanistic role of Cyclophilin D in the mitochondria
Authors: Adegbite Oluwatobi Samuel , Adegbite Yetunde Ifeoma , David Criddle , Lian Lu‐Yun
Published: June 2021

Introduction

Cyclophilin D (CypD) is an 18 kda protein encoded by the nuclear ppif gene and transported to the mitochondria. Being the only member of the cyclophilin family localized in the mitochondria, its known function is the regulation of the mitochondrial permeability transition pore (MPTP) which usually precedes mitochondrial dysfunction during calcium overload and oxidative stress. However, the mechanism of MPTP induction by CypD is still unknown, therefore, this study is aimed at understanding the generic role of CypD in the mitochondria.

Method

CypD of murine liver mitochondria was quantitatively determined from recombinant CypD standard curve using densitometric analysis programme, image J. Difference in CypD level between liver and heart tissue mitochondria was quantified. Modulation of the binding stability of recombinant CypD by some mitochondrial metabolites (in vitro) was carried out by dynamic scanning fluorimetry (DSF). Mitochondrial swelling and NAD(P)H fluorescence were monitored spectrophotometrically at 540 nm and 355/450 (Ex/Em) respectively. Metabolic differences between CypD WT and KO murine tissues were carried out using HNMR spectroscopy.

Results

CypD was observed to constitute about 0.1% of the total mitochondrial protein. It was also observed to be more than 2‐fold higher in the liver than heart tissue mitochondria. MPTP activation experiments revealed 2‐fold higher calcium induced swelling in liver than heart tissue mitochondria. Mitochondrial NAD(P)H fluorescence was observed to be highest at 100 uM calcium in the presence of CsA while a massive loss of NAD(P)H was observed when CypD was uninhibited at this concentration. Succinyl CoA and alphaketoglutarate, TCA cycle intermediates enhanced calcium induced mitochondrial swelling however, DSF reveals that Succinyl CoA negatively regulates CypD binding interaction with CsA while alphaketoglutarate had no effect. Global metabolite differences were observed in CypD genotypes and increased level of succinate and fumarate in were observed in ppif KO when compared with WT heart tissues.

Conclusion

These findings give further insight into the role of CypD in the mitochondria as it revealed differences in MPTP between tissues and impact on cellular reductive equivalents which are associated with the differential amount of CypD in the mitochondria of these tissues. It also reveals the influence of CypD on global metabolism especially in the liver which is the majour site of metabolism and that some of these may play direct or indirect role in MPTP activation.

Support or Funding Information

OSA acknowledge Osun State University, Nigeria for financial support.YIA is grateful to University of Liverpool for a Postgraduate Bursary

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
posttranslational arginylation enzyme Ate1 regulates cell death through a mitochondrial‐dependent pathway
Authors: Zhang Fangliang , Kumar Akhilesh , Birnbaum Michael , Moorthy Balaji
Published: June 2021

Arginyltransferase1 (Ate1) is an evolutionarily conserved enzyme that mediates posttranslational addition of one extra arginine to a protein substrate in a process called arginylation. Recent evidence suggests that Ate1 is essential for normal stress response. The level of Ate1 and total arginylation activity appear to increase follow acute stress and leads to cell death. Vice versa, the removal or reduction of this enzyme minimized cellular sensitivity to high doses of common cellular stressors such as oxidants, radiations, heavy metal, and high temperature. All these evidence suggest that Ate1 is a master regulator of cell death/growth. However, how does Ate1 accomplish this role remain unknown. In this study, we found that Ate1 regulates cell death in a mitochondrial‐dependent manner. By using S.cerevisiae, which contains a conserved Ate1 gene, we found that during acute stress, Ate1 is highly recruited to mitochondria. By using recombinant Ate1 that is bound to cell membrane, we found that the ability of locating to mitochondria is essential for Ate1‐mediated cell death. By screening of dozens of other players with known roles in cell death, we discovered that Ate1‐mediated cell death appears to be independent of the pan‐caspase in yeast, the mitochondrial membrane potential, or the ubiquitin‐proteasome system in general. Instead, the Ate1‐mediated cell death is dependent on the integrity of the mitochondrial pore complex and the existence of apoptosis‐inducing factor (AIF), a mitochondrial protein that is often released into cytosol during mitochondrial stress. Consistently, the Ate1‐mediated cell death can be at least partially blocked by the over‐expression of mammalian pro‐survival protein bcl2, which is known to block the formation of mitochondrial permeability transition pore. Finally, in mammalian cells, where the intrinsic and extrinsic cell‐death pathways are well‐separated, we found that the absence of Ate1 only has impacts on intrinsic pathway that is mainly mediated by mitochondria, but has little effects on cell‐death triggered by death receptors such as TNF‐R.

In summary, our study has discovered a novel cell death mechanism that is regulated by a posttranslational modification enzyme.

Support or Funding Information

NIGNMS R01 GM107333

DoD (CDMRP), Idea Award, PC140622

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Curcumin Induces Apoptosis via the Capase‐8 Activated Extrinsic Pathway in MDA‐MB‐231 Breast Cancer Cells
Authors: Ali Mohammed , Smiley Rebecca
Published: June 2021

Aim

Chemotherapiesfor breast cancer are associated with resistance development, serious side effects, and morbidity. Natural compounds hold promise in both cancer prevention and therapy. Curcumin, a natural polyphenolic compound found inturmeric, has potential cancer treatment efficacy, including apoptosis‐induction in many cancer cell types. Cancer cells are characteristically anti‐apoptotic, highly proliferative and continue to live indefinitely. Control of cell cycle progression and initiation of apoptosis in breast cancer cells is considered to be a potentially effective strategy for the prevention/treatment of the disease. The triple‐negative breast cancer cell line MDA‐MB‐231 does not express progesterone, estrogen, or human epidermal growth factor receptor, is highly aggressive and difficult to treat. This study aims to elucidate the mechanism of curcumin‐induced apoptosis and its cell‐cycle effects in the MDA‐MB‐213 breast cancer cell line.

Methods

Apoptosis was measured by flow cytometry, fluorescence microscopy, and Western blotting. The cytotoxic activity of curcumin was determined by a sulphorhodamine B assay. Caspase cascade was studied by measuring the activities of caspase‐3, caspase‐8, and caspase‐9 using chromogenic substrates. Cell cycle arrest and cell proliferation were measured by the expression levels of p21Waf‐1 and PCNA, respectively. Mitochondrial involvement of apoptosis was investigated following the translocation of Cytochrome C by Western Blot.

Results

Flow cytometry data suggested that curcumin induced both early and late stage apoptosis in MDA‐MB‐231 cells, which was confirmed by PARP cleavage, fluorscence microscopy as well as activation of caspases. While there were no changes in the level of PCNA, a robust increase was observed in p21Waf‐1 for curcumin treated cells. These results suggest that curcumin might cause cell cycle arrest by increasing the levels of p21 Waf‐1, resulting in an accumulation of cells at G1 phase, leading to G1/S check point arrest. Our data show that curcumin significantly induces caspase‐8 and caspase‐3 activity without modulating caspase‐9 activity, indicating an extrinsic apoptotic pathway. Curcumin did not provoke Cytochrome c release from mitochondria, thus indicating no mitochondrial involvement.

Conclusion

In MDA‐MB‐413 cells, curcumin induces apoptosis following an extrinsic pathway via caspase‐8 activation, and cell cycle arrest occurs at the G1 phase. Our findings will aid in the development of futureth erapeutic strategies, including curcumin as a novel treatment supplement.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Individual and Combined Toxicity of Ochratoxin A, Acrylamide on Oxidative Stress in Human Proximal Tubule HK‐2 Cells
Authors: Pyo Min Cheol , Shin Hye Soo , Lee Hyun‐Sun , Bae Jung Min , Lee Kwang‐Won
Published: June 2021

Ochratoxin A (OTA) is a toxic secondary metabolite produced by fungi species, such as Aspergillus and Penicillium. Since OTA can take place in various human foodstuffs such as cereals, wine, coffee, nuts, and dairy products, people are commonly exposed to OTA in everyday life. Acrylamide (AA) has been applied in many industries and laboratories and is commonly found in carbohydrate‐rich food heated at high temperatures. AA is a potential carcinogen that has attracted a great deal of attention recently. Heat treatment of food contaminated with OTA can lead to the production of AA. OTA and AA are known to cause oxidative stress and are also likely to cause carcinogenesis. Therefore, it can be assumed that the interaction between OTA and AA may exist. To understand the combinatorial toxicity of OTA and AA, HK‐2 cells were exposed to OTA and AA alone or combination. Mono‐exposure and co‐exposure treatments were established to determine individual and binary combined toxicity on oxidative stress. Factorial analyses were performed to determine interactions between OTA and AA. When OTA and AA alone were exposed to a slight concentration of toxicity, oxidative stress was not induced in HK‐2 cells. However, exposure to the combination of OTA and AA increased biomarkers of oxidative stress and mRNA expression of enzymes associated xenobiotic mechanism. The findings of an interaction between OTA and AA might have an impact on the potential human health threat of this mixture.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microtubule Involvement with the WRAMP Structure, a Mechanism for Rear Membrane Retraction in Mammalian Cells
Authors: Murillo Bryan , Miller Suzannah , Connacher Mary Katherine , Ahn Natalie
Published: June 2021

Cellular locomotion involves coordination between membrane protrusions and attachments to extracellular matrix at the front, and membrane retraction and disassembly of attachments at the rear. While Rac‐ and Cdc42‐dependent polymerization of F‐actin to push lamellipodia and filopedia protrusions have been well studied, rear end retraction during cell locomotion is still poorly understood. Our lab has discovered a complex of interacting proteins and membranes known as the Wnt5a‐receptor‐actin‐myosin‐polarity (WRAMP) structure, which forms dynamically and transiently, and is associated with rear membrane retraction and directional cell migration. A remarkable feature of this complex is that it involves endosomes containing the cell surface glycoprotein, melanoma cell adhesion molecule (MCAM, aka CD146/MUC18), suggesting that organelle positioning links to rear‐end retraction. Live images of MCAM‐GFP show that WRAMP structures relocalize intracellularly before cells change direction, suggesting that their placement helps guide the new direction of movement. We hypothesize that microtubules mediate the delivery of endosomes to WRAMP structures, through microtubule binding proteins that contact the plasma membrane. Preliminary experiments using indirect immunofluorescence indeed show colocalization of microtubules with MCAM and F‐actin within WRAMP structures. Ongoing studies are investigating the potential involvement of plus end tracking proteins (+TIPs) such as EB1, and its binding partners, IQGAP1 and ezrin, as potential membrane anchors. Conceivably, recruitment of cortical microtubules via +TIPs may traffic endosomes to the plasma membrane, forming WRAMP structures which in turn control the new direction of migration. In this way, WRAMP structures may provide a mechanism allowing the orientation of cells and direction of movement to be guided from the rear.

Support or Funding Information

This work is supported by the National Institutes of Health (R01GM105997 and T32GM008759).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the Role of Protein Interactions at WRAMP Structures in Directional Migration
Authors: Miller Suzannah , Murillo Bryan , Connacher Mary Katherine , Ahn Natalie
Published: June 2021

Cell migration is a critical process in development, tissue regeneration, and cancer cell invasion, but mechanisms that establish the location of the cell rear and activate rear contractile forces are still poorly understood. Our lab previously discovered a protein network named the “Wnt5a‐receptor‐actomyosin‐polarity (WRAMP) structure” which may serve as a novel mechanism for rear‐directed control of migration. The WRAMP structure forms transiently in response to the signaling ligand Wnt5a and is characterized by polarization of F‐actin, myosin‐IIB, and melanoma cell adhesion molecule (MCAM). Cells with WRAMP structures migrate in a persistent direction for longer periods of time compared to cells without WRAMP structures, and the location where WRAMP structures form may determine the position of the cell rear and direction of migration. However, the specific interactions of proteins at WRAMP structures that activate actomyosin contraction are unknown. To address this question, we have generated plasmids to express previously identified WRAMP structure members MCAM and moesin fused to the engineered peroxidase APEX2 or the promiscuous biotin ligase BirA(R118G) for use in the proteomics techniques APEX and BioID, respectively. In these methods, proteins in close proximity to the bait protein are biotinylated and subsequently isolated on streptavidin beads for identification by mass spectrometry. Ongoing goals of this project are to compare APEX and BioID methods in melanoma cells to build up a WRAMP structure “interactome,” prioritized by interactions that increase in the presence of Wnt5a treatment. In addition, we are examining the role of RhoA/Rho kinase (ROCK) signaling in activation of actomyosin contraction using immunofluorescence for activated substrates of ROCK at WRAMP structures, and we will test a literature‐based model for RhoA activation through MCAM‐moesin interactions using an MCAM mutant which lacks moesin binding. These findings will help define the spatial organization of WRAMP structures and identify key interactions and signaling pathways involved in control of cell migration.

Support or Funding Information

This work is supported by funding from the National Institutes of Health (R01GM105997 and T32GM008759).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pasteurella multocida PfhB2 toxin displays a novel unconventional cysteine protease fold
Authors: Mattoo Seema , Kumar Sanjeev
Published: June 2021

Pasteurella multocida causes respiratory tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multi‐domain toxin, called PfhB2, which contains a YopT‐like putative cysteine protease domain at its C‐terminus. PfhB2's YopT domain contains a well‐conserved Cys‐His‐Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. PfhB2 is implicated in virulence. Its FHA (filamentous hemagglutinin) and Fic (filamention induced by cAMP) domains have been shown to provide protective immunity in turkeys and to adenylylate Rho‐GTPases in vitro, respectively. In contrast, little is known about PfhB2's YopT‐like domain. Here, we show that PfhB2‐YopT does not display cysteine protease activity in a manner similar to Yersinia YopT or any other characterized YopT family members. We, therefore, opted to characterize PfhB2‐YopT structurally. Using a catalytically inactive C3733S mutant of PfhB2‐YopT that provides enhanced protein stability, we determined the crystal structure of PfhB2‐YopTC3733S solved by experimental phasing with Selenomethionine derivative crystals at 2.2Å resolution. Our structure shows that PfhB2‐YopT exists as four molecules arranged as an asymmetric unit consisting of two dimers. Importantly, the Cys from each molecule of the dimer coordinates with the His and Asp from the other molecule to complete the catalytic triad. Such an unconventional orientation of the catalytic triad has never been observed for any class of Cysteine proteases. Thus, our PfhB2‐YopT structure represents a novel cysteine protease fold, and provides critical insights for the function of Cys‐His‐Asp containing domains that are often associated with large, multi‐domain bacterial toxins.

Support or Funding Information

This work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying New Regulators of Insulin‐Stimulated GLUT4 Exocytosis
Authors: Crisman Lauren , Gulbranson Daniel , Miller Jessica , Yu Haijia , Shen Jingshi
Published: June 2021

GLUT4 is an insulin responsive glucose transporter responsible for the maintenance of blood glucose homeostasis. At basal conditions GLUT4 is sequestered inside the cell in specialized storage vesicles. After a meal, insulin signaling causes translocation of GLUT4 from these storage vesicles to the plasma membrane where it aids in the removal of glucose from the blood. Misregulation of this system is a major contributor to the global rise in rates of insulin resistance and metabolic disease. While the physiological importance of this pathway is obvious, the underlying molecular mechanisms remain poorly understood. This difficulty in elucidating the pathway is due to several factors, such as the complicated nature of vesicle transport pathways and technical barriers that have existed with using mammalian cells for genetic screens. The advent of CRISPR‐Cas9 genome editing has allowed us to overcome this second obstacle. Many labs have shown its utility in dissecting pathways for cell viability and growth; our lab has adapted it for vesicle trafficking studies. Using the CRISPR‐Cas9 system in conjunction with our trafficking assay, we performed an unbiased genome wide knockout screen to search for previously unknown regulators of this complex and important metabolic pathway. Specifically, the screen was designed to find regulators whose knockout causes a decrease in GLUT4 translocation after insulin stimulation. The screen identified several known regulators of GLUT4 exocytosis, such as RABIF and Rab10, suggesting that the screen was successful. Many of the hits, however, are not known to be involved in the GLUT4 exocytic pathway, such as TRAF3 and ANTRXL.

Support or Funding Information

R01 DK095367

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of antioxidant and anti‐inflammatory activity of quercetin, isoquercitrin and rutin against alcohol‐induced liver injury in HepG2 Cells
Authors: Song Jinhee , Kim Younghwa , Lee Junsoo
Published: June 2021

The dietary flavonoids and their glycosides are the most vital phytochemicals in diets and are of great general interest due to their diverse bioactivities. The natural flavonoids almost all exist as their O‐glycoside or C‐glycoside forms in plants. Quercetin and quercetin glycosides (isoquercitrin, rutin) are strong polyphenolic antioxidants abundant in plants and human diets. The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damages. In this study, we compared the hepatoprotective activity of quercetin, quercetin‐3‐glucoside and rutin on alcohol‐induced liver damage in HepG2 cells. Treatment with quercetin and its glycosides prevented alcohol‐induced alterations in hepatic aminotransferases, hepatic lipid peroxides, antioxidant enzymes and pro‐inflammatory factors as well as ROS generation. Nuclear factor erythroid 2‐related factor 2 (Nrf2), the main regulator of endogenous antioxidant enzymes was significantly induced by quercetin. These results indicate that quercetin and its derivatives might be beneficial in mitigating alcohol‐induced liver diseases through the enhancement of phase II and antioxidant gene expressions via Nrf2 activation; thereby modulating the oxidative stress and inflammatory responses in hepatocytes. Furthermore, it was found that quercetin showed higher hepatoprotective activity than quercetin glycosides.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of Glycosaminoglycans in the Ang/Tie Signaling Axis
Authors: Griffin Matthew E. , Miller Gregory M. , Sorum Alexander W. , Hsieh‐Wilson Linda C.
Published: June 2021

Cell‐surface glycosaminoglycans (GAGs) dictate cellular function through their interactions with soluble ligands and transmembrane receptors. However, their importance in signal transduction is often overlooked due to the difficulty in identifying new protein‐glycan binding events. Using a combination of biochemical, computational, and chemical biology techniques, we have examined the possible roles of GAGs to modulate the Ang/Tie signaling axis, a key regulator of vascular development and maintenance. Our combined approach revealed that heparin/heparan sulfate (HS) binds to both the Ang1 and Ang4 ligands and potentiates downstream activation through the formation of a ternary complex with the Tie2 receptor. Moreover, we discovered that HS also interacts with the orphan receptor Tie1, the first evidence of a ligand for this receptor since its discovery nearly 25 years ago. Together, these results reveal novel mechanisms by which sulfated polysaccharides modulate an important endothelial signaling pathway.

Support or Funding Information

National Institutes of Health (R01‐GM093627); National Science Foundation Graduate Research Fellowship (DGE‐1144469)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.