bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

IntroductionCyclophilin 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.MethodCypD 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.ResultsCypD 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.ConclusionThese 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 InformationOSA acknowledge Osun State University, Nigeria for financial support.YIA is grateful to University of Liverpool for a Postgraduate BursaryThis 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 InformationNIGNMS R01 GM107333DoD (CDMRP), Idea Award, PC140622This 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

AimChemotherapiesfor 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.MethodsApoptosis 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.ResultsFlow 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.ConclusionIn 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 InformationNoneThis 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 InformationThis 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 InformationThis 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 InformationThis 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 InformationR01 DK095367This 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 InformationNational 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.