bluuebunny/crossref_metadata_2025_split huggingface dataset:
Energetics and Interfacial Interactions of Spliceosomal Protein Dib1 Predicted with MD Simulations
Authors: Goldstein Rachel , Cheng Sara , Orr Gabby , Schreib Christian , Cheng Kwan , Maeder Corina 
Published: June 2021

The spliceosome is the highly dynamic macromolecular complex responsible for catalyzing the excision of introns from pre‐messenger RNA. The spliceosome consists of five small nuclear ribonucleoproteins as well as several additional proteins. As part of a complex assembly pathway, the essential protein Dib1 must leave the spliceosome. Experimental analysis has found a temperature sensitive phenotype of Dib1 (TS‐Dib1, F85A) that hinders splicing activity. This phenotype has also been hypothesized to perturb spliceosome assembly. Our current work uses atomistic molecular dynamic (MD) simulations to study the molecular interactions in the region of the spliceosome around Dib1 in order to determine what energetic and structural changes result from this point mutation.Starting with recently published cryo‐EM structures, the spliceosome was reduced to only include components in close proximity to Dib1 (either native or F85A). The reduced Dib1 structure consists of three snRNAs (U4, U5, and U6) and four proteins (Prp6, Prp8, Brr2, Prp31). Both systems, containing either native Dib1 or mutant Dib1, were simulated using atomistic MD simulations to identify energetic differences between native Dib1 and mutant TS‐Dib1. The simulation conditions mimicked the biological conditions of previous bulk splicing assays, with an aqueous solvent, 0.1 M salt, and temperature differences that have experimentally shown TS‐Dib1 inactivation. The MD simulations revealed a lower global binding energy in the TS‐Dib1 system compared to the native Dib1 system. Additionally, interfacial hydrogen bonding patterns displayed increased hydrogen bonding among Prp6, Prp8, and Dib1 in the native Dib1 system that were not present in the TS‐Dib1 system, which could contribute to the lower global binding energy. The results from the MD simulations support the experimentally observed temperature sensitivity in the TS‐Dib1 system and could help explain the role of Dib1 in spliceosome assembly.Support or Funding InformationNIH R15GM120720, Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidants prevent cytotoxic and mutagenic effects of hexavalent chromium on human and bacterial cell cultures
Authors: Mayotte Timothy , Himes Ryan 
Published: June 2021

Cr(VI) (hexavalent chromium) has recently been found in the drinking water of over 250 million Americans. It is a powerful oxidizing agent, and is known to cause cancer, although the specific mechanism has yet to be elucidated. There is no known preventative treatment for Cr(VI) exposure, and the US EPA is currently determining what concentration of Cr(VI) in drinking water can be safely tolerated. This study sought to test the hypothesis that Cr(VI) cytotoxicity can be prevented by various antioxidants. We tested this hypothesis by exposing human embryonic kidney and human intestinal epithelial cells to Cr(VI), with and without the presence of the antioxidants vitamin C, or epigallocatechin gallate (EGCG). Cr(VI) was found to be significantly toxic to human cell cultures at concentrations of 200 parts per billion (ppb) and higher. This effect was blocked by 10 parts per million (ppm) vitamin C and by 15 ppm EGCG. No cytotoxic effects were observed on cells treated with vitamin C or EGCG alone. We also used an Ames test to determine if these antioxidants can prevent Cr(VI)‐induced mutagenesis on bacterial cells. The Ames test revealed that Cr(VI) causes DNA mutations in bacterial cells at concentrations of at least 20 ppb. This effect was blocked by 20 ppm vitamin C. No mutagenic effect was observed with vitamin C alone. Taken together, the results suggest that Cr(VI) is toxic to human cells through an oxidative mechanism at a concentration of at least 200 ppb. Additionally, Cr(VI) may also cause DNA mutations at concentrations of 20 ppb or more. These effects can be abrogated with antioxidant co‐treatment. While further study is merited, these results should help to inform government agencies tasked with monitoring drinking water quality.Support or Funding InformationThis research was made possible by funding from the Craighton T. and Linda G. Hippenhammer Faculty Scholarship Grant, the Elbert Pence and Fanny Boyce Undergraduate Summer Research Fund, and the Olivet Nazarene University Honors Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RECK suppresses interleukin‐17/TRAF3IP2‐mediated MMP‐13 activation and human aortic smooth muscle cell migration and proliferation
Authors: Mummidi Srinivas , Das Nitin A , Carpenter Andrea J , Yoshida Tadashi , Mostany Ricardo , Izadpanah Reza , Higashi Yusuke , Sukhanov Sergiy , Delafontaine Patrice , Siebenlist Ulrich , Randy Scott Rector , Bysani Chandrasekar 
Published: June 2021

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular proliferative/occlusive disorders. The proinflammatory cytokine interleukin (IL)‐17 signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various transcription factors critical for inflammatory responses, including AP‐1 and NF‐κB. Here we investigated whether IL‐17A/TRAF3IP2 signaling promotes MMP‐13‐dependent human aortic smooth muscle cell (SMC) migration and proliferation, and whether RECK overexpression blunts its mitogenic and migratory potential. Indeed, IL‐17A treatment induced (i) AP‐1, NF‐κB, CREB and p38 MAPK activation, (ii) miR‐21 induction, (iii) miR‐27b and miR‐320 inhibition, (iv) MMP‐13 expression and activation, (v) RECK suppression, and (vi) SMC migration and proliferation, all in a TRAF3IP2‐dependent manner. RECK (REversion‐inducing‐Cysteine‐rich protein with Kazal motifs) is a membrane‐anchored MMP inhibitor, and forced expression of TRAF3IP2 suppressed its expression, but induced MMP‐13 expression and activation. Importantly, forced expression of RECK promoted IL‐17‐mediated MMP‐13/RECK physical association, reduced MMP13 activity and inhibited MMP‐13‐dependent SMC migration and proliferation. Further supporting its causative role in vascular proliferative diseases, addition of recombinant MMP‐13 stimulated SMC migration and proliferation, in part via ERK activation. These results indicate that increased MMP‐13 and decreased RECK contribute to IL‐17A/TRAF3IP2‐mediated SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.Support or Funding InformationVA‐I01‐BX002255IK6BX004016‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium status influences selenoprotein H interactions with other selenoproteins in the response to oxidative stress
Authors: Cao Lei , Lee Sanggil , Cheng Wen‐Hsing 
Published: June 2021

Selenium (Se) is an essential trace element exerting its biological functions mainly through selenoproteins. Evidence suggests that selenoprotein H (SELONOH), a nuclear protein suppressing oxidative stress and cellular senescence, is the most prominent selenoprotein responding in parallel to dietary Se deficiency and age in telomere‐humanized mice. On the other hand, SELENOH is one of the few low‐hierarchy selenoproteins whose expression is very sensitive to fluctuations in Se status. The objective of this study was to explore impacts of SELENOH on the expression of other selenoproteins and redox regulation upon changes in Se status. A plasmids expressing wild‐type SELENOH was constructed and transfected into human HEK293T cells. Adding Se (Na2SeO3, 0–200 nM) to cell culture medium dose‐dependently increased protein levels of overexpressed SELENOH and other selenoproteins including glutathione peroxide 1 and thioredoxin reductase 1; however, their mRNA transcripts were not affected by Se status and SELENOH suppressed protein expression of the other two selenoproteins. Supplementing Se (100 nM) enabled overexpressed SELENOH to protect against paraquat‐induced senescence and declines in cellular viability. Analyses of SELENOH‐associated proteins through LC‐MS‐MS co‐identified nucleolar and glycosylation proteins. Altogether, these results suggest that SELENOH could compete with other selenoproteins for available Se but protects against oxidative stress together with them.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Establishing the Role of Rieske Protein Reduction Potential in the Formation of Reactive Oxygen Species in Complex III
Authors: Henderson Victoria , Hunsicker‐Wang Laura 
Published: June 2021

The factors that modulate the reduction potential of complex III's catalytic Rieske metalloprotein subunit (RP) have been the subject of much study. During the bifurcated Q‐cycle, RP transports one electron from quinol to cytochrome c1. Quinol's other electron is passed from heme bL to heme bH, which reduces a quinone bound at the QN site. If the Q‐cycle is disrupted, electrons may interact with molecular oxygen to produce reactive oxygen species (ROS). For example, if RP's reduction potential is too low, it may never accept quinol's electron or do so inefficiently. The latter scenario may shift Q‐cycle equilibria against RP movement towards cytochrome c1, instead favoring RP electron release and interaction with molecular oxygen to produce ROS. Thus, we propose that a mismatch in reduction potential between RP and quinol may serve as a possible source of ROS generated from complex III. Understanding the sources of ROS in the electron transport chain may lead to better treatments for diseases that arise from high levels of ROS.We are investigating this hypothesis by (1) measuring the reduction potential of isolated Saccharomyces cerevisiae (yeast) RP expressed in Escherichia coli (E. coli) cells (ScRieske) and (2) measuring the ROS produced by intact yeast complex III expressed in yeast cells (ScComplexIII). We created a wild‐type ScComplexIII yeast strain (JPJ1‐RIP1) by restoring respiratory function to an anaerobic RP‐deletion strain (JPJ1) via complementation with a RP expression vector. Now, we are measuring JPJ1‐RIP1 and JPJ1 ROS production by fluorometry using dichlorofluorescein (DCF) as a ROS indicator. We have also expressed and purified wild‐type ScRieske in E. coli and have characterized its structure using circular dichroism (CD) and UV‐visible spectroscopy. Furthermore, we have probed the electronics of ScRieske's [2Fe‐2S] cluster via chemical modification experiments with diethyl pyrocarbonate (DEPC). Lastly, we have obtained E. coli and JPJ1‐RIP1 expression vectors for ScRieske mutants Y185F, S183A, and S183T. Ultimately, we will use our ROS assay to quantify ROS production of ScRieske mutants with modulated reduction potentials to study the relationship between RP reduction potential and ROS production in complex III.Support or Funding InformationTrinity Chemistry and Neuroscience Departments, Welch Foundation, The San Antonio Area Foundation, 2017 Murchison Summer Research Fellowship, 2017 ASBMB Undergraduate 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.
Implication of Cholesterol in Regulating the Pore‐formation Mechanism of Vibrio Cholerae Cytolysin, a β‐barrel Pore‐forming toxin
Authors: Kathuria Reema 
Published: June 2021

Vibrio cholerae cytolysin (VCC) is a β‐barrel pore‐forming toxin with potent cell‐killing cytolytic/cytotoxic activity. To exert it's cytolytic effect, VCC binds to the target cell membranes and forms transmembrane oligomeric β‐barrel pores. Earlier studies have indicated that cholesterol might play a critical role in regulating the mode of action of VCC. However, exact mechanistic basis of the process still remains unknown. In the present study, we have explored and examined the roles of cholesterol in the different steps of VCC mode of action. Employing the membrane lipid bilayer of lipid vesicles or liposomes containing varying amount of cholesterol, we have shown that cholesterol governs the efficient interaction of the toxin with the membrane lipid bilayer that in turn facilitates subsequent oligomeric pore‐formation process. We have also studied the physiological relevance of membrane cholesterol in the context of the biomembranes of the eukaryotic cells. Our results have shown that the depletion of membrane cholesterol from erythrocytes compromises the pore‐forming activity of VCC. In the biomembrane of erythrocytes, where accessory interactions are available, binding of VCC is not affected to any significant extent. However, upon depletion of membrane cholesterol in the erythrocytes membranes, membrane‐bound fraction of VCC remains trapped in the form of abortive oligomeric assembly, and does not form the functional pores. Our study has also revealed that VCC shows a marked tendency to associate with the cholesterol‐rich membrane micro‐domains on erythrocytes that, in turn, appears to facilitate cholesterol‐dependent pore‐formation mechanism of the toxin. In sum, our results suggest that the presence of cholesterol in the target membranes play critical role for the pore‐formation mechanism of VCC.Support or Funding InformationWe acknowledge the support through funding from the Department of Biotechnology (DBT), Govt. of India [DBT grant no. BT/HRD/NBA/37/01/2014 (x)] and also the support through funding under the Centre of Excellence (COE) in Frontier Areas of Science and Technology (FAST) program of the Ministry of Human Resource Development, Govt. of India, in the area of protein science, design, and engineering (to K.C.). We also thank the Indian Institute of Science Education and Research Mohali for the 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.
Handover mechanism of the growing pilus by the bacterial outer‐membrane usher FimD
Authors: Du Minge 
Published: June 2021

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host‐cell receptors. Type 1 pili are assembled via the conserved chaperone usher pathway. The outer‐membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N‐terminal domain of the usher. Subunit translocation through the β‐barrel channel of the usher occurs at the two C‐terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone–subunit complex bound to the N‐terminal domain is handed over to the C‐terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo‐electron microscopy to capture a pilus assembly intermediate (FimD–FimC–FimF–FimG–FimH) in a conformation in which FimD is in the process of handing over the chaperone‐bound end of the growing pilus to the C‐terminal domains. In this structure, FimF has already polymerized with FimG, and the N‐terminal domain of FimD swings over to bind CTD2; the N‐terminal domain maintains contact with FimC–FimF, while at the same time permitting access to the C‐terminal domains. FimD has an intrinsically disordered N‐terminal tail that precedes the N‐terminal domain. This N‐terminal tail folds into a helical motif upon recruiting the FimC‐subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N‐terminal and C‐terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits.Support or Funding InformationR01GM062987Van Andel Institute Graduate SchoolThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Pharmacology of a Conformational‐Specific Extracellular Nanobody Against CXCR4
Authors: Soave Mark , Heukers Raimond , Smit Martine J , Briddon Stephen J , Hill Stephen J 
Published: June 2021

Camelid single‐domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain[1]. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes[2]. Here, we have used the nanobody VUN400[3], which recognises the second extracellular loop of the human CXCR4 chemokine receptor, as a probe to monitor specific CXCR4 conformations. VUN400 was fused on its C‐terminus to the 11‐amino acid HiBiT tag (VUN400‐HiBiT) which, in the presence of the LgBiT protein, complements to form a full length functional NanoLuc luciferase. Here, we use complemented luminescence to detect binding of the VUN400‐HiBiT to specifically recognise N‐terminally tagged LgBiT‐CXCR4 expressed in living HEK293 cells (KD 59.1±11.2 nM; n=4). The binding of VUN400‐HiBiT to CXCR4 could be prevented by the orthosteric ligand CXCL12 and allosteric antagonists AMD3100 and IT1t (Table 1), allowing VUN400‐HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular‐targeted nanobodies can be utilised to probe receptor pharmacology.Support or Funding InformationThe authors would like to thank the MRC (grant code MR/N020081/1) for financial 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.
CP‐25 Inhibits Methotrexate‐Induced Apoptosis in Renal Tubular Epithelial Cells
Authors: Wang Chun , WU Yi‐jin , TANG Hao , WANG Bin , WANG Yong , SUN Wei , XIAO Feng , WEI Wei 
Published: June 2021

ObjectiveMethotrexate (MTX), known as nephrotoxic drug, frequently causes renal injury via inducing apoptotic death in human renal tubular epithelial cells(RTEC). Paeoniflorin‐6′‐O‐benzene sulfonate (CP‐25) is active compound obtained by modifying the structure of Paeoniflorin. We have previously showed CP‐25 inhibited chronic inflammation and effectively improved symptoms of arthritis and Sjogren syndrome in experimental animal models. This study was to investigate effects of CP‐25 on MTX‐induced apoptosis in RTEC lines in vitro and the related mechanisms.MethodsNRK‐52E and NK‐2 were treated with various concentration of MTX in the presence or absence of CP‐25. After treatment, the proliferation of cells were measured by MTT assay, and apoptotic death of the cells were detected by Annexin V/PI dual staining. The expression of apoptotic related protein were measured by western blotting.ResultsTreatment of MTX significantly inhibited proliferation of NK‐2, NRK‐52E in dose‐and time‐dependant manners. Moreover, MTX could significantly induce apoptotic death in NK‐2 and NRK‐52E cells. Further study revealed that MTX exposure to the cells could activate intrinsic and extrinsic apoptotic signal pathways, which was evidenced by increased expression of Bax, cyt‐c, clea‐caspase‐3, clea‐caspase‐8, and clea‐caspase‐9, and decreased expression of Bcl‐2 and XIAP. Interestingly, we found that co‐treatment of MTX with CP‐25 could significantly reduce MTX‐induced apoptotic death in the cells. As expected, CP‐25 inhibited MTX‐induced activation of intrinsic and extrinsic apoptotic pathways via regulating abnormal expression of apoptosis‐related proteins. Finally, we found MTX exposure to the cells significantly increased nuclear translocation of NF‐κB coupled with increase in phosphorylated Iκ‐B, which was significantly inhibited by CP‐25.ConclusionCP‐25 could inhibit MTX‐induced pro‐apoptotic effects on RTEC. The underlying mechanism is associated with inhibitory effects of CP‐25 on the activation of intrinsic/extrinsic apoptotic pathways and NF‐κB nuclear translocation. Our data indicate CP‐25 maybe a promising therapeutic drug for the treatment of MTX‐induced renal damage.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Rutaecarpine on the Benzo(a)pyrene‐Induced DNA Damage In Vitro
Authors: Li You , Park Miki S. , Chan William K. , Livesey John , Zhou Zhu 
Published: June 2021

Benzo[a]pyrene (BaP) is one of the most potent mutagens and carcinogens known. The metabolic pathway of BaP involves several steps initiated by cytochromeP450 (CYP) enzymes such as CYP1A1 to give the ultimate carcinogen benzo(a)pyrene‐7,8‐diol‐9,10‐epoxide (BPDE). BPDE is responsible for genotoxic effects by alkylating the DNA at the N2 position of guanine bases. Rutaecarpine, a pentacyclic indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been used in traditional medicine to treat various diseases. It has been reported as a CYP1 family inducer. Our objective is to determine if co‐administered BaP and rutaecarpine would increase the formation of BPDE, which will ultimately increase the DNA damage. We utilized ethoxyresorufin‐O‐deethylase (EROD) assay to measure CYP1A1 enzyme activity and Muse™Multi‐Color DNA Damage Kit to examine DNA damage levels. The results showed that BaP (5 μM) and rutaecarpine (1.25–10 μM) significantly induced CYP1A1 enzyme activity (p<0.005) when administered separately in mucoepidermoid pulmonary carcinoma (H292) cells, with induction by rutaecarpine occurring in a concentration‐dependent manner. Interestingly, co‐administration of rutaecarpine, especially at a high concentration (10 μM), with BaP caused induction of CYP1A1 to a lesser extent as compared with either compound alone. Similar results were observed in hepatocellular carcinoma (Hep3B) cells. It was subsequently confirmed by the analysis of DNA damage in both H292 and Hep3B cells, in which the percent of DNA damage decreased by more than 2‐fold after combined concurrent administration of rutaecarpine and BaP, compared with BaP alone. In summary, our data supports the possibility of a protective effect of rutaecarpine against BaP‐induced DNA damage in vitro.Support or Funding InformationThis research was supported by a Scholarly and Artistic Activity Grant from University of the Pacific.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.