bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of ethanol extract of peros Punica granatum L. peel on the oxidative damage induced by diabet in rats
Authors: ÖMÜR Ali Dogan , Yıldırım Betül Dogan Apaydın
Published: June 2021

This study is aimed to evaluate the effects of ethanol extract of peros Punica granatum L. peel on the oxidative damage induced by diabet in rats. In present study used twenty‐eight male Sprague–Dawley rats were randomly divided into four equal groups containing of seven rats per group. Group 1 kept as a control group, Received only vehicle 0.5 %CMC (peros), Group 2; streptozotocin group: Streptozotocin 60 mg/kg/ip single dose, Group 3; Punica granatum L peel extract (PGE) Group: PGE 10 mg/kg/20days/po, Group 4; Streptozotocin+PGE group: Streptozotocin as 60 mg/kg/ip single dose+PGE 10 mg/kg/20days/po. At the end of these processes, the animals were sacrificed, testis tissues were excised by dissection and evaluated as biochemical. Glutathione, malondialdehyde, catalase, glutathione peroxidase and superoxide dismutase were measured in the plasma and testis tissues. Punica granatum L peel extract was shown that the activity of catalase, superoxidase, glutathione peroxidase and glutathione activities increased and decreased levels of malondialdehyde in diabetic rats. As a result, the results showed that oral administration of Punica granatum L peel extract reduced the levels of lipid peroxidation and improved the antioxidant activity in plasma and testis tissues in rats treated with Punica granatum L. peel extract as compared with the diabetic and control rats. These results suggested that the Punica granatum L. peel extract enhanced the antioxidant defence under hyperglycaemic conditions hence protected the testes cells.

Support or Funding Information

Ataturk University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quorum sensing‐disrupting molecules to control virulence of Vibrio vulnificus, a fulminating foodborne pathogen
Authors: Choi Sang Ho , Kim Byoung Sik , Choi Garam , Jang Song Yee , Kim Myung Hee
Published: June 2021

Bacterial pathogenicity involves numerous virulence factors required for successful survival in host environments as well as invasion and destruction of host tissues. The pathogenic marine bacterium Vibrio vulnificus is a causative agent of food‐borne diseases such as gastroenteritis and life‐threatening septicemia. It has been reported that expression of V. vulnificus virulence factors is well‐coordinated and regulated by sophisticated mechanisms consisting of many global regulatory proteins. Among them, SmcR, a quorum‐sensing master regulator and homologue of V. harveyi LuxR, has been identified and found to play a critical role in the regulation of the V. vulnificus virulence factors. The crystal structure of SmcR reveals that SmcR belongs to the TetR family protein possessing putative ligand‐binding pockets. To identify the small molecules which bind to and inhibit SmcR, chemical libraries were screened and an SmcR inhibitor exhibiting the most effective SmcR‐inhibition activity was selected and named QStatin. The lack of reduction of intracellular levels of SmcR in QStatin‐treated V. vulnificus suggested direct binding of the chemical to SmcR. Isothermal titration calorimetric (ITC) and crystallization analyses confirmed the chemical binding of QStatin into the pockets of SmcR. Although no big conformational changes were observed in the crystal structure, binding of QStatin reduced the DNA binding affinity of SmcR, and thereby changed the expressions of SmcR regulon similar to those of SmcR mutants. Accordingly, QStatin significantly inhibits expression of V. vulnificus virulence genes, leading to the decreased mortality of a brine shrimp infected with V. vulnificus. Most interestingly, QStatin attenuates pathogenicity of V. vulnificus, but does not inhibit growth itself at all, unlike existing antibiotics. Consequently, the combined results indicate that QStatin could be a novel lead compound to control QS‐mediated pathogenesis of V. vulnificus with the lowest possibility of the appearance of resistant strains.

Support or Funding Information

This work was supported by the National Research Foundation of the Ministry of Science, ICT, and Future Planning (2015R1A2A1A13001654 to S.H.C.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Advantages and Disadvantages of Circadian Rhythms: A Tale of Two Microbes
Authors: Rust Michael
Published: June 2021

Circadian clocks generate reliable ~24‐h rhythms despite being based on stochastic biochemical reactions. The circadian clock in Synechococcus elongatus uses a post‐translational oscillator that cycles deterministically in a test tube. Because the volume of a single bacterial cell is much smaller than a macroscopic reaction, we asked how clocks in single cells function reliably. Here, we show that S. elongatus cells must express many thousands of copies of Kai proteins to effectively suppress timing errors. Stochastic modeling shows that this requirement stems from noise amplification in the post‐translational feedback loop that sustains oscillations. The much smaller cyanobacterium Prochlorococcus expresses only hundreds of Kai protein copies and has a simpler, hourglass‐like Kai system. These altered dynamics are likely due to the loss of the KaiA protein in the Prochlorococcus system, which acts as a key node for the negative feedback loop in the system. Using advanced sampling techniques and molecular dynamics simulations, we find that KaiA acts as a nucleotide exchange factor by destabilizing the hexameric structure of the C‐terminal domain of KaiC. In KaiA‐less versions of this system, nucleotide exchange is presumably constitutive, potentially allowing the KaiC hourglass to act by measuring the ATP/ADP ratio in the cell. We show that this hourglass strategy can outperform a free‐running clock if internal noise is significant. This conclusion has implications for clock evolution and synthetic oscillator design, and it suggests hourglass‐like behavior may be widespread in microbes.

Support or Funding Information

This work was supported by NIH training grant T32‐GM007281, NIH F30 fellowship F30‐GM117962 (to J.C.), NIH R01‐GM107369 and a Pew Biomedical Scholars award (to M.J.R.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elimination of Wnt secretion from hepatic stellate cells impairs hepatocyte proliferation after partial hepatectomy
Authors: Ko Sungjin , Kim Min‐wook , Preziosi Morgan E. , Monga Satdarshan Pal
Published: June 2021

Wnt‐dependent β‐catenin activation contributes to hepatocyte proliferation following 2/3 partial hepatectomy (PH) through regulation of Cyclin‐D1 (Ccnd1) expression, as we have previously shown using β‐catenin or Wnt coreceptors low‐density lipoprotein receptor‐related protein 5/6 conditional knockouts (KO). However, given the redundancy between the 19 mammalian Wnt proteins and various cell sources for Wnt secretion, the precise mechanisms of Wnt signaling in liver regeneration (LR) are not completely understood. Recently, using endothelial cell (EC)‐specific deletion of Wntless (Wls), a cargo receptor required for Wnt secretion, we have reported that sinusoidal ECs secrete Wnts (Wnt2 and Wnt9b) to activate β‐catenin in hepatocytes and enhance Ccnd1 expression, which is indispensable for proliferation of hepatocytes during LR after PH. Alternatively, conditional deletion of Wls in hepatocytes or cholangiocytes did not impact LR, whereas Wls deletion in macrophages only marginally affected LR post‐PH. Here, to investigate the roles for hepatic stellate cell (HSC)‐secreted Wnts in LR, we have characterized HSC‐Wls‐KO, generated by interbreeding Wls‐floxed and Lecithin Retinol Acyl Transferase (LRAT)‐driven Cre (Lrat‐cre) transgenic mice. HSC‐Wls‐KO mice showed no effect on liver weight or β‐catenin‐dependent pericentral gene expression at baseline. However, hepatocyte‐specific Cyclin‐D1 expression was abolished in baseline KO liver, whereas Cyclin‐D1 expression in non‐parenchymal cells was intact in KO liver. After PH, HSC‐Wls‐KO showed diminished protein and mRNA expression of Ccnd1, Ccnb1 and Ccna2 at 24–40 hour (h) post‐PH, but displayed slight increases at 72h, which led to a lower number of BrdU+ hepatocytes at 24 and 40h but a rebound increase by 72h. Interestingly, among several Wnts, HSC‐Wls‐KO livers at 24h post‐PH showed decrease of Wnt3 and Wnt9b mRNA compared to controls, although expression became comparable at 40–72h post‐PH.

Conclusion

At baseline, HSCs secrete Wnt ligands essential for Cyclin‐D1 expression in midzonal hepatocytes but not for β‐catenin‐dependent hepatic zonation. Following PH, HSCs‐derived Wnts also contribute to Cyclin‐D1‐dependent hepatocyte proliferation during LR.

Support or Funding Information

The work was supported by NIH grants to S.P.M. (DK62277, DK100287, CA204586). S.P.M. is an Endowed Chair for Experimental Pathology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of expression of SG2NA isoforms in human cancer cell lines
Authors: Jain Buddhi Prakash , Goswami Shyamal K
Published: June 2021

The Striatin family members (Striatin, SG2NA and Zinedin) have characteristic protein‐protein interaction domains as a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at its N‐terminus and WD40 domain at carboxyl end. SG2NA was first reported in Lung and Bladder cancer patient serum as a Nuclear Autoantigen and its expression was found to be up regulated during the S and G2 phase of cell cycle. Mouse SG2NA generates five isoforms (87, 82, 78, 52 and 35kDas) by alternative splicing, intron retention and RNA editing. SG2NA isoforms are differentially expressed in mouse tissues viz., brain, heart, lung, liver and skeletal muscle. Besides alternative splicing and polyadenylation, sg2na expression is also regulated by post‐translational stability as its dephosphorylated form is more stable than the phosphorylated form. Further its role in Cancer progression and survival was established by its knockdown study. In the present study two splice variants (78 kDa and 87 kDa) of human sg2na were characterized. Further the expression of SG2NA isoforms in different human cancer cell lines (HEK293T, A549, H1299, HepG2, Hep3B, DU145, HELA) were assessed at the protein and RNA level which shows dyanamic expression. The expression of SG2NA isoforms varies among different cancer cell lines. In case of human cancer cell lines the expression of larger isoform i.e. 87 kDa was prominent unlike mouse which suggests that this SG2NA isoform in human is not undergoing proteasomal degradation. The ambiguity between mRNA and protein level in different cancer cell lines indicate its regulation at the post‐transcriptional or post‐translation level. There was no effect of proteasome inhibition on the stability of SG2NA in all the cells tested. The post‐transcriptional regulation study of SG2NA in different cancer cell lines would be a useful to show its expression and role in cancer progression.

Support or Funding Information

DBT‐BUILDER, Government of India, awarded to the Jawaharlal Nehru University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Disulfide Isomerase Inhibits and Reverses Alpha‐Synuclein Fibrillization
Authors: Ragusa Joey , Evangelista Baggio , Serrano Albert , Qiao Xin , Kim Yoon‐Seong , Chen Bo , Teter Ken
Published: June 2021

Protein disulfide isomerase (PDI) is a ubiquitously expressed member of the thioredoxin family with related but independent oxidoreductase and chaperone properties. As a chaperone, PDI plays a crucial role in preventing the aggregation of misfolded proteins. Notably, PDI is known to inhibit alpha‐synuclein (SYN) fibrillization, a pathological hallmark of Parkinson's disease, but the mechanism by which PDI blocks the formation of SYN fibrils is unknown. Here, we report for the first time that PDI not only prevents SYN aggregation but is able to reverse aggregation both in vitro and in cultured cells. Assays using Thioflavin‐T fluorescence and transmission electron microscopy revealed that PDI is able to break down nascent but not mature fibrils. Furthermore, PDI was able to inhibit and reverse cellular fibrillization in HEK293T cells as assessed by protein‐fragment complementation and non‐reducing SDS‐PAGE. These data suggest a novel disaggregase function for PDI that disrupts the SYN fibrils most commonly found in Parkinson's disease. Current treatments for PD are limited to palliative care, but strategies focused on targeted therapeutic intervention are becoming popular. Thus, our work suggests the disaggregase property of PDI may lend itself to therapeutic development for Parkinson's disease.

Support or Funding Information

This work was supported, in part, by a LIFE at UCF Richard Tucker Gerontology Applied Research Award and a UCF College of Medicine competitive research grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Incorporation of Unnatural Amino Acids via Proteomically Informed Selective Pressure Techniques
Authors: Hill Austin Reece , Brown Amy , Boles Jeffrey Oakley
Published: June 2021

Determining the three‐dimensional structure of a protein using x‐ray crystallography can be enhanced by complete or partial incorporation of unnatural amino acids into a target protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons, therefore, heavy atom derivatives of target proteins are required. The Incorporation of unnatural metalloid containing amino acids provides a clear solution to the problem. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionine and methionine exposed cultures were analyzed with the objective of identifying differentially expressed proteins. The analysis was carried out using 2D separation with isoelectric focusing serving in the first dimension and SDS polyacrylamide gel electrophoresis in the second. In‐gel tryptic digestion followed by peptide mass fingerprinting using nano‐ESI/qTOF/MS allowed identification of differentially expressed proteins. A few of the differentially expressed proteins identified as down‐regulated in systems exposed to TeMet were UPase, ZnuA, phosphoserine aminotransferase, and fructose‐bisphosphate aldolase. Understanding the biological roles of differentially expressed proteins was used to improve methods of TeMet bioincorporation using the selective pressure technique.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DART‐ID Increases Single‐Cell Proteome Coverage
Authors: Chen Albert Tian , Franks Alexander , Slavov Nikolai
Published: June 2021

Analysis by liquid chromatography and tandem mass spectrometry (LC‐MS/MS) can identify and quantify thousands of proteins in microgram‐level samples, such as those comprised of thousands of cells. Identifying proteins by LC‐MS/MS proteomics, however, remains challenging for lowly abundant samples, such as the proteomes of single mammalian cells. To increase the identification rate of peptides in such small samples, we developed DART‐ID. This method implements a data‐driven, global retention time (RT) alignment process to infer peptide RTs across experiments. DART‐ID then incorporates the global RT‐estimates within a principled Bayesian framework to increase the confidence in correct peptide‐spectrum‐matches. Applying DART‐ID to hundreds of samples prepared by the Single Cell Proteomics by Mass Spectrometry (SCoPE‐MS) design increased the peptide and proteome coverage by 30 – 50% at 1% FDR. The newly identified peptides and proteins were further validated by demonstrating that their quantification is consistent with the quantification of peptides identified from high‐quality spectra. DART‐ID can be applied to various sets of experimental designs with similar sample complexities and chromatography conditions, and is freely available online.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Colocalization of the Rho GTPase TCL/RhoJ with Coronin Proteins at Vesicular Membranes
Authors: McRoss Jasmine , Anliker Clair , Grigoryants Angelina , Samuelson Chelsea , Tader Brooke R , Hamann Michael J
Published: June 2021

The Rho‐family GTPase TCL/RhoJ has been shown to enhance pro‐angiogenic and pro‐metastatic cellular processes; however, less is known about the signaling pathways and protein binding partners regulating its activity. TCL is part of the Cdc42 subfamily of Rho GTPases and bears a short, unique N terminus that is important for GTP‐loading and localization to the plasma membrane. Additionally, we have evidence that TCL is held to vesicular membranes through a protein/protein interaction when it is specifically in the GDP‐loaded confirmation. As several coronin family proteins bind other Rho family GTPases when they are in a GDP‐loaded conformation, we are evaluating whether coronins also selectively and spatially interact with GDP‐loaded TCL. To determine if the coronins co‐localize with TCL at vesicular membranes, mCherry‐fused versions of several Homo sapiens coronin proteins will be transfected into HeLa cells along with YFP‐tagged versions of TCL bearing constitutively active and dominant negative mutations. These TCL mutations will mimic GTP and GDP bound forms of TCL and help identify nucleotide dependent interactions with coronin proteins. Given the importance of TCL in tumor‐associated angiogenesis, these results will provide valuable insights into additional binding partners of TCL and may help clarify its role in pro‐angiogenic processes.

Support or Funding Information

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

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of miR‐144‐3p enhances Nrf2 and protects against retinal degeneration
Authors: Jadeja Ravirajsinh N. , Thounaojam Menaka C. , Jones Malita A , Abdelrahman Ammar A. , Powell Folami L. , Bartoli Manuela , Martin Pamela M.
Published: June 2021

Retinal pigment epithelial (RPE) cells are consistently exposed to high levels of pro‐oxidant and inflammatory stimulation. As such under normal conditions, the antioxidant machinery in this cell type is one of the most efficient in the entire body. In aging and disease however, these defense mechanisms often fail, leaving RPE susceptible to damage, which contributes, to degeneration of the outer retina. Thus, understanding better the mechanisms that regulate antioxidant responses is critically important. Oxidative stress increases the expression of several miRNAs, known as oxidative stress‐responsive miRNAs however, the mechanisms by which these miRs regulate expression of antioxidant target genes and related signaling is not completely understood. In the present study, we evaluated the role of the redox sensitive microRNA, miR‐144‐3p, in antioxidant signaling in RPE cells. In our cell culture model system, miR‐144‐3p expression was up‐regulated in response to pro‐oxidant stimuli. Over‐expression of miR‐144‐3p attenuated expression of Nrf2 and related antioxidant genes thereby increasing the susceptibility of RPE cells to further oxidative damage. Thus, we hypothesized that inhibiting miR‐144‐3p might be protective against RPE/retinal damage. To test this, 8‐wk old male C57BL/6J mice were treated with single dose of 50 mg/kg sodium iodate (i.p.) to induce retinal degeneration. However, sub‐retinal injection of miR‐144‐3p antagomir (5μg/kg) preserved retinal integrity, decreased oxidative stress, limited apoptosis and enhanced the expression of antioxidant genes in this mouse model of retinal degeneration. Our present study establishes inhibition of miR‐144‐3p as a potential therapeutic target for prevention and treatment of degenerative retinal diseases in which RPE is prominently affected.

Support or Funding Information

This study was supported by NIH Grant EY022704.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization and Structure Determination of the Perilipin 5 N‐Terminal Domain
Authors: Juarez Manning Erick C , Wei Derek T , Tansey John T
Published: June 2021

Many countries, including the United States, have been afflicted with a group of lipid‐related diseases including type II diabetes, obesity and non‐alcoholic fatty liver disease. The commonality behind these disorders is lipid storage and lipid storage is mediated in part by the perilipin proteins. The perilipin family are a group of five conserved genes that differ with respect to size of the proteins they encode, tissue expression and transcriptional regulation. The only member of the perilipin family to have significant protein structural data collected is perilipin 3, and this is only on the carboxyl terminus. The C‐terminus of perilipin 5 has a high sequence homology to that of perilipin 3. Less is known about the amino terminus. The central focus of this study is to characterize and determine the structure of the N‐terminal domain of perilipin 5. The amino terminal 104 amino acids of perilipin 5 were expressed with a 6‐His tag in E. coli. Optimization of the expression of the protein fragment was ascertained using an immuno‐dot blot that contained cell samples treated with various ITPG concentrations and temperatures. Isolation and purification of the protein will then be performed using nickel affinity chromatography for further structural analysis. Secondary and tertiary structures of the N‐terminal fragment were predicted using Phyre2 and RaptorX. These computer models predicted a predominantly alpha helical structure with the presence of two 4‐helix bundles. Overall, determination of the N‐terminal structure for the perilipin 5 protein will increase our understanding of the perilipin family and their functions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of PINK1 Accelerates and Exacerbates High‐fat‐diet Induced Cardiomyopathy
Authors: Mu Jing
Published: June 2021

Rationale

Mitochondrial dysfunction is a central event in the development of diabetic cardiomyopathy. Damaged mitochondria are a major source of reactive oxygen species (ROS) and pro‐apoptotic factors. Therefore, selective removal of dysfunctional mitochondria via autophagy (i.e., mitophagy) is an important mechanism to maintain mitochondrial homeostasis and preserve cell viability. Although mitochondrial dysfunction is implicated in diabetic cardiomyopathy, the molecular mechanisms by which diabetes impairs mitochondrial function remain elusive. PTEN‐induced putative kinase 1 (PINK1), a mitochondrial serine/threonine‐protein kinase encoded by the Pink1 gene, protects cells from stress‐induced mitochondrial dysfunction by activating mitophagy pathway.

Objective

This study was designed to evaluate the role of PINK1 in the development of cardiomyopathy in mice fed with high fat diet (HFD).

Results

We observed that PINK1 protein levels were markedly reduced in HFD‐treated mouse heart. HFD‐treated mouse developed obvious cardiomyopathy, demonstrated by impaired diastolic function, which was evidenced by a decreased E/A‐wave ratio (E‐wave and A‐wave represent the left ventricle early filling and atrial contraction filling, respectively). Moreover, ejection fraction and fractional shortening were all decreased, indicating an impaired systolic function. The cardiac dysfunction was concurrent with increased cardiac fibrosis and apoptotic cells. Pink1−/− mouse also exhibited abnormal cardiac structure and function. Importantly, PINK1 ablation accelerated the development of diastolic cardiac dysfunction, as evidenced by a decreased E/A ratio, and exacerbated cardiomyopathy, as revealed by decreased ejection fraction, fractional shortening, E/A ratio, cardiomyocyte apoptosis and cardiac fibrosis. Autophagy‐dependent clearance of mitochondria was drastically reduced with a much more pronounced effect in Pink1−/− mouse, resulting in the accumulation of damaged mitochondria in the hearts. Thus, HFD feeding caused more severe mitochondrial injury, ROS generation, and apoptotic cell death in Pink1−/− mouse.

Conclusion

Taken together, our results suggested that PINK1‐mediated mitophagy plays an important role in the development of HFD‐induced cardiomyopathy.

Support or Funding Information

This work is supported by NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Physical Model of Mycobacterium tuberculosis MazF‐mt6 Illustrates the Catalytic Residues Needed for 23S rRNA Cleavage via a Proton‐Relay Mechanism
Authors: Dobbins Aaron , Guzman Karen , Herm Cayla , Phillips Evan , Russell KayLee
Published: June 2021

Toxin‐antitoxin systems, which are commonly found in prokaryotes, regulate cell growth in response to environmental stressors. When the labile antitoxin is degraded, the toxin is released to exert its inhibitory effects. The Mycobacterium tuberculosis toxin, MazF‐mt6, is a small globular protein composed of two β‐sheets and three α‐helices. This particular protein regulates growth by cleaving the 23S rRNA at helix 70 on the large 50S ribosomal subunit, thus inhibiting translation. The 3D structure of MazF‐mt6 was recently solved by the lab of Dr. Christine Dunham. Their work demonstrated that Asp‐10, Arg‐13, and Thr‐36 are critical residues needed for cleavage within the sequence 5′ UU↓CCU 3′ located at positions 1939 to 1943. These catalytic residues perform endoribonuclease cleavage through a proton‐relay mechanism, where threonine stabilizes the transition state. Lending support to the location of the active site, a negatively charged sulfate ion, which can mimic the negatively charged phosphate backbone of RNA, is found bound adjacent to the residues that appear to participate in the cleavage reaction. We have designed a physical model to illustrate the 3 catalytic residues of the active site, as well as the bound sulfate ion. As is true of any model, physical and computer models provide a means of visualizing protein structure that is useful both as an aid to hypothesis generation and as an instructional tool.

Support or Funding Information

Campbell University Department of Biological SciencesCBM: NSF‐IUSE #1725940

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and functional consequences of SMCHD1 mutations associated with arhinia and muscular dystrophy
Authors: Kim Susan Ok , Pedersen Lars , Inoue Kaoru , Perera Lalith , Shaw Natalie
Published: June 2021

SMCHD1 (Structural Maintenance of Chromosomes Flexible Hinge Domain Containing‐1) encodes an epigenetic repressor that silences a subset of X‐linked and autosomal genes. SMCHD1 contains a conserved N‐terminal GHKL‐type ATPase domain and a C‐terminal SMC‐hinge domain important for dimerization and chromatin association. Heterozygous missense mutations in SMCHD1 cause two extremely rare disorders: Bosma arhinia microphthalmia syndrome (BAMS), which is characterized by arhinia, the absence of an external nose, as well as ocular and reproductive defects, and facioscapulohumeral muscular dystrophy type 2 (FSHD2), a late onset neuromuscular disorder. Patients with both BAMS and FSHD2 show hypomethylation at SMCHD1 binding sites (e.g., the 4q35 D4Z4 macrosatellite repeat array), but it is unknown how point mutations in SMCHD1 perturb protein structure and function at the molecular level and ultimately result in these diverse disease phenotypes.

To begin to address this question, we solved the crystal structure of a dimeric human N‐terminal SMCHD1 construct. We discovered two novel functional domains and observed ATP‐dependent dimerization in the absence of the SMC‐hinge domain. BAMS and FSHD2 missense mutations mapped either to the protein surface, dimer interfaces, domain interfaces, or to hydrophobic pockets with complete sparing of the enzyme active site. As previously observed in studies that used the monomeric form of SMCHD1, we found that mutations in the dimeric form had variable and subtle effects on SMCHD1 ATPase activity and ATP binding affinity. Thermal stability and dimerization were generally preserved in BAMS mutant constructs, as determined by differential scanning fluorimetry and chemical cross‐linking.

In summary, we find that human disease mutations map to multiple functional domains of SMCHD1 yet have only modest effects on the two most well‐described activities of this protein: ATP hydrolysis and dimerization. Work is ongoing to determine the effect of disease mutations on chromatin association, SMCHD1 interactome, and the dynamics underlying monomer‐dimer equilibrium.

Support or Funding Information

This work was supported by the Intramural Research Program of the NIH/NIEHS and equipment grants from the NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of OAT2 as Endoplasmic Reticulum Membrane Transporters in Bumetanide Glucuronidation
Authors: Arakawa Hiroshi , Araya Karin , Masuo Yusuke , Wakayama Tomohiko , Kato Yukio
Published: June 2021

The glucuronidation reaction, which is catalyzed by the UDP‐glucuronosyltransferase (UGT) superfamily of enzymes, plays an important role in metabolism of numerous endogenous and exogenous compounds. Since the active site of UGT resides in the lumen of the endoplasmic reticulum (ER), permeation of substrates across ER membranes is necessary for its glucuronidation. The aim of the present study is to clarify the involvement of ER transporters in drug transport across the ER membranes and investigate the role in drug glucuronidation. Time‐dependent increase of bumetanide glucuronide formed was observed up to 30 min in mouse primary cultured hepatocytes. Glucuronidation of bumetanide was inhibited by anionic inhibitor nateglinide with IC50 value of 45.4 μM, while its glucuronidation in mouse liver microsomes was not inhibited. Moreover, uptake of bumetanide was inhibited by nateglinide with IC50 value of 365 μM. These results suggest active ER transport of bumetanide. Moreover, knockdown of OAT2 in HepaRG cells reduced bumetanide glucuronide without inhibition of bumetanide uptake into the cells. Immunohistochemical study revealed that mOat2 was localized in intracellular compartment in mouse liver, and that myc‐tagged OAT2 was localized in the ER in HEK293 cells. In the western blotting, a signal for mOat2 was abundantly detected in mouse liver microsome fraction. In conclusion, transporters inhibited by nateglinide are likely to participate in bumetanide glucuronidation after uptake process of bumetanide across plasma membranes in mouse primary cultured hepatocytes and HepaRG cells. OAT2 localized in ER may be involved in drug transport across the ER membranes and glucuronidation of various UGT substrates.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Capillary Lymphatic Venous Malformations are caused by Endothelial‐Specific Gain‐of‐Function Mutations in the PIK3CA Gene
Authors: Lakes Nora , Goines Jillian , Pastura Patricia , Le Cras Timothy , Boscolo Elisa
Published: June 2021

Capillary lymphatic venous malformations (CLVM) are complex congenital vascular malformations characterized by aberrant vessels and soft tissue overgrowth (figure 1). CLVM are first detected in utero during late embryonic development and enlarge with time. Previous studies show that somatic, mosaic mutations in the 110‐kD catalytic □‐subunit of phosphoinositide‐3 kinase (PIK3CA) are present in CLVM lesions. These hotspot mutations are known to promote cell proliferation and angiogenesis in cancer. Since targeted molecular therapies for CLVM are lacking, patients require repeated sessions of sclerotherapy and surgical debulking over their lifetime. Therefore, there is a significant need to understand the pathogenesis of CLVM in order to develop therapies that target etiology rather than manage symptoms.

This study uses patient‐derived cell lines from affected tissue to accomplish two aims: 1) Characterize disease pathogenesis by studying proliferation and signaling in the cell type expressing PIK3CA mutations and 2) Generate a patient‐derived xenograft model which recapitulates the histology of patient lesions.

We isolated cells from the resected CLVM tissue of nine patients and cultured them in endothelial growth medium. Cells were separated into CD31+ (endothelial) and CD31‐ (non‐endothelial) populations via immunomagnetic bead sorting. By Sanger sequencing for PIK3CA hotspot mutations, we found that mutations are localized to endothelial cells (EC) in 8/9 patient lesions. This novel finding suggests that PIK3CA‐mutated EC drive CLVM pathogenesis and therefore are the cell‐of‐interest in therapeutic studies.

To test this hypothesis, we evaluated AKT, a downstream effector of PI3K, via immunoblotting. We found that CLVM‐EC exhibit constitutive and significantly elevated phosphorylation of AKT (p<0.05) compared to healthy donor EC. Additionally, proliferation studies revealed that CLVM‐EC proliferate 5‐fold faster compared to donor EC, even in the absence of growth factors (p<0.05).

Finally, we generated a xenograft murine model of CLVM by injecting patient‐derived CLVM‐EC (suspended in Matrigel□) subcutaneously into the flanks of immunocompromised mice. Healthy donor EC served as a control. Lesions were dissected at day 10, fixed, embedded in paraffin, and stained with hematoxylin and eosin. We found that patient‐derived CLVM‐EC form enlarged vascular and lymphatic channels in the xenograft model, and xenograft lesions resemble the histology of patient lesions.

This pre‐clinical xenograft model will be important in identifying FDA‐approved inhibitors that selectively attenuate CLVM growth and may be advanced to clinical trial.

Support or Funding Information

NIH‐R01HL117952, Ohio Physiological Society

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A mathematical model for the assembly of ion channel clusters in cardiac and smooth muscle cells
Authors: Matsumoto Collin , Sato Daisuke , Hernández‐Hernández Gonzalo , Moreno Claudia M. , Dixon Rose E. , Navedo Manuel F. , Binder Marc D. , Clancy Colleen E. , Santana Luis Fernando
Published: June 2021

Various membrane ion channels have been shown to cluster in excitable cells. However, the mechanism of spatiotemporal organization of cluster complexes is unknown. We used super resolution microscopy to study the time course of membrane expression of CaV1.2 and TRPV4 channels in transfected tsA‐201 cells. Channel clusters vary in size but can be fit exponentially at all time points. Similarly, mean cluster size and cluster density increase rapidly, but eventually reach a plateau around 24 hours post transfection. Using our findings, we devised a model for stochastic cluster assembly based on probabilities of three factors: cluster nucleation, growth and removal. This model reproduced distributions from previous studies of various channels in different tissues and suggests that a feedback process is responsible for determining a setpoint of cluster density and size.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Tail Region of Pre‐messenger RNA Spliceosomal Protein Dib1
Authors: Lee Grace , Meeks Garrison Armbruster
Published: June 2021

Pre‐messenger RNA splicing is carried out by a complex and dynamic molecular machine called the spliceosome. Assembly of the spliceosome is a dynamic, complex process in which there are several RNA‐RNA and RNA‐protein rearrangements. As part of this process, Dib1 is an essential protein involved in the assembly of the spliceosome on a pre‐messenger RNA. In yeast, Dib1 is a small, 17 kDa protein that has a thioredoxin‐like fold. Previous studies have found that Dim1, the human homolog of Dib1, possesses protease activity and has the ability to cleave its own C‐terminal tail region. However, how this proteolytic cleavage works as well as the role of this proteolytic activity in splicing is unknown. The purpose of this project is to biochemically characterize the in vitro auto‐cleavage mechanism in order to further understand the unidentified mechanism. Additionally, we aim to determine if truncations of the tail region of Dib1 impact pre‐messenger RNA splicing. The effects of the truncations will be assessed in Saccharomyces cerevisiae. Plasmids were constructed to express Dib1 mutants in yeast to conduct growth assays as well as in E. coli to purify mutant proteins. The effects of mutations on protein stability will be analyzed by circular dichroism and then assessed in auto‐cleavage studies. Our preliminary studies have found that truncations of the Dib1 tail have the most significant effect when the entirety of the tail that is removed in auto‐cleavage is deleted.

Support or Funding Information

San Antonio Area Foundation NIH R15GM120720

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Rps2 in Pre‐Initiation Complex Stability Using an In Vitro Assay for mRNA Recruitment
Authors: DiNardo Acadia L , Aitken Colin Echeverría
Published: June 2021

The translation of mRNA into protein is highly regulated, with misregulation often observed in viral infections, cancer, neurodegenerative disease, and other maladies. Translation initiation is the most regulated step of this process, and requires the participation of at least twelve protein initiation factors in eukaryotic cells. Eukaryotic translation initiation factor 3 (eIF3) plays a central role in recruitment of mRNA to the pre‐initiation complex (PIC) and is known to interact with the PIC near the mRNA entry and exit channels of the ribosome. Structural analysis has shown that eIF3 binds to the solvent face of the small ribosomal subunit (40S) and projects arms into each of these channels, surrounding the PIC. Previous research has analyzed the effects of mutations to the eIF3 complex on discrete interactions and events during initiation utilizing an in vitro reconstituted system. These studies revealed that mutations to eIF3 disrupt PIC interactions and affect the rate and efficiency of mRNA recruitment. mRNA recruitment experiments with model mRNAs that control the number of nucleotides present in both the entry and exit channels of the PIC demonstrate that eIF3 plays a critical role in the stabilization of mRNA in the exit channel. These experiments also suggest that eIF3 may contribute to the mechanistic events underlying the rate of recruitment at the entry channel. Consistent with previous work, this contribution may involve collaboration with the 40S latch elements Rps2/3. Indeed, mutations to Rps3 destabilize mRNA bound by the PIC in a manner that exacerbates its dependence on eIF3 for this interaction. We are now interrogating the effect of mutations to Rps2 within a similar in vitro reconstituted system using five distinct model mRNAs. Previous work has identified Rps2 mutants that may affect conformational changes of the PIC in response to mRNA binding and start‐codon recognition, and thereby destabilize its binding to mRNA. We are now investigating the effects of these mutations on mRNA recruitment. We will also further interrogate the interaction between eIF3 and the 40S latch by asking whether mutations to Rps2 or Rps3 are sensitive to mutations within elements of eIF3 thought to interact the latch.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designing a Stapled Peptide Inhibitor of CHP‐NHE1 Protein‐Protein Interactions
Authors: Bell Isaac , Gufstason Jeffrey , Provost Joseph
Published: June 2021

The Sodium Hydrogen Exchanger (NHE1) is a membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. NHE1 plays a critical role interacting with, directing, and regulating proteins that drive cell motility. We have shown that NHE1 plays a pivotal role in pH homeostasis and migration of lung tumor cells. The Calcineurin homologous B protein isoforms 1 and 2 (CHP1/2) interact with NHE1 at the same 40aa domain (CHP binding domain (CBD)) both impacting NHE1 function. CHP1 is ubiquitously expressed and its interaction with NHE promotes basal activity. While CHP2 expression is limited to the gut in non‐diseased tissue, it is highly expressed in lung and other tumor cells. When CHP2 or NHE1 expression is diminished, tumor cell growth is nearly abrogated. This supports that CHP2‐NHE1 interactions have potential as an anticancer target. Models of both CHP proteins bound to NHE1 show each bind in the CBD of NHE but with subtle structural differences. To analyze the potential differences between CHP‐NHE binding, recombinant proteins were generated and used to determine the thermodynamics of CHP/NHE interaction through CD thermal melts. Recombinant CHP2 bound to NHE1 exhibited a lower free energy of binding (−1.08 kJ mol−1) compared to CHP1 (−0.48 kJ mol−1), a ΔΔG of 0.60 kJ mol−1. Supporting this are in‐vitro ligand binding assays where we show a 2.23 fold greater affinity of CHP2‐NHE than CHP1‐NHE1. In order to create an effective peptide inhibitor, Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) computations were carried out using know crystal structures of each CHP isoform bound to NHE1. From these computations CHP1 was determined to have a two‐state binding interaction, whereas CHP2 had a one‐state binding interaction with NHE1. Unique initial binding contacts of NHE1 were determined for each CHP isoform. Stapled peptides mimicking the CHP‐NHE1 binding region were synthesized via a sulfenylation reaction with tryptophan amino acids. Peptides were then screened using a competitive fluorescent plate binding assay in which equal amounts of tagged CHP1/2 in the presence of NHE1. These peptide inhibitors will allow for increased CHP1‐NHE1 activity in targeted (cancer) cells while simultaneously reducing CHP2‐NHE1 activity.

Support or Funding Information

Supported in part by NSF Che 1664565

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitoylation‐Dependent Regulation of RhoGTPase Signaling and Cardiac Pathophysiology
Authors: Brody Matthew J , Vagnozzi Ronald J , Molkentin Jeffery D
Published: June 2021

S‐palmitoylation is a reversible lipid modification that regulates protein trafficking, localization, and function by providing a lipid tag that serves as a molecular instruction for association with specific cellular membranes. S‐palmitoylation is catalyzed by the zinc finger Asp‐His‐His‐Cys (zDHHC) family of S‐acyl transferases, however the role of these enzymes in the regulation of signal transduction in cardiomyocytes and its effects on cardiac pathophysiology are not well understood. To identify zDHHC enzymes with important regulatory functions in the heart, we used AAV9 to screen for the effects of overexpression of different zDHHC enzymes in the heart. Overexpression of the Golgi‐localized enzyme zDHHC3 resulted in severe cardiomyopathy, an effect that was not observed with overexpression of plasma membrane‐localized zDHHC5, endoplasmic reticulum‐localized zDHHC6, or Golgi‐localized zDHHC13. These data suggest important signaling mechanisms regulated by zDHHC3‐mediated palmitoylation at the cardiomyocyte Golgi apparatus. We next generated mice with cardiomyocyte‐specific overexpression of zDHHC3 to further investigate signaling mediated by this enzyme in the heart. Overexpression of zDHHC3 resulted in cardiomyopathy and congestive heart failure that was preceded by an unprecedented induction of all Rho family small GTPases, including RhoA, Rac1, Cdc42, and RhoGDI. Importantly, transgenic mice overexpressing an enzymatically‐dead mutant of zDHHC3 did not induce RhoGTPase signaling or develop cardiac disease. Quantitative palmitoyl proteomics in zDHHC3 overexpressing cells identified Rac1 as a novel substrate of zDHHC3. Rac1 palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts, implicating zDHHC3‐mediated palmitoylation of Rac1 as an important signaling circuit in cardiomyocytes. Taken together, these data indicate that zDHHC3 functions as a novel effector of Rho family of small GTPases and suggest that inhibition of zDHHC3 acyl transferase activity may represent a novel therapeutic strategy to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

Support or Funding Information

This work was supported by NHLBI K99 HL136695 (MJB) and funding from the NIH and Howard Hughes Medical Institute (JDM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating Mammalian Anabolic Three‐Carbon Metabolism by Liquid Chromatography‐High Resolution Mass Spectrometry
Authors: Doan Mary T , Jiang Helen , Snyder Nathaniel W
Published: June 2021

The metabolism of propionate, a 3‐carbon short chain fatty acid to 2‐methyl‐2‐pentenoic acid (2M2PE), a 6‐carbon intermediate, occurs in humans via propionyl‐Coenzyme A. Anabolic metabolism of propionate in humans is unexplored and the enzymology, control, and further metabolism of this pathway remains unclear. To elucidate and quantify this pathway, we employed both targeted and untargeted liquid chromatography‐mass spectrometry in combination with stable isotope labeling. Kinetic and dose response studies in human hepatocellular carcinoma HepG2 cells indicated that 2‐methyl‐2‐pentenoic acid was generated in a propionate‐dependent manner and underwent further metabolism over time. Fibroblasts cells from apparently healthy controls, as well as propionic acidemia and methylmalonic acidemia patients demonstrated similar results confirming this pathway is not dependent on entrance of carbon into the Krebs cycle. In these experiments, surprisingly, simultaneous untargeted metabolomics did not detect any propionate derived metabolites larger than 2M2PE. Rotenone, a complex I inhibitor, increased the production of 2M2PE suggesting that this three‐carbon pathway likely functions opposing anaplerosis. However, co‐incubation with glutamine reduced 2M2PE‐CoA, suggesting other metabolic influences on this pathway. To extend this work to study human metabolism, we also found that 2M2PE formed in human myocardial tissue incubated with propionate.

Support or Funding Information

NIH grants R03HD092630 and K22ES026235

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