bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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

RationaleMitochondrial 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.ObjectiveThis study was designed to evaluate the role of PINK1 in the development of cardiomyopathy in mice fed with high fat diet (HFD).ResultsWe 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.ConclusionTaken together, our results suggested that PINK1‐mediated mitophagy plays an important role in the development of HFD‐induced cardiomyopathy.Support or Funding InformationThis 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 InformationCampbell University Department of Biological SciencesCBM: NSF‐IUSE #1725940This 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 InformationThis 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 InformationNIH‐R01HL117952, Ohio Physiological SocietyThis 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 InformationSan Antonio Area Foundation NIH R15GM120720This 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.