bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the impact of splicing protein Dib1 on the pre‐messenger RNA interactions with the spliceosome
Authors: Potts Camille , Goldstein Rachel , Pittner Nicholas , Widom Julia , Duran Elizabeth , Walter Nils , Maeder Corina
Published: June 2021

In eukaryotic organisms, pre‐messenger RNA splicing facilitates the removal of non‐coding regions of RNA to create a functional mRNA. Pre‐mRNA splicing is catalyzed by the spliceosome, a large, dynamic, molecular machine composed of five small nuclear ribonucleoproteins (snRNPs). Dib1, a key protein of the spliceosome, resides in the U4/U6.U5 triple‐snRNP. Dib1 is essential for cell viability and is conserved from yeast to humans. Dib1 must depart the splicing complex before the catalytic steps of splicing can occur; however, how Dib1's departure is facilitated is unknown. Dib1 and pre‐mRNA are in close proximity in the pre‐catalytic spliceosome (Plaschka et al. Nature 2017); characterizing the interactions between Dib1 and pre‐mRNA may be necessary to understand Dib1's departure. We are characterizing potential interactions between Dib1 and pre‐mRNA using single molecule fluorescence colocalization experiments in collaboration with Dr. Nils Walter at the University of Michigan. An open question is how and whether Dib1 and pre‐mRNA are co‐localized in the splicing machinery. We are addressing this question using Cy5‐labeled Dib1 and Cy3‐labeled pre‐mRNA. We have labeled purified Dib1 with a Cy5 fluorophore and have been able to splice Cy3‐labeled pre‐mRNA. Progress and results of the single molecule fluorescence colocalization between Dib1 and pre‐mRNA will be discussed. Another area of interest is how temperature‐sensitive mutant Dib1, which causes a loss of splicing function in Saccharomyces cerevisiae, may affect the pre‐mRNA throughout the splicing cycle. To this end, we are characterizing pre‐mRNA conformation in S. cerevisiae extracts containing wild type or mutant Dib1 protein. We are using single molecule fluorescence resonance energy transfer (smFRET) to analyze the conformation of pre‐mRNA labeled with a Cy3 fluorophore at the 5′ splice site and a Cy5 fluorophore at the branch point. Comparison of the smFRET analysis will show whether mutant Dib1 causing loss of function is associated with a change of RNA conformation. Progress and results of the smFRET analysis of pre‐mRNA will be discussed.

Support or Funding Information

NIH R15GM120720; Robert A Welch Foundation Grant W‐1905

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding to the ribosome by eIF4B drives yeast translational control in response to membrane stressors
Authors: Walker Sarah E , Liu Xiaozhuo E , Moshiri Houtan E
Published: June 2021

The eIF4 group of eukaryotic translation initiation factors promotes mRNA binding to the ribosome. Increased levels or activities of these factors have been associated with a number of human diseases, highlighting the need for additional mechanistic information for these proteins. Our previous work indicated that ribosome binding by the N‐terminal domain of yeast eIF4B is critical for robust initiation in vitro and in vivo. In contrast, RNA‐binding activities by the RRM domain of eIF4B were dispensable. Our work left open the possibility that the RRM of eIF4B could allow for enhanced translation under stress conditions. Here we have compared the effects of disrupting RNA and ribosome binding of yeast eIF4B under ~1400 different growth conditions. The RRM was dispensable for vegetative growth in all conditions tested, but we found that ribosome binding promotes growth in response to a number of stressors through changes in translation. In particular, the NTD confers a strong growth advantage in the presence of the denaturing reagent, Urea and a number of other osmolytes that require robust cellular integrity for survival. Ribosome profiling of cells with and without the ribosome‐binding NTD of eIF4B reveals gross changes in translation of mRNAs containing longer than average and highly structured 5‐prime untranslated regions in response to Urea stress. These changes are dependent on the ribosome‐binding activity of eIF4B, which suggests eIF4B regulates mRNA recruitment as a part of the ribosomal preinitiation complex rather than by activating isolated mRNPs prior to ribosome binding. This analysis indicates the cellular response to urea in yeast includes a translational component, driven by increased translation of proteins associated with the cellular periphery. The results of these studies highlight the importance of the general translation factor eIF4B in adapting to external conditions, and allow us to tie the mechanical functions of this translation factor to specific cellular phenotypes.

Support or Funding Information

This work is supported by NIH grant R00GM119173 and startup funds from SUNY at Buffalo‐College of Arts and Sciences.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monofunctionalization with dextrans endows cell‐penetrative properties to proteins
Authors: Kilgore Henry Ralph , Ressler Valerie T , Raines Ronald T
Published: June 2021

Dextrans have proven to be a widely useful class of polysaccharides, with applications ranging from antithrombotic agents to enhancers of vaccine stability. We have examined an unprecedented property of dextrans: the capacity to convey cargo into the cytosol. Mono‐functionalized dextran–flourogenic probe and dextran–protein conjugates were prepared to assess the ability of dextran to mediate cellular delivery. We show that dextran conjugates are taken up in an energy‐dependent process culminating in cytosolic access. Several cell types were examined, indicating the generality of this uptake pathway. These observations motivated the preparation of dextran–cytochrome C conjugates. Release of cytochrome C from the intermembrane space activates the intrinsic apoptosis pathway. Cytotoxicity measurements of dextran–cytochrome C conjugates thus provide a straightforward metric for cytosolic access of dextran–protein conjugates. We verify that cytochrome C mediates cell death by measuring activation of downstream caspases 3 and 7, indicating that dextran–cytochrome C conjugates promote formation of the apoptosome. These experiments document a new and exciting frontier for polysaccharide bioconjugates and support the existence of an uptake pathway for 1,6‐linked polysaccharides.

Support or Funding Information

[This work was supported by Grant R01 GM044783 (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.
Optimizing Lipid Extraction in Anabaena
Authors: Menning Hope Deann , Mazzer Paula Deann
Published: June 2021

Lipidomics provides a great snapshot technique to determine how the lipids work in a cell. We do not know a lot about the lipids of cyanobacteria. We set out to optimize an extraction protocol, to figure out the different lipids in a type of blue green algae, Anabaena. Our best extraction by mass is with a 10‐minute sonication step with a modified Bligh and Dyer technique. After extraction, analysis was conducted using an FTICR‐MS and derivatization of fatty acid methyl esters. We have developed the baseline lipidome for Anabaena as a starting point for future research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MK‐STYX Impacts Autophagy and Microtubule Dynamics
Authors: Mattei Andrew M. , Christian Patrick S. , Hinton Shantá D.
Published: June 2021

Macroautophagy (autophagy) is a critical process that degrades cytoplasmic components to maintain homeostasis and which requires microtubule interactions to function. The autophagosomes (double membrane vesicles) envelop substrates and then are transported along microtubules through motor proteins to fuse with degradative lysosomes. The formation of the autolysosome allows the breakdown and degradation of various cellular targets such as stress granules (SG; stalled mRNA). We reported that the pseudophosphatase MK‐STYX [MAPK (mitogen‐activated protein kinase) phosphoserine/threonine/tyrosine‐binding protein] inhibits SG. Since autophagy and MK‐STYX each negatively affect stress granule assembly, we sought to determine whether MK‐STYX has a role in autophagy and microtubule dynamics. Our studies show that MK‐STYX causes cytosolic TFEB (Transcription factor E‐Box; the autophagy “master switch”) to become nuclear and form peripheral nuclear aggregates independently of nutrient status. Whereas, MK‐STYXactive (active mutant in which catalytic activity has been “restored”), only increased TFEB cytosolic aggregates in serum starved cells. TFEB also localizes to the lysosomal surface, acting as negative regulator of lysosomal and autophagosomal biogenesis. MK‐STYX alters lysosomal and autophagosomal dynamics; we observed oversized lysosomes in the presence of MK‐STYX. Furthermore, autophagosomes localized to the distal ends of HEK‐293 cellular extensions in the presence of MK‐STYX. In addition, MK‐STYX increases the phosphorylation of the essential autophagosome constituent LC3 (microtubule‐associated proteins 1A/1B light chain 3B). To determine MK‐STYX's effects on microtubule function, we over‐expressed MK‐STYX in HEK‐293 cells in the presence or absence of nocodazole, which disrupts microtubules. MK‐STYX did not affect microtubules dynamics directly, suggesting that further analysis of the motor proteins involved in stress granule regulation and/or autophagy is needed. Investigating MK‐STYX as a player in autophagic mechanisms is an intriguing scientific trajectory. Autophagy plays a major role in prominent cellular processes ranging from initial developmental stages to the onset of progressive human pathologies such as neurodegenerative diseases and cancer. These studies suggest that MK‐STYX may have an important role in autophagy, illuminating the importance of pseudoenzymes in regulating critical cellular pathways.

Support or Funding Information

Support for this research was provided by two Howard Hughes Medical Institute Research Fellowships and a National Science Foundation 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.
Live‐Cell Fluorescent Visualization of T3SS Needle and Its Dynamics
Authors: Cheng Dorothy , Wimmi Stephan , Diepold Andreas
Published: June 2021

The Type III Secretion System (T3SS) is a complex transmembrane apparatus used by many Gram‐negative pathogens for protein translocation and host invasion. An essential T3SS component is the needle, a hollow substructure extending from the bacterial membrane, spanning the space from the bacterium to the host cytosol. The needle is formed by helical polymerization of a single small protein, which is secreted by the T3SS and assembled outside the bacterium. Visualization of the needle previously depended on immunofluorescence, which requires cell fixation. We hereby describe a simple methodology for T3SS needle fluorescent tagging and visualization, which enables live‐cell microscopy and investigation of needle kinetics and dynamics.

Support or Funding Information

This study was supported in part by the DAAD RISE Scholarship, Reed College Biology Undergraduate Student Travel Award, Reed College Summer Opportunity Fellowship, Reed College Undergraduate Research Opportunity Grant and Galakatos funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skin Lipid Abnormalities in Patients with a History of Eczema Herpeticum Indicate the Involvement of the Sphingosine‐1‐Phosphate Signaling System in HSV Viral Replication
Authors: Berdyshev Evgeny , Goleva Elena , Bronova Irina , Jung John , Bronoff Anna S. , Norquest Kathryn , Richers Brittany , Leung Donald YM
Published: June 2021

Rationale

Atopic dermatitis (AD) with eczema herpeticum (EH) can result in systemic infections and can be life‐threatening. The purpose of this study was to identify differences in non‐lesional skin between AD with a history of recurrent EH (ADEH+), AD without EH (ADEH−), and non‐atopic (NA) controls to gain insight into mechanisms involved in EH pathogenesis using a novel minimally invasive skin tape strip (STS) methodology and mass spectrometric approaches.

Methods

STS layers 5–6 from 13 NA, 13 ADEH−, and 15 ADEH+ adult subjects were collected from non‐lesional skin. AD subjects were balanced by gender, age and disease severity; STS were subjected to lipidomic and filaggrin breakdown products analyses by liquid chromatography tandem mass spectrometry (LC‐MS/MS). For the in vitro experiments, differentiated primary human keratinocytes were transfected with control or sphingosine‐1‐phosphate (S1P) lyase (SGPL), sphingosine kinase‐1 (SPHK1), or sphingosine kinase‐2 (SPHK2) siRNA for 48h and infected with HSV‐1 (0.1 MOI). The effect of enzyme silencing on sphingolipidome was evaluated via the LC‐MS/MS. Viral replication was assessed in 24h with qPCR and plaque assay.

Results

ADEH+, as compared to ADEH− and NA, subjects had a significantly increased proportion of short‐chain over long‐chain NS‐ceramides (sphingosine N‐acylated with non‐hydroxy fatty acids) (p=0.002), and increased levels of sphingomyelins (p=0.006). In contrast to ADEH−, ADEH+ subjects demonstrated increased level of free C18‐ and C17‐sphingosines (p=0.007 and 0.052, correspondingly) that suggests upregulated skin sphingosine‐1‐phosphate (S1P) metabolism and signaling. A comparison of the effect of silencing of major enzymes that directly regulate S1P levels (SPHK1, SPHK2, SGPL) in primary human keratinocytes in vitro revealed that only the silencing of SGPL resulted in increased viral replication after keratinocyte inoculation with HSV‐1 (Mean±SE, 0.55±0.09 vs. 0.25±0.05 for S1PL vs. control siRNA transfected keratinocytes, p<0.01, as detected by qPCR).

Conclusions

STS from ADEH+ subjects and in vitro studies indicate involvement of the S1P signaling system in EH and suggest novel potential therapeutic targets to treat EH.

Support or Funding Information

This study was funded by the Atopic Dermatitis Research Network supported by NIH/NIAID grant U19 AI117673.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PCID2 influences BRCA1/BARD1 Localization and Centrosome Duplication through its functions in Nuclear Protein and mRNA Export
Authors: Resendes Karen K , Arrigo Abigail , Arrigo Alexis
Published: June 2021

Nuclear protein export has an emerging role in the regulation of centrosome duplication, a function that involves interaction of centrosomal proteins with the Crm1 nuclear export factor. The protein PCID2, while originally identified as an mRNA export factor, is also known to function in Crm1 mediated protein export, with PCID2 siRNA knockdown slowing the rate of export. In addition, PCID2 has been localized to the centrosome. Despite the fact that PCID2 is poised to be a cell cycle regulator through potential combination of it's centrosomal and nuclear export roles, the function of PCID2 at these locations has not been well characterized. We sought to elucidate a potential link between these roles of by exploring PCID2's involvement in the export of particular cargo known to transit between these the nucleus and centrosome. Our studies focus on BRCA1 and BARD1 as potential PCID2 nuclear to centrosomal cargo, as these DNA damage repair proteins transit from the nucleus to the centrosome in a Crm1 dependent manner, particularly in times of cellular stress. We found that knockdown of PCID2 caused a 10% increase in the nuclear retention of BRCA1, with a concomitant 40‐fold decrease in centrosomes exhibiting BRCA1 localization. These results suggest a role for PCID2 in the export of the BRCA1 protein from the nucleus and in it's subsequent localization to the centrosome. Conversely, BARD1 nuclear levels decreased by nearly 20% in the absence of PCID2, which was accompanied by a 40‐fold decrease in BARD1 positive centrosomes. These effects appear to stem from a 50% drop in BARD1 protein levels in PCID2 knockdown cells, suggesting a potential role for PCID2 in the export of BARD1 mRNA rather than protein, with failure to export contributing to loss in protein expression. Additionally, we discovered that altered transport of BRCA1/BARD1 due to PCID2 knockdown had an unexpected effect on centrosome duplication. Rather than the increase in centrosome number seen in the absence of the BRCA1/BARD1 interacting protein OLA1, PCID2 knockdown led to a four‐fold reduction in cells with an aberrant number of centrosomes (3 or more) as visualized by yTubulin staining. This variation is potentially due to the use of non‐breast cancer cells in this work. Our ongoing studies seek to identify CRM1‐PCID2‐BRCA1 export complexes and determining the effect of PCID2 knockdown on the CRM1‐BRCA1 interaction. In addition, we are investigating the role of PCID2 in BARD1 mRNA export. Determining how PCID2 influences the cellular localization of the BRCA1/BARD1 complex will help to elucidate the mechanism of PCID2's effect on centrosome duplication and therefore determine it's potential impacts on cell cycle regulation and tumor development.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Common Non‐East Asian Aldehyde Dehydrogenase 2 Deficiencies; Potential Drug Target for Alzheimer's Disease and Mitochondrial Dysfunction
Authors: Li Sin‐Jin , Stevens Matthew C. , Chen Che‐Hong , Joshi Amit U. , Mochly‐Rosen Daria
Published: June 2021

Aldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation and detoxification of aliphatic aldehydes, including acetaldehyde. About 40% of East Asians carry a single point mutation in ALDH2*2 (E504K) that contributes to a severe enzyme deficiency and accumulation of toxic reactive aldehydes. We previously identified Alda‐1 (an aldehyde dehydrogenase activator) that reduces aldehydic load by increasing the catalytic activity and stability of wild type enzyme and by correcting the structural defect in ALDH2*2, in vitro and in vivo. Using the ExAC genomic database, we identified and characterized new ALDH2 variants in several non‐East Asian populations. Nine other ALDH2 variants with a single point mutation were identified among Latino, African, and Finnish ethnic groups at a frequency of >1/1000. We focused on three variants, two with a reported frequency of 2.5% and one with a reported frequency of 1.2%. All three newly‐identified ALDH2 mutants exhibited a significant reduction in activity as compared with wild type enzyme (ALDH2*1/*1; WT) but differed in their sensitivity to Alda‐1‐induced activation as determined in vitro and in 3T3 cells overexpressing each of the variants. Acetaldehyde‐induced toxicity (mitochondrial dysfunction and cell death) was also higher in these cells relative to 3T3 cells overexpressing WT ALDH2. Recent evidence suggests a correlation between increased aldehydic load and Alzheimer's disease. In line with this evidence, there was a greater mitochondrial dysfunction and cell toxicity induced by oligomeric amyloid beta1‐42 (oAb1‐42) in 3T3 cells expressing inactive variants of ALDH2 relative to WT enzyme. These defects were reduced by Alda‐1 co‐treatment. Ethanol, which is consumed by ~80% of the world's population, increases aldehydic load. Using ALDH2*2 knockin mice, we found that chronic exposure to ethanol (1g/kg/day) for 11 weeks increased oxidative stress, apoptosis, and Ab1‐42 accumulation in their brains relative to WT mice. Co‐treatment with Alda‐1 (10mg/kg/day) significantly reduced these toxic effects in both WT and ALDH2*2 mice. Finally, treatment of Alzheimer's disease patient‐derived fibroblasts with 0.1% ethanol resulted in a higher mitochondrial dysfunction and cytotoxicity relative to fibroblasts of healthy subjects. These effects were reduced by co‐treatment with Alda‐1, even in cells overexpressing ALDH2*2.

In conclusion, ALDH2 deficiencies are much more common than previously thought, possibly affecting >10% of people worldwide. As aldehydic load contributes to many chronic diseases, including Alzheimer's disease, drugs that activate ALDH2 and correct the functional deficiency may provide benefit to those at risk.

Support or Funding Information

Supported by NIAAA11147 R37 Merit award to DM‐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.
Redox Deficient Cysteine Residues Impair β2‐Adrenergic Receptor Function
Authors: Rambacher Kalyn Marie , Moniri Nader H.
Published: June 2021

Inhaled β2‐adrenergic receptor (β2AR) agonists are clinically used to elicit rapid bronchial dilation, primarily for the treatment of asthma attacks. Asthmatic tissue is characterized by high levels of reactive oxygen species (ROS), over‐expression of the ROS generating enzyme NADPH oxidase, and reduced expression of the ROS metabolizing enzyme superoxide dismutase 2. However, most pharmacological data on the β2AR has been derived from receptor evaluation in a non‐oxidative environment, which fails to address the potential impact ROS may have on the β2AR itself. Cysteine residues of the β2AR undergo transient first order oxidation, a functionally significant post translational modification known as cysteine‐S‐sulfenation. Despite the vast literature explaining the physiology of β2AR and the known interplay between β2AR and ROS, the actual impact of ROS on β2AR has thus far been overlooked. HEK293 cells overexpressing β2AR and CALU3 endogenously expressing β2AR were utilized in these studies. Redox dependent β2AR alterations in ligand binding were evaluated with membrane and whole cell preparations by utilizing tritiated alprenolol. Functional G protein signaling was examined with the use of a cAMP. Functional G protein independent signaling was looked at via confocal microscopy and receptor internalization. Receptor oxidation increases ligand binding, an effect reversed in redox deficient receptors. Redox deficient receptors have a diminished G protein signaling cascade, reduced agonist mediated β‐arrestin recruitment and a blunted internalization response. This research is the first to address β2AR function based on oxidation state, which implicates altered physiology dependent upon redox state. Given asthmatic tissue has elevated ROS levels and β2AR agonists are clinically utilized for the treatment of asthmatic attacks, our findings provide evidence that β2AR function in an oxidizing environment may be different from the physiology described in current literature. Furthermore, this research highlights the effects of a novel post translation modification on one of the most well understood GPCRs, further indicating we may still have much to learn about β2AR function and physiology.

Support or Funding Information

This work was funded by the NIH/NHLBI grant HL138603 to NHM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Profiling Gαq Cancer Mutations Using a Novel BRET‐based Biosensor
Authors: Maziarz Marcin , Leyme Anthony , Patel Prachi P. , Garcia‐Marcos Mikel
Published: June 2021

Heterotrimeric G proteins are critical signal transducers and dysregulation of their activity causes many diseases, including cancer. For instance, activating mutations in the Gα subunit Gαq are frequently found in uveal and meningeal melanomas. A current challenge in the field of cancer in general and oncogenic G protein signaling in particular is that the rate of discovery of cancer‐associated mutations clearly outpaces our ability to analyze their functional consequences. This limits our ability to discern driver mutations from passenger mutations, and to elucidate possible mechanisms by which they favor cancer progression. Here, we set out to develop and implement a novel approach to rapidly profile cancer‐associated mutations that impact Gq‐dependent signaling. We envisioned the design of a Gq activity biosensor to detect GTP‐bound Gαq in live cells based on the principle of Bioluminescence Resonance Energy Transfer (BRET). First, we validated that our sensor specifically detects Gαq activity, and that it can faithfully report its modulation by G protein‐coupled receptors (GPCRs) and by Regulators of G protein signaling (RGS) in real time with sub‐second resolution. After benchmarking our sensor against well‐characterized Gαq oncogenic mutants, we implemented it in a battery of cancer‐associated Gαq mutants that had not been characterized before. Our findings reveal that a hotspot mutation present in >10% of hepatocellular carcinomas does not give rise to a hyperactive Gαq, whereas other infrequent mutations enhance Gαq activity. More specifically, the latter mutations are insensitive to deactivation by GTPase activating proteins (GAPs) of the RGS family, which sensitizes Gαq activation and delays inactivation upon GPCR‐mediated modulation. In conclusion, we have developed a G protein activity biosensor that directly monitors the formation of Gαq‐GTP, and showed that it is useful to rapidly profile disease‐associated G protein mutants and reveal novel mechanisms of aberrant activation.

Support or Funding Information

Funding: R01GM130120, R21MH118745

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Submicroscopic cAMP/PKA Compartmentalization: Ion flux at the Cardiomyocyte Plasmalemma
Authors: Musheshe Nshunge , Surdo Nicoletta C , Schmidt Martina , Zaccolo Manuela
Published: June 2021

In the heart cyclic AMP‐dependent Protein Kinase A (PKA) mediates the catecholaminergic control over the force and frequency of cardiac contraction via phosphorylation of proteins that are involved in excitation‐contraction coupling (ECC). As part of the ECC machinery, catecholamine dependent activation of PKA leads to phosphorylation of the L‐type Ca2+ channels (LTCC) and phospholamban (PLB), resulting in increased systolic Ca2+. PKA also phosphorylates phospholemman (PLM), a regulator of the cardiac Na+/K+ ATPase (NKA) which, by setting the Na2+ gradient for Na2+/Ca2+ exchanger, promotes Ca2+ extrusion from the cytosol. How cAMP/PKA signaling coordinates these apparently opposing effects on intracellular Ca2+ remains unclear.

The aim of this study was to investigate cAMP/PKA signaling at the A Kinase Anchoring Protein 79(AKAP79)/150 which binds PKA that phosphorylates LTCC and at the PLM/NKA complex and determine whether the two sites are under distinct cAMP pools which could explain the resulting opposing effects on Ca2+ ion flux.

Fluorescence Resonance Energy Transfer (FRET)‐based sensors for cAMP (CUTie) and PKA‐dependent phosphorylation (AKAR4) targeted to PLM/NKA and AKAP79/LTCC nanodomains at the plasmalemma were used for real time monitoring of cAMP levels and PKA activity. Experiments were conducted in rat ventricular myocytes.

A heterogeneous cAMP response was observed at the two sites on β‐AR stimulation with Isoproterenol (Iso), with the cAMP increase at PLM/NKA being significantly lower than that at AKAP79/LTCC. We demonstrated that this differential local regulation of cAMP response is due to phosphodiesterases (PDEs) which degrade cAMP and contain its increase at the PLM/NKA site. We found that PDE2 and PDE8 play a major role in selectively shielding the PLM/NKA complex from cAMP generated on activation of the b‐adrenergic receptor. However, despite the difference in the amplitude of the cAMP response to catecholamines at the AKAP79/LTCC and PLM/NKA complexes, we found that the PKA‐dependent phosphorylation of local targets is similar at the two sites. This apparent discrepancy appears to depend on a more robust phosphatase activity at AKAP79 compared to PLM. Our findings show profound differences in local handling of cAMP levels and phosphatase activity at these two plasmalemma sites and reveal that adrenergic regulation of Ca2+ flux across the plasmalemma relies on submicroscopic compartmentalization of cAMP/PKA signals.

Support or Funding Information

This research was funded by the British Heart Foundation, (PG/10/75/28537 and RG/17/6/32944), the BHF Centre of Research Excellence, Oxford (RE/13/1/30181), the Graduate School of Science and Engineering (GSSE), and Groningen Research Institute of Pharmacy (GRIP)

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‐kinase anchoring proteins define cAMP compartments in TGF‐β1/cigarette smoke induced lung EMT
Authors: Schmidt Martina , Zuo Haoxiao , Musheshe Nshunge , Nikolaev Viacheslav
Published: June 2021

Aims

Epithelial‐to‐mesenchymal transition (EMT) plays a critical role in chronic obstructive pulmonary disease (COPD), a disorder induced by air pollution and cigarette smoke (CS). EMT is characterized by loss of epithelial and acquisition of mesenchymal marker. The phosphodiesterase (PDE)4 inhibitor rolipram inhibits TGF‐β1 mediated EMT in A549 cells. Thus, cAMP might be worthwhile to target to attenuate EMT‐associated lung diseases. A central feature of cAMP signaling is the compartmentalization via A‐kinase anchoring proteins (AKAPs). Functional studies on the role of AKAPs and their therapeutic value to target the process of EMT in the lung are still lacking.

Methods

TGF‐β1 and cigarette smoke were used to induce EMT in BEAS‐2B and in primary epithelial cells. Expression (gene, protein) of E‐cadherin and collagen 1α1 were analyzed. Expression of Ezrin, AKAP95 and Yotiao was examined by qPCR (gene), western blotting and immunofluorescence (protein). β2‐Agonist fenoterol (0.01–10μM), PDE4 inhibitor rolipram, PDE3 inhibitor cilostamide, adenylyl cyclase activator forskolin (each 10 μM), dibutyryl‐cAMP (db‐cAMP, 10–100 μM) and 30μM st‐Ht31 were used 30 min before TGF‐β1. The TGF‐β1 sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Migration was analyzed using both a wound healing assay and xCELLigence transwell migration. CS induced release of TGF‐β1 was measured by ELISA. CS induced reduction in E‐cadherin was blocked by an TGF‐b1 neutralizing antibody. Phosphorylation of SMAD2 and SMAD3 served as control.

Results and conclusions

TGF‐β1 changed cell morphology, downregulated E‐cadherin and upregulated collagen 1α1 in BEAS‐2B cells. TGF‐β1 decreased Ezrin but increased AKAP95 and Yotiao gene expression. St‐Ht31 alone led to a strong decrease of E‐cadherin (mRNA, protein), but further augmented TGF‐β1‐induced E‐cadherin (protein) decrease and prevented collagen 1α1 (protein) upregulation. Co‐silencing of Ezrin, AKAP95, Yotiao diminished collagen upregulation, wound closure and transwell migration induced by TGF‐β1. CS exposure increased phospho‐SMAD2, TGF‐β1 release, and augmented cell migration, linking CS to TGF‐β1. Elevation of cAMP in AKAP silenced cells pointed to AKAP defined cAMP compartments.

AKAP family members Ezrin, AKAP95 and Yotiao regulate EMT by TGF‐β1, a process potentially involving CS induced TGF‐β1 release. AKAPs seem to define the ability of the β2‐agonist fenoterol, and PDE inhibitors rolipram and cilostamide to modulate the EMT process. Our data point to novel strategies in COPD treatment involving members of the AKAP superfamily.

Support or Funding Information

Ubbo Emmius Programme, GRIP, FSE, DFG and the Gertraud und Heinz‐Rose Stiftung.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Heat Therapy Increases Murine Hepatic Mitophagy
Authors: Von Schulze Alex , Deng Fengyan , McCoin Colin , Thyfault John , Geiger Paige
Published: June 2021

Hepatic steatosis impacts ~25% of the global population and contributes to the etiology of type II diabetes and cardiovascular disease. As there are currently no pharmacologic interventions available for hepatic steatosis, research has focused on elucidating novel molecular targets that can be leveraged for drug discovery. We have previously shown that in vitro heat therapy (HT) reduces hepatic lipid accumulation, however the mechanism by which this effect occurs remains unknown. The purpose of this investigation was to determine whether HT impacts hepatic steatosis by improving hepatic mitochondrial function in vivo via increased mitophagy. Male C57BL/6J mice were divided into HT and sham treatment groups (n=8/group). Mice in the HT group were heated to a core temperature of 41°C for 20min, while mice in the sham group were heated to 37°C for 20min. To measure mitophagy flux, all groups were treated with the autophagy inhibitor leupeptin (LEU, 40mg/kg) immediately following the 20min intervention and 4h prior to sacrifice. All animals were sacrificed 24h post HT. HT significantly increased heat shock protein 72 (HSP72) and mitophagy related proteins sequestosome 1 (p62) and microtubule‐associated proteins 1A/1B light chain 3B‐ phosphatidylethanolamine (LC3II) in hepatic mitochondrial isolates. HT also increased total ubiquitin in the mitochondrial isolates, but the E3 ubiquitin ligase parkin was not altered. Interestingly, we found that the E3 ubiquitin ligase, carboxy terminus of Hsp70‐interacting protein (CHIP) was significantly higher in the hepatic mitochondrial isolates post HT. Subsequent co‐immunoprecipitation assays suggest that HT causes CHIP, HSP72, and HSP25 physically interact in the mitochondrial fraction. While this CHIP‐HSP complex is only thought to facilitate the proteasomal and autophagosomal degradation of soluble cytoplasmic proteins, we believe that it can also facilitate the targeted degradation of damaged mitochondria through autophagy, or mitophagy. This mitophagy pathway may provide a novel target to maintain mitochondrial quality control under nutrient overload, potentially reducing the risk for hepatic steatosis.

Support or Funding Information

NIH P30GM122731; Madison and Lila Self Graduate Fellowship (The University of Kansas)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle‐derived exosome/miRNA‐26a attenuates skeletal muscle wasting and cardiomyopathy in chronic kidney disease (CKD) mice
Authors: Wang Bin , Liu Bi cheng
Published: June 2021

Introduction

Uremic cardiomyopathy and muscle atrophy contribute to CKD‐induced morbidity and mortality. Exosomes, natural carriers of many signal molecules including microRNA (miR), mediate organ to organ communication. We hypothesized that miR‐26 would benefit both CKD‐induced muscle wasting and cardiomyopathy through exosome‐mediated muscle‐heart crosstalk.

Methods

We used an engineered exosome vector which contains an exosomal membrane protein gene Lamp2b fused with muscle specific surface peptide for targeting delivery. Exosome encapsulated miR‐26a precursor RNA (Exo/miR26) were generated in muscle satellite cells and injected into the tibialis anterior (TA) muscle of CKD mice (5/6 subtotal nephrectomy) for 12 weeks. NanoSight instrument was used to quantify exosomes. A miR deep sequencing assay and qPCR were used to identify microRNA expression. Cardiac ultrasound was used to detect heart size and function. In‐Vivo Xtreme camera system was used to detect exosome in vivo.

Results

miR‐26a was decreased in skeletal muscle and heart of CKD mice. Uremic serum enhanced secretion of miR‐26a exosomes in cultured C2C12 skeletal and H9C2 cardiac muscle cells. The intervention of Exo/miR26a increased the expression of miR‐26a in skeletal muscle and heart, as well as increased muscle cross‐section area and decreased CKD‐induced upregulation of atrogin‐1 and MuRF1. Curiously, cardiac fibrosis lesion was partially depressed, and FoxO1, α‐SMA, connect tissue growth factor (CTGF), fibronectin and collagen1α were decreased in CKD mice with intramuscular injection of Exo/miR‐26a. Echocardiography showed that the percentage of ejection fraction was increased in CKD mice treated with Exo/miR26a. Using fluorescence dye labeled Exo/miR26a, we found that the fluorescence intensity in heart was correlated with skeletal muscle, examined by linear regression. We found that miR‐26a directly inhibits FoxO1 and CTGF, which provided mechanism for inhibition of muscle atrophy and cardiac fibrosis by Exo/miR26a.

Conclusions

Overexpression of miR‐26a in muscle prevents CKD‐induced muscle loss and attenuates cardiac fibrosis via exosome‐mediated muscle‐heart crosstalk.

Support or Funding Information

National Natural Science Foundation of China (81700618), National Key Research Program (2018YFC130046, 2018YFC1314000).

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postganglionic Parasympathetic but Not Postganglionic Sympathetic Neuron Derived Exosomes Inhibit Hyperglycemia Induced Apoptosis in H9c2 Cells
Authors: Singla Reetish Kumar , Garner Kaley , Singla Dinender K. , Cheng Zixi (Jack)
Published: June 2021

Diabetic cardiomyopathy involves the two main types of cell death: apoptosis and necrosis. It is unclear, however, on whether apoptosis induced by hyperglycemia in a cell culture model can be inhibited by postganglionic parasympathetic neuron derived exosomes (PPN‐Exos). Postganglionic parasympathetic and postganglionic sympathetic neurons were isolated, cell cultured, and exosomes were isolated. H9c2 cells were utilized to induce apoptosis via hyperglycemia, which were then divided into 4 groups: 1. Control, 2. H9c2 + Glucose, 3. H9c2 + Glucose + PPN‐Exos, 4. H9c2 + Glucose + postganglionic sympathetic neuron‐derived exosomes (PSN‐Exos). An MTT assay kit was utilized to determine cell proliferation and viability, while apoptosis was determined using TUNEL staining and cell death detection (CDD) ELISA kit. The H9c2 cells that were exposed to glucose displayed significantly (p<0.05) reduced cell viability, and a significant (p<0.05) increase in TUNEL staining and CDD ELISA was present in hyperglycemic H9c2 cells compared to controls and this reduced cell viability and increased apoptosis was reversed with PPN‐Exos but not with PSN‐Exos, suggesting a cell protecting components are present in these PPN‐Exos. Our data on pro‐apoptotic gene expression of caspase‐3 and BAX was significantly (p<0.05) increased in glucose treated H9c2 cells compared with control. These pro‐apoptotic genes were attenuated with PPN‐Exos treatment. Anti‐apoptotic protein Bcl‐2 levels were significantly (p<0.05) reduced in hyperglycemia group and this decrease in Bcl‐2 was improved following PPN‐Exos treatment. In conclusion, our data demonstrate that apoptosis is present in hyperglycemic H9c2 cells mediated through caspase‐3 and BAX pathway is inhibited with PPN‐Exos but not with PSN‐Exos. Our data also suggest that PPN‐Exos must be containing a unique set of protective factors that needs further investigation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histones Activate Toll‐Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Cells
Authors: Cui Zong Jie , Guo Hai Yan
Published: June 2021

In acute pancreatitis histones are released by infiltrating neutrophils, but how histones modulate pancreatic acinar cells has not been investigated. We have examined histone modulation of rat pancreatic acini and pancreatic acinar tumor cell AR4‐2J by calcium imaging. Histones were found to have no effect on calcium in pancreatic acini but blocked calcium oscillations induced by CCK or ACh. Both mixed (Hx) and individual (H1, H2A, H2B, H3, H4) histones induced calcium oscillations in AR4‐2J. RT‐PCR and Western blot verified expression of histone‐targeted TLR2, 4, 9. Immunocytochemistry identified TLR2/TLR4 on apical plasma membrane and TLR9 in zymogen granule regions in pancreatic acini. TLR2 was found on neighboring and TLR9 on peripheral plasma membranes but TLR4 was in nucleus in AR4‐2J clusters. Neither TLR2 agonist zymosan‐A nor TLR4 agonist lipopolysaccharide had any effect on calcium, but TLR9 agonist ODN1826 induced calcium oscillations; TLR9 antagonist ODN2088 blocked H4‐induced calcium oscillations in AR4‐2J, which also disappeared after treatment of AR4‐2J with glucocorticoid dexamethasone (Dex), with concurrent TLR9 migration from plasma membrane to cell interiors. TLR9 down regulation with siRNA completely suppressed H4‐induced calcium oscillations. These data together suggest that extracellular histones activate plasma membrane TLR9 to trigger calcium oscillations in AR4‐2J cells. TLR9 protein shuttling between plasma and endosomal membranes determines whether calcium signaling is triggered. Ongoing work will outline the signaling pathway from plasma membrane TLR9 activation to cytosolic calcium oscillations and its correlation with the canonical MyD88‐dependent pathway of TLR9 activation.

Support or Funding Information

This work was supported by The Natural Science Foundation of China (31670856).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Matrix Fibrous Proteins and Cells in Central Vein Fibrosis of Cadaveric Livers
Authors: Mak Ki M
Published: June 2021

The central vein (CV) is defined as a hepatic vein at the center of the classic liver lobule. It has a cross‐sectional diameter not exceeding 125 μm. The endothelium lining of the vein is thin and has openings that drain every sinusoid of a liver lobule. The rim or wall of the CV is less than 2 μm in thickness with sparse matrix fibrous proteins and cells. Fibrosis of the CV (CVF), also is called perivenular fibrosis, is marked by thickening of the rim of the vein. The fibrotic tissue deposited in the CV may act like a “pervenular fibrous dam” that obliterates the sinusoidal openings into the vein, resulting in sinusoidal portal hypertension. CVF occurs early in hepatic fibrogenesis of rats induced by CCl4 or porcine serum administration. CVF often accompanies perisinusoidal fibrosis of the centrilobular area in alcoholic liver disease and nonalcoholic fatty liver disease. In the liver of elderly cadavers (mean age, 82.1±10.4), we have observed a 49.2 % incidence (31/63 livers) of CVF, but the matrix components of the fibrotic veins are not fully characterized. This study assessed fibrous proteins and cells in CVF of cadaveric livers. A total of 72 fibrotic veins out of 315 veins from 63 livers were studied. The diameters of the veins ranged from 25.4 to 123.7 μm (mean, 75.5 ± 18.3 μm). CVF was considered present when the rim thickness exceeded 9 μm (determined on Sirius red stained sections) coincident with fibrous tissue extension into the perivenous parenchyma. Immunoperoxidase staining revealed that the CV endothelium was positive for factor VIII‐related antigen, a vascular endothelial phenotypic marker. It also stained for CD31 and D34. Staining of basement membrane collagen IV was continuous around the CV endothelium, while laminin, another principal basement membrane protein, was not detected in the CV matrix. Thus, the CV appeared to lack a continuous endothelial basement membrane and a diffusion barrier between the CV circulation and perivenous parenchyma. Fibrillar collagens I, III and V and filamentous collagen VI were robust in the rim of the fibrotic vein. The codistribution of these collagens rendered thickening of the CV as well as provided strength to the CV matrix scaffold. Elastic fibers were noted occasionally among collagen bundles. Myofibroblasts, identified by a‐smooth muscle actin, were the dominant fibrogenic cells producing the fibrous matrix. Few mononuclear cells were also present. The deposition of collagenous fibrous tissue in the rim could occlude the CV endothelial openings, reducing the efficiency and increasing the resistance of sinusoidal blood flow into the CV. This may cause sinusoidal hypertension, impair hepatocyte functions and enhance disease susceptibility of the aged people.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP2C9 and CYP2C19: Massively Parallel Characterization of the Function of Genomic Missense Variants
Authors: Zhang Lingxin , Sarangi Vivekananda , Moon Irene , Devarajan Sandhya , Reid Joel M. , Wang Liewei , Weinshilboum Richard
Published: June 2021

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. Because of the very large number of missense variants in ORFs, it is difficult to link the genotypes of these variants to their phenotypes using “one at a time” expression systems. Therefore, the functional characterization of variants of unknown significance in clinically important pharmacogenes remains a major challenge in pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that make it possible to analyze hundreds of different missense variants in a parallel and scalable fashion. Engineered “landing pad” HEK 293T cells were used as a platform to integrate pooled variant libraries, resulting in one variant per cell for further assessment. We adapted this DMS system to analyze the function of missense variants in the ORFs of CYP2C9 and CYP2C19. The ORFs of CYP2C9 and CYP2C19 were fused to green fluorescent protein, and individual variants with different fluorescence intensities were used to indicate different levels of protein expression. Pooled variant libraries were generated by nicking mutagenesis, and we then used recombinase to integrate the libraries into landing pad cells, one per cell. Multiplexed functional selection performed with Fluorescence Activated Cell Sorting separated cells into different bins by fluorescence readout at the single–cell level. Amplicon sequencing of DNA collected in each bin, followed by computational analysis of the frequency of variants appearing in each bin, was used to determine levels of protein expression. To identify potentially severely damaging variants for these two important pharmacogenes, we studied 243 missense variants in the CYP2C9 and CYP2C19 ORFs that had been identified by the Exome Aggregation Consortium Project and by the Mayo Clinic RIGHT 1K Study. We found that 13 of 115 CYP2C9 and 25 of 128 CYP2C19 missense variants displayed less than approximately 25% of the wildtype protein expression, a level that may have clinical relevance for drug metabolism. Variant calling by the DMS method for the severely damaging variants was compared with the results of individual variant assay by western blot and with the predictions of computational algorithms such as Polyphen and SIFT. Our results suggest that DMS is an efficient method for the high‐throughput identification of damaging ORF variants that might have potential clinical application in pharmacogenomics.

Support or Funding Information

Funded by NIH grant U19 GM61388 (The Pharmacogenomics Research Network), R01 GM28157, R01 GM125633, T32 GM08685 and by the Mayo Clinic Center for Individualized Medicine

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