bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationSupported by NIAAA11147 R37 Merit award to DM‐RThis 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 InformationThis 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 InformationFunding: R01GM130120, R21MH118745This 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 InformationThis 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.
NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium and the modulatory mechanism of D39
Authors: Kou Junping , Zhai Kefeng , Zhang Yuanyuan , Yu Boyang 
Published: June 2021

Non‐muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor‐α (TNF‐α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose‐dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF‐κB p65 nuclear translocation and I B degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3 and suppressed NF‐κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF‐α‐stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model. Moreover, D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior venacava‐ligated mice dose‐dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β–NF‐κB signalling pathways. Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders. In addition, we identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.Support or Funding InformationThe present research was supported by the National Natural Science Foundation of China (Grant No. 81274131 and 81601034), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.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

AimsEpithelial‐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.MethodsTGF‐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 conclusionsTGF‐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 InformationUbbo 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 InformationNIH 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.