bluuebunny/crossref_metadata_2025 huggingface dataset:
PGRMC1 stabilizes CYP1A2 and other cytochromes P450
Authors: McGuire Meredith R.
Published: June 2021

Cytochromes P450 are a family of monooxygenase enzymes required for biosynthetic reactions in the body and for the metabolism of pharmaceutical drugs. Compared to our understanding of P450 enzymology, little is known about the post‐translational control of P450 activity. Progesterone Receptor Membrane Component 1 (PGRMC1) is a membrane‐bound hemoprotein that is highly expressed in the liver and binds >20 P450s in vivo. Dap1p/PGRMC1 binds and promotes the activity of CYP51A1 in yeast and cultured cells. To understand the function of PGRMC1 in relation to P450 biology, we generated whole‐body Pgrmc1 KO mice. Proteomic and transcriptomic data in Pgrmc1−/Y mouse livers showed that 81% of detectable P450s are downregulated at the protein level (17/21), while 83% of detectable P450s are unchanged at the transcript level (66/80). The constitutively expressed cytochrome P450 Cyp1A2, which is homologous to human CYP1A2, follows this trend. Consistent with a reduction in protein level, the Vmax of the Cyp1A2‐dependent O‐deethylation of 7‐ethoxycoumarin is 57% lower in Pgrmc1−/Y liver microsomes. In PGRMC1 KO human fibroblasts, PGRMC1 doubles the half‐life of CYP1A2 in a heme‐independent manner. These proteomic, transcriptomic, and turnover data establish PGRMC1 as a novel post‐translational stabilizer of Cyp1A2. Given that our proteomic data identified 17 P450s that are downregulated in Pgrmc1−/Y liver, these data suggest a broad role for PGRMC1 in post‐translational cytochrome P450 stability.

Support or Funding Information

NIHF31HL131185 / AHA15PRE25250008 / NIHT32GM007445 / NIHRO1HL077588

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP synthase: A potential molecular drug target
Authors: Ahmad Zulfiqar
Published: June 2021

Background

According to the Review on Antimicrobial Resistance, antibiotic resistance will result in 10 million additional deaths worldwide per year by 2050 surpassing cancer. Fast‐encroaching antibiotic resistance by microbes in general and E. coli in particular is the main reason for this situation. Thus, finding alternative ways to kill microbes is of paramount importance. ATP synthase is the principal source of cellular energy in almost all organisms. Selective inhibition of microbial ATP synthase may deprive microbes of required energy leading to cell death. A wide variety of inhibitors including phytochemicals and peptides bind and inhibit ATP synthase. These phytochemicals and peptides bind to the explicit binding sites on ATP synthase. Although binding sites are highly conserved, they are flanked by variable amino acids, which participate in selective binding of inhibitors. Our lab is interested in identifying the selective inhibitors of ATP synthase using Escherichia coli as a model organism.

Method

Wild type, null, and mutant E. coli strains are used to select the potent inhibitors of ATP synthase. Growth of E. coli strains is measured on fermentable and non‐fermentable carbon sources. Membrane bound F1Fo ATP synthase is isolated by harvesting cells in the minimal media, breaking cells by French Press, and separating the enzyme by multiple high‐speed ultracentrifugation. A variety of phytochemical and peptide inhibitors are used to inhibit membrane bound ATP synthase. Inhibitory potency of inhibitors is amplified through addition or subtraction of functional groups.

Results

Natural inhibitors caused variable degree of inhibition. Structural modifications resulted in augmented inhibition. Re‐positioning of −OH, NH2, PO4 groups in phytochemicals or addition of a c‐terminal NH2 group in peptides augmented the inhibitory effects by about 100‐fold. Phytochemicals and peptides also showed synergistic inhibitory effects on ATP synthase.

Conclusions

Phytochemicals and peptides potently kill E. coli cells by binding at the F1 sector of ATP synthase. Thus, ATP synthase can be used as a potent molecular drug target to combat antibiotic resistant microbial infections.

Support or Funding Information

A.T. Still University Warner/Fermaturo Research Grant, grant 501–605 to ZA.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Secretome and Transmembrane Proteins of Non‐Professional Phagocytes
Authors: Calikyan Anoush , Serizier Sandy , Mondragon Albert , Silvestrini Melissa , Peterson Jeanne , Emili Andrew , Kwan Julian , McCall Kim
Published: June 2021

Apoptosis and the engulfment of dead cells are essential processes for the proper development and maintenance of an organism. While apoptosis is well‐characterized, many modulatory signals that control apoptotic cell engulfment have not been well described. We use the Drosophila ovary as a model for cell death and clearance, where follicle cells act as non‐professional phagocytes and engulf the dying germline. In this system, several transmembrane receptors, such as Draper, have been found to be essential for nurse cell clearance by follicle cells. However, large‐scale approaches have not been used to determine the signaling molecules of follicle cells that promote cell clearance. We hypothesized that many of the proteins involved in this network of signaling are secreted or are transmembrane proteins. This project, thus, focuses on identifying the “secretome” and transmembrane proteins of engulfing follicle cells by labeling proteins in the endoplasmic reticulum (ER) with a biotin phenol tag. Horseradish peroxidase (HRP) catalyzes the biotinylation of all proximal proteins. When fused to an ER retention signal (KDEL) the expression of HRP is targeted to the ER. We generated flies carrying this HRP‐KDEL construct under UAS control, and then expressed it in desired follicle cell populations. We demonstrated that HRPKDEL localizes around the follicle cell nucleus with immunohistochemistry, suggesting it is expressed appropriately in the endoplasmic reticulum. Moreover, the stains probing for biotinylated proteins and the HRPKDEL construct, respectively, co‐localize, indicative of proximal protein biotinylation. Protein biotinylation was also confirmed by western blot analysis. Ongoing experiments will identify and characterize the biotin‐labeled proteins by mass spectrometry to determine the secretome of follicle cells. By characterizing the secretome of the follicle cell population, we can identify new proteins that regulate the engulfment of apoptotic cells.

Support or Funding Information

The Undergraduate Research Opportunities Program at Boston University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Hexavalent Chromium on Dictyostelium Chemotaxis and Proteome
Authors: Daniel Hanix Emeral , Moen Rebecca
Published: June 2021

Hexavalent chromium (Cr6+) is a common environmental pollutant extensively used in industrial processes like chrome‐plating, leather tanning, and paint making that is capable of accumulating in aquatic vascular plants and tissue of animals. The uneven geographical distribution of chromite mining makes this issue a global one and indicates that further study of Cr6+ may lead to targeted treatment in individuals who ingest contaminated water and wildlife or suffer from direct skin contact with contaminated soil. Dictyostelium discoideum is a soil‐dwelling eukaryote that contains many genes that are homologous to human genes while exhibiting a simple lifecycle, restricted cell types and behaviors, and rapid growth. Dictyostelium is widely regarded as a good model organism for studying eukaryotic cell response to toxic compounds. The aim of the study is to determine how hexavalent chromium affects cytoskeletal proteins, specifically actin, and the movement of Dictyostelium in response to Cr6+. A focus is placed on actin and its associated proteins because it has been shown to be a key player specialized stress signaling pathways, and its associated proteins may be multifunctional and may be implicated in a variety of conditions. Chemotaxis, cell movement in response to an external stimulus, was assayed following cell treatment to determine how cell signaling changes as a result of Cr6+ exposure altered the assembly of the actin network. Differences in the actin network of both the treatment and control groups were visualized using fluorescence microscopy. Electrophoresis was then performed on control and treatment groups to resolve proteins and identify specific changes in the expression of actin and its associated proteins.

Support or Funding Information

Minnesota State University, Undergraduate Research Center.

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 Conserved C‐terminal Region of Gα12 and Gα13 Harbors Unique Determinants of SRF Signaling and RhoGEF Binding
Authors: Mull Makenzy L. , Fleming Todd L. , Quick Courtney R. , Meigs Thomas E.
Published: June 2021

Heterotrimeric G proteins of the G12/13 subfamily play key signaling roles in cell growth, oncogenic transformation, migration, and invasion. The G protein alpha subunits, Gα12 and Gα13, share 67% amino acid identity and several downstream signaling pathways, including transcriptional activation via Serum Response Factor (SRF). Gα12 and Gα13 have multiple, unique binding partners, suggesting divergent signaling mechanisms within this subfamily. To identify different structural features required for growth signaling in Gα12 and Gα13, we utilized amino acid substitutions from non‐SRF signaling invertebrate homologs. Replacing the N‐terminal region of these mammalian α subunits with invertebrate sequence caused no disruption of SRF signaling. Conversely, invertebrate substitution of a highly conserved sequence downstream of the Switch III region abolished SRF signaling by Gα12 and Gα13. In this “Post‐Switch region” of Gα12, a single amino acid substitution disrupted its SRF signaling, while the same mutation in Gα13 had no effect. Replacement of additional residues in this region uncoupled Gα13 from SRF activation, suggesting divergent growth signaling mechanisms in the G12/13 subfamily. In addition, invertebrate substitution in the Post‐Switch region disrupted Gα13 interaction with RhoGEFs, whereas the corresponding SRF‐uncoupled Gα12 retained binding to these effector proteins. To further explore differences in the growth signaling mechanisms of Gα12 and Gα13, we engineered invertebrate chimeras within the C‐terminal α5 helix. These substitutions partially disrupted Gα12‐mediated SRF signaling but were inconsequential for Gα13. Our findings reveal novel C‐terminal motifs in Gα12 and Gα13 that are necessary for their non‐overlapping mechanisms of RhoGEF binding and SRF‐mediated cell growth.

Support or Funding Information

We acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, and the UNC Lineberger Comprehensive Cancer Center.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding G Protein Selectivity of Dopamine Receptors Using Computational Methods
Authors: Portillo Jennifer , Abrol Ravinder , Sakkiah Suguna
Published: June 2021

Dopamine is important in physiological processes associated with the rewards and pleasure system. Its function is facilitated by five G Protein – Coupled Receptors (GPCRs). The coupling that occurs between the dopamine receptor and G protein dictates the signaling cascade. The dopamine receptors D1 and D5 couple to the Gs protein while the dopamine receptors D2, D3, and D4 couple to the Gi protein. Dysregulation of the dopamine receptors can lead to neuropsychiatric disorders such as schizophrenia. These disorders are difficult to treat due to the side‐effects that transpire. Understanding the coupling that occurs between the dopamine receptors and the G proteins can further assist with the development of pharmaceutical drugs by helping to alleviate some of the side‐effects. Using computational biophysical methods, the coupling between the dopamine receptors and G protein is being studied to understand the structural mechanisms observed in G protein selectivity of these receptors. The dopamine receptor structures for D1 and D2 in complex with the two G proteins (Gs and Gi) have been relaxed in the membrane environment. A structural analysis of those complexes in combination with structural bioinformatics methods has elucidated receptor residues playing a role in the G protein coupling, which has led to the design of receptor mutants with altered G protein selectivities. These mutants are now being characterized by computational biophysical methods before they will be tested in biochemical assays for altered G protein preferences.

Support or Funding Information

National Institutes of Health (NIH) Building Infrastructure Leading to Diversity (BUILD) #5TL4GM118977

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA Exclusion Mechanism of the Cytidine Deaminase APOBEC3G
Authors: Solomon William C , Myint Wazo , Hou Shurong , Kanai Tapan , Schiffer Celia A , Matsuo Hiroshi
Published: June 2021

APOBEC3G (A3G) is a potent antiretroviral protein that specifically targets and deaminates cytidine nucleotides to uracil in single stranded DNA (ssDNA) intermediates produced during HIV replication. A3G displays high selectivity for specific polynucleotide contexts targeting 5′‐CC sites for deamination. A3G binds strongly to RNA via the N‐terminal non‐catalytic domain, which is crucial for recruitment of A3G to the virion, yet A3G displays no deamination activity on the cytidines in single stranded RNA. Our recent structure of A3G bound to ssDNA shows the preferred substrate conformation but does not reveal the mechanisms of substrate selection and non‐substrate exclusion. Here, we report NMR and molecular dynamics (MD) simulation based analysis of the interactions between the C‐terminal catalytic domain of A3G (A3Gctd) and multiple substrate and non‐substrate ssDNAs, which are coupled with deamination assays. The extent of interaction with residues located in helix1 and loop1 (T201‐L220) distinguishes substrate ssDNAs from non‐substrate ssDNAs, revealing the difference between catalytic and non‐catalytic ssDNA‐binding modes of A3Gctd. Using fluorine substituted ribonucleotides we show that 2′‐endo sugar pucker of the target cytidine is an essential factor for effective deamination. Furthermore, MD simulations show that ribose cytidine does not stay at catalytic position during 100ns of simulation time whereas deoxy‐ribose cytidine does. Trajectories of the MD simulation suggest that 2′‐hydroxyl group of RNA forms a hydrogen bond with His257 that chelates Zn2+, which results in loss of two hydrogen bonds between the target cytidine and A3Gctd, dislocating cytosine base from the catalytic position. This study provides mechanistic models of the catalytic selectivity of A3G against RNA.

Support or Funding Information

Institute for Molecular Virology Training Program (NIH grant T32 AI83196) University of Minnesota

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of Plasminogen Activator Inhibitor Type‐2 (PAI‐2) in modulating venous thrombus resolution
Authors: Johnson Tierra A , Mukhopadhyay Subhradip , Duru Nadire , Buzza Marguerite S , Antalis Toni M
Published: June 2021

Deep Vein Thrombosis (DVT) which can arise from vascular endothelial injury, venous stasis, and/or alterations in blood hypercoagulability, affects patients of increased age who have limited to no mobility, paralysis, cancer, surgery and/or trauma. DVT is characterized by thrombi that form on the surface of activated endothelium in both the vein valve pockets and dilated sinuses of the lower limbs and is a highly inflammatory process. Macrophages and neutrophils are known to play key roles, being major producers of inflammatory cytokines, chemokines, and proteases. However, the precise mechanisms and key players that modulate venous thrombosis and its resolution are poorly understood. Plasminogen activator inhibitor type‐2 (PAI‐2) or SerpinB2, a Clade B serine protease inhibitor (SERPIN) was originally identified as an inhibitor of the serine protease, urokinase‐plasminogen activator(uPA) and has also been found to possess unique properties associated with inflammation and cell survival. Utilizing a clinically relevant mouse model of DVT, we discovered that genetic deletion of PAI‐2 accelerates venous thrombus resolution in vivo. PAI‐2 deficiency leads to enhanced venous thrombus resolution, increased proteolytic activity of uPA, and modulation of inflammatory processes. Recent work in our lab has focused on the primary effector cells of thrombus formation and resolution, neutrophils and macrophages respectively. PAI‐2 expression can be found in both resident and activated, neutrophils and macrophages, and may modulate how these cells function on fibrin and collagen matrices found within the thrombus microenvironment. Clarifying the mechanism by which PAI‐2 contributes to venous thrombus resolution has potential to lead to new therapies that accelerate venous thrombus resolution.

Support or Funding Information

Department of Physiology, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine and VA Maryland Health Care System, Department of Veterans Affairs, Baltimore, Maryland

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kindlin‐3 recruitment to the plasma membrane in neutrophils precedes high affinity integrin activation
Authors: Wen Lai , Roy Payel , Kiosses William B. , Ginsberg Mark , Ley Klaus
Published: June 2021

Integrin‐mediated adhesion of circulating leukocytes to the blood vessel wall starts by arrest from rolling. Activation of integrins by inside‐out signaling involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is critically required for leukocyte adhesion. Mutations of kindlin‐3 cause leukocyte adhesion deficiency type III (LAD‐III) syndrome, characterized by severe bleeding (a platelet adhesion defect) and recurring infections (a neutrophil adhesion defect). The clinical significance of kindlin‐3 has been extensively studied; however, the molecular mechanism by which kindlin‐3 regulates the β2 integrin activation is unknown. Kindlin‐3 can bind the β2 integrin cytoplasmic tail at a site distinct from the talin‐1 binding site. It remains unclear whether kindlin‐3 affects individual integrin conformation and/or integrin clustering. Here we monitored the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil rolling, arrest and spreading under flow (6 dyn/cm2) using high‐resolution quantitative dynamic footprinting (qDF) microscopy in combination with an integrin conformation‐specific antibody reporting assay and kindlin‐3 fusion reporters. We find that kindlin‐3 is recruited to the plasma membrane during neutrophil adhesion prior to induction of H+ β2 integrin conformation, indicating that kindlin‐3 is required for H+ integrin conformation. Indeed, deletion of kindlin‐3 in neutrophil‐like HL‐60 cells completely abolishes high affinity β2 integrin. Together, our data suggest that kindlin‐3 is permissive for the high affinity β2 integrin conformation.

Support or Funding Information

This work was supported by funding from the National Institutes of Health, USA (NIH, P01‐HL078784).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Testosterone Modulates the Multiplication and Viability of C2C12 Cells Cultivated In Vitro
Authors: Camargo Anne Marielle , Alves Eduardo Donatto , Achcar Jorge Alberto , da Costa Iemma Monica Rosas , Montrezor Luis Henrique
Published: June 2021

The use of C2C12 cell cultures is strategic for the study of functional mechanisms of skeletal striated muscle, as well as their possible correlations with other tissues and organ systems, in physiological and pathological situations. The objective of this work was to evaluate the effects of different concentrations of testosterone (T) on the multiplication and viability of C2C12 cells, in the absence and presence of flutamide (F), an androgen receptor inhibitor. The cells were cultured (in triplicates) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fungicide, streptomycin, penicillin, and 2% equine serum, in 48‐well plates (5000 cells / 200 μL‐day zero). The cells were incubated (37 °C, 5% CO2, 95% atmospheric air) for up to 10 days, in the absence (control) and presence of T (10−8 M and 10−4 M, mimicking normo‐ and hyperandrogenemia), and in the absence and presence of F (100 nM). The medium was exchanged every 48 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter. The MTT (3‐ (4,5‐dimethylthiazol‐2‐yl) ‐2,5‐diphenyltetrazolium) reduction method was used to determine cell viability. Statistical analysis of the data employed ANOVA and Fisher's test. Cell multiplication. After 5 days of culture, cell multiplication was inhibited by T at 10−4 M, in the presence of F. After 10 days of culture, the cell count showed inhibition in the presence of T at 10−4 M, in the absence of F, and stimulation in the presence of T at 10−8 M, in the presence of F. Cell viability. After 5 days of culture, cell viability was stimulated by T at 10−8 M, with and without flutamide, and by T at 10−4 M, in the absence of F. After 10 days of culture, cell viability decreased in the presence of T at 10−8 M, without flutamide, and in the presence of T at 10−4 M, with F. The results suggested that the culture time influenced the cellular responses analyzed. In addition, there was a dual effect of T on multiplication and cell viability, in the absence and presence of flutamide, for both culture times studied. Interestingly, when T, in the absence or presence of flutamide, stimulated or inhibited cell multiplication, the cell viability was unaffected by the treatments, and vice versa. The findings suggested that T directed the cellular mechanisms towards multiplication, and sometimes towards metabolic activity. This cell culture model is shown to be feasible for studies of the effects of T mediated by androgen receptors; however, molecular studies are needed in order to obtain a better understanding of the mechanisms involved.

Support or Funding Information

Funding: CNPq (Grant 159893/2017‐8)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioinformatics analysis for heart failure with preserved ejection fraction
Authors: Zhang Wenxi , Huo Yunlong , Tan Wenchang
Published: June 2021

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure but little is known about the underlying mechanisms. Dahl salt‐sensitive (Dahl‐SS) rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF and controls were fed a normal‐salt diet (0.3% NaCl). Echocardiograms and electrocardiography (ECG) was performed to assess cardiac function. Diastolic dysfunction and preserved ejection fraction along with signs of heart failure was showed and QTc interval on ECG was prolonged (p<0.001) at 14 weeks of age. Hearts were harvested for gene array and label‐free quantitative proteomics. Bioinformatics analysis using DAVID website, GSEA software and BinGO plugin in Cytoscape environment. GO analysis showed microtubule binding and calcium dependent phospholipid binding protein were significant downregulated while oxidative stress response protein were upregulated (p<0.05, q<0.05). GSEA results indicated that gap junction, focal adhesion and actin cytoskeleton regulation gene sets were concentrated in upregulated transcripts (p<0.01, q<0.25) while cardiac muscle contraction and oxidative phosphorylation pathway were downregulated in proteomics (p<0.001, q<0.001). Western blot and PCR experiments showed significant changes in the expression of Dynamin‐1, Integrin‐α9 and MFF, which had important functions including cell stiffness regulation. Our study provides valuable information on the molecular functions and regulatory network of HFpEF, as well as new perspectives on the relationship between diastolic dysfunction and calcium stimulate, cytoskeleton stiffness and oxidative stress.

Support or Funding Information

Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of corylin in regulating NLRP3 inflammasome activation in macrophages
Authors: Lai Yi‐Xin , Wu Hsin‐En , Li Chia‐Yang
Published: June 2021

Chronic inflammation is associated with several diseases and cancers. The activation of inflammasomes has been indicated as a main role in chronic inflammation, which is a group of cytosolic protein complexes, classically consisting an NOD‐like protein (NLR), the adaptor apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and caspase‐1. Activation of inflammasome results in assembly of the inflammasome, caspase‐1 activation as well as IL‐1β and IL‐18 secretion. A number of studies have reported that increased concentration of IL‐1β protein in tumor tissues is associated with poor prognosis for cancer patients.

Corylin is a main compound isolated from Psoralea corylifloia L., a common Chinese herbal medicine. The Psoralea corylifloia L. has been used in the treatment of skin diseases and osteoporosis. The extracts of this plant has been found to have anti‐oxidative, anti‐bacterial, anti‐diabetic, neuroprotective and immunomodulatory effects. Our previous study demonstrated that corylin inhibits the production of TNF‐α, IL‐6 and NO by murine macrophages through inhibiting the activities of MAPKs and NF‐κB. In the present study, we will examine the effect of corylin in regulating NLRP3 inflammasome activation in macrophages. We used murine macrophages (J774A.1 cells) and human monocytes (THP‐1 cells), and treated with LPS/ATP to induce the activation of NLRP3 inflammasome, then gave different doses of corylin to investigate its effects. The supernatant is used to determine the level of IL‐1β by enzyme‐linked immunosorbent assay (ELISA), and the lysates is used to investigate the level of NLRP3 inflammasome associated protein by Western blot. Our experimental results indicated that corylin inhibits the activation of NLRP3 inflammasome in both murine and human macrophages. We will further examine the effect of corylin on the polarization of macrophages.

Support or Funding Information

This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant No. MOST 107‐2320‐B‐037‐019).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Cl−in signaling and ion transport by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways
Authors: Vachel Laura , Shcheynikov Nikolay , Yamazaki Osamu , Fremder Moran , Ohana Ehud , Son Aran , Shin Dong Min , Yamazaki‐Nakazawa Ai , Yang Chin‐Rang , Knepper Mark A. , Muallem Shmuel
Published: June 2021

IRBIT is a multifunctional protein that also controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters, including NBCe1‐B, CFTR and Slc26a6, by unknown mechanisms. With the ubiquitous NBCe1‐B, IRBIT interacts with the autoinhibitory domain (AID), exposing two cryptic GXXXP motifs to confer regulation by Cl−in. Here, we report that IRBIT‐dependent combinatorial dephosphorylation of S232/S233/S235 controls NBCe1‐B close and open states by determining autoinhibition and regulation by Cl−in. The pS233/pS235 conformer is fully active, no longer activated by IRBIT or inhibited by Cl−in. The pS232/pS235 conformer is similar to wild‐type NBCe1‐B, while the pS232/pS233 conformer is partially active, but retains inhibition by Cl−in. IRBIT then recruits the kinases SPAK and CaMKII to control phosphorylation of S65 and S12, respectively, which determine inhibition by Cl−in at the 32GXXXP36 and 194GXXXP198 motifs. Recruitment by IRBIT of PP1 and calcineurin dephosphorylates S65 and S12, respectively, and relieves inhibition by Cl−in. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which has important implications for epithelial fluid and HCO3− secretion.

Support or Funding Information

All the work was supported by NIH funding

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 Alcohol Exposure‐Induced let‐7a Inhibition Exacerbates Hepatic Apoptosis by Regulating Caspase‐3 in Mice
Authors: Beane Kaleigh , Pan Jeong Hoon , Kim Hyunjin , Tang Jingsi , Park Jeen‐Woo , Kong Byungwhi C , Shin Eui‐Cheol , Lee Jin Hyup , Kim Jae Kyeom
Published: June 2021

Alcohol consumption is a critical risk factor for hepatic pathogenesis, including alcoholic liver diseases (ALD), but implications of alcohol induced dysregulation of microRNA (miRNA) in ALD pathogenesis are poorly understood. In this study, we aimed to explore effects of dysregulated miRNA profile on ALD conditions in mice. C57BL/6J male mice were treated with either saline (CON; oral gavage; n=8) or alcohol (EtOH; 3 g/kg·body weight; oral gavage; n=8) for 7 days. The ALD conditions were assessed by a serum AST/ALT ratio and hepatic fat accumulation. A total of 599 miRNA, and 158 key mRNAs of fatty liver and hepatotoxicity pathway were measured in liver tissues using NanoString miRNA expression array and quantitative real‐time PCR arrays, respectively. The array datasets were utilized to predict interactions with miRNAs. The predicted interactions of miRNA‐mRNA pairs were further validated using in vitro miRNA transfection experiments using AML12 mice hepatocytes. As results, miR‐21 and let‐7a were significantly decreased in EtOH group, and Ccng1, Rb1, and Casp3 mRNAs were predicted as their target genes. In addition, p53 expression was increased in the EtOH group, which is consistent with increased Casp3 activity, cPARP expression, and TUNEL positive cells by alcohol treatment. Similarly, in vitro suppression of let‐7a increased apoptosis by upregulating Cas‐3 protein expression in AML12 hepatocytes. Although there was no direct interaction between let‐7a and Rb1 protein expression, MDM2/p53 axis was also influenced by let‐7a inhibition. Therefore, alcohol‐induced inhibition of let‐7a may increase hepatic apoptosis by both directly targeting Casp3, and indirectly modulating MDM2/p53 axis.

Support or Funding Information

This work was supported by the University of Arkansas, VPRED Start‐up fund (JKK, and JHP). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF‐2015R1C1A1A01053746; JHL) as well as the China Scholarship Council (JT).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐Term High‐Fat Diet Decreases Hepatic Iron Storage Associated with Suppressing TFR2 and ZIP14 Expression in Rats
Authors: Jiang Shuxia , Yan Kai , Sun Bo , Gao Shixing , Yang Xiaojing , Ni Yingdong , Ma Wenqiang , Zhao Ruqian
Published: June 2021

High fat (HF) diet‐induced obesity is known to disturb hepatic iron metabolism in a time‐dependent manner. The mechanism of decreased hepatic iron store induced by long‐term high fat diet remains unclear. In this study, twenty‐four 6‐week‐old male Sprague‐Dawley rats were fed a high fat diet for 16 weeks and hepatic iron metabolism was examined. HF diet feeding considerably decreased hepatic iron contents, enhanced transferrin (TF) expression, reduced ferritin heavy chain (FTH), ferritin light chain (FTL), transferrin receptor 2 (TFR2) and ZRT/IRT‐like protein 14 (ZIP14) expression in the liver of rats. Down‐regulation of hepatic TFR2 coincided with DNA hypermethylation on the promoter and repressing expression of transcription factor hepatocyte nuclear factor 4α (HNF4α). MiR‐181 family expression was markedly increased and verified to regulate Zip14 expression using the dual‐luciferase reporter assay. These results indicate that the long‐term high fat diet decreases hepatic iron content, which is closely associated with inhibition of hepatic iron uptake via TFR2 and ZIP14‐dependent pathway.

Support or Funding Information

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0500500), the National Natural Science Foundation of China (31302053), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combination of Traction Force Microscopy with Adhesive Protein Micropattern to Measure E‐cadherin‐based Intercellular Adhesion
Authors: Lu Chuan‐Fen , Tsou Chieh‐Chung , Chu Yeh‐Shiu , Lo Chun‐Min
Published: June 2021

Cellular mechanical forces can be generated intrinsically and extrinsically; these physical cues regulate a wide variety of critical cellular functions such as cell proliferation, differentiation, and migration. Mechanical forces generated by cell‐cell interaction are able to transmit to the interior of the cell through the coordination of cortical actomyosin networks. Following that, physical signals can be converted to chemical ones through transducing process. E‐Cadherins (E‐Cdh) are prototypical adhesive molecules which can construct strong adhesive interfaces between cells. Currently E‐Cdh complexes comprised of β‐catenin and α‐catenin are suggested to associate with cortical F‐actin in a dynamic manner; however, the mechanistic steps of mechanotransduction in E‐Cdh‐mediated cell adhesion remain elusive. In this study, we attempt to establish a novel protocol for measuring intercellular forces generated by two adhering cells on 2D flexible substrate with a better temporal and spatial control. A combination of protein micropattern and traction force microscopy (TFM) is therefore developed. Pair micropattern is photolithographed on polyacrylamide gel (Young's modulus = 9 kPa) with two triangle forms, spaced 8 μm apart, to confine cell spreading area (1600 μm2). EpH4 cells of mouse breast epithelial origin are allowed to form the cell doublets on micropattern and then calcium switch is proceeded to initiate the E‐Cdh mediated cell adhesion. Pilot study has demonstrated that the intercellular strengths are measureable with calcium sensitivity and cadherin specificity. A series of CRISPR clones targeted primarily on E‐Cdh and p120‐catenin are prepared to decipher the novel mechanotransduction pathway of E‐Cdh. Thus, we describe an integrated mechanobiological method for the analysis of intercellular forces, which may provide deep insights on the biophysical principle governing mechanotransduction of E‐Cdh‐mediated intercellular adhesion.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of DNA ligation
Authors: O'Brien Patrick
Published: June 2021

Humans have three DNA ligases with distinct structural and biochemical features. Together, these enzymes are responsible for completing the majority of replication, repair, and recombination pathways. Deficiencies in the different enzymes cause different diseases and engineered cell lines lacking DNA ligases individually or in combination have challenged traditionally assigned roles of these enzymes. Quantitative mechanistic approaches with reconstituted enzymes and defined DNA breaks provide new insights into the substrate specificity and catalytic mechanisms of the different ligases that helps to define their normal biological roles and their compensatory roles when the other enzymes are compromised. These findings provide insight into the genomic instability of cancer cells and the immune deficiencies associated with defects in DNA ligases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Physiological Basis of No‐Go Decay Using sod1Δ Strains in Saccharomycescerevisiae
Authors: Lobb Amber , Cianflone Emily , Champine Madison , Bailey Lindsey , Seaberg Lucas , Segal Scott P
Published: June 2021

Chromium VI (Cr(VI)) induces oxidative stress in cells, resulting in altered gene expression, increased apoptosis and cell death. Previous work showed that Cr(VI) exposure results in the formation of 8‐oxo(G) bases in mRNA. The presence of 8‐oxo(G) bases can lead to ribosome stalls during translation and activation of no‐go decay. During no‐go decay, the Dom34p/Hbsp1p complex acts to remove stalled ribosomes and subsequent endonucleolytic cleavage of the damaged mRNA at the stall site. Although oxidative agents and other non‐physiological substrates have been used to activate no‐go decay, very little work exists to elucidate the true physiological role of this pathway.

Sod1p (superoxide dismutase 1) acts to scavenge free oxygen species by catalyzing the partitioning of superoxide (O2− ) into O2 and H2O2. Cells that lack functional Sod1p were observed to have increased oxidative stress resulting in increased P‐body assembly. Yet, global translation was only attenuated. To determine whether no‐go decay is being activated in strains lacking Sod1p, double mutant strains were created in which SOD1 was knocked out in conjunction with one of the effectors of no‐go decay. Consistent with no‐go decay being activated in response to oxidation, the hbs1Δsod1Δ strain resulted in a decrease in P‐body assembly, as compared to wild‐type, even in the presence of Cr (VI).

Interestingly, Stm1p is thought to potentially to aid in the dissociation and recycling of the ribosomal subunits. In the stm1Δ strain, P‐body assembly was mildly decreased as compared to the wild‐type upon treatment with Cr(VI). This is consistent with Stm1p functioning to recycle ribosomes downstream of no‐go decay. Additionally, a stm1Δ can can suppress sod1Δ growth defects on Cr(VI) containing media.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Versatile Tools Toward Real‐time Single‐molecule Biology
Authors: Barbetsea Kassandra , Simpson Trey , Raja Ali , Candelli Andrea
Published: June 2021

Biological processes performed by proteins interacting with and processing DNA and RNA are key to cell metabolism and life. Detailed insights into these processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go awry.

The next scientific breakthrough consists in the actual, direct, real‐time observations and measurements of the individual mechanisms involved, in order to validate and complete the current biological models.

Single‐molecule technologies offer an exciting opportunity to meet these challenges and to study protein function and activity in real‐time and at the single‐molecule level.

Here, we present our efforts for further enabling discoveries in the field of biology and biophysics using the combination of optical tweezers with single‐molecule fluorescence microscopy (C Trap™).

We show the latest applications of these technologies that can enhance our understanding not only in the field of DNA/RNA‐protein interactions but also in the fields of molecular motors, protein folding/unfolding, cell membranes and genome structure and organization.

These experiments show that the technological advances in hybrid single‐molecule methods can be turned into an easy‐to‐use and stable instrument that has the ability to open up new venues in many research areas

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Reductase‐Chaperone Activity of the Epoxidase Component of Styrene Monooxygenase
Authors: Ma Chen Patrick , Gassner George
Published: June 2021

Styrene Monooxygenase (SMO) is a two‐component flavoprotein composed of a NADH dependent reductase (SMOB) and FAD specific epoxidase (SMOA) that catalyzes the enantioselective epoxidation of vinyl side‐chains of styrenes. The chemical versatility of SMO makes it a desirable biocatalyst for the environmentally friendly synthesis of pharmaceuticals and for the removal of styrene as a toxic contaminant of terrestrial and aqueous environments. Previous thermodynamic and kinetic studies from our lab and others have demonstrated that we can stabilize the reduced, SMO(FADred), peroxide, SMO(FADOOH) or hydroxyflavin, SMO(FADOH), intermediate states by establishing steady‐state reaction conditions in the presence of NADH alone, NADH and the substrate analog, benzene, or NADH and the substrate, styrene. In addition to its oxygenase function, SMOA has been demonstrated to have a chaperone‐function that stabilizes SMOB and greatly increases the catalytic half‐life of the reductase. The nature of this interaction remains an area of current investigation.

In the present work, the protein‐protein interaction of SMOA and SMOB was evaluated by a series of pulldown assays under various conditions of catalytic turnover. In this approach, the N‐terminally histidine‐tagged SMOA (NSMOA) and SMOB protein components were prepared in buffer systems designed to stabilize the aforementioned intermediate states and loaded onto a miniature nickel (Ni2+)‐affinity column. After loading, the column was first washed with buffer containing 10 mM imidazole to remove non‐specifically bound SMOB and then washed with buffer containing 250 mM imidazole to remove SMO bound to the column by the N‐terminal histidine tag of NSMOA. The composition of eluted protein was evaluated by UV spectroscopy and polyacrylamide gel electrophoresis. We find that holoSMOB with oxidized FAD bound (SMOBFADox) passes directly through the column and does not significantly interact with NSMOA immobilized on the Ni2+ column. In the presence of NADH, FADred is efficiently transferred from SMOB to NSMOA bound to the Ni2+‐resin. Under reaction conditions designed to stabilize FADred, FADOOH, and FADOH intermediate states of SMOA, a significant fraction of the resulting apoSMOB is observed to be bound to NSMOA. The fraction of apoSMOB not retained by NSMOA on the column is observed to lose activity and aggregate in the absence of the chaperone‐like activity of SMOA with a half‐life of minutes. These results support a reaction model in which the components of SMO interact weakly with each other when oxidized FAD is bound to SMOB. A stronger binding interaction occurs between apoSMOB and the reduced, peroyiflavin, and hydroyflavin intermediates of NSMOA. These interactions serve to stabilize SMOB in a catalytically active state.

Support or Funding Information

NIH GM008574

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Germ Cell Apoptosis and Oxidative DNA Damage in Testicular Ischemia Reperfusion Injury: Survivin’ the Lipoxygenases
Authors: Al‐Maghrebi May
Published: June 2021

Objective

Testicular ischemia reperfusion injury (tIRI) is widely accepted as the underlying mechanism for the pathophysiology of testicular torsion and detrorsion (TTD) in young children that could increase the risk of male infertility. The aim of this study is to examine the role of the pro‐oxidative lipoxygenases (LOXs)‐induced testicular oxidative stress in the etiology of tIRI‐induced germ cell apoptosis (GCA) and oxidative DNA damage using its potent selective inhibitor nordihydroguaiaretic acid (NDGA).

Materials and methods

Sprague‐Dawley male rats (n = 36, 8 weeks old, 250–300g) were randomly assigned to three groups: Sham, unilateral tIRI and tIRI + NDGA (25 mg/kg). Rats underwent 1 hour ischemia followed by 4 hours reperfusion. Harvested testes were evaluated for spermatogenic damage by H&E staining, oxidative DNA damage by biochemical assays, GCA, and tissue immunoexpression of the three LOXs (LOX‐5, ‐12 and ‐15). The induction of GCA and the involvement of the apoptosis pathways survivin/ASK‐1/JNK and was investigated by immunofluorescence staining.

Results

Our data showed that tIRI caused disorganization of the seminiferous tubules layers and significant spermatogenic damage. Increased LOX immunoexpression but not mRNA expression was calculated as a result of tIRI. Augmented levels of LOX‐induced protein, lipid, and DNA oxidative damage were significantly elevated during tIRI. In addition, tIRI activated the ASK‐1/JNK signaling pathway, which was associated with the down regulation of survivin, an inhibitor of apoptosis. Selective inhibition of LOXs by NDGA diminished the above tIRI‐induced changes.

Conclusion

The study outcomes implicates the role of LOXs in the generation of reactive oxygen species, which partly contributed to the tIRI‐induced oxidative damage that also led to spermatogenic damage, oxidative DNA damage and induction of the apoptosis pathway survivin/ASK‐1/JNK. This new finding provides a good understanding to the tIRI/TTD mechanism in the field of translational medicine worldwide.

Support or Funding Information

Kuwait Foundation for the Advancement of Science (KFAS), Kuwait – Project Code: P116‐13MB‐01.

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