bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell survival during complete nutrient deprivation depends on lipid droplet‐fueled β‐oxidation of fatty acids (577.3)
Authors: Cabodevilla Ainara , Sánchez‐Caballero Laura , Picatoste Fernando , Gubern Albert , Claro Enrique
Published: June 2021

Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down‐regulation of ceramide kinase. Nutrient deprivation also induced β‐oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β‐oxidation of fatty acids that requires biogenesis and mobilization of LD.
In vitro testing of putative Escherichia coli MurA inhibitors (582.1)
Authors: Frost Andrea , Boone Benjamin , Song Vanessa , Muzyka Jennifer , Haile January
Published: June 2021

Antibiotic resistance necessitates the continuous development of novel inhibitors targeting a variety of bacterial pathways. The pathway for the synthesis of peptidoglycan, a necessary component of the bacterial cell wall, is an ideal target for antibiotic development for two reasons: 1) the cell wall is a prokaryote‐specific structure, and 2) the cell wall is necessary for bacterial survival. MurA, the target of current study, is the enzyme that catalyzes the first step in peptidoglycan biosynthesis. Our objective is to develop novel inhibitors that competitively bind to the MurA catalytic sight for UDP‐N‐acetylglucosamine. In an effort to study the effects of various putative inhibitors on MurA, the active enzyme was purified in high concentrations. In vitro tests of the known inhibitor fosfomycin and other potential inhibitors were conducted using the purified enzyme. The Malachite green assay as previously described was used to determine the IC50 values for a number of potential inhibitors. Of the compounds assayed, two salts, VS5 and BCB33b, exhibited consistent IC50 values of 160 uM and 300 uM respectively. The IC50 values of the two putative inhibitors are the lowest we have seen out of all the tested compounds; therefore, VS5 and BCB33b will be tested further in vitro, in vivo, and in silico in their salt and acid forms for comparison against the known inhibitor fosfomycin.

Grant Funding Source: Faculty Development Committee, Centre College
Cloning of a 1‐pyrroline‐5‐carboxylate dehydrogenase from Taiwanofungus camphorata (583.1)
Authors: Wen Lisa , Lai Chin‐Chun , Huang Jenq‐Kuen , Lin Chi‐Tsai
Published: June 2021

A cDNA (1890 bp) encoding a putative 1‐pyrroline‐5‐carboxylate dehydrogenase (P5CD) was cloned from Taiwanofungus camphorata. The DNA sequence encodes a protein of 547 amino acid residues with calculated molecular mass of 59.4 kDa. The deduced amino acid sequence is conserved among the reported P5CDs. To characterize the T. camphorata P5CD, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged TcP5CD was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a major band of ~59.4 kDa by 12 % SDS‐PAGE. The Michaelis constant (KM) value for 3,5‐dimethoxybenzaldehyde with NAD+ as cofactor was 0.56 mM. The enzyme’s half‐life of deactivation at 45oC was 4.4 min, and its thermal inactivation rate constant kd was 1.23 x 10‐2 min‐1. The enzyme was most active at pH 7.0. The enzyme’s preferred substrate is veratraldehyde. It can also use other aldehyde as substrates including acetaldehyde and propionaldehyde.

Grant Funding Source: National Science Council of the Republic of China under grant NSC95‐2313‐B‐019‐007 to C‐T. L.
Leucine‐aminopeptidase activities in heavy‐metal contaminated soils from brownfields of Liberty State Park (583.6)
Authors: Mathieu Carolyn , Hagmann Dianne , Krumins Jennifer , Goodey Nina
Published: June 2021

The brownfields of Liberty State Park served as rail yards for the Central New Jersey Terminal until the 1960s. These areas contain heavy metals such as Cd and Pb, which vary along a gradient. Previous research, such as the study done by Lee and Baek on a general shooting ranging in South Korea, has concluded that initial addition of heavy metals can inhibit enzymatic activities in soils. However, there was no known ongoing‐addition of heavy metals in our study sites since the closing of the rail station; concentrations of the heavy metals continued to persist in the soil for over the past 50 years. The purpose of this research is to determine the effect of persistent heavy metals concentrations on the activity of leucine‐aminopeptidase in the different brownfield sites at Liberty State Park. Fluometric analysis was used to determine the amount of the product amino‐7‐methylcoumarin (AMC) present in a given soil sample. The substrate analog leucine‐amino‐7‐methylcoumarin (LAMC) was added to the soil samples and enzymatic activities was determined by the amount of AMC produced over time. Interestingly, results showed an increase in the enzymatic activity of leucine‐aminopeptidase in the soil samples of the high heavy metal concentration sites compared to those of the lower heavy metal concentration sites
Hemoglobin binding and catalytic heme extraction by the IsdB receptor of Staphylococcus aureus (583.7)
Authors: Bowden Catherine , Verstraete Meghan , Eltis Lindsay , Murphy Michael
Published: June 2021

The Isd (Iron‐regulated surface determinant) system is a multi‐protein transporter that enables Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall‐anchored surface protein that contains 2 NEAT domains and rapidly extracts heme from Hb. Using a series of recombinant IsdB constructs which included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB only extracted heme from oxidized metHb although it also bound oxyHb and the Hb‐CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed heme transfer from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 μN and a kcat of 0.22 ± 0.01 s‐1. The latter is consistent with the transfer of heme from metHb to IsdB as being the rate‐limiting step.

Grant Funding Source: Supported by CIHR grant MOP‐49597
Investigating structural determinants of maleamate amidohydrolase (NicF) substrate specificity (584.3)
Authors: Kondow Helena , Snider Mark
Published: June 2021

NicF catalyzes the hydrolysis of maleamate to maleate in the degradation of nicotinate to fumarate. Kinetic assays, using 1H NMR and UV spectroscopy, were developed to test substrate analogs for reactivity with NicF. Although (Z)‐methyl 4‐amino‐4‐oxobut‐2‐enoate, E‐maleamate, and succinamate showed evidence to be slow substrates, the first was shown to be hydrolyzed to maleamate and the second was potentially isomerized to maleamate. To quantify the thermodynamic basis of succinamate as a slow substrate, temperature dependence of the NicF‐catalyzed reaction with maleamate, and with succinamate, were determined. An observed difference of ΔΔH‡ = 0.6 kcal/mol and TΔΔS‡ = ‐3.1 kcal/mol at 25°C in the reaction of succinamate compared to that of maleamate indicates that the presence of a double bond in the natural substrate appears to play a larger role in productive substrate binding than in potential electronic effects to enhance the electrophilicity of the substrate. Three NicF variants (F34A, F83A, and T146A) were engineered to further test hypotheses concerning the substrate specificity and chemical mechanism of the NicF‐catalyzed reaction.

Grant Funding Source: Supported by a HHMI Undergraduate Science Education Program Award at the College of Wooster
Probing the role of active site residues in an antibiotic‐resistance methyltransferase (584.9)
Authors: Hernandez Jennifer , Kuiper Emily , Conn Graeme
Published: June 2021

Many antibiotics target the bacterial ribosome to inhibit the translation of bacterial proteins, resulting in decreased bacterial growth. As pathogenic bacteria become progressively resistant to antibiotics, the need for in‐depth understanding of resistance‐conferring mechanisms becomes more critical. Streptomyces azureus expresses thiostrepton‐resistance methyltransferase (Tsr), an enzyme that confers resistance by modifying its antibiotic binding site, specifically catalyzing 2’‐hydroxyl‐methylation of adenosine 1067 on 23S rRNA. Six amino acids surrounding the S‐adenosyl‐L‐methionine (SAM) cofactor, conserved in SAM‐dependent methyltransferases, are believed to play a critical role in cofactor binding and/or catalysis. Mutating these residues was hypothesized to disrupt methylation by Tsr. Residues in the catalytic domain were individually mutated using site‐directed mutagenesis. Tsr mutants were purified using fast protein liquid chromatography (FPLC). The formation of Tsr‐cofactor complex and the catalytic activity of Tsr were evaluated with isothermal titration calorimetry and a methylation assay respectively. All Tsr mutants showed reduced methylation, but similar binding affinity to the cofactor product as wild‐type Tsr. Therefore, the major effects of the mutated amino acids are on the Tsr catalytic mechanism. Elucidating the molecular mechanisms by which Tsr recognizes and methylates its substrate will provide insight into how Tsr and similar methyltransferases confer resistance to antibiotics.
Evaluation of new cathepsin K inhibitors (585.4)
Authors: Ellison Amanda , Wen Lisa , Huang Jenq‐Kuen , McConnell Rose
Published: June 2021

Cathepsin K is a lysosomal cysteine protease that is predominantly expressed in osteoclasts. It has also been found in elevated levels in people with rheumatoid arthritis, prostate cancer, and breast cancer. Due to its strong collagenase activity, cathepsin K has been described as a major enzyme responsible for the turnover of extracellular matrix proteins and plays a fundamental role in bone resorption. Cathepsin K is a novel drug target for osteoporosis, osteoarthritis, and bone metastasis. Here, we report screening and evaluation of five new synthetic inhibitors against recombinant cathepsin K. The recombinant human procathepsin K was overexpressed in E. coli, purified from inclusion bodies, and activated by pepsin treatment. Cathepsin K inhibition assay was performed spectrophotometrically with N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride as the chromogenic substrate. Data analysis of the effectiveness of these inhibitors will be used to foster future development of newer effective inhibitors for the treatment osteoporosis and possibly rheumatoid arthritis and cancer.

Grant Funding Source: National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1) and WIU
Structural and biological characterization of NOS inhibitors with antimicrobial properties (585.5)
Authors: Holden Jeffrey , Li Huiying , Richo Jerry , Lim Nathan , Jing Qing , Kang Soosung , Huang He , Silverman Richard , Poulos Thomas
Published: June 2021

Nitric oxide produced by bacterial nitric oxide synthase (bNOS) protects Gram‐positive pathogens Bacillus anthracis and Staphylococcus aureus against oxidative stress and a wide variety of antibiotics. Recently, we demonstrated that co‐treatment with NOS inhibitors increases the killing efficacy of a common antimicrobial(1). Taking an interdisciplinary approach using microbial in vitro assays, spectroscopic binding analyses, enzyme kinetics and X‐ray crystal structures we have identified a series of lead inhibitors for future structure‐based drug design. Crystal structures of lead bNOS inhibitors have revealed an unprecedented mode of binding to the bacterial NOS that has not been previously observed in mammalian homologs. Continued exploitation of this novel‐binding mode will be imperative for rationally designing a potent and selective bNOS inhibitor.

Grant Funding Source: National Institute of Health
Biochemical response to acute temperature change in Atlantic killifish, Fundulus heteroclitus (586.1)
Authors: Biederman Amanda , Williams E. Eugene
Published: June 2021

It is documented that after temperature acclimation, the killifish Fundulus heteroclitus shows differences in the activities of a number of enzymes including lactate dehydrogenase (LDH), an important catalyst in anaerobic respiration. Less well understood is the temporal pattern of change in activity from one temperature to another. We acclimated fish to 22°C, and shifted the temperature such that an equilibrium was reached at 4°C after 9 hours. During the shift, we collected liver and white muscle samples and assayed homogenates for the specific activity of LDH. After three hours, LDH activity in liver increased from 22.2 µmol·min‐1·mg protein‐1 to 75.1 µmol·min‐1·mg protein‐1 , but then dropped by nine hours to 25.4 µmol·min‐1·mg protein‐1. Conversely, LDH activity in muscle decreased from 154 µmol·min‐1·mg protein‐1 to 61.3 µmol·min‐1·mg protein‐1 and continued to decrease at a slowed rate. These data demonstrate that F. heteroclitus adjusts LDH activity extremely rapidly in response to temperature change. We are currently determining the response to the opposite temperature shift (4 to 22°C), and we plan to assess several enzymes associated with aerobic respiration and other aspects of metabolism. Together, these analyses will help us determine which major metabolic pathways are most critical to this species during rapid temperature change.

Grant Funding Source: Supported by the Guerrieri Foundation and the Salisbury University Department of Biological Sciences
Complex regulatory interplay of γ‐catenin and β‐catenin in liver cells (59.10)
Authors: Zhou Lili , Li Zongfang , Monga Satdarshan Singh
Published: June 2021

β‐catenin, an adherens junction (AJ) component and key Wnt pathway effector, regulates numerous developmental processes, and its aberrant activation is observed in hepatocellular carcinoma (HCC). The β‐catenin homologue γ‐catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. We have reported hepatocyte‐specific β‐catenin knockout (KO) mice to lack any cell‐cell adhesion defects due to increased γ‐catenin especially at the AJs. To further elucidate the role and regulation of γ‐catenin‐β‐catenin relationship, we modulated these molecules in HCC cells. β‐Catenin knockdown (KD) in HCC cells led to increased γ‐catenin, which was associated with its increased serine/threonine phosphorylation. γ‐Catenin KD led to increased β‐catenin levels and transactivation. Overexpression of γ‐catenin led to β‐catenin downregulation, with increased Serine‐45 phosphorylation and increased GSK3β activity. Transfection of γ‐catenin plasmid did not change TopFlash reporter activity in Hep3B cells. In fact, levels of cyclin‐D1, a known β‐catenin target, decreased notably. Overexpression of γ‐catenin also decreased E‐cadherin protein levels. Thus, complex regulatory interplay between γ‐catenin and β‐catenin is observed, which may have important biological implications in hepatic pathobiology.
Mutations and cavities in choline acetyltransferase (591.4)
Authors: Michael Demetria , Rogers David
Published: June 2021

Choline Acetyltransferase (ChAT) synthesizes acetylcholine, the neurotransmitter that carries signals across the neuromuscular junction. Point mutations in ChAT cause motor disorders, apparently by disrupting its active site structure, which is unstable due to many cavities in the enzyme core. One of the large cavities in ChAT was targeted for study and was “filled” by mutating a nearby residue. ChAT with a specific congenital mutant alone as well as ChAT with this congenital mutant plus the cavity‐filling mutation were constructed. The mutants were expressed and purified, thermal stability measured through a thermal shift assay, and activity measurements made using a coupled enzyme activity assay. It was discovered that the induced point mutation did rescue both the thermal stability and overall activity of the enzyme. Also, both the thermal stability assay and activity assay showed that the cavity‐filling mutation surpassed the thermal stability and activity of the wild type enzyme. It was concluded that this cavity is a good target for studying potential ligand binding in hopes of developing therapeutics for the disease caused by certain ChAT mutations.
A computational evaluation of the steric and electronic contributions to the stability of the structures of α− and β−D‐glucopyanose Part 1: conformational analysis of monosubstituted cyclohexanes and pyrans (593.2)
Authors: Brutofsky Christian , Abdulsalam Maryam , Baello Rofe , Balilli Rahih , Kasner Marc
Published: June 2021

Geometry optimizations at a variety of computational levels were carried out on a series of substituted cyclohexanes (‐CH3 ‐OH and ‐CH2OH) and similarly 2‐substituted tetrahydro‐2H‐pyrans. By comparing the relationship between structure and energy of the cyclohexane series to the tetrahydro‐2H‐pyran series, it is possible to isolate the steric factors that are the principle contributor to the energy in the cyclohexanes from the combined steric and electronic factors that are present in pyrans.

The optimized geometries and minimum energies for each compound were determined using HF, DFT and MP2 methods with increasingly complex basis sets. Thermal correction factors were applied to the conformational energies and the conformational free energy from the B3LYP/6‐311G++(d,p) optimizations closely reflected the available experimental values. Relaxed scans (also at the B3LYP/6‐311G++(d,p) level) of rotations of the exocyclic substituents helped to identifiy the steric contributions for different orientations of the rotamers. Analysis of the optimized geometries non‐bonded distances bond lengths, and bond angles contributed to an understanding of the steric contributions. Additionally, analysis of bond lengths and bond angles showed changes that reflected possible hyper‐conjugation (electronic) contributions to the stability of some configurations. The series of compounds selected in this study provide a series of model compounds that lead to modeling the steric and electronic contributions to the free energies energies of D‐glucopyranose.
Photocleavable dimerizer for the rapid reversal of molecular trap antagonists (597.3)
Authors: Ahmed Shubbir , Xie Jun , Horne David , Williams John
Published: June 2021

Developing a molecular tool that can rapidly turn on and off an acute antagonist will be immensely helpful to understand the initiation of a cellular program. Herein, we report the development of a photocleavable analog of AP20187, a cell permeable molecule used to dimerize FKBP fusion proteins and initiate biological signaling cascades, gene expression or disrupt protein‐protein interactions. We demonstrate that this reagent in conjugation with a molecular trap permits the ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g., endosome dispersion). More importantly, photocleavage of the analog permits the unique ability to follow the cell’s return to homeostasis. Herein, we demonstrated the fine temporal control of endosome dispersion and restoration using an LC8 trap. We posit that this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways (e.g., kinases). In toto, the combination of a molecular trap and the photocleavable analog opens up new avenues to study signaling events within a single cell in a whole organism.

Grant Funding Source: Supported by the generous support of R21NS071166 and also by P30 CA033572 from the NCI
Cholesterol binding is a determinant of canonical Wnt signaling activity of Dishevelled (598.3)
Authors: Xin Yao
Published: June 2021

The Wnt cell‐cell signaling pathway plays a critical and evolutionarily conserved role in directing cell fates through the β‐catenin‐dependent canonical pathway and other non‐canonical pathways like planar cell polarity (PCP) pathway. Dishevelled (Dvl) determines the direction of Wnt and PCP signaling, however, it is still unknown how Dvl directs the two pathways. Here, we show that cholesterol in the plasma membrane serves as a regulatory lipid, selectively driving the progression of canonical Wnt signaling over PCP signaling by specifically interacting with the PDZ domain of Dvl. Single Molecule imaging shows that cholesterol is essential for sustained association of Dvl with Axin in response to a canonical Wnt ligand, but not for the formation of the PCP signaling complex component like Damm1. After the treatment with the cholesterol oxidase, it shows a tremendously decrease of Dvl and Axin interaction. Single molecule tracking also shows local enrichment of cholesterol near the Frizzle receptor upon its binding to a canonical Wnt ligand through the Frizzle co‐localization with the cholesterol binding domain, D4. Collectively, our results suggest a new regulatory role of cellular cholesterol and a potential link between cellular cholesterol levels and canonical Wnt signaling activities.
Role of the desmosmal adhesion molecule desmoglein 2 in Crohn’s disease (60.4)
Authors: Spindler Volker , Vigh Balázs , Meir Michael , Flemming Sven , Schlegel Nicolas , Waschke Jens
Published: June 2021

The barrier function established by intestinal epithelial cells is essential for gut homeostasis and is compromised in inflammatory bowel disease (IBD). This function is dependent on junctional complexes comprising of a set of intercellular junctions, namely tight junctions, adherens junctions, and desmosomes. Previously, we have shown that intercellular adhesion mediated by the desmosomal adhesion molecule desmoglein2 (Dsg2) is required for the integrity of tight junctions. Here, we demonstrate that Dsg2 levels are dramatically reduced in biopsies of patients with Crohn’s disease. Furthermore TNFa, a central cytokine in IBD, led to loss of cell cohesion, reduced Dsg2 expression, and increased permeability in Caco‐2 and HCT116 cells. This was paralleled by loss of Dsg2 immunostaining at cell borders with concomitant reduction of the tight junction protein claudin1. These effects were mediated at least in part by increased activity of p38MAPK, since inhibition of this kinase restored intercellular adhesion and blunted the permeability increase induced by TNFa. Importantly, stabilizing desmosomal adhesion via a Dsg‐specific peptide ameliorated loss of barrier functions. Thus, our data indicate an important role of p38MAPK‐mediated regulation of desmosomal adhesion for epithelial permeability and suggest a contribution of reduced Dsg2 expression to the pathogenesis of Crohn’s disease.
CAR‐like membrane protein negatively regulates the intestinal barrier and promotes neutrophil transepithelial migration (60.6)
Authors: Luissint Anny‐Claude , Bennett Ashley , McCall Ingrid , Nusrat Asma , Parkos Charles
Published: June 2021

CAR‐Like Membrane Protein (CLMP) is expressed in the intestinal epithelium and has been shown to be essential for embryonic intestinal development in Humans and Zebrafish; however, the role of CLMP in adult intestinal homeostasis is unknown. In analyses of CLMP expression in the gut, we observed upregulation of CLMP under inflammatory conditions in human and murine colonic epithelium. In human intestinal epithelial cell lines (IEC), CLMP localized at contacts between adjacent cells, and soluble recombinant proteins consisting of the CLMP extracellular domain bound specifically to IEC expressing CLMP, suggesting that CLMP may engage in homophilic interactions. Interestingly, permeability as assessed by transepithelial resistance, was decreased after siRNA‐mediated loss of CLMP, while its overexpression enhanced permeability. CLMP overexpression in IECs also resulted in delayed wound healing, suggesting that CLMP may alter epithelial repair. Further expression analyses revealed surface expression of CLMP in human and murine neutrophils (PMN). Therefore, we evaluated whether CLMP may play a role in mediating PMN migration during inflammation and observed that anti‐CLMP antibodies inhibited PMN transepithelial migration in vitro. Altogether, our findings suggest that CLMP is an adhesion molecule that negatively controls intestinal epithelial homeostasis and promotes immune cells response.

Grant Funding Source: Supported by PR121194 Department of Defense, Peer Reviewed Medical Research Program
Pilin SpaC‐mediated epithelial adhesion is required for Lactobacillus rhamnosus GG‐induced probiotic effects (60.9)
Authors: Ardita Courtney , Mercante Jeffery , Kwon Young Man , Jones Rheinallt , Powell Domonica , Neish Andrew
Published: June 2021

Lactobacillus rhamnosus GG (LGG) is a widely used probiotic, and the strain’s salutary effects on the intestine have been extensively documented. We have demonstrated this strain can modulate inflammatory signaling, as well as epithelial migration and proliferation via stimulation of Nox1 dependent reactive oxygen species. However, how this strain mediates these effects is largely unknown. Here, we report that LGG’s probiotic benefits are dependent on the SpaC pilin subunit. By comparing LGG to an isogenic mutant that lacks SpaC (LGGΩSpaC) in a variety of in vitro and in vivo assays, we establish that SpaC is necessary for LGG to adhere to gut mucosa, that SpaC contributes to LGG’s ability to induce the epithelial generation of ROS, and that SpaC plays a role in LGG’s capacity to stimulate the phosphorylation of ERK in enterocytes. In addition, we show that SpaC is required for LGG‐mediated stimulation of cell proliferation, and protection against radiologically‐inflicted intestinal injury. The identification of a critical surface protein required for LGG to mediate its probiotic influence furthers our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat inflammatory bowel diseases and other intestinal disorders.

Grant Funding Source: Supported by NIH RO1 AI 64462
The regulation of the cell surface proteome by AMP‐activated protein kinase (604.5)
Authors: Ross Eden , Antonescu Costin
Published: June 2021

The many proteins at the cell surface (collectively, the cell surface proteome) control numerous cellular functions. The amount of each protein at the cell surface is dynamically controlled by endocytosis and recycling. AMP‐activated protein kinase (AMPK) is an important metabolic regulator that responds to energetic stress, such as increased cellular AMP:ATP and certain hormones. Studies examining a small number of cell surface proteins suggest that AMPK may have an important role in regulating endomembrane traffic. How AMPK may more broadly regulate the cell surface proteome is not known. We have refined a method to isolate the cell surface proteome from cultured cells, involving biotinylation of surface‐exposed proteins in intact cells and purification of biotinylated proteins, which we coupled to quantitative mass spectrometry to identify novel AMPK‐regulated membrane traffic phenomena. We find that activation of AMPK results in a decrease in the cell surface abundance of the β1‐integrin, revealing a mechanism by which cellular metabolism may regulate cell adhesion and migration. Our results provide novel insight into the regulation of the cell surface proteome by metabolic stress signaling.
cAMP stimulates exocytic delivery of vesicle‐associated membrane protein 2 in renal epithelial cells (604.6)
Authors: Caceres Paulo , Ortiz Pablo
Published: June 2021

Soluble NSF Attachment Protein Receptors (SNAREs) drive membrane fusion in eukaryotic cells. SNAREs of the Vesicle Associated Membrane Protein (VAMP) family reside in vesicles and mediate vesicle fusion. After fusion, VAMP remains inserted in the plasma membrane until retrieved for another round of fusion. Thus, exocytic delivery of VAMP could be used to measure VAMP‐mediated exocytosis. In renal epithelia like the collecting duct and the thick ascending limb (TAL), cAMP stimulates exocytic delivery of apical transporters (aquaporin‐2 and NKCC2) and this is mediated by VAMP2. However, the effect of cAMP in VAMP2 exocytic delivery has not been studied. We hypothesize that cAMP stimulates VAMP2 exocytic delivery in renal epithelia. We studied cultured TALs and LLC‐PK1 cells transduced with VAMP2‐GFP where GFP faces the vesicle lumen and the extracellular space after exocytosis. We labeled surface VAMP2‐GFP with an extracellular GFP antibody to differentiate it from intracellular VAMP2‐GFP. We observed a clustered distribution of VAMP2 at the surface by confocal microscopy (n = 5). Stimulation of cAMP with forskolin+IBMX enhanced the rate of VAMP2‐GFP exocytic delivery from 4.8 ± 0.9 to 8.7 ± 1.4 exocytic events/ min (81% increase, p<0.05, n = 4) as measured by surface biotinylation. To monitor VAMP2‐GFP exocytic delivery in real time, we quantified the number of exocytic events by total internal reflection fluorescence (TIRF) microscopy. We observed that cAMP increased VAMP2‐GFP exocytic events from 10 ± 4 to 87 ± 8 events/min/area (p<0.01, n = 3). We conclude that VAMP2 is distributed in surface clusters in renal epithelial cells and cAMP stimulates VAMP2 exocytic delivery to the plasma membrane.

Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Depicting a novel regulatory Bcl2/SHP/lncRNA H19 network in bile acid homeostasis (605.12)
Authors: Zhang Yuxia , Tsuchiya Hiroyuki , Smalling Rana , Cox James , Delker Don , Hagedorn Curt , Wang Li
Published: June 2021

Objective: The aim of this study is to investigate a novel function of the anti‐apoptotic protein Bcl2 in bile acid (BA) homeostasis. Methods: Bcl2 was overexpressed using adenoviruses in male C57BL6 livers. After two weeks, BA homeostasis, liver morphology, fibrosis, and inflammation were analyzed. RNA‐seq and GC/MS were used to identify changes in gene expression and metabolites, respectively. Results: Hepatic overexpression of Bcl2 largely induced serum BA and FGF15 levels and decreased total BA pool size and fecal BA output. Severe hepatocyte necrosis, fibrosis, and Kupffer cell activation were observed, which was accompanied by a marked induction of long non‐coding RNA H19. RNA‐seq identified significant downregulation of genes in bile acid synthesis and transport, but upregulation of genes in collagen formation and inflammatory response by Bcl2. The increases in TCA cycle and lipid metabolites were the most drastic metabolomics changes. Using a series of cell based biochemistry and molecular biology approaches, we found that Bcl2 induced rapid nuclear receptor SHP protein degradation via activation of the caspase 8‐caspase 3 pathway. Furthermore, bile acid directly induced H19 expression in cultured primary hepatocytes that was independent of Bcl2 activation by bile acid. Importantly, Bcl2 and H19 were both dramatically increased in human cirrhotic livers and in mouse cholestatic liver fibrosis models. The dysregulation of the Bcl2/SHP/H19 network resulted in the disruption of bile acid homeostasis which contributed to the development of cholestatic liver fibrosis. Conclusions: Our results uncovered a unique metabolic regulatory network that integrated Bcl2 and SHP with lncRNA H19 to control BA homeostasis.

Grant Funding Source: Supported by DK080440 (L.W).
Stearoyl‐CoA desaturase‐2 deficiency protects mice against high fat diet‐induced adiposity (605.16)
Authors: O'Neill Lucas , Ntambi James
Published: June 2021

Stearoyl‐CoA desaturase‐2 (SCD2) is the second most abundant Δ9 desaturase in mouse and is strongly induced in adipose tissue by high fat feeding. Remarkably, SCD2 deficient mice (SCD2KO) are protected against high fat diet induced adiposity. Compared to wild‐type controls, SCD2KO mice fed a high fat diet exhibit reduced adipose triglycerides, smaller adipocytes, and improved glucose tolerance. Currently, it is unclear why deletion of SCD2 plays such an important role in the development of adiposity given the presence of SCD1. Both SCD2 and SCD1 synthesize monounsaturated fatty acids (MUFAs), predominately oleate and palmitoleate, from saturated fatty acids, predominately stearate and palmitate. We hypothesize that in adipose tissue, SCD2 plays a differential role from SCD1 and that either the SCD2 protein itself or SCD2 derived MUFAs regulate the biosynthesis of triglycerides, the main form of energy storage in adipose tissue.

Grant Funding Source: NIH, ADA, MBTG, and SciMed GRS
cAMP stimulates diacylglycerol kinase‐θ phosphorylation in adrenocortical cells (605.18)
Authors: Cai Kai , Sewer Marion
Published: June 2021

Diacylglycerol kinase theta (DGKθ) is an intercellular lipid kinase that coordinates nuclear phospholipid metabolism and cortisol production in the adrenal cortex by generating phosphatidic acid (PA), an agonist of the nuclear receptor steroidogenic factor 1 (SF1). We previously found that cAMP signaling induces the transcription of DGKθ and increases nuclear DGK activity. Moreover, mass spectrometric analysis of DGKθ isolated from H295R human adrenocortical cells has revealed that DGKθ is a phosphoprotein. However, the functional significance of DGKθ phosphorylation is unclear. Additionally, the mechanism by which cAMP signaling regulates DGKθ activity is not known. We show that dibutyryl cAMP increases the phosphorylation of DGKθ in H295R human adrenocortical cells. Alanine substitution at Ser‐354 reduces cAMP‐stimulated phosphorylation impairs the ability of DGKθ to activate SF1‐dependent transcription of CYP17. Cycloheximide sensitivity assays revealed that the S354A mutant is more stable than the wild type. Notably, we also identify Ser‐241 as a second site that is phosphorylated, where the S241A mutant abrogates DGKθ‐stimulated activation of a CYP17 reporter gene. Since Ser‐241 lies near to two proline residues, Pro‐244 and Pro‐245, that have been identified as key for optimal catalytic activity, we postulate that mutation of these prolines also alters the ability of DGKθ to be phosphorylated at this site. Indeed, P244L and P245L impair SF1‐dependent CYP17 luciferase activity. In summary, we identify DGKθ as an effector in the cAMP signaling pathway and a target of protein kinase A.

Grant Funding Source: Supported by NIH DK084178.
Deletion of the cardiolipin‐specific phospholipase Cld1 rescues growth and lifespan defects in the tafazzin mutant: implications for Barth syndrome (605.4)
Authors: Ye Cunqi , Lou Wenjia , Li Yiran , Chatzispyrou Iliana , Hüttemann Maik , Lee Icksoo , Houtkooper Riekelt , Vaz Frédéric , Chen Shuliang , Greenberg Miriam
Published: June 2021

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects and, in humans, results in the life‐threatening disorder Barth syndrome (BTHS). Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL‐specific phospholipase, Cld1, which can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin‐deficient cells is caused by the decreased CL/MLCL ratio, not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the HAP transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth, and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1‐mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.

Grant Funding Source: the Barth Syndrome Foundation
Homology modeling of perilipin 5 (606.8)
Authors: Sarr Aboubackrine , Bennett Jennifer , Niday Zach , Tansey John
Published: June 2021

Lipid storage droplets (LSD) regulate the storage of triacylglycerols and cholesteryl esters in the eukaryotic cell. The Perilipin family is a group of five conserved genes coding for LSD‐binding proteins. At least one perilipin has been found in the coat of all currently known LSD. The goal of the project is to generate a threaded model of perilipin 5 from existing structures in the Protein Data Bank, identify putative conserved domains or folds in perilipin 5, and identify the location of important residues (such as phosphorylation sites) in the proposed model . The amino acid sequence of perilipin 5 was entered into threading programs RaptorX and itasser in attempt to find conserved domains. Predicted structures are indicative of perilipin 5 being approximately 80% α‐helical for the analyzed segment. Associated QMEAN4 scores ranged from 0.50‐0.83, indicating the homological models are reliable predictions. The interaction of perilipin 5 with the lipase ATGL and its co‐lipase CGI‐58 occurs in the carboxy‐terminus, which substantiates the need for further structural characterization of this domain.
A novel protein family controls trafficking and metabolism of glycosphingolipids (606.9)
Authors: Jovic Marko , Kim Yeun Ju , Toth Balazs , Tuymetova Galina , Tsiomenko Arnold , Balla Tamas
Published: June 2021

Glycosphingolipid (GSL) trafficking and metabolism are closely linked with cell fate determination, balancing signaling cascades controlling proliferation, apoptosis and autophagy. Altered cellular levels of complex glycosphingolipids often correlate with cancer progression, yet little is known about the principles that guide trafficking of these lipids.

Here we identified a family of proteins, consisting of five mammalian homologues of the yeast Sfk1 protein that are involved in ganglioside transport. These six‐transmembrane domain proteins are widely expressed, each showing a unique cellular localization spanning the ER, Golgi, lysosomes and plasma membrane. One of these proteins, DNA‐damage regulated autophagy modulator 1 (DRAM1), was recently described as a p53‐inducible lysosomal regulator of autophagy. However, the cellular function(s) of the Sfk1 homologues remains unknown. Here we describe that mammalian Sfk1 proteins control the cellular distribution and metabolism of complex GSLs and one of them, DRAM2, specifically regulates transport of ganglioside GM3 from the plasma membrane to the Golgi and lysosomes. DRAM2‐mediated trafficking of GM3, in turn, was found to be critical for processing GM3 into higher ganglioside species.

We propose that the Sfk1/DRAM family of proteins recognize GSLs within membranes and facilitate their sorting and hence their metabolic fate within the cell.

Grant Funding Source: Supported by the NICHD Intramural research program
O‐GlcNAcomic profiling reveals altered O‐GlcNAcylation of mitochondrial proteins in diabetes (608.4)
Authors: Ma Junfeng , Liu Ting , Wei An‐Chi , O'Rourke Brian , Hart Gerald
Published: June 2021

As a dynamically regulated post‐translational modification, O‐linked β‐D‐N‐acetylglucosamine addition (O‐GlcNAcylation) plays critical roles in various physiological events [1]. Aberrant O‐GlcNAcylation is involved in numerous diseases including diabetes and diabetic complications [2]. However, mitochondrial O‐GlcNAc proteins have far from being well characterized and their functions are largely unknown.

To this end, a refined β‐Elimination followed by Michael Addition with Dithiothreitol approach was combined with reversed phase liquid chromatography (RPLC) fractionation followed by tandem mass spectrometry, yielding dozens of O‐GlcNAcylated proteins from cardiac mitochondria. The identified O‐GlcNAc proteins are involved not only in energy metabolism (i.e., oxidative phosphorylation, the Krebs cycle, and beta‐oxidation) but also in many other processes (e.g., heme biosynthesis, and amino acid metabolism). Moreover, by incorporating isotopic dithiothreitol reagents, the relative site‐specific O‐GlcNAc occupancy was also uncovered in control and diabetic hearts.

Of note, multiple respiratory chain proteins have been found differentially O‐GlcNAcylated, as confirmed by western blots with O‐GlcNAc‐specific antibodies. Moreover, the O‐GlcNAcylation of respiratory chain proteins appears to be closely related to the alteration of mitochondrial dysfunctions including respiratory rate and production of reactive oxygen species. These results suggest that mitochondrial protein O‐GlcNAcylation underlies the etiology and progression of diabetic cardiomyopathy, providing a novel insight and, more importantly, new treatments to diabetes.

Grant Funding Source: Supported by NIH R01CA42486, R01DK61671, N01‐HV‐00240, P01HL107153.
Proteomic characterization of signal transduction by P2X7 in regulatory T cells (609.8)
Authors: Tedeschi Gabriella , Maffioli Elisa , Negri Armando , Romagnani Andrea , Grassi Fabio
Published: June 2021

ATP released from CD4+helper T cells upon TCR stimulation contributes to the activation of MAPK signalling through purinergic P2X receptors. T regs produce lower amounts of ATP than conventional CD4+ T cells, although the gene encoding P2X7 receptor is comprised in T regs signature genes. Activation of purinergic P2X7 receptor by ATP mediates T regs conversion to pro‐inflammatory T cells, thus promoting autoimmunity. We identified and characterized the phosphoproteome triggered by BzATP as P2X7 agonist both in stimulated and non‐stimulated T regs employing a SILAC‐based proteomic approach. C57BL/6 wild‐type (WT) and p2rx7‐/‐ T regs labelled with different isotopes (R0K0 or R10K8, respectively) in SILAC media were stimulated with BzATP. After Tregs stimulation, cells were lysed and combined prior to enzymatic digestion. Following fractionation by HILIC and further enrichement on a Titansphere Phos‐TiO resin, the phosphopeptides were analyzed by an LTQ‐Orbitrap velos. For identification and quantification of phosphopeptides, MaxQuant 1.3.0.5 was used
AKAP13 coordinates cAMP signaling and glucocorticoid production in H295R human adrenocortical cells (612.2)
Authors: Escajadillo Tamara , Sewer Marion
Published: June 2021

The cAMP pathway activates steroid hormone biosynthesis in the adrenal cortex by regulating the activity of various proteins. We have previously identified a macromolecular protein complex that modulates cortisol biosynthesis by facilitating communication between the endoplasmic reticulum (ER) and mitochondria. One of the proteins in this complex is A‐kinase anchoring protein (AKAP)‐13. AKAPs play a critical role in the spatiotemporal compartmentalization of cAMP signaling, with AKAP13 being implicated in integrating Rho and cAMP‐dependent protein kinase A (PKA) signaling. AKAP13 has also been shown to regulate extracellular‐signal regulated kinase (ERK) and protein kinase D (PKD) activity in other cell types. We hypothesized that AKAP13 regulates cortisol production by coordinating RhoA and PKA signaling at the mitochondrial‐ER interface, and that disruption of AKAP13 leads to mislocalized cAMP signaling. We show that silencing AKAP13 in H295R human adrenocortical cells impairs cAMP‐stimulated RhoA, PKA and PKD phosphorylation and decreases cortisol and DHEA production. Consistent with previous findings, silencing AKAP13 had no effect on ERK activation or on the activation of hormone sensitive lipase. We conclude that AKAP13 is critical for the relay of cAMP signaling for cortisol production.

Grant Funding Source: Supported by NIH DK094151.
Modulation of ERK and breast cancer cell growth by estrogen receptors (613.3)
Authors: Rofelty Angela , Schmitt John
Published: June 2021

Estrogen Receptors participate in both genomic and non‐genomic signaling pathways that affect breast cancer cell growth and proliferation. The growth‐enhancing actions of estrogen (E2) in MCF‐7 breast cancer cells are mediated through Estrogen Receptor alpha (ERα), activating a kinase pathway involving CaM KK, CaM KI, and ERK. Conversely, overexpression of Estrogen Receptor beta (ERβ) has been shown to restrict the transcriptional activity of ERα and inhibit breast cancer cell growth. Our goal was to explore the roles of ERα and ERβ in ERK activation and cell growth in MCF‐7 and MDA‐MB‐231 breast cancer cells. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERα antagonist MPP and siRNA directed against ERα. Inhibition of ERβ with its antagonist PHTPP did not reduce ERK activity following stimulation with E2. Interestingly, pretreatment of MCF‐7 cells with the ERβ agonist, FERb, prevented the activation of ERK by E2. Treatment of MCF‐7 cells with E2 stimulated an increase in cell growth that was blocked by pretreatment with MPP, but not by pretreatment with PHTPP. In both MCF‐7 and MDA‐MB‐231 cells, stimulation with FERb reduced cell growth slightly below control levels. Treatment of MDA‐MB‐231 cells with FERb rapidly increased ERK phosphorylation and this effect was blocked by pretreatment with PHTPP. Our data suggest ERβ differentially regulates ERK in MCF‐7 and MDA‐MB‐231 cells. While activation of ERβ inhibits the growth of both cell lines, ERβ prevents ERK phosphorylation in MCF‐7 cells, but enhances ERK phosphorylation in MDA‐MB‐231 cells.

Grant Funding Source: Supported by M.J.Murdock Charitable Trust #2011267