bluuebunny/crossref_metadata_2025 huggingface dataset:
Divalent metal ion effect upon Rv0045c esterase enzymatic activity
Authors: Hoops Geoffrey Charles , Savas Christopher , Johnson R. Jeremy
Published: June 2021

The Rv0045c protein from Mycobacterium tuberculosis has previously been characterized as an esterase enzyme. Protein sequence alignments with other known esterases indicated the presence of a serine esterase catalytic triad. Surprisingly, the enzymatic activity of Rv0045c was found to be sensitive to specific divalent metal cations. Enzymatic activity for the Rv0045c protein, overexpressed in Eschericia coli host as a His‐tag fusion, was measured via fluorescence emission spectroscopy using a fluorogenic substrate (kcat/KM = 140 M−1‐sec−1). Ni2+ was found to activate the protein >;5‐fold (kcat/KM = 1050 M−1‐sec−1) while Zn2+ was found to deactivate the protein (kcat/KM = <3 140 M−1‐sec−1), with other metals (Co2+, Ca2+, K+) having no significant effect. Removal of the His‐tag by thrombin cleavage did not significantly change the Ni2+ effect upon catalytic activity. The starkly contrasting effects of Ni2+ and Zn2+ cations therefore appear to be due to specific interactions with the native protein.
Determination of Allosteric Residues Involved in Ligand Selectivity in the Dihydrofolate Reductase Family
Authors: Patel Seema , Egbert Emily , Little Michael , Goodey Nina
Published: June 2021

Dihydrofolate Reductase (DHFR) is an essential enzyme for most organisms, ranging from bacteria to humans. It has essential functions in DNA biosynthesis and cell replication and cell growth can be diminished by inhibition of DHFR. It is critical to understand inhibitor specificity of DHFR homologs from different organisms. To design compounds that inhibit DHFR from infectious organisms while leaving human DHFR alone. A computational analysis was previously developed to predict residues away from the active site that are significant in ligand specificity in the DHFR enzyme family. Allosteric residues H38, A39, I86, W85, Q84, T125, F126, and Y127 (human DHFR numbering) were predicted to be key residues in ligand specificity in the DHFR enzyme family. To validate these predictions, mutations (H38N, A39Y, I86A, T125A, F126L, and Y127A) have been introduced into the B. Strearothermophilus DHFR gene and the mutants have been expressed and purified. We have measured the binding constants to select ligands (Trimethoprim, Raltitrexed, and Pyrimethamine) by equilibrium binding titrations using Fluoromax‐4. Comparisons on the ligand binding profiles of the mutants to those of the wild type enzyme have revealed that some of the mutated amino acid positions do have an effect on ligand selectivity and this work is still going on. Turnover (kcat) values have also been determined for the mutants and wildtype DHFR and showed that mutations do not affect catalytic activity, with the exception of the I86A mutant. Further comparison of the ligand specificity of double mutants with two mutated positions is currently in progress to investigate synergy between different positions in ligand binding specificity in this enzyme family.
The X‐ray crystal structure of the Acinetobacter‐Derived Cephalosporinase, ADC‐7, at 1.7 Å
Authors: Porambo Alex , Florek Nicholas , Powers Rachel , Wallar Brad
Published: June 2021

β‐Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. The Acinetobacter‐Derived Cephalosporinases (ADCs) are class C β‐lactamases found in A. baumannii and other Acinetobacter species that are responsible for resistance to penicillins, cephalosporins, and β‐lactam‐β‐lactamase inhibitor combinations. In order to probe the mechanism of substrate turnover, as well as to design novel β‐lactam antibiotics, it was important to elucidate the protein structure of an ADC enzyme. Here, we report the successful purification, crystallization, and determination of the crystal structure of ADC‐7 at 1.7 Å. This complete structure allows for the critical comparison of the overall structure and active site architecture of ADC‐7 with the known cephalosporinase, AmpC. Hopefully, our work will contribute to the development of a structure/function relationship for ADC‐7 that will provide insight into bacterial antibiotic resistance.
The N152T mutation in the class C beta‐lactamase, AmpC, can serve as a substrate selectivity switch
Authors: Baggett Vincent , Docter Brianne , Powers Rachel , Wallar Brad
Published: June 2021

The class C β‐lactamase, AmpC, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. While some active site residues have been characterized to be involved in the recognition and breakdown of the substrate, many of their specific roles in substrate selectivity are not fully understood. Previous work on the Enterobacter cloacae P99 cephalosporinase demonstrated that a mutation of a highly conserved residue in the active site (N152) can result in substrate selectivity changes. Here, we investigate the role of the active site residue asparagine‐152 (N152) in E. coli AmpC by mutating it to a threonine (T) residue and examining the effect that this mutation has on kinetic and structural properties with different β‐lactam drugs. We discovered that although the N152T mutation caused higher Km values with all substrates, N152T exhibits over 150 fold higher kcat value against cefotaxime. In addition, the N152T mutation provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC. To probe the mechanism behind the observed substrate selectivity switch, the crystal structure of AmpC N125T was determined at 2.05 Å. In comparison to the wild type AmpC structure, the small structural differences in the active site have been associated with the changes in the Km and kcat kinetic values as a way to uncover the specific role of N152 in the function of AmpC.
Protein Epistasis Revealed from Thermostability Profiles of Nicotiana tabacum 5‐epi‐Aristolochene Synthase
Authors: Crenshaw Charisse , Aljadeff Johnatan , Fernandez Irma , Laurendon Caroline , Defernez Marianne , Koo Hyun Jo , O'Maille Paul E. , Sharpee Tatyana , Noel Joseph P.
Published: June 2021

The limits of protein evolution are ultimately defined by biophysical constraints. Specifically, the maintenance of protein folding and stability is essential for the emergence novel enzyme function in the face of accumulating mutations. This fundamental requirement necessitates a viable mutational pathway towards beneficially altered function, but the frequency of such pathways has never been measured. To measure the biophysical constraints on the evolution of terpene biosynthesis, we performed a quantitative assessment of fold thermostability along all possible mutational pathways linking a wild type terpene synthase from Nicotiana tabacum (tobacco) to its catalytically evolved nonuple (9) mutant. We measured the thermal unfolding profiles of a 512 mutant (29=M9) library using a high throughput fluorescence‐based assay. These unfolding profiles revealed a broad range of stability phenotypes across the library. Co‐variation analyses across the folding and stability landscapes of the 9 mutant combinations identified several statistically significant non‐additive effects and distinct thermal unfolding profiles. Together, these computational and experimental approaches uncovered additional levels of protein epistasis beyond the previously identified catalytic landscapes of the M9 library [1].
Suppression of adipocyte differentiation and adipogenesis by extracts of sweet potato leaves
Authors: Lin Chia‐Yu , Chiang Tzu‐Yuan , Yin Mei‐Chin
Published: June 2021

Obesity is a major health concern due to its strong association with a number of diseases and metabolic abnormalities, including diabetes, dyslipidemia, and heart disease. Inhibition of adipocyte differentiation is an important strategy to inhibit obesity. In this study, we investigated the effects of sweet potato leaves (SPL) on the inhibition of adipocyte differentiation and adipogenesis in 3T3‐ L1 cells. SPL at 5mg/ml~0.5 mg/ml impaired 3T3‐L1 cell proliferation as indicated by MTT assay. To determine whether SPL reduces proliferation through cytotoxic effects, we measured LDH leakage into the culture medium in response to SPL treatment. SPL was found significantly to promote adipocyte cell death (P<0.05). To examine the effect of SPL on differentiation and adipogenesis, the postconfluent 3T3‐L1preadipocytes were exposed to differentiation medium in the presence or absence of SPL for 9 days. The inclusion of SPL in the media remarkably inhibited adipocyte differentiation and fat accumulation, evidenced by both Oil Red O staining and the measurement of intracellular triacylglycerol concentrations in a dose‐dependent manner (P<0.05). Moreover, SPL treatment also reduced ROS generation during adipocyte differentiation and adipogenesis. Our results suggest that SPL might be the potential agent for obesity therapies.
Leptin regulates surface expression of β ‐cell ATP‐sensitive potassium channels via an AMPK/PKA/Cofilin signaling cascade
Authors: Chen Pei‐Chun , Shyng Show‐Ling
Published: June 2021

An adipoinsular axis is a dual hormonal feedback loop in which insulin produced by pancreatic β‐cells increases leptin production from adipose tissue and leptin released from adipose tissue in turn down regulates insulin secretion. Leptin has been shown to reduce insulin secretion at least in part by increasing KATP channel conductance, a key regulator of β‐cell membrane potential in response to glucose metabolism. However, the mechanism by which leptin increases KATP currents is unknown. We report here that leptin increases KATP channel expression in the β‐cell plasma membrane. Using a combination of pharmacological and molecular genetics approach, we show that this effect is mediated by a signaling cascade involving activation of AMPK via phosphorylation at Thr‐172 and activation of PKA, which then leads to phoshorylation of the actin binding protein cofilin and actin depolymerization. Imaging and surface biotinylation pulsechase experiments show that increased surface KATP channel abundance in response to leptin is primarily a consequence of increased KATP channel trafficking to the cell surface and that this trafficking event is tightly correlated with actin depolymerization. Taken together, our findings suggested that leptin inhibit insulin secretion by recruiting KATP channels to the β‐cell plasma membrane via a AMPK/PKA/cofiln/F‐actin depolymerization signaling mechanism. (This work was supported by NIDDK057699.)
Can BPA leaching from baby bottles and take‐out containers contribute to the obesity epidemic?
Authors: Tarasenko Alexandra , Benitez Benedetta Sampoli
Published: June 2021

The prevalence of obesity has increased dramatically in the last two decades. Several studies suggest a link between Bisphenol‐A (BPA) exposure and obesity. Although BPA is banned now in baby bottles, many children could have been exposed to large amounts of this chemical by its leaching in baby bottles upon heat stress. BPA is commonly found in polycarbonate plastics and behaves as an endocrine disruptor at concentrations well below toxic levels. This study looks at the amount of BPA leaching in various baby bottles, upon microwave heating and boiling. Our samples include baby bottles from different years and foreign markets and BPA amounts were determined by an ELISA assay specific for BPA. Our results show that the baby bottles used to leach a concerning amount of BPA and that this leaching became more significant with repeated heating. When baby bottles were heated by water‐bath instead of microwaving, the amount of leaching was considerably less. A possible explanation for this phenomenon will be presented. Take‐out containers were also found to leach BPA. The amount of BPA leeched was found to be significant; however, this amount was not as high as in baby bottles non‐marketed as BPA‐free. In conclusion, the amount of leaching in baby bottles was high enough to suggest a possible source of BPA linked to the development of obesity. Research supported by the congressionally directed grant #P116Z10068A.
Palmitate‐induced impairment of melanocortin‐4 receptor
Authors: McDaniel Faith Kennedy , Baldini Giulia
Published: June 2021

Obese patients, and diet induced obese rodents, exhibit elevated palmitate in the blood stream and hypothalamus. Elevated palmitate has been found to attenuate hypothalamic insulin signaling, and this effect is thought to be mediated through endoplasmic reticulum (ER) stress. Here we investigated the role of elevated palmitate on melanocortin‐4 receptor (MC4R) expression and signaling. Treatment with 200 μM palmitate (highp‐hysiological) and 400 μM palmitate (supraphysiological) for 24 hours in N42 immortalized hypothalamic neurons lead to a loss of MC4R signaling concurrent with a decrease in cholesterol content. While treatment with 200 μM palmitate did not induce an increase of GRP78/BiP or apoptosis, treatment with 400 μM palmitate did. In Neuro2a cells stably transfected to express the reporter HAMC4R‐ GFP we observed similar effects of palmitate treatment on MC4R signaling and cholesterol. Through the reporter we observed a loss of up to 70% of the MC4R protein, and a decreased rate of internalization which lead to a redistribution of the receptor to the plasma membrane. Repletion of cholesterol reversed the accumulation of MC4R at the plasma membrane, but did not recover MC4R expression. We conclude that elevated palmitate induces a loss MC4R expression and signaling before there is an overt ER stress. This research was supported by National Institutes of Health GrantsR01‐DK080424 (to G. B.)
Optimizing the binding affinity of a peptoid antagonist of VEGFR2 through backbone modifications
Authors: Cox Jacqueline Arely , Rubin Garret , Aditya Animesh , Kodadek Thomas
Published: June 2021

Activation of vascular endothelial growth factor receptor‐2 (VEGFR2) by VEGF is a critical step in angiogenesis. Antagonists of this process are employed clinically to treat certain cancers and wet macular degeneration. The best antagonists are anti‐VEGF antibodies such as Avastin®, but antibody‐based medicines are expensive, difficult to produce and are not orally bioavailable. The purpose of this project was to identify a synthetic VEGFR2 antagonist with selectivity for VEGFR2 comparable to that of an antibody as a first step in the development of better anti‐angiogenic drugs. Previously, a one‐bead one‐compound peptoid library was screened and a hit from the screen, named GU40C, was found to bind to VEGFR2 with a KD of 2 μM. To improve the binding of this antagonist, this experiment focused on modifying GU40C by incorporating various heterocyclic building blocks into the GU40C backbone. Three heterocyclic carboxylic acid derivatives were used in solid phase synthesis of backbone modified GU40C analogs. The binding affinity of these modified peptoids was measured by a ligand displacement assay on an ELISA platform. This experiment revealed positions that were more tolerant to modifications than others, generating several analogs with a higher binding affinity than the original GU40C lead. The synthesis and biochemical evaluation of backbone‐modified GU40C analogs will be presented. (NHLBI‐N01‐HV‐00242)
Carbon monoxide (CO)‐dependent modulation of polymorphonuclear leukocyte (PMN) adhesion to human cerebrovascular endothelial cells in an in vitro model of endotoxemia
Authors: Serizawa Fukashi , Patterson Eric , Potter Richard F , Fraser Duglas , Cepinskas Gediminas
Published: June 2021

CO liberated from a water‐soluble CO‐releasing molecule (CORM‐3) suppresses sepsis‐induced inflammation. However, the exact mechanisms are poorly understood.

In this study we examined the mechanisms of CORM‐3‐dependent modulation of PMN adhesion in an in vitro model of endotoxemia employing human‐derived cerebrovascular endothelial cells, hCMEC/D3 (provided by Dr. P. Couraud, INSERM, Paris). hCMEC/D3 were stimulated with LPS (1μg/mL) for 6 hrs. In some experiments hCMEC/D3 were: 1) pre‐treated, 2) co‐treated, or 3) treated with CORM‐3 or inactive CORM‐3 (iCORM‐3) (200μM) before/during/following stimulation with LPS, respectively.

PMN rolling/adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7dyn/cm2). In parallel, MAPK‐signaling (phosphorylation of p38, ERK1/2 and JNK1/2; Western Blot) and expression of adhesion molecules (E‐selectin, ICAM‐1 and VCAM‐1; RT‐PCR) were also assessed.

CORM‐3 significantly reduced PMN adhesion to hCMEC/D3 with the most profound effects seen in “pre‐treatment” group. This was accompanied by decreased levels of JNK1/2 Tyr‐phosphorylation and suppressed expression of E‐selectin and VCAM‐1.

The results indicate that CORM‐3 interferes with activation of JNK1/2 phosphorylation and expression of hCMEC/D3 pro‐adhesive phenotype, thus potentially could be used to reduce systemic inflammation (HSFO‐393, HSFO‐NA6914, IRF‐04–79)
Contribution of E‐selectin Ligands and the Chemokine CCL20 to Th17 Lymphocyte Rolling and Transendothelial Migration
Authors: Grodecki‐Pena Anna , Knapp Andrew , Salvador Ane Miren , Alcaide Pilar
Published: June 2021

IL17 producing T helper lymphocytes (Th17 cells) are often recruited at sites of tissue inflammation. This requires interactions with the vascular endothelium, and these interactions are quantitatively different from other T cell subsets (e.g, Th1 cells) and highly dependent on E‐selectin, ICAM‐1 and the chemokine CCL20. CD44 and CD43 cooperate with PSGL‐1 to mediate Th1 cells E‐selectin rolling, but their role in Th17 cells is unclear. Similarly, the role of CCL20 in Th17 cell transendothelial migration (TEM) has not been explored. We examined the contribution of the E‐selectin ligands PSGL‐1, CD44 and CD43 to Th17 cell adhesion to E‐selectin using wild type (WT), CD44−/−/PSGL‐1−/− and CD43−/−/PSGL1−/− deficient mice. Flow cytometry revealed that Th17 cells expressed all three ligands, with CD44 and PSGL‐1 at levels comparable to Th1 cells, and CD43 at significantly higher levels. Very few CD43−/−/PSGL1−/− Th17 cells accumulated on E‐selectin under shear flow conditions compared with WT or CD44−/−/PSGL‐1−/− cells. Blot rolling studies on Th17 lysates revealed a main reactive E‐selectin rolling area corresponding to CD43. Using WT and ICAM‐1−/− endothelial cells, we also examined the ability of CCL20 to induce Th17 cell TEM and whether this was dependent on ICAM‐1. Our data shows that Th17 cell rolling on E‐selectin is mainly mediated by CD43 whereas Th17 cell TEM requires CCL20 and ICAM‐1. NIH K99‐HL097406.
Computerized simulation and integration of biosystems based on the mass‐action law algorithms
Authors: Chou Ting‐Chao
Published: June 2021

Physico‐chemical principle of the mass‐action law has been merged with mathematical induction/deduction to derive the general “median‐effect equation” that can be used for deriving Michaelis‐Menten eq., Henderson‐Hasselbalch eq., Hill eq. and Scatchard eq. (Chou TC. Pharmacol. Rev.58: 621–681, 2006; http://pharmrev.aspetjournals.org/cgi/reprint/58/3/621). The “median” is the common‐link for the first order and higher order dynamics, the universal reference point for single entity parameters and for multiple entities interaction, and for synergism or antagonism simulation and quantification (Chou, Cancer Res. 70: 440–446, 2010; Am. J. Cancer Res. 1: 925–954, 2011). Median is also the harmonic mean of kinetic constants. All dose‐effect curves whether hyperbolic or sigmoidal, single entity or multiple entities can be linearlized into straight lines by the median‐effect plot and thus the theoretical minimum requires only “two data points” for drawing its dose effect “curve”. This insight offers an efficient, quantitative approach for “econo‐green bio‐research” using small size experimentations. Applying this theory in this author's laboratory (personnel ≤ 5 for decades) has published 253 articles that have been cited 13,716 times in over 523 different biomedical journals with h‐index of 55 (Thomson Reuters Web of Science, www.researcherid.com/rid/B‐4111–2009) along with 30 US patents. Thus, the mass‐action law based theory and algorithms couple with computer simulation (CompuSyn, www.combosyn.com) have proven to facilitate an efficient and quantitative biomedical research and new drug R&D, as illustrated in Chou, Integr. Biol. 3: 548–559, 2011; DOI: 10.1039/c0ib00130a.
Reflections on the use of protease inhibitor cocktails for preserving protein in tissue lysates
Authors: Hoegler Carl S , Murphy‐Goldberg Gabriela , Ponessi Louis , Blando‐Hoegler Charlene F
Published: June 2021

Commercially‐available protease inhibitor cocktails (PI) are routinely included in tissue lysates to insure that there is no loss of protein integrity of tissue samples. In view of the paucity of published research, our study investigated whether such inclusion was efficacious. We examined the effects of four protease inhibitor cocktails on rat skeletal muscle protein lysates. Total protein band analysis was performed after polyacrylamide gel electrophoresis (PAGE); in addition, myosin light chain (MLC) banding was compared after Western blot. Our results showed that, although most of the tested PI's do not appear to alter the protein content or band pattern (from 10 to 250kD) of the samples, anomalies were evident. One PI significantly reduced the measured amount of MLC. In another experiment, muscle (biceps femoris) lysates were treated with protease (trypsin) and incubated at 37°C for one hour. The enzyme treatment eliminated all of the protein bands (including MLC). In trypsin‐treated lysates containing PI's, most protein bands were rescued from hydrolysis to varying degrees. Restoration of MLC bands varied with the PI. Our conclusion from these studies is precautionary; we recommend that investigators reflect on their routine use of any PI. (Support from SURE Program at MSMC and Pace Scholarly Research)
Luciferin analogs for improved genetic reporter assays
Authors: Kovic Yumi , Rosenberg Justin C. , Behney Curran E. , Southworth Tara L. , Branchini Bruce R. , Woodroofe Carolyn C. , Meisenheimer Poncho L. , Klaubert Dieter H.
Published: June 2021

In an effort to produce new firefly luciferin analogs for application to dual reporter assays, we prepared and evaluated novel substrate analogs. The benzothiazole ring structure of the natural substrate that produces light with firefly luciferase and ATP was replaced with the benzimidazole, benzofuran, benzothiophene, benzoxazole and indole rings. In vitro testing with P. pyralis luciferase revealed that the benzothiophene‐containing analog BtLH2 had particularly interesting kinetic, bioluminescence and fluorescence properties. Particularly, bioluminescence reactions initiated with BtLH2 displayed slow decay glow kinetics that resulted in the substrate emitting more than four times the light compared to the natural substrate. In addition, the substrate sustains a blue‐shifted emission maximum at 523 nm over a pH range of 6.2 to 9.4 compared to its natural substrate counterpart, which shifts from green to red with increasing pH. The stable light emission signal and absence of color shift observed with BtLH2 demonstrates a potential for improved dual luciferase‐based genetic reporter applications using available red emitting luciferins.
Metabolic Lysosomal Enzyme Probes
Authors: Naleway John Joseph , Harlan Fiona Karen , Longo Nicola , Pasquali Marzia , Stein Jason Derek
Published: June 2021

Lysosomes are acidic cytoplasmic organelles that are present in nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of diseases including Parkinson's, Tay‐Sachs, Sandhoff, Krabbe and Gaucher's syndromes. New lysosomal staining probes useful for labeling lysosomes in a live‐cell format and capable of monitoring lysosomal metabolic activity have been developed. These new targeted substrates are based upon fluorescent probes that have a low pKa value for optimum fluorescence at the lower physiological pH values found in lysosomes, contain specific enzymatically‐cleavable functions for specific enzyme activities as well as targeting groups to direct their accumulation to the lysosomes using a live‐cell staining format. Application to the staining of cells derived from blood samples of patients with Metachromatic Leukodystrophy, Krabbe, Gaucher I, II and III, Tay‐Sach's and Sandhoff Diseases as well as healthy human fibroblast and leukocyte control cells are presented. In addition the ability to monitor the effect of secondary therapeutic agents on enzyme activity is presented. This work was supported by NIH Grant 5R44NS073225–04
Structure‐based engineering of an infrared fluorescent protein marker
Authors: Puetz Stephanie Lynn , Meraz Jacqueline , Varela Angela , Igova Brigitte , Baker Anna , Schirmer Aaron E. , Stojkovic Emina A. , Kennis John T.M.
Published: June 2021

Infrared fluorescent proteins (IFPs) emit a signal less prone to absorbance by hemoglobin and water, making them ideal candidates for mammalian imaging. Bacteriophytochromes (Bphs), bacterial red‐light photoreceptors, are an optimal source of IFPs. The required cofactor for Bph photochemistry, biliverdin (BV), exists naturally in mammalian cells as a product of heme metabolism. Recently, RpBphP2 (P2) from Rhodopseudomonas palustris was successfully engineered as an IFP marker for noninvasive mammalian imaging. RpBphP3 (P3), which shares a 52% sequence identity with P2, is naturally fluorescent due to an unusually long excited‐state lifetime. Comparative sequence and structural analyses of Bphs identified a highly conserved Asp residue essential for stabilization of BV in the chromophore‐binding domain. Using site‐directed mutagenesis we introduced a single amino acid substitution, D216H, effectively disrupting the photochemistry of P3 as observed by UV‐vis absorption spectroscopy. In addition, the P3 D216H mutant displays a higher fluorescence quantum yield relative to wild type P3. We cloned P3 D216H into the mammalian expression vector, Adtrack‐CMV. Stability of this protein marker will be characterized using in vitro comparison studies of protein maturation with the P2‐derived IFP. Subsequent research goals include expression and visualization of the P3 mutant in HEK293 and HeLa cell lines.
Antioxidants in your diet: a new method to measure antioxidant effectiveness
Authors: Bartow‐McKenney Casey , Belli Stuart , Caruso Francesco , Rossi Miriam
Published: June 2021

The superoxide radical is formed as a byproduct of aerobic respiration in biological systems. It is very reactive and can cause cellular damage leading to several degenerative diseases. Although biological systems are equipped with defenses against superoxide, dietary antioxidants are thought to aid in cell protection. Our goal was to develop a method to measure the effectiveness of exogenous antioxidants at sequestering and neutralizing the superoxide radical. We used cyclic voltammetry (CV) to both generate and measure the concentration of superoxide radical in solution. Antioxidant efficacy is then tested by quantifying the amount of radicals removed. A key challenge was correlating the measured signal, electrical current, with superoxide concentration. Superoxide is so unstable that accurately known standards are impossible to obtain, so we developed a titration with iodine solution using cyclic voltammetry as the end‐point indicator. The titration generates a standard calibration curve correlating electrical current (peak height on the CV) with the amount of radical in a solution of dissolved potassium superoxide. This curve also provides a way of measuring the concentration of superoxide radicals that have been electrogenerated from dissolved dioxygen via CV reduction, a straightforward and safer method of producing the radicals than earlier methods. Supported by HHMI 52006322 and VC URSI 2012.
Analysis of the Binding of Metals to Riboflavin Binding Protein
Authors: Hall Morgan , Snyder Marissa , Murray Tracey Arnold
Published: June 2021

Riboflavin binding protein (RBP) is a protein used primarily for the transport and storage of riboflavin, also known as Vitamin B2, in avian and reptilian eggs. Recent studies have shown the binding of copper (II) to RBP in a 1:1 molar ratio after dialyzing the RBP with copper, suggesting that riboflavin transport and storage may not be RBP's only function. Dialysis of RBP against additional metals at both a 1:1 molar ratio and physiological ratios was used to test the binding of other cations to RBP. The RBP concentration of the resulting samples was determined using UV‐Vis spectrophotometry and the bound metal ion concentration was determined using atomic absorbance spectroscopy. Results confirm the binding of other metals to RBP, and several of these metals are shown to outcompete copper (II) for RBP binding. These results also suggest that RBP may have another function, such as the transport, storage, and controlled release of essential metals inside the egg.
Study of the “structure‐biological activity” relationship in a number of flavonoids of plants of Kazakhstan
Authors: Zeinuldina Aizhan , Gabbasova Anar , Kazbekova Ainagul , Abduldayeva Aigul
Published: June 2021

The aim of the work was the study of the biological activity of technologically available flavonoid isolated from Salsola collina Pall. and Serratula coronata L. plants growing on the territory of Kazakhstan, as well as some modified derivatives of flavonoid quercetin and crude extracts. The method of acute hypoxia was applied in experiment to determine antioxidant activity of modified flavonoid and plant extracts. The results of in vivo bio screening on antioxidant activity have shown that quercetin and dry extracts of Serratula coronata L., as well as the extracts of Salsola Collina Pall. show remarkable antioxidant activity. It was found that the concentration of malondialdehyde in the brain and liver tissues of laboratory rats, which were preventively treated with flavonoids and extracts, is lower than in the control animals and is close to that of the intact group. Quantitative comparison showed that antioxidant effect is more significant in the case of extract of Serratula coronata L. Explanation of the high antioxidant effect in the studied plant of Serratula species lies in the composition of the extract. Feature of the whole extract of Serratula coronata L is the presence of molecules with a diphenyl structure, which opens up the possibility of participation in the reconstruction of biomembranes, lipid‐sensitive processes.
MPV17, DGUOK and Mitochondrial Depletion Disorders
Authors: Venkataraman Chantel M. , Field Martha S. , Stover Patrick J.
Published: June 2021

Mutations in DGUOK, a gene encoding a protein catalyst of the mitochondrial salvage pathway, and MPV17, a gene encoding an inner mitochondrial membrane protein of unknown function, have been shown to cause the hepatocerebral form of mitochondrial DNA depletion syndrome. MPV17 has been postulated to function as a nucleotide transporter. HeLa cell culture models were established using siRNA to model the mutations found in these patients. Following qPCR analysis, it was found that mtDNA copy number modestly decreased in both MPV17 and DGUOK siRNA‐treated cells cultured in nutrient replete medium. Surprisingly, MPV17 siRNA‐treated cells cultured in nutrient deplete medium exhibited a modest increase in mtDNA copy number. Individual nucleotide supplementation experiments were conducted, and the results indicate that pyrimidine nucleotide availability increases mtDNA copy number in MPV17 siRNA‐treated cells as much as 10‐fold.
Involvement of Seipin in Directing Localized PA for Lipid Droplet Formation
Authors: Han Sungwon , Chang Yu‐Fang , Cartwright Bethany , Binns Derk , Goodman Joel M
Published: June 2021

A loss of function of seipin, an endoplasmic reticulum (ER) membrane protein causes a severe congenital generalized lipodystrophy in humans. In yeast, deletion of seipin, FLD1, results in aberrant lipid droplets. However, the molecular mechanism of seipin is still unknown. Fei et al. (2011) reported that the deletion of FLD1 results in an increase in membrane phosphatidic acid (PA) concentration. In this study, we use Opi1p, a transcription repressor involved in phospholipid synthesis, as a PA sensor. Opi1p is known to bind to PA as well as the ER resident protein Scs2p. We report that Opi1p‐GFP forms puncta in the absence of FLD1. These puncta, usually next to droplets, are more pronounced with an Opi1p mutant (Opi1pFFAT) that cannot bind to Scs2p but still binds to PA. In order to test whether lipid droplets are necessary for the Opi1p‐GFP puncta in fld1Δ cells, we expressed Opi1p‐mCherry both in a mutant that cannot generate neutral lipids nor droplets (4KO; are1are2lro1dga1 KO), and one which also lacks FLD1 (5KO). Puncta were attenuated in these strains compared to fld1Δcells. In combination with the finding that Opi1p transiently localizes to droplets during inositol starvation (Chang et al, poster in this meeting), our data suggest that PA flux is important for droplet formation and that Fld1p facilitates utilization of PA during lipid droplet biogenesis. Supported by NIH grant GM084210.
Distinct roles of three acyl carrier proteins (ACPs) in Pseudomonas aeruginosa
Authors: Chen Wei , Gruber Jordon , Wang Bo , Zhang Yong‐Mei
Published: June 2021

Acyl carrier protein (ACP) is a key component in bacterial fatty acid biosynthesis by shuttling acyl intermediates to different enzymes. The human opportunistic pathogen, Pseudomonas aeruginosa, possesses three ACP homologues, Acp1, Acp3 and AcpP, whose roles have not been determined. In this study, we showed that only AcpP but not Acp1 and Acp3 complemented the AcpP‐null mutant of Escherichia coli, indicating AcpP is the canonical ACP essential for membrane fatty acid biosynthesis and survival. Consistently, knock out of acp1and acp3 did not affect cell growth and the membrane fatty acid composition, suggesting that Acp1 and Acp3 are involved in other processes than membrane biogenesis. Gene expression analysis using qRT‐PCR demonstrated that transcription of both acp1 and acp3 were activated by quorum‐sensing (QS) regulators LasR and RhlR. Considering Acp1 and Acp3 are unique for P. aeruginosa among all Pseudomonas species and fatty acids are substrates for molecules important for virulence including QS signals, we hypothesized that Acp1 and Acp3 were associated with QS signal biosynthesis and pathogenicity. To test this, we determined the concentrations of secreted QS signals and found that acyl homoserine lactones (AHLs) were significantly reduced in acp1 and acp3 mutants, but increased in Acp1 and Acp3 overexpression strains. However, major virulence factors, including pyocyanin, rhamnolipids and elastase, did not change significantly in vitro. Surprisingly, acp1 and acp3 deletion mutants exhibited more toxicity in both Caenorhabditi elegans and mouse infection models. Taken together, our results showed that Acp1, Acp3 and AcpP exhibit distinct roles in P. aeruginosa: AcpP is essential for membrane fatty acid synthesis whereas Acp1 and Acp3 are involved in virulence pathways.
Characterizing Mitochondrial Phostidylserine Decarboxylase 1
Authors: Onguka Ouma , Claypool Steven M
Published: June 2021

With the ability to synthesize phosphatidic acid, cardiolipin (CL), and phosphatidylethanolamine (PE), the mitochondrion is a major producer of cellular phospholipids. In yeast, there are two enzymes that make PE de novo, one of which, Psd1p, resides in mitochondria. In contrast, mammals only have the mitochondrial pathway for the de novo synthesis of this important cellular phospholipid. Psd1p decarboxylates phosphatidylserine (PS) to form PE. Knowledge about Psd1p is fragmentary. Psd1p is a nuclear encoded protein that is imported into the mitochondrion post‐translationally, where it undergoes proteolytic processing to become an active enzyme. The biogenesis of Psd1p involves three distinct processing events, with the final autocatalytic events generating the active alpha subunit. In my experiments I have shown that two of the processing steps are mediated by the matrix processing peptidase (MPP) and Oct1p. Oct1p is a soluble monomeric metalloprotease localized in the mitochondrial matrix that recognizes and cleaves substrates after initial processing by MPP. Oct1p proteolysis has recently been implicated as an important determinant of protein stability. Interestingly, Psd1p autocatalysis, the final processing step, does not appear to depend on the upstream activity of Oct1p raising the possibility that Oct1p processing is additionally important for Psd1p function. I hypothesize that Oct1p is required for Psd1p activity and/or stability, and in its absence aberrantly processed Psd1p has a reduced half‐life and/or less associated decarboxylase activity. Understanding the biogenesis of Psd1p may uncover potential regulatory steps in Psd1p biogenesis which may be exploited to modulate cellular PE content.
Identification of UAS domain as a motif polymerizing upon interaction with unsaturated fatty acids
Authors: Kim Hyeonwoo , Ye Jin
Published: June 2021

In cells fatty acids(FAs) are required for the synthesis of membrane phospholipid components and energy generation. However, overaccumulation of FAs is toxic. Accumulation of FAs prevents their further synthesis by stabilization of Insulin‐induced gene 1(Insig‐1) that inhibits proteolytic activation of sterol regulatory element binding protein‐1(SREBP‐1) that activates all genes required for FA synthesis. This stabilization is achieved through disrupting the interaction between Insig‐1 and UBX domain‐containing protein 8(Ubxd8). Here, we identified Ubxd8, a protein that does not contain any previously recognized domains that bind FAs, as a sensor for unsaturated FAs. In cultured cells, we demonstrated that unsaturated FAs but not saturated FAs stimulated polymerization of Ubxd8 through bimolecular fluorescence complementation assays. In vitro, unsaturated but not saturated FAs also stimulated polymerization of purified recombinant Ubxd8. The effect of different FAs and their derivatives on polymerization of Ubxd8 in vitro correlated with their effect on stabilization of Insig‐1 in cultured cells. Ubxd8 contains 3 domains, namely UBX, UBA and UAS domain. Deletion analysis showed that UAS domain was necessary and sufficient for polymerization of the protein in response to unsaturated FAs. Point mutations in UAS domain that disrupted its interaction with unsaturated FAs in vitro also impaired the ability of full length Ubxd8 to stabilize Insig‐1 in response to unsaturated FAs in cultured cells. In addition to Ubxd8, the only other protein expressed in mammalian cells that contains a UAS domain similar to that of Ubxd8 is Fas‐associated factor 1(FAF1). We showed that unsaturated FAs also specifically induced polymerization of FAF1, and this polymerization was mediated by the UAS domain. The identification of UAS domain as a motif polymerizing upon interaction with unsaturated FAs should provide more insights into cellular responses to FAs. [the National Institute of Health (HL‐20948/Jin Ye)]
Flippase function is required for signaling competence in the mating pheromone response pathway
Authors: Sartorel Elodie , Barrey Evelyne , Thorner Jeremy
Published: June 2021

In the plasma membrane (PM) of eukaryotic cells, phospholipid asymmetry appears to be important for cell signaling and cytokinesis. The outer leaflet of the PM contains predominantly phosphatidylcholine and sphingolipids, whereas the inner leaflet is enriched in phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth) and phosphatidylinositol and its phosphorylated derivative (PIP2). Members of the P‐type 4 ATPases (Flippase) maintain membrane asymmetry by translocating PtdSer and PtdEth from the outer leaflet to the cytosolic leaflet of the PM. In S. cerevisiae, five genes (DNF1, DNF2, DNF3, DRS2 and NEO1) encode flippases. In MATa cells responding to α‐factor, we found that Dnf1, Dnf2 and Dnf3, and a flippase activator (Fpk1 kinase), localize at the shmoo tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone MAPK cascade) is recruited. Moreover, a MATadnf1Δ dnf2Δ dnf3Δ triple mutant exhibits a reduction in its ability to respond to α‐factor due to a pronounced reduction in the stability of Ste5 resulting from an elevated rate of its Cln2·Cdc28 degradation. By contrast, a MATadnf1Δ dnf3Δ drs2Δ mutant also displays a reduction in its ability to respond to α‐factor, but in these cells the defect was attributable to inefficient recruitment of Ste5 to the PM because of severe mislocalization of the PtdIns4P and PIP2 pools.
VPA inhibits myo‐inositol phosphate synthase activity by inhibition of Gsk3
Authors: Yu Wenxi , Greenberg Miriam
Published: June 2021

Valproic acid (VPA) has been used for decades for the treatment of bipolar disorder. Yet, the therapeutic mechanism of VPA is not understood, hindering the development of more effective drugs. VPA causes inositol depletion in the human brain and in yeast cells. A similar degree of inositol depletion was observed in the yeast gsk3Δ mutant, in which the four glycogen synthase kinase 3 (GSK3) homologs were deleted. VPA was shown to inhibit Gsk3 in mammalian cells. These findings suggest that Gsk3 may play a role in inositol metabolism, and that VPA may cause inositol depletion by inhibiting Gsk3. The rate limiting enzyme in yeast inositol de novo synthesis is myo‐inositol phosphate synthase (MIPS). We hypothesize that Gsk3 is required for optimal MIPS activity. Consistent with this, the gsk3Δ mutant exhibited decreased MIPS activity. Although VPA inhibited MIPS activity in wild type cells, it did not inhibit MIPS in gsk3Δ cells, suggesting that VPA‐induced inositol depletion occurs by inhibition of Gsk3, leading to decreased MIPS activity. Of the four GSK3 homologs, we show that MCK1 is required for optimal inositol synthesis. mck1Δ exhibited multiple features of inositol depletion, including inositol‐dependent growth and decreased intracellular inositol. We conclude that inhibition of MIPS by VPA is dependent on Gsk3.

Funding: NIH‐DK081367 & Enhancement Award for Doctoral Student Travel
Investigating the Molecular Basis of cPLA2α Membrane Bending
Authors: Ward Katherine E , Ropa James P , Stahelin Robert V
Published: June 2021

Signal transduction mediates disease through key molecular targets in the cell. As protein‐lipid interactions have been examined in the literature, their role in cellular signaling has become more prevalent. Lipid‐binding proteins have become high impact drug targets in cancer, inflammation and viral egress. One such target, termed cytosolic phospholipase A2 α (cPLA2α), has been shown to play a key role in the production of inflammatory mediators. A novel function of the protein was recently discovered in our lab showing that the cPLA2α C2 domain bends zwitterionic membranes, a process that is mediated by cPLA2α's ability to deeply penetrate the lipid bilayer. Others in the literature have reported cPLA2α to participate in Fc mediated phagocytosis, intra‐Golgi trafficking and endosomal trafficking further supporting cPLA2α's ability to bend membranes in cellular processes. In addition, direct evidence has been reported in the literature using siRNA showing that cPLA2α induced vesiculation in cells. These results translate into our cellular system as cells transfected with EGFP‐cPLA2α form cytoplasmic vesicular structures. We have preliminary evidence showing cPLA2α membrane bending is mediated by oligomerization. The origin of oligomerization is currently under further investigation using both in vitro and cellular techniques.

This research is funded by AHA 11PRE7640028 and NIH T32GM075762
Mature VLDL Particles Exit the Golgi in Distinct Post‐Golgi VLDL Vesicles
Authors: Siddiqi Shadab A , Hossain Tanvir , Siddiqi Shaila
Published: June 2021

Post‐Golgi transport of VLDL is crucial for maintaining normal triacylglycerol (TAG) homeostasis of hepatocytes, however, nothing is known about the exit of VLDLs from the Golgi. To study this important process, we established an in vitro Golgi‐budding assay to examine the formation of Golgi‐derived VLDL vesicles using hepatic Golgi containing [3H]TAG as a marker for VLDL. Post‐Golgi vesicles (PGV) containing TAG‐rich VLDL were distributed in the low‐density portion of sucrose density gradient. Consistent with the biogenesis of ER‐derived VLDL transport vesicle (VTV), the generation of PGV required cytosolic proteins, ATP and incubation at 37 OC. PGVs were found to concentrate ApoB100, Apo‐AIV and Apo‐AI. The presence of Apo‐AI in PGVs, which is not present in the VTVs, suggested the differences in protein composition of two vesicles. Proteinase K treatment did not affect the PGV‐cargo proteins, apoB100, Apo‐AIV and Apo‐AI, indicating that PGVs were sealed. PGVs were able to fuse with plasma membrane as determined by in vitro PGV‐plasma membrane fusion assay. Electron microscopy revealed that PGVs were larger (~280–340 nm) than VTVs (~100–120 nm). Further biochemical and morphological characterization revealed that PGVs are different than VTVs and facilitate the transport of VLDL to the plasma membrane. This study was supported by NIDDK, NIH R01 DK081413 to SAS.
The molecular determinants of channelrhodopsin‐2 ion conductance
Authors: Dempski Robert E , Richards Ryan , Gaiko Olga
Published: June 2021

Channelrhodopsin‐2 (ChR2) is a microbial‐type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono‐ and divalent cations. Although this protein shares a high sequence homology with other microbial‐type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non‐covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. Furthermore, we used cysteine scanning mutagenesis combined with methanethiosulfonate labeling to examine the permeation pathway of channelrhodopsin‐2. An analysis of our experimental results provide important insight into the cation permeation pathway of channelrhodopsin‐2.
Critical Residues in the Extracellular Loop of the Epithelial Sodium Channel
Authors: Buck Michael Taylor , Whisenant Ty , Booth Rachell E
Published: June 2021

The epithelial sodium channel (ENaC) is responsible for maintaining sodium homeostasis in the body and is composed of 3 subunits: α, β, and γ. Each subunit has two membrane spanning regions, a large extracellular loop, and short cytoplasmic terminal tails. Although intracellular regulation of ENaC has been well characterized, the extracellular region has not. Our goal is to elucidate the intra‐ and inter‐subunit interactions needed in folding and assembly of the extracellular loop. Error‐prone PCR was used to generate random mutations in an extracellular region of αENaC. The resulting PCR products, which undergo recombination with a yeast expression vector, were transformed into BY4742 yeast cells and dilution pronging assays were used to monitor mutant ENaC function (i.e. salt sensitivity). We identified several residues including V1358, S1781, S1245, N1314, and G1790 within the extracellular region of mouse αENaC that appear to be critical for ENaC. Future studies will include surface biotinylation to assess the level of mutant ENaCs within the plasma membrane. Funding provided by NIH (R15GM086798) and Welch Foundation (Dept Grant A10048)