bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking protein myristoylation inhibits prostate cancer progression
Authors: Cai Houjian , Kim Sungjin , Alsaidan Omar Awad
Published: June 2021

Objective of the study

Protein N‐myristoylation enables localization to membranes and helps maintain protein conformation and function. N‐myristoyltransferases (NMT) catalyze co‐ or post‐translational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. The goal of this study is 1) to demonstrate that N‐myristoylation is essential for the tumorigenic potential of oncogenioc proteins such as Src kinase; 2) to identify novel lead compounds to inhibit the NMT enzymatic functions; 3) to demonstrate that the compound exhibits its inhibitory function on cancer progression.

Statement of Methods

NMT1/2 proteins were purified for the evaluation of their enzymatic functions. The proteins were further crystalized for study their structure. The myristoylation of Src kinase was detected by Click chemistry. Protein fractionation was evaluated by immunoblotting. A low throughput screening was performed to identify an myristoyl‐CoA analog to inhibit NMT enzymatic function. TCGA database was used for the analysis of the expression correlation. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate regeneration assay and xenograft tumor models were applied for in vivo studies.

Summary of the results

We provide genetic and pharmacological evidence that inhibiting the N‐myristoyltransferase NMT1 suppresses cell cycle progression, proliferation and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl‐CoA analog B13 as a small molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localizaiton to the cytoplasmic membrane, attenuating Srcmediated oncogenic signaling. B13 exerted its antiinvasive and antitumor effects against prostate cancer cells with minimal toxic side‐effects in vivo. Structural optimization based on structure‐activity relationships enabled the chemical synthesis of LCL204 with enhanced inhibitory potency against NMT1.

Conclusion

Our study demonstrates that B13, a myristoyl‐CoA analog, inhibits NMT enzymatic functions and Src mediated tumorigenesis. Our results offer a preclincal proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression.

Support or Funding Information

NIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatocyte growth factor receptor (HGFR) as a potential lung cancer target
Authors: Ahmed Haidar , Skouta Rachid
Published: June 2021

Lung cancer is the leading cause of cancer‐related death in the world affecting both sexes, with more than 222,500 new cases each year in the U.S, and poor survival rate. Due to low efficacy of current therapies, recent studies are focusing on discovering new targets that generates better and more effective therapies. While most of the current drugs target the epidermal growth factor receptor (EGFR) and KRAS, other targets have not been very well studied. c‐Met, also known as tyrosine‐protein kinase Met or hepatocyte growth factor receptor (HGFR), belonging to the tyrosine kinase family play a major role in signaling transduction pathways and has been a potential target of lung cancer. For this reason, kinase receptors have been the target of many modern drugs. In solid tumors, such as lung cancer, upregulation of c‐Met occurs as mechanism to promote proliferation, survival, metastasis and angiogenesis. Here, we present a low cost drug discovery approach to identify a novel effective inhibitor of c‐Met using structure based virtual screening (SBVS). We obtained c‐Met top crystal structures from the Protein Data Bank (PDB ID: 3BUX, 3F66, 3ZCL, 3DKC, 4R1V). Potential binding site were screened using Schrodinger software package to generate receptor grids. Using the obtained receptor grids a drug library was docked using Glide. After this docking results were compared by the free energy of binding. Using Phase e‐pharmacophore and “create consensus” top ligands were further screened to understand ligand‐protein interaction to improve efficacy of drug. Future directions is to adapt top compound to in vitro.

Support or Funding Information

This lab is supported in part by the USDA‐NIFA‐HSI grant 2012‐38422‐19910 and NIH Grant #5G12MD007592 from the National Center for Research Resources to the UTEP Border Biomedical Research Center. And by the Lung Cancer Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Localization of the Mutant SepG1 Protein to Sites of Cell Division in the Filamentous Fungus Aspergillus nidulans
Authors: Ugwu Uchenna , Jackson‐Hayes Loretta , Hill Terry William
Published: June 2021

Cell division (cytokinesis) in filamentous fungi involves inward growth of the plasma membrane at sites of crosswall (septum) formation, under the guidance of a contractile actomyosin ring (CAR). The process involves the coordinated activities of multiple proteins, most of which are poorly characterized or wholly undiscovered. A forward‐genetics approach to identifying proteins with unrecognized roles in cell division has been to artificially mutagenize cells and isolate viable progeny defective in septum formation (septation), followed by identification of mutation sites at the molecular level. Prior work in this laboratory has localized a classic temperature‐sensitive septation mutation, termed sepG1, in the model fungus Aspergillus nidulans to a gene locus predicted to encode an IQGAP homologue. Reduced expression of the wild type gene under the regulatable AlcA promoter blocks septation, and a GFP‐tagged version of the wild type protein localizes to sites where septa are forming. We have also succeeded in GFP‐tagging the protein encoded by the mutant sepG1 allele and have demonstrated that it too localizes to septation sites, even under restrictive conditions that prevent completion of septation, indicating that the sepG1 defect is not in protein targeting. Multiple attempts using Mendelian methods to create double‐mutant strains expressing both GFP‐tagged SepG1 protein plus RFP‐tagged versions of actin or myosin have not been successful, preventing investigation of whether the SepG1 protein associates normally with the CAR. Therefore, we have generated riboflavin‐auxotrophic strains containing untagged sepG1 mutant alleles along with RFP‐tagged wild‐type alleles of actin or myosin, with the goal of tagging the sepG1 allele in these strains via direct genetic transformation. We have constructed a plasmid‐based cassette containing sequences that, after integration, will allow the simultaneous expression of GFP‐tagged SepG1 protein in the same strains that are also expressing RFP‐tagged actin or myosin.

Support or Funding Information

National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Undergraduate biology students view scientific models as easy‐to‐learn simple explanations
Authors: Trujillo Caleb , Bennett Steve , Long Tammy
Published: June 2021

The inclusion of modeling in undergraduate science classrooms is warranted so that students engage in authentic scientific practices to understand the world. This study investigates students' perceptions of modeling by asking: what attributes do introductory biology students associate with scientific models? We conducted an exploratory mixed methods study with participants recruited from model‐based introductory biology courses across two interviews. During the first interview, students provided attributes of models including definitions, purposes, and qualities. An inductive analysis revealed that most students viewed models as explanatory visual representations that aid learning. During the second interview one year later, the same students assigned identified attributes to different representations in a sorting task. A correspondence analysis revealed that learners closely associate scientific models to simplistic easy‐to‐learn explanations, but not to data or predictions. The results suggest that students associate scientific models with explanations that aid learning by making complex content simple and comprehensible. Conversely, students perceive the attributes of empirical support and predictive power as distinct from scientific models. This study furthers an understanding of students' perspectives of models in a biology classroom and informs potential changes to model‐based instruction.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. DRL 1420492.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
S‐Succinylcysteine Breakdown – How Bacillus subtilis Utilizes an Inevitable Product of Metabolite Damage
Authors: Niehaus Thomas Daniel , Folz Jacob , McCarty Donald R. , de Crécy‐Lagard Valérie , Moraga David , Fiehn Oliver , Hanson Andrew D.
Published: June 2021

Cellular thiols such as cysteine and glutathione spontaneously and readily react with the respiratory intermediate fumarate to form stable S‐succinyl adducts. Succination of thiols by fumarate increases in certain tumors and in response to glucotoxicity associated with diabetes, thus S‐succinyl adducts such as S‐succinylcysteine (SSC) are considered to be on cometabolites and biomarkers for human disease. No plausible disposal routes for S‐succinyl compounds have been demonstrated. Here, we show that Bacillus subtilis has a pathway to metabolize SSC to cysteine and that this pathway is encoded by the yxe operon. The first pathway step is N‐acetylation mediated by the yxeL gene product, most probably follow‐ed by oxygenation and elimination of oxaloacetate to yield N‐acetylcysteine, which is then deacetyl‐at‐ed. In support of this breakdown pathway, knockouts of the predicted B. subtilis genes involved in each catabolic step of SSC breakdown lose the ability to grow on SSC as a sulfur source and accumulate the predicted upstream metabolite(s). We further show that N‐acetylation reduces SSC toxicity. SSC disposal substantiates the emerging concept that cells often handle toxic metabolites by pathways that begin with acetylation and end with deacetylation, in direct analogy to the blocking‐deblocking strategies used in organic chemistry.

Support or Funding Information

This work was supported by U.S. National Science Foundation grant MCB‐1153413 and by an endowment from the C.V. Griffin Sr Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Rising of Acylsugar Diversity: Metabolic Innovation in Tomato Trichomes through BAHD Enzyme Promiscuity and Pathway Evolution
Authors: Fan Pengxiang , Miller Abigail M. , Jones A. Daniel , Liu Xiaoxiao , Last Robert L.
Published: June 2021

In cultivated (Solanum lycopersicum) and wild tomatoes, glandular trichomes produce structurally diverse acylsugars. These insect protective metabolites are mixtures of aliphatic esters of sucrose and glucose, with acylchains varying in length from C2 to C11. We are analyzing acylsugar biosynthesis in the cultivated tomato, along with the biochemical and evolutionary mechanisms that generate acylsugar diversity across wild tomatoes (1, 2). Previous studies identified four BAHD family Acylsucrose Acyltransferase (ASAT) enzymes. These catalyze consecutive reactions from sucrose and acyl‐CoA substrates to produce the full set of cultivated tomato acylsucroses in vitro (3). Recently we identified a modified ‘upside down’ acylsugar pathway in the wild tomatoes S. pennellii and S. habrochaites that produces unusual types of acylsucroses, namely triacylsucroses with all three chains on the pyranose ring (P‐type) (4). This is in contrast with the furanose ring acylated acylsucroses (F‐type) produced in cultivated tomatoes and a variety of wild tomato species. Biochemical analysis demonstrates that the acyl acceptor substrate specificities were changed in two S. pennellii ASAT orthologous enzymes, which reversed their enzymatic activity order, leading to the ‘flipped pathway’. A phylogeny‐based structure‐function analysis of the ASAT2 and ASAT3 enzymes led to identification of a small number of amino acid changes that switch their activities between the F‐ and P‐ acylsucrose pathways (4, 5). Investigation of these pathways in other tomato species allowed inference of the evolutionary events giving rise to divergence of F‐ and P‐type acylsucroses across tomato species. This work demonstrates multiple evolutionary mechanisms in biochemical pathway evolution, including gene duplication and loss, amino acid substitution, and how the emergence of enzyme promiscuity restructured a specialized metabolic pathway and led to metabolic product innovation.

Support or Funding Information

This work was funded by National Science Foundation grants IOS‐1025636 and IOS‐PGRP‐1546617 to A.D.J. and R.L.L. Abigail Miller was supported by NSF REU grant DBI‐1358474 in the summer of 2014 and an American Society of Plant Biologists Summer Undergraduate Research Award during the summer of 2015. A.D.J. acknowledges support from the USDA National Institute of Food and Agriculture, Hatch project MICL‐02143.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipidomic Profiling Reveals Soluble Epoxide Hydrolase As a Therapeutic Target of Obesity‐induced Colonic Inflammation
Authors: Wang Weicang , Yang Jun , Zhang Jianan , Wang Yuxin , Hwang Sung Hee , Wan Debin , Sanidad Katherine Z. , Yang Haixia , Liu Jun‐Yan , Park Yeonhwa , Liu Zhenhua , Hammock Bruce D. , Zhang Guodong
Published: June 2021

Obesity is associated with enhanced colonic inflammation, which is a major risk factor of colorectal cancer. Considering the obesity epidemic in United States, there is an urgent need to identify novel therapeutic targets for obesity‐induced colonic inflammation. Eicosanoids are endogenous lipid signaling molecules involved in regulating inflammation and immune responses. Using a LC‐MS/MS‐based lipidomics approach, we found that obesity‐induced colonic inflammation is associated with increased expression of soluble epoxide hydrolase (sEH) and its eicosanoid metabolites, termed fatty acid diols, in colon tissue. To study the functional role of sEH, we treated mice with sEH inhibitors, TPPU or t‐TUCB, in high fat diet (HFD)‐induce obesity model, and found that pharmacological inhibition of sEH attenuated obesity‐induced colonic inflammation. Administration of sEH inhibitors diminished obesity‐induced infiltration of immune cells and expressions of pro‐inflammatory cytokines in colon tissue. Furthermore, we examined the sEH−/− mice in obesity model, and found genetic ablation of sEH reduced colonic concentrations of fatty acid diols and decreased the expressions of pro‐inflammatory cytokines. Finally, HFD treatment increased expression of phosphorylated GSK3β in colon tissue, while such effect was attenuated in sEH−/− mice, or by treatment with sEH inhibitors, suggesting that pharmacological inhibition and genetic ablation of sEH attenuated HFD‐induced activation of Wnt signaling in colon. Together, these results support that sEH could be a novel therapeutic target for obesity‐induced colonic inflammation and associated diseases.

Support or Funding Information

This work was supported by USDA NIFA grants 2016‐67017‐24423 and 2014‐67017‐21762, NIEHS grant R01 ES002710 and NIEHS Superfund Research Program P42 ES004699, and National Natural Science Foundation of China (NSFC) grants 81702832, 81672938 and 81470588.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYTOCHROME P450 EPOXYGENASE EICOSANOID PATHWAY AS NOVEL BIOMARKER AND THERAPEUTIC TARGET OF COLORECTAL CANCER
Authors: Zhang Guodong , Wang Weicang , Yang Jun , Edin Matthew , Hammock Bruce , Zeldin Darryl
Published: June 2021

In US, there are ~130,000 new cases and ~50,000 deaths resulted from colorectal cancer (CRC) every year, making CRC the second leading cause of cancer‐related deaths. There is an urgent need to identify novel biomarkers and/or therapeutic targets of CRC, in order to reduce the risks of CRC. Here we report that the expressions of cytochrome P450 (CYP) epoxygenases and their eicosanoid metabolites (fatty acid epoxides) are dramatically increased in cell culture model, animal model, and human cancer patients of CRC; in addition, pharmacological inhibition or genetic ablation of CYP epoxygenases suppresses development of CRC in mice, illustrating that the previously unappreciated CYP epoxygenase pathway could serve as novel biomarkers and/or therapeutic targets of CRC. In a well‐established azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced CRC model in C57BL/6 mice, LC‐MS/MS showed that CYP‐derived fatty acid epoxides were the only eicosanoid metabolites which were significantly increased in both plasma and colon tissues of tumor‐bearing mice. RT‐PCR and immunoblotting analysis demonstrated that the expressions of CYP (including CYP2C38, 2C39, 2C50, 2C65, 2C70, and 2J6) were significantly increased in colon tumors. Consistent with the animal results, LC‐MS/MS showed that the serum levels of CYP‐derived fatty acid epoxides were significantly increased in patients clinically diagnosed with CRC. To study the functional roles of CYP in CRC, we tested whether pharmacological inhibition or genetic ablation of CYP affects CRC development in mice. We found that treatment with SKF‐525A and clotrimazole, which are two different inhibitors of CYP epoxygenases, suppressed AOM/DSS‐induced CRC in mice. We further showed that compared with wild‐type (WT) control mice, the AOM/DSS‐induced CRC was reduced in Cyp2c gene cluster knockout mice. To elucidate the specific metabolites involved in the pro‐CRC effects of CYP, we treated mice with epoxyoctadecenoic acids (EpOMEs) which are metabolites of linoleic acid produced by CYP, and found that EpOMEs promoted development of colonic inflammation in mice. Together, our studies showed that the previously unappreciated CYP epoxygenase pathway plays critical roles in promoting progression of CRC, in part through formation of pro‐inflammatory EpOMEs. These results suggest that the circulating levels of CYP‐derived fatty acid epoxides could serve as novel diagnostic biomarkers of CRC, and the CYP enzymes could be promising therapeutic targets of CRC.

Support or Funding Information

This work was supported by USDA NIFA 2016‐67017‐24423 (to G.Z.). In addition, the research is supported in part by NIEHS R01 ES002710 and Superfund Research Program P42 ES04699.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ibuprofen increases resistance to exertional heat stroke in female mice
Authors: Garcia Christian Kyle , Alzahrani Jamal , Mattingly Alex , Laitano Orlando , Robinson Gerard , Murray Kevin , Clanton Thomas
Published: June 2021

The impact of nonsteroidal anti‐inflammatory drugs (NSAIDs) on the severity or susceptibility to exertional heat stroke (EHS) is unknown. Previous studies have demonstrated that in humans, thermogenic responses to endurance exercise are suppressed by NSAIDs and that NSAIDs increase the endurance time to exhaustion in rats. However, NSAIDs have also been shown to contribute to organ damage in both severe exercise alone and in conditions of heat stress alone, which may limit performance.

METHODS

In this study, we determined the effects of ibuprofen administration (IBU) on exercise performance and resistance to heat stroke in male and female mice in an established model of exertional heat stroke (EHS). Two days prior to EHS, the mice were given continuous IBU‐loaded chow (ad libitum) at 375 mg/kg (N = 7–8) compared to controls (N =44). To induce EHS, the mice ran a preprogrammed incremental exercise protocol using a forced running wheel in an environmental chamber at 37.5°C/35% RH, until a symptom‐limited max was reached (loss of consciousness). The EHS protocol was defined by an incremental exercise phase (IP) and a steadystate exercise phase (SP). During the IP, wheel speeds were increased every 10 min until core temperature (Tc) = 41°C. This was followed by a SP phase, in which speed was maintained until a symptom‐limited endpoint.

RESULTS

All groups ran to nearly identical peak Tcs (42.2–42.4°C). IBU‐treated female mice exercised longer in heat, prior to collapse, compared to controls (223 vs. 176 min; P = 0.0018), but there was no significant effect in males (133 min vs 124 min). Time in the IP was higher in the IBU‐female mice compared to controls (181 vs 131; P=0.0018). Though the duration of the SP phase did not change in any group, female IBU mice ran at significantly higher power outputs during the SP phase compared to controls (females: 34.5 mW vs 27.4 mW, P=0.0004) demonstrating a lower rate of heat accumulation for a given rate of external power expended.

CONCLUSIONS

These data demonstrate that in female mice, short‐term IBU treatment improves exercise performance in the heat and delays the onset of EHS. The results are consistent with the hypothesis that a component of the temperature elevation seen in EHS arises from endogenous pyrogen production that can be blocked by IBU. The lack of similar responsiveness in males may reflect greater sensitivity to EHS in male vs. female mice and predominance of other mechanisms limiting performance in the heat. Authors' views not official US Army or DoD policy. W81XWH‐15‐2‐0038

Support or Funding Information

Authors' views not official US Army or DoD policy. Department of Defense Grant W81XWH‐15‐ 2‐0038

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermoregulation Deficiencies in Mice with a Deletion in the Muscle Protein Titin
Authors: Miyano Carissa Ashley , Buck Charles Loren , Nishikawa Kiisa
Published: June 2021

The muscular dystrophy with myositis (mdm) mouse carries a deletion in the muscle protein titin. A previous study found that mdm mice shiver at a lower than expected frequency for their body size, are mostly heterothermic with a narrow thermoneutral zone beginning at 34C, and have reduced active muscle stiffness in vivo compared to their wildtype siblings. Impairment in heat production (shivering thermogenesis) could be due to the N2A deletion in the titin protein leading to a more compliant muscle and a lower shivering frequency. The ability of mdm mice to use nonshivering thermogenesis via Uncoupling Protein 1 in brown adipose tissue to generate heat during cold stress may also be impaired and contributing to their heterothermy. The purpose of this study was to evaluate mechanisms for the inability of mdm mice to maintain homeothermy during cold stress and to further understand their metabolic properties. We hypothesized the inability of mdm mice to maintain homeothermy is due to an impairment in heat production (e.g. shivering thermogenesis and nonshivering thermogenesis) and that metabolic rate will reach a maximum at a higher ambient temperature than in wildtype mice. In a temperature experiment, mice were placed in a mouse cage that used indirect calorimetry to measure metabolic rate at four different temperatures (34C, 29C, 24C, 19C). Nonshivering thermogenesis was stimulated by administering 1.2 mg kg−1 of norepinephrine subcutaneously. In the temperature experiment, there was a significant interaction between genotype and temperature, with significant differences in metabolic rates at the lowest temperatures of 19C and 24C (Two‐Way ANOVA and Tukey HSD, p<0.05. Mdm mice had significantly lower body temperatures than wildtype mice at 29C, 24C, and 19C, and reached their maximum metabolic rate at 24C, suggesting that a temperature between 19–24C is the minimum at which mdm mice are able to sustain thermogenesis. Area under the curve (AUC) using the trapezoidal rule was used to calculate total thermogenic capacity following norepinephrine injection. Mdm mice had a lower overall AUC (Welch's T‐Test, p=0.03) as well as a lower overall time that they increased metabolic rate after norepinephrine injection (Welch's T‐Test, p=0.02). Our results suggest that in addition to a deficiency in shivering frequency, mdm mice also have a reduced capacity for nonshivering thermogenesis. The deficiency in shivering thermogenesis and nonshivering thermogenesis combined likely lead to inability to maintain euthermic body temperatures below 34C.

Support or Funding Information

NSF IOS‐0742483, NSF IOS‐1025806, and NSF IOS‐1456868, the W. M. Keck Foundation, and the Technology Research Initiative Fund of Northern Arizona University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lower Diffusing Capacity of the Lungs for Carbon Monoxide in Women with a Patent Foramen Ovale
Authors: Lovering Andrew , Schallerer Annalisa , Olson Travis , Davis James
Published: June 2021

The test of diffusing capacity of the lungs for carbon monoxide (DLco) assumes that all of the cardiac output flows through the pulmonary circuit. Right to left blood flow across a patent foramen ovale (PFO) allows blood to bypass the pulmonary circuit. The shunted red blood cells are not exposed to alveolar gas and thus, would not bind any carbon monoxide with a DLco test. This may result in a lower carbon monoxide uptake and therefore a lower measured DLco. Consequently, we hypothesized that subjects with a PFO (PFO+) would have a lower DLco than subjects without a PFO (PFO−). We retrospectively analyzed historical data from our lab that included 77 PFO+ subjects (Ages:18–35; 31 Female) and 55 PFO− subjects (Ages 18–38; 24 Female). DLco was measured using the single‐breath, breath‐hold technique and ATS/ERS standards. We compared DLco absolute, DLco% predicted and DLco/Alveolar volume (VA) absolute and DLco/VA %predicted. There were no significant differences between PFO+ and PFO− subjects as a group. There were no significant differences between PFO+ and PFOmen. However, PFO+ women had DLco and DLco/VA % predicted values that were 10% lower than PFO− women, though these values were still >100% predicted. Median and mean degree of resting PFO shunt scored with transthoracic saline contrast echocardiography were identical between men and women. In conclusion, these data suggest the degree of blood flow through the PFO under resting conditions may not explain the reason for the lower DLco observed in PFO+ women.

Support or Funding Information

Department of Defense Grant W81XWH‐10‐2‐0114

American Heart Association Scientist Development grant 2280238

American Physiological Society's Giles F. Filley Memorial Award for Excellence in Respiratory Physiology and Medicine

American Lung Association in Oregon and administered through American Thoracic Society Grant C‐10‐014

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multisubunit Multiactive Site DNA Polymerase Complexes with Coordinated Activities
Authors: Trakselis Michael A. , Cranford Matthew , Chu Aurea M.
Published: June 2021

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. However, whether those interactions are stable or stochastic is currently being tested. These enzymatic abilities include high processivities, fidelities and plasticities to ensure faithful genome maintenance in spite of many obstacles to DNA synthesis. Many multisubunit enzyme subcomplexes have been identified within the replisome and most stabilize enzyme complexes on DNA for continued and processive enzymatic action over long stretches of the genome. One of the most well characterized subcomplexes is the elongation complex minimally consisting of the DNA polymerase holoenzyme which includes accessory proteins known to stabilize the association of the polymerase and direct repeated catalysis.

In particular, the stability of the DNA polymerase holoenzyme is sensitive to stable secondary DNA structures, protein/DNA complexes, and various DNA damage. High fidelity (HiFi) DNA polymerases in complex with their accessory proteins are responsible for the bulk of genome synthesis, however, most organisms encode multiple separate specialized DNA polymerases to aid in overcoming genomic obstacles with translesion synthesis (TLS). The presence of anywhere from 2 to 18 separate DNA polymerases (depending on the organism) all with identical specificities for a 3′‐OH primer/template DNA creates a complex multiequilibrium within a cell. Moreover, many of these DNA polymerases have been shown to directly or indirectly interact to enhance catalysis. For example, a HiFi trimeric DNA polymerase complex has increased kinetics and processivity explained through dynamic active site switching and exchange. Other direct DNA polymerase interactions allow for coordinated synthesis across DNA lesions through a concerted switch of active sites. Oligomeric DNA polymerase active site coordination will be shown using a variety of presteady‐state kinetic and thermodynamic assays. These and other DNA polymerase interactions and equilibria properties will be put into context and compared across different domains of life to illustrate DNA synthesis mechanisms that maintain a high degree of fidelity and plasticity in spite of a multitude of genomic obstacles.

Support or Funding Information

Baylor UniversityAmerican Cancer Society (RSG‐11‐049‐01‐DMC)National Science Foundation (NSF1613534)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic Regulations of Genes Related to Lipid Metabolism by MicroRNA in Mice Fed High Fat Diet
Authors: Tang Xinyu , Xu Guanying , Pan Yuan‐Xiang , Chen Hong
Published: June 2021

Background

High fat diet impacts lipid metabolism by altering the transportation, oxidation, and storage of fatty acids. Lipoprotein lipase (LPL) plays a critical role in lipid metabolism by catalyzing triglyceride hydrolysis and lipoprotein uptake in multiple tissues. Previous study reported that miR‐29b negatively regulated LPL expression in mammary epithelial cells transfected with miR‐29b mimics. The present study investigated changes in LPL expression and epigenetic mechanisms by miR‐29 family in different tissues in mice fed a high fat diet.

Method

Five‐week old male CBA mice were fed with either a control diet (Con group, 10% kcal from diet) or a high fat diet (HF group, 45% kcal from diet) ad libitum for 11 weeks.

Results

LPL mRNA was increased in adipose, muscle and colon in response to high fat diet. However, LPL mRNA expression decreased in the liver by high fat diet as well as hepatic lipase (HL). The results also showed the highest expression level of LPL mRNA in adipose tissue, followed by muscle, colon, and liver. Cpt1a mRNA, an important gene related to beta oxidation, was increased in adipose, muscle, colon, and liver in the HF group. Moreover, Cpt1a mRNA expression was the highest in liver followed by adipose, muscle, and colon. Meanwhile, high fat diet reduced the expression of miR‐29a, a predicted suppressor of LPL from miR‐29 family, in adipose tissue.

Conclusion

The present results indicated that the triglyceride hydrolysis could be activated by high fat diet in other tissues than the liver and fatty acid transportation could be induced in mitochondria. Overall, LPL and Cpt1a mRNA changed differently in multiple tissue in response to high fat diet and is potentially post‐transcriptionally regulated by microRNA.

Support or Funding Information

Source of research support (in a separate textbox): Campus Research Board of University of Illinois at Urbana‐Champaign and the USDA Cooperative State Research, Education and Extension Service, Hatch project numbers # ILLU‐698‐923 and ILLU‐698‐379

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Insights into the Assembly Mechanism of an RNA Polymerase III‐Specific Transcription Complex on a Drosophila U6 snRNA Gene Promoter
Authors: Hurlburt Ann Marie , Verma Neha , Phan Phuc , Wolfe Angela , Stumph William
Published: June 2021

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).

Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.

In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.

A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.

Support or Funding Information

This work was supported by the National Science Foundation and by the California Metabolic Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changing the Ligand Specificity of a Riboswitch from Guanine to Hypoxanthine
Authors: Garcia Jeremy George , Morse Daniel Paul
Published: June 2021

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Previously, we modified several riboswitches for use as sensitive and specific sensors for their ligands. To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We initially tested our strategy by attempting to change the specificity of a guanine riboswitch from guanine to hypoxanthine. Our first selection experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. For our second experiment, we reduced the abundance of the wild‐type riboswitch by increasing the degree of sequence randomization. This experiment resulted in isolating RNAs with many sequence changes and deletions that failed to detect hypoxanthine. We reasoned that these aberrant RNAs arose during PCR with Taq DNA polymerase. Here, we describe the results of a new selection experiment in which we used a proofreading DNA polymerase and a redesigned partially‐randomized RNA pool.

Support or Funding Information

Defense Threat Reduction Agency (DTRA)

Office of Naval Research (ONR)

Chemistry Department, United States Naval Academy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing Protein Kinase C Interactions with Rho4 and Bud3 in the Filamentous Fungus Aspergillus nidulans
Authors: Betton Brianna , Hobson Joanna , Olsen Elisabet , Hill Terry W. , Jackson‐Hayes Loretta
Published: June 2021

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, the GTPase Rho4, and its guanine nucleotide exchange factor (GEF) called Bud3. In previous work we showed that PkcA both physically and functionally interacts with SepA. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. In A. nidulans Rho4 is likely the GTPase required for SepA activation. Reports from another group have shown that Rho4 and Bud3 work as a module that is required for septation to occur. The current study focuses on the relationship of the Bud3/Rho4 partnership with PkcA and SepA during polarized growth. Using bimolecular fluorescence complementation (BiFC) and overexpressing PkcA in Bud3‐ and Rho4‐null strains, we examined the relationships between PkcA and Rho4 and PkcA and Bud3. Through BiFC we found that PkcA and Rho4 interact at septation sites. Overexpressing PkcA in a Rho4‐null mutant strain increased colony diameter and improved sporulation. However, PkcA overexpression in a Bud3‐null mutant had no effect on growth and sporulation. These results suggest that there are physical and functional interactions between PkcA and Rho4 that are important in polarized growth.

Support or Funding Information

National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preferential Interaction of Beryllium Ion with Carboxylate‐Rich Peptides
Authors: De Silva Bhagya , Gary Ronald K.
Published: June 2021

Glycogen synthase kinase 3β (GSK3β) regulates a number of cellular pathways including glycogen metabolism, the Wnt signaling pathway, and cell proliferation. Hyperactivity of GSK3β has been hypothesized to be potentially related to Alzheimer's disease, bipolar disorder, type II diabetes, and some cancers, therefore GSK3β is of interest as a target for therapeutics. Lithium ion (Li+) is a classical inhibitor of GSK3β, but beryllium ion (Be2+) is approximately 1000‐fold more potent. We are interested in the inhibitory mechanism and thermodynamic characteristics for the metal‐protein binding of Be2+ and GSK3β. Be2+ is believed to inhibit GSK3β by competing for magnesium ion (Mg2+) binding sites that feature aspartic acid (D) or other acidic side chains. As a first step toward understanding how Be2+ could outcompete Mg2+ in metal‐protein interactions, we used model chelating compounds and simple peptides that might partially mimic a carboxylate‐rich binding pocket, and compared their binding properties towards Be2+ and Mg2+. Isothermal titration calorimetry (ITC) binding studies for nitrilotriacetic acid, 4,5‐imidazole dicarboxylic acid, and salicylic acid all showed higher binding affinity for Be2+ compared to Mg2+. ITC binding studies were used to compare Be2+ and Mg2+ interactions with three short peptides: DDDD, GGGG and KKKK. Under the conditions employed, Be2+ exhibited binding with the carboxylate‐rich DDDD peptide while Mg2+ displayed no binding. The neutral tetrapeptide GGGG and the polybasic tetrapeptide KKKK served as negative controls; no metal ion interactions were observed with these peptides in the ITC binding experiments. Further analysis with Be2+ and peptides GDDD and GGDD showed reduced binding affinity compared to DDDD. These results demonstrate that Be2+ has stronger affinity for negatively‐charged carboxylate‐rich peptide sequences compared to Mg2+. This strong affinity may lead to conformational changes that distinguish the inactive Be2+‐GSK3β complex from the active Mg2+‐GSK3β complex. Because GSK3β regulates multiple cellular pathways, the ideal therapeutic inhibitor would lead to selective downstream effects. Investigations using an unusual class of inhibitor like Be2+ might provide insight into the variety of modes potentially available for controlling GSK3β activity.

Support or Funding Information

This work was supported by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant numbers W911NF‐15‐1‐0043 and W911NF‐15‐1‐0216.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin
Authors: Chintapalli Sree V. , Anishkin Andriy , Adams Sean H.
Published: June 2021

Our previous studies confirmed the interaction of different chain lengths of fatty acids and their derivatives (acylcarnitines) with Oxy‐ and Deoxy‐Myoglobin, using both molecular dynamic simulations and Isothermal Titration Calorimetry experiments. However, those studies were limited to testing only the binding sites predicted from comparative studies of fatty acid binding protein homologs and pre‐calculated binding predictions using AutoDock grid maps. Also, those methods could not address the dynamic processes of the ligand entry into the binding pocket of the protein and the residues involved. To analyze the entry mechanisms of the lipid ligands into myoglobin, and to assess binding specificity at the proposed binding site, we have carried out 80 different molecular dynamic simulations (total length 4.4 microseconds) of murine Oxy‐ and Deoxy‐Mb structures with either the palmitate or palmitoylcarnitine initially placed at different positions near the protein surface. The simulations indicated that both ligands quickly enter the Oxy‐Mb structure through a dynamic area called the “portal region” near the heme group, which forms the entry point for small molecule gaseous ligands like O2, CO and NO. The same phenomenon is not observed with Deoxy‐Mb where the lipid ligands move away from the protein surface, due to a quick compaction of the entry portal and the heme‐containing crevice in the Mb protein upon O2 removal. On the other hand, random starting points for the ligands on the protein surface have shown an “alternate” interaction site which is common for both Oxy‐ and Deoxy‐Mb. The latter observations strongly suggest that spontaneous and “non‐specific” binding of long‐chain fatty acids and acylcarnitines to Mb is driven by hydrophobic forces, in contrast with the “portal region” crevice being a primary and specific binding spot that can be precluded by deoxygenation. Considering the importance of lipid metabolism and trafficking in oxidative, Mb‐rich tissues such as type 1 skeletal muscle and cardiomyocytes, identifying the mechanisms of entry, binding and off‐loading of long‐chain fatty acids and acylarnitines on Mb may have important ramifications for muscle management of bioactive lipids.

Support or Funding Information

USDA‐ARS Project 6026‐51000‐010‐05S

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding How Distal Residues Play a Role in Parkin Activity
Authors: Winters Jenifer , Beuning Penny , Makowski Lee , Ondrechen Mary Jo
Published: June 2021

Partial Order Optimum Likelihood (POOL) is a machine learning method that predicts catalytically important residues based on the tertiary structure of the protein, with computed chemical and electrostatic properties of the residues as input features. POOL has predicted spatially extended active sites, where residues that are not in direct contact with the substrate still contribute to catalysis for many enzymes, including for parkin. Parkin is an E3 ubiquitin ligase protein involved in the ubiquitin‐proteosome system that mediates the targeting of proteins for degradation. Mutations in parkin lead to the accumulation of toxic substrates that damage dopaminergic neurons, causing the autosomal recessive form of Parkinson's disease (PD). POOL has predicted a dozen residues in parkin that are important for catalytic activity. Interestingly, half of these residues are positions that when mutated lead to the onset of PD. Enzyme variants were created through single‐site directed mutagenesis to further look into how these residues may play a role in catalysis through biochemical and biophysical techniques.

Support or Funding Information

Supported by NSF MCB‐1517290

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.