bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating Carrier Domain Positioning During Catalytic Turnover in Pyruvate Carboxylase
Authors: Hakala Joshua , St. Maurice Martin
Published: June 2021

A central debate in protein biochemistry focuses on the mechanism by which ligand binding correlates with protein conformational change. Two primary hypotheses describe this process: the induced‐fit hypothesis states that ligand binding induces a conformational change in the protein, while the conformational ensembles hypothesis states that the protein pre‐exists in multiple conformational states, with ligand binding shifting the equilibrium towards a preferred conformation. Pyruvate carboxylase (PC) is a multi‐domain, swinging‐arm enzyme that coordinates reactions between two remote active sites and serves as a structurally and biophysically accessible paradigm to study the relationship between ligand binding and protein conformational change. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. The biotin cofactor on the biotin carboxyl carrier protein (BCCP) domain is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then translocates to the carboxyltransferase (CT) domain on an opposing subunit, where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate.

In order to better understand the mechanism of carrier domain translocation, the equilibrium positioning of the BCCP domain was directly observed using site‐specific cross‐linking to trap the BCCP domain in the putative exo‐binding site on the opposing subunit. This approach permits a detailed analysis of BCCP domain positioning in response to various substrates and allosteric effectors. Samples were first analyzed using SDS‐PAGE and densitometry to measure BCCP cross‐linked dimers as reporters for equilibrium positioning changes. Given that BCCP cross‐linking inactivates the enzyme, rates of inactivation were measured to assess changes in BCCP domain positioning in the presence of substrates and effectors. Finally, intrinsic tryptophan fluorescence changes were measured on fast timescales to more accurately determine the effect of ligands on the rates of BCCP domain repositioning. These studies reveal that substrates and effectors shift the BCCP domain positioning from one domain to another and offer the first evidence that the carrier domain exists in a dynamic equilibrium between active sites.

Support or Funding Information

This work was supported by the National Institutes of Health grant GM117540.

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 Life and Times of a Carboxysome: Tracking a Single Protein Complex Over Multiple Generations
Authors: Hill Nicholas , Cameron Jeffrey C.
Published: June 2021

Cyanobacteria are abundant primary producers in the biosphere that require only CO2 and light as carbon and energy sources to grow. Cyanobacteria contain an icosahedral proteinaceous nanostructure known as the carboxysome, which functions by increasing the concentration of CO2 around the carbon‐fixing enzyme Rubisco, thereby maximizing carbon fixation rates. While this core functionality of these macromolecular protein complexes is understood, dynamic activities of individual carboxysomes are largely unknown due to a lack of insight at the single molecule level. Since the average cyanobacterium contains 3–4 carboxysomes per cell, it is easy to obtain an average carbon fixation rate per cell, but no way to determine the precise activities of each individual carboxysome through population‐level experiments. As carboxysomes last for multiple generations, it is likely that their carbon‐fixing ability decreases with time, which would have a substantial impact on carbon fixation, and subsequently, overall cell fitness and physiology.

To understand precisely how a single carboxysome affects cell growth and fitness, we have created a strain of cyanobacteria where carboxysome expression is controlled via an inducible system, thus enabling us to reduce the number of carboxysomes per cell to one. Since only a single carboxysome is present in each cell, all of carbon fixation and growth can be accredited to that individual carboxysome. By tracking single cells with single carboxysomes, it is possible to estimate carboxysome functionality over multiple generations by measuring cell growth rates. The results from this experiment can be used to quantify the in vivo functional lifetime and stability of individual carboxysomes. In addition to understanding how carboxysome age affects function, real‐time visualization of carboxysomes in single cells will allow for analysis of their spatiotemporal dynamics and potentially the identification of a novel carboxysome degradation pathway.

Understanding these dynamic properties of carboxysomes is critical for an accurate model of cyanobacterial growth and physiology. It is also valuable information for industrial and biotechnological applications that involve optimizing growth and carbon‐fixation rates, and is critically important for engineering more stable and active carboxysomes. This is the first example of tracking the enzymatic activity of a single protein complex in vivo over its entire lifetime and across multiple generations. Basic properties discovered here are likely to apply to diverse bacterial microcompartments, and possibly to entirely unrelated large macromolecular complexes.

Support or Funding Information

Funding for Nicholas Hill is provided by the NIH T32 Training Grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Catalytic Role of a Conserved Non‐Active Site Residue in Triosphosphate Isomerase
Authors: Park Julie Heejin , Chang Timothy , Schwans Jason
Published: June 2021

Understanding enzyme function often begins with examination of the active site, but important catalytic roles for residues surrounding the active site have been shown. The active site residues of triosephosphate isomerase (TIM), K12, H95, and E165, have been extensively studied, but the role of the highly conserved E97, situated adjacent to the active site, is not well understood. The proximity of E97 and K12 led to a model where the glutamate positions the active site lysine, and the E97D and E97Q mutations in TIM from Plasmodium falciparum (PfTIM) showed a 100‐ and 4000‐fold decrease in activity, respectively, suggesting an important catalytic role for this residue. In contrast, the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity, challenging E97 as a catalytically critical residue. To investigate further the basis for how the same mutations in essentially superimposable structures give different mutational effects, we studied the effects of mutating E97 in TIM from Trypanosoma brucei brucei (tbbTIM) and Saccharomyces cerevisiae (yTIM). Mutants were constructed and the enzymes were recombinantly expressed and purified. Kinetics, circular dichroism (CD), and fluorescence studies were performed to evaluate the effects of the mutations on enzyme function and structure. A new expression system for generating yTIM mutants was developed to overcome prior challenges in obtaining pure enzymes. Michaelis‐Menten kinetics showed the E97D and E97Q mutants led to an ~three‐fold decrease in activity, a modest effect compared to the 102–103‐fold effect in PfTIM. CD studies showed the mutants contain ɑ‐helical contents of 42% (E97D) and 37% (E97Q), similar to the 42% ɑ‐helical content in wild type. CD and fluorescence chemical denaturation studies showed the midpoint concentration of denaturant was within 0.2 M for the mutants relative to the corresponding wild‐type enzymes, suggesting the mutants have little effect on protein stability. A model for the different functional effects is that variations of the surrounding residues in the TIM isozymes from different organisms allow for such a range of deleterious mutagenic effects. These results highlight the complexity of interactions surrounding the active site. A better understanding of the variations in residues surrounding the active site and their role in positioning residues may aid in the design of enzymes with new functions.

Support or Funding Information

This project was supported by a Single‐Investigator Cottrell College Science Award from the Research Corporation for Science Advancement (22489).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of Caspase Allostery and Enzyme Specificity
Authors: Clark Clay , Grinshpon Robert , Thomas Melvin E. , Yao Liqi , Shrestha Suman
Published: June 2021

We used phylogenetic, structural, and biophysical studies to examine the evolution of enzyme specificity and allosteric regulation in caspases. Caspase‐3 activation and function has been well defined during programmed cell death, but caspase activity, at low levels, is also required for cell development. We established a database, the CaspBase, for phylogenetic and ancestral reconstruction of caspases, which consists of >1,500 caspase genes. From this database, we resurrected ancestors of the caspase‐3, ‐6, ‐7 subfamily, and the common ancestor displays enzyme specificity similar to the initiator caspase subfamily from which it evolved. The specificities of the caspase‐3/‐7 and caspase‐6 enzymes evolved early, after the two lineages split. Structural studies of the ancestral enzymes identify changes in the active sites that result in changes in substrate selection. In contrast to enzyme activity, several allosteric sites evolved later in the lineages. We define interaction networks that facilitate the allosteric regulation in caspase‐3, and we show that, within a conserved loop, one site of phosphorylation evolved with the apoptotic caspases, while a second site is a more recent evolutionary event in mammalian caspase‐3. Localized changes in the loop propagate to the active site of the same protomer and disrupt substrate hydrolysis by facilitating transient ionic interactions with the catalytic histidine. In contrast, a cluster of hydrophobic amino acids in the dimer interface connects the conserved loop to the active site of the second protomer. The presence of the second modification in the conserved loop introduces a “kill switch” in mammalian caspase‐3. The data reveal how evolutionary changes in a conserved allosteric site result in both a common pathway for lowering activity during cell development as well as introducing a more recent cluster‐specific switch to abolish activity.

Support or Funding Information

UT Arlington

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
What is the origin of partial agonist activity of CBt‐PMN for hRXRα?
Authors: Shimizu Kanade , Miyashita Yurina , Matsuo Noya , Yamamoto Yuta , Nakano Syogo , Ito Souhei , Numoto Nobutaka , Ikura Teikichi , Ito Nobutoshi , Kakuta Hiroki , Tokiwa Hiroaki
Published: June 2021

Introduction

Nuclear receptors are involved in the control of a variety of physiological processes. In particular, retinoid X receptor (RXR) agonists are well‐known to be effective in the treatment of various diseases but they sometimes cause side effect including elevation of blood triglyceride, weight gain, hepatomegaly and hypothyroidism. We have developed human RXRα partial agonist to reduce these side effects with keeping main effects. Recently we have reported CBt‐PMN (1) shows a potent glucose‐lowering effect without causing serious adverse effects. However, the origin of molecular basis of the partial agonistic activity for hRXRα is still controversial. In this research, we aimed to analyze crystal structure and molecular simulation results of the ligand binding domain of hRXRα in complex with CBt‐PMN, to elucidate molecular mechanism of partial agonist activity.

Methods

The cording sequence of hRXRα‐LBD (residues 227–462) was cloned to the vector pET15b, and Escherichia coli strain BL21(DE3) was used for protein expression. Complexes of CBt‐PMN and hRXRα‐LBD (residues 227–462) were crystalized and X‐ray diffraction data was collected. Fragment Molecular Orbital (FMO) method and Molecular Dynamics (MD) simulation were performed using the obtained crystal structure.

Results & Discussion

Crystal structure of hRXRα‐LBD/CBt‐PMN was determined at 2.4 Å and it showed a tetrameric hRXRα‐LBD oligomerization with CBt‐PMN bound to each hRXRα monomer. There were two types of CBt‐PMN binding conformation (form I and II ). By the FMO method, interaction between hRXRα/CBt‐PMN of form I is primarily contributed by the hydrophilic interactions through its carboxyl group, whereas that of form II is contributed by hydrophobic interactions. CBt‐PMN bound form shows more flexible nature at activation function‐2 (AF‐2) interface than that of bexarotene, full agonist for hRXRα by MD simulation.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genomics of physiological adaptations of two milkweed species (Asclepias) in their hybrid zone
Authors: Selberg Avery Grace Ann , Olson‐Manning Carrie
Published: June 2021

Speciation is the source of diversity on the planet, but the genomic mechanisms that lead to the reproductive isolating barriers between populations that cause speciation are not well understood. Here we are studying the genomic differentiation between two species of milkweed that grow in the Western (Asclepias speciosa) and Eastern United States (A. syrica). In addition to different geographic locations, these species have diverged in a number of important phenotypes, including: production of secondary metabolites, flower morphology, and drought resistance. In the central US, these species have come into secondary contact and sometimes form hybrids in the wild. We are interested in quantifying the amount of gene flow between these species and identifying genomic locations that may be indicative of local adaptation within populations and reproductive isolation between species.

To compare these species of milkweed, we isolated DNA from A. speciousa, A. syrica, and individuals that displays intermediate phenotypes from the hybridization zone. We extracted and isolated genomic DNA and sequenced their genomes with Oxford Nanopore technology. With the aligned genomes, we will be able to compare polymorphisms in these species and begin to quantify the amount of gene flow between them. We will also identify polymorphisms in candidate genes and pathways that may have led to ecological specialization between these species.

Support or Funding Information

South Dakota EPSCoR IIA‐1355423

South Dakota BRIN P20GM 103443

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FXR Inhibits NLRP3 Inflammasome Activation by ER Stress
Authors: Kim Ayoung , Han Chang Yeob , Rho Hyun Soo , Kim Tae Hyun , Kim Sang Geon
Published: June 2021

Chronic endoplasmic reticulum (ER) stress is closely associated with hepatic inflammation, which may contribute to liver injury and fibrosis. However, the potential regulators that act against ER stress‐mediated inflammasome activation in hepatocytes is largely unknown. This study investigated the role of farnesoid X receptor (FXR) in the regulation of NLRP3 inflammasome activation by ER stress. The degree of NLRP3 inflammasome activation was inversely correlated with hepatic FXR level in patients with HBV‐associated hepatic failure or non‐alcoholic fatty liver disease. The inverse relationship between FXR and NLRP3 was confirmed in animal models with liver injury. In mice, FXR deficiency exacerbated the effect of ER stress inducer on NLRP3 and TXNIP expression levels. Consistently, FXR agonist treatment had an opposite effect, alleviating liver injury. In addition, FXR activation specifically inhibited PERK pathway among canonical UPR pathways in hepatocytes, and CHOP was necessary for NLRP3 and TXNIP induction by ER stress. Moreover, FXR activation abolished the ability of ER stress to decrease the expression of Nck1, leading to the inhibition of inflammasome activation and cell death. MiR‐186 was identified as a repressor of Nck1 and involved in ER stress‐mediated NLRP3 inflammasome activation. Overall, our results demonstrate that FXR acts as a novel regulator of hepatic ER stress and consequential NLRP3 inflammasome activation through PERK‐CHOP signaling pathway, which may depend on miR‐186 regulation of Nck1.

Support or Funding Information

NRF‐2015R1A2A1A10052663, 2017R1D1A1B03028272

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tannic Acid Downregulates Angiotensin Type 1 Receptor in Aortic Smooth Muscle Cells Through EGFR Dependent Phosphoinositide 3‐kinase Pathway
Authors: Iyer Laxmi , Zhang Yong , Thekkumkara Thomas
Published: June 2021

Tannic acid (TA), a hydrolysable plant polyphenol commonly found in food and beverages, possesses several cardioprotective effects through its anti‐inflammatory and anti‐oxidant activities. In this study, we aimed to investigate the role of TA on the angiotensin type 1 receptor (AT1R) a major hypertension target and its associated signaling pathways in primary rat aortic smooth muscle cells (ASMCs). In the presence of TA (10 μg/ml for 20 h), cells exhibited down‐regulation of AT1R specific [3H] angiotensin II (AngII) binding compared to untreated control (3628.32±289.42 vs. 7244.66 ± 40.26 DPM/mg protein, P < 0.001). TA mediated AT1R receptor downregulation in a dose dependent (0–40 μg/ml) and a time‐dependent (0–24 hours) manner, and was reversible. The reduction in AngII binding in TA‐treated cells was due to decreased receptor density rather than a change in receptor affinity as demonstrated by competition binding assay (Kd 1.302±0.152nM for TA treated vs. Kd 1.391±0.048 nM for untreated). Consistent with downregulation of AngII binding, AT1R mRNA was inhibited upon TA treatment (49.63% compared to control). TA mediated AT1R downregulation was associated with phosphorylation of extracellular signal regulated kinases (ERKs) p42/44. Pretreatment with MEK inhibitor PD98059 (20 mM) blocked TA induced ERKs p42/44 phosphorylation and AT1R downregulation. Moreover, AG1478 (5μm), an EGFR specific inhibitor, blocked TA mediated ERKs activation and down‐regulation of AT1R. Under similar conditions, treating cells with EGF (0.5 mg/ml) alone mediated ERKs activation and AT1R downregulation independent of TA (EGF; 3788.04±206.51 vs. Control; 6549.22±115.48 DPM/mg protein, P < 0.001). LY294002 (10 mM), an inhibitor of phosphoinositide 3‐kinase (PI3K), inhibited TA and EGF mediated EGFR induced ERKs phosphorylation and reversed AT1R downregulation suggesting that TA mediated AT1R downregulation is through EGFR induced PI3K dependent ERK p42/44 pathway. Consistent with the receptor downregulation, quantitative calcium analysis revealed significant inhibition of AngII induced intracellular calcium release in TA treated cells, which was reversed when cells were pretreated with either MEK or EGFR inhibitor. Collectively, our study demonstrates for the first time that TA induced EGFR, PI3K and ERK p42/44 signaling axis is essential for downregulation of AT1R expression in ASMCs. Furthermore, the study highlights the significance of TA rich diet for the prevention of hypertension and associated cardiovascular disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vitamin D and CaM Kinase Phosphatase Mediate Breast Cancer Proliferation
Authors: Morrow Quinlan B. , Schmitt John Michael
Published: June 2021

The family of enzymes known as CaM Kinases have been shown to participate in cell signaling pathways in a variety of cell types including cancer cells. Ionomycin, carbachol and estrogen (E2) may increase intracellular calcium resulting in the activation of either CaM Kinase II (CaM KII) or CaM Kinase Kinase (CaM KK) and their substrates. Several agonists including E2 trigger calcium‐responsive pathways that regulate a number of transcription factors such as Elk‐1 leading to cell growth. CaM KK has been shown to activate numerous enzymes including CaM KI and ERK. In contrast, hormones Vitamin D (VitD) and thyroid hormone (T3) have been suggested to antagonize ERK activation and growth of certain cells, although the mechanism remains unknown. CaM Kinase Phosphatase (CaM KP) inactivates CaM KK and CaM KI, so we hypothesized that it may mediate the inhibitory effects of VitD and T3 in our cells. To the best of our knowledge, the specific role for CaM KP in E2 signaling in breast cancer cells has not been examined. Our results suggest that calcium‐mediated activation of CaM KK, CaM KI, and ERK leads to the growth of MDA‐MB‐231 (MDA) and MCF‐7 cells. VitD and T3 appear to inhibit E2 stimulation of CaM KI and ERK activity in MCF‐7 cells. Carbachol‐ and E2‐mediated cell growth was decreased by pretreatment of cells with VitD or T3. Interestingly, inhibition of CaM KP via siRNA knockdown reversed the inhibitory effect of VitD in our system. These results suggest that VitD and T3 may utilize CaM KP to antagonize calcium signaling through CaM KK and ERK in MDA and MCF‐7 cells.

Support or Funding Information

Jach Holman Endowment Fund at George Fox 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.
Calcium Control of MDM2 in Breast Cancer Cells
Authors: Nguyen Gia Han N. , Schmitt John Michael
Published: June 2021

The hormone estrogen (E2) can activate a series of intracellular signaling pathways, one of which being the calcium/calmodulin‐dependent kinases (CaM Kinases) that have been implicated in cancer cell signaling including breast cancer cells. CaM Kinase Kinase (CaM KK) can directly phosphorylate and activate CaM KI, CaM KIV or Protein Kinase B (AKT), which promote cell growth, differentiation and survival. AKT can directly phosphorylate the pro‐survival enzyme murine/human double minute 2 (M/HDM2) that regulates the amount of p53 present in breast cancer cells. The purpose of this study was to examine the ability of calcium‐elevating agents carbachol and E2 to stimulate MDM2 phosphorylation and survival of breast cancer cells. In addition, we focused on examining whether CaM KK and AKT may be mediating MDM2 phosphorylation downstream of calcium signaling. Treatment of MDA‐MB‐231 (MDA) cells or MCF‐7 cells to increase intracellular calcium levels resulted in phosphorylation and activation of ERK and MDM2. Treatment of MDA and MCF‐7 cells with CaM KK and ERK inhibitors, STO‐609 and U0126 respectively, decreased MDM2 phosphorylation and cell survival, suggesting the involvement of CaM KK and ERK in this pathway. Interestingly, pre‐treatment of cells with the AKT inhibitor, AKT‐X, followed by E2 stimulation did not decrease phosphorylation of MDM2. Transfection of cells with shRNA directed against CaM KK decreased MDM2 and ERK phosphorylation. In summary, the data obtained suggests that carbachol and E2 activate a novel CaM KK, ERK and MDM2 pathway in breast cancer cells.

Support or Funding Information

Paul and Evelyn Richter Memorial 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.
Ligand‐Mediated Mitochondrial Translocation of the Transforming Growth Factor‐β Type I Receptor and Hexokinase 2
Authors: Kang Jeong‐Han , Jung Mi‐Yeon , Yin Xueqian , Andrianifahanana Mahefatiana , Choudhury Malay , Leof Edward B.
Published: June 2021

Transforming growth factor beta (TGF‐β) plays a significant role in diverse biologic processes, including the regulation of cell growth, survival, differentiation, development, inflammation, immunity, hematopoiesis, and tissue remodeling. TGF‐β is essential for wound healing, stimulates matrix molecule deposition, and has been implicated in the pathogenesis of a variety of fibrotic disorders, including idiopathic pulmonary fibrosis, scleroderma, radiation‐induced pulmonary fibrosis, and asthma. Furthermore, activation of profibrotic TGF‐β signaling has a central role in glucose‐induced cell hypertrophy, induces glucose transporter 1, and regulates mitochondrial energetics. However, the mechanisms by which TGF‐β induces specific metabolic alterations in the fibrogenic process have not been fully defined. In this study, we demonstrate that TGF‐β stimulates type I TGF β receptor (TβRI) translocation to the mitochondria and induction of hexokinase 2 (HK2) via both Smad‐dependent and –independent pathways. Also, we showed that TbRI association with HK2 and the mitochondrial voltage‐dependent ion channel (VDAC). These findings suggest that TβRI plays a novel role in controlling mitochondrial function and regulation of glucose metabolism.

Support or Funding Information

This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from the National Institutes of General Medical Sciences.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Steroid Sulfatase activates the integrin signaling pathway in human cervical cancer cells
Authors: Ye Dong‐Jin , Kwon Yeo‐Jung , Baek Hyoung‐Seok , Chun Young‐Jin
Published: June 2021

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a crucial function in the production of estrogens and androgens that induce the growth of hormone‐dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not elucidated clearly. To investigate the function of STS in cancer cell growth, we examined whether STS is able to enhance the integrin signaling pathway. We observed that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), the major metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. STS overexpression and DHEA treatment induced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Furthermore, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS enhances the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up‐regulation of integrin β1 expression and activation of FAK.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blue light‐excited retinal delocalizes plasma membrane bound PIP2 and triggers stress responses challenging the cellular integrity
Authors: Ratnayake Kasun , Payton John L. , Karunarathne Ajith
Published: June 2021

The photoreceptor chromophore, retinal, an aldehyde derivative of vitamin A, harvests light to generate visual perception. Upon photon absorption, the receptor bound 11‐cis‐retinylidene (11CR) isomerizes to all trans retinal (ATR), changing the electrostatic environment in the chromophore‐binding site, subsequently transforming the inactive receptor to a momentary active state. The subsequent release of ATR allows the receptor to be relaxed to the basal state, enables a new 11CR binding and continuous photon reception. Due to the defects in retinal clearance mechanisms, higher concentrations of 11CR and ATR can be found in the retina, and a fraction of excess retinal can enter the circulation, exposing the rest of the body to retinal as well, leading to pathophysiological effects. Nevertheless, visible light induced, photoreceptor independent signaling activities of retinals and mechanisms of retinal phototoxicity are less explored. Here, we show that, retinal, in the presence of blue light, irreversibly remove the plasma membrane (PM) bound phosphatidylinositol 4,5 bisphosphate (PIP2) in photoreceptor and non‐photoreceptor cells. Interestingly, addition of retinals alone without blue light exposure as well as exposing cells to only blue light without retinal, did not show any detectable PIP2 dislodging from PM. Additionally, even in the presence of retinal, the other wavelengths of lights failed to show any effect on PM bound PIP2. Moreover, compounds that are structurally similar to retinal such as retinol and retinoic acid did not show any effect on PIP2 in the presence of blue light. Data further show that, this PIP2 removal from the PM is not due to regular enzymatic hydrolysis of PIP2, but as a result of lipid peroxidation induced by the generation of singlet oxygen and reactive oxygen species. The resultant PIP2 dislodging is followed by cellular stress responses and cell death. Clinically used photodynamic therapeutic agents (photosensitizers), also showed a unique PIP2 dislodging effect from PM in the presence of blue light as well as in other wavelengths of light, similar to blue light excited retinal. This further confirms retinal's ability to harvest light to induce photosensitization that leads to lipid peroxidation which suggests its future potential in photodynamic therapeutics in cancer as well as identification of new molecular targets for phototoxicity. Overall, here we show that, retinal exhibit light sensitivity in non‐photoreceptor cells and trigger crucial signaling pathways altering the cellular fate.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IQGAP Proteins Do Not Regulate Mitogen‐Activated Protein Kinase (MAPK) Signaling through Direct Interaction with Ras
Authors: Morgan Chase J. , Li Zhigang , Hedman Andrew C. , Sacks David B.
Published: June 2021

The Ras family of small GTPases modulates numerous essential processes, including development and tissue repair. Activating Ras mutations occur in approximately 30% of human malignancies and understanding the molecules that modulate Ras function is key to elucidating its role in oncogenesis. The IQGAP family of scaffold proteins regulates numerous signaling proteins, including members of the MAPK pathway and the Rho family GTPases RAC1 and CDC42. However, it is controversial whether IQGAP proteins interact with Ras family GTPases. While initial reports showed no binding between IQGAP1 and Ras proteins (EMBO J 1996; 15:2997), subsequent studies claim that IQGAP1 and IQGAP3 bind K‐Ras (Biochem Biophys Res Commun. 2014; 444:360) and H‐Ras (Nat Cell Biol 2008; 10:971), respectively. To resolve these discrepancies, we performed careful and thorough binding analyses. Here we demonstrate that neither IQGAP1 nor IQGAP3 interacts with the major Ras isoforms, namely H‐, K‐, and N‐Ras. No IQGAP1 or IQGAP3 was detected when flag‐tagged, constitutively active mutant (G12V) H‐, K‐, or N‐Ras proteins were immunoprecipated from HEK293 cells. Similarly, IQGAPs were not detected in pulldown experiments from HEK293 lysate with wild type or constitutively active (G12V) GST‐Ras proteins. By contrast, we immunoprecipitated Myc‐IQGAP3 from HEK293 cells and show for the first time that IQGAP3 binds B‐Raf, an established binding partner of IQGAP1 (Proc Natl Acad Sci U S A 2007; 104:10465). It is possible that IQGAP and Ras do not interact directly in cells, but rather are in a complex with effectors such as B‐Raf, which could explain the positive binding results at over‐expressed, nonphysiological protein concentrations. Knowledge of the binding partners of Ras is of considerable biological and medical importance, as the direct interactors of Ras provide potential therapeutic targets for Ras‐driven cancers.

Support or Funding Information

This work was supported by the National Institutes of Health Intramural Research Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Follicle Stimulating Hormone Stimulation of a Murine Monocyte Cell Line
Authors: Leamon Annie B. , Cohen Brian D.
Published: June 2021

Osteoporosis, an illness resulting in weak and brittle bones, will soon cause over $25 billion dollars of economic damage to the US economy each year. For decades, menopausal osteoporosis was thought to be the result of estrogen loss due to depletion of ovarian follicles. Recent research, however, has shown that the hormone primarily responsible for estrogen synthesis, follicle stimulating hormone (FSH), can contribute to bone degeneration. What is unclear is the specific mechanism by which FSH has this effect. We hypothesized that FSH may contribute to monocyte differentiation to osteoclasts. To test this, we used the RAW 246.7 murine monocyte cell line which can differentiate into osteoclasts in the presence of RANKL. We were able to find evidence of FSH‐dependent signaling in cells FSH treated cells with or without RANKL. In particular, time dependent activation of the cyclic AMP Response Element Binding Protein (CREB) was observed. Interestingly, RANKL treatment changed the pattern of phosphorylation of CREB and the related transcription factor ATF1. Since CREB partially regulates osteoclast differentiation and function this could be one part of the mechanism by which FSH stimulates osteoclastogenesis and bone demineralization. With this in mind, future therapies targeting FSH action could be developed to prevent the onset of osteoporosis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis of the Strigolactone Signaling through Plant SCF E3 complex
Authors: Hinds Thomas Richard , Shabek Nitzan , Mao Haibin , Zheng Ning
Published: June 2021

Strigolactones (SL) are plant hormones that play an essential role in the control of plant branching, and act as rhizospheric signals for communication with symbiotic fungi and parasitic plants. MORE AXILLARY GROWTH2 (MAX2/D3) and DWARF14 (D14), are crucial signalling components that regulate SL‐repressed plant branching. D14 is a α/β hydrolase that hydrolyses SL into an inactive intermediate. MAX2/D3 is an ubiquitin ligase that has a leucine‐rich (LRR) F‐box region, which interacts with D14 in a SL‐dependent fashion. Once formed the D14‐D3 complex can bind to the repressor DWARF53 (D53) for subsequent ubiquitination and proteasomal degradation. The D3‐D14‐D53 complex is proposed to be involved in SL signaling. However, the recruitment of D53 by the D3‐D14 complex and the role of SL perception and hydrolysis remains largely unclear. In this study, we determine the structures of rice MAX2/D3 and D14‐SL complexes. In addition we measure the hydrolytic activity of D14 with and with out its binding partners D3 and D53. We also measured the binding of these three components using ALPHA technology. Our structural and biochemical studies shed light on SL sensing mechanism that is largely regulated by MAX2/D3 ubiquitin ligase.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the effects of novel positive allosteric modulators on the functionality of the α7 nicotinic acetylcholine receptor (nAChR)
Authors: Reilly‐Andujar Francis , Lasalde José A. , Colón José O.
Published: June 2021

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in the development of various neurodegenerative disorders such as Alzheimer's, Parkinson's, Schizophrenia, and a series of inflammatory disorders. In recent years, there has been an increased interest in the pharmacological modulation of receptor function as a therapeutic target. However, these efforts have been limited due to poor temporal and tissue selectivity. Therefore, there is a growing need for the synthesis of new modulators that can address these problems. Positive allosteric modulators (PAMs) for the α7‐nAChR have grown in significance and are currently being developed by many pharmaceutical companies. The California Northstate University has provided us with three novel PAMs (PAM‐2, OCF‐3, CF‐3) in order to test their functionality on the α7‐nAChR. Previous tests on these compounds have shown indirect PAM activity through calcium influx tests using fluorescence microscopy. However, the direct effects of these drugs on the α7‐nAChR have not been tested. Taking advantage of the Xenopus oocyte expression system we are studying the direct effects of these drugs on the α7‐nAChR using Two‐Electrode Voltage Clamp (TEVC) electrophysiology. Our results show that pre‐application of 20 μM PAM‐2, OCF‐3, or CF‐3 on oocytes expressing the α7‐nAChR causes a significant increase in response amplitude compared to controls. Also, we observe a dose dependent increase in current amplitude for PAM‐2 and OCF‐3. These results are consistent with the idea of positive allosteric modulation. Reverse potentials were performed to verify the effects of PAM‐2 and OCF‐3 on calcium permeability of the α7‐nAChR. We have observed that for PAM‐2 the shift in Vrev (ΔVrev) is similar to that of our control. However, for OCF‐3 we observe a reduction in ΔVrev in a low vs high calcium solution. Further studies of these compounds will give us a better insight into their potential use as therapeutic targets of the α7‐nAChR.

Support or Funding Information

This project was funded by the MARC Program at the University of Puerto Rico, Rio Piedras NIGMS Grant #5T34GM007821‐38.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redox Modulation of NQO1
Authors: Siegel David , Dehn Donna , Quinn Kevin , Backos Donald , Di Francesco Andrea , Bernier Michel , Reisdorph Nichole , de Cabo Rafael , Ross David
Published: June 2021

NQO1 is a FAD containing NAD(P)H‐dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C‐terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β‐lapachone which caused rapid oxidation of NAD(P)H, NQO1 could be readily pulled‐down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β‐lapachone demonstrating that under non‐denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)+/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co‐localize with acetyl α‐tubulin and the NAD+‐dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle, NQO1 may provide NAD+ for Sirt2‐mediated deacetylation of microtubules. Overall, the data suggest that NQO1 can act as a redox‐dependent switch where the protein responds to the NAD(P)+/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.

Support or Funding Information

This work was supported by NIH/NDDK grant 1R01DK109964‐01A.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Globally Monitoring Allosteric Coupling in the A2A Adenosine Receptor by NMR in Solution
Authors: Eddy Matthew , Jacobson Kenneth , Stevens Raymond , Wuthrich Kurt
Published: June 2021

In human G protein‐coupled receptors (GPCRs), drug binding near the extracellular region drives conformational changes over 30 Å away at the intracellular surface, leading to complex formation with partner proteins that initiate signaling. GPCR crystal structures have revealed the need for deeper understanding of conformational dynamics to describe mechanisms of allosteric coupling between the extracellular and intracellular surfaces.

Using nuclear magnetic resonance (NMR) in solution, we provide a structural basis for allosteric coupling in the human A2A adenosine receptor (A2AAR), a validated drug target for Parkinson's disease and cancer co‐immunotherapies. Production of A2AAR in Pichia pastoris allowed stable‐isotope labeling with 2H,15N, and 13C, yielding reproducible high‐quality NMR correlation spectra. Assignment of endogenous tryptophans and glycines provided numerous probes distributed over the receptor intracelluar surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The “toggle switch” Trp 2466.48 was especially sensitive to variable drug efficacy due to the changes in relative orientation of the nearby aromatic ring of Phe 2426.44 in the highly conserved Pro5.50‐Ile3.40‐Phe6.44 (P‐I‐F) activation motif, providing a highly responsive probe of changes in structure and dynamics upon agonist binding. The novel NMR probes also enabled us to observe ligand efficacy‐related local conformational polymorphisms near the intracellular surface.

The distributed NMR probes were utilized to monitor the impact of a key mutation in the A2AAR allosteric center at Asp522.50. Amino acid replacement of Asp522.50 leads to immediate loss of intracellular signaling. In NMR studies, this mutation caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at Trp 2466.48 and the P‐I‐F motif, demonstrating an interplay between the allosteric center and Trp 2466.48, and providing novel insights into a signaling mechanism that may be applicable to a wide range of class A GPCRs.

Support or Funding Information

American Cancer Society (MTE)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Homogenization Methods for Extraction of Live Bacteria and Recombinant DNA in Soil
Authors: Proctor Caleb , Atwood James , Easparro Brandon , Morehouse Zachary
Published: June 2021

In many soil types, ubiquitous bacteria are present very in low quantities due to the inherent challenges of microorganism survival in environmental conditions. While, in some cases, only present in low quantity, soil microorganisms are a critical component of our ecosystem. Soil microbiome studies aim to characterize the microbial repertoire and to correlate the microbial population with soil and ecological functions. While many studies involve culturing and isolation of bacteria harvested from the soil, some organisms are hard to propagate in the laboratory or require the sample to be processed directly to maintain analyte integrity. Given time involved and complexity of culture based enrichments, many modern studies involve direct cell lysis and DNA recovery from soil followed by PCR for target enrichment and detection. In order to infer absolute taxa abundance non‐conserved, targeted gene products are quantified by qPCR. While this approach is rapid and robust, qPCR is subject to inhibitory effects of soil based compounds such as humic acids and polysaccharides.

In this study, we compare the recovery and qPCR detection of a modified bacterial standard from multiple types of spiked soil samples. The standard was composed of E. coli, Bacillus subtilis, and Pseudomonas fluorescens which were genetically modified with green florescent protein (GFP). The modified organism allowed the absolute subtraction of background signal originating from native organisms found in the soil samples. The soil samples were spiked with decreasing quantities of the standard. The samples were extracted through a combination of bead milling and silica spin column based DNA purification. The DNA was then quantified by qPCR against the GFP gene and the resulting data established limits of recovery for the combination of organisms. These limits were evaluated side by side with a common incubation lysis to evaluate the effects of mechanical lysis on bacterial cells in soil samples that vary in resistance to lysis. Along with the limits of recovery, cycle threshold values (ct values) were compared to evaluate the impact of PCR inhibitor release.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lee University's Integrated Math and Science Scholars Program; Cohort 1 Results
Authors: Daft Sheila , Mitchum Jessica , Schlosser Sarah , Kasper Sherry
Published: June 2021

Lee University's integrated Math and Science Scholars (iMASS) program began in the spring of 2015 as a part of the Scholarships in Science, Technology, Engineering, and Mathematics (S‐STEM) funded by the National Science Foundation (NSF). The purpose of this program is to introduce STEM students to industry and research opportunities in the region, cultivate a learning community by cohorting them in interdisciplinary groups with biology, chemistry, biochemistry and mathematics majors, and increase retention of the participants. The iMASS program seeks to include academically talented, financially eligible students, with a focus on those who are minority or first – generation. Thus far, we are on target to achieve many of our primary goals. In these first three years, we have recruited and selected talented and financially eligible students to participate in the program, retaining at least 90% of these scholars into each successive year. We have also provided these scholars with increased educational opportunities in and outside of the classroom. These opportunities include a seminar class featuring resume building, interview tips, and speakers from regional industries (Bayer and Oak Ridge National Labs) and graduate schools (University of Tennessee and University of Alabama at Birmingham). Additionally, we have provided industry and graduate school tours at different points throughout the academic year, resulting in successful internships for some of the scholars and thereby meeting one of Lee University's Quality Enhancement Plan goals. Through these educational opportunities and supports, the retention rate and persistence in STEM of our first cohort exceeds that of their peers in STEM at Lee (Cohort 1 at 100% vs. all STEM at 71%). This program is an example of the success made possible from introducing students to successful mentors in the community and the type of power that learning communities can have on students from a variety of backgrounds.

Support or Funding Information

National Science Foundation S‐STEM

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