bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationFunding 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 InformationThis 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 InformationUT ArlingtonThis 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

IntroductionNuclear 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.MethodsThe 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 & DiscussionCrystal 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 InformationThis 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 InformationSouth Dakota EPSCoR IIA‐1355423South Dakota BRIN P20GM 103443This 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 InformationNRF‐2015R1A2A1A10052663, 2017R1D1A1B03028272This 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 InformationJach Holman Endowment Fund at George Fox UniversityThis 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 InformationPaul and Evelyn Richter Memorial FoundationThis 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 InformationThis 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.