bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and thermal stability of a coral metabolic enzyme
Authors: Wolfe Jacob A , Qian Yiwen , Perez Astrid M , Schermerhorn Janse T , Cela Bekim , Kalinowski Cody R , Largoza Garrett E , Fields Peter A , Brandt Gabriel
Published: June 2021

Increased ocean temperatures can lead to coral bleaching, as the symbiotic relationship between coral and photosynthetic algae breaks down. The molecular details of this temperature‐dependent disruption are not well understood. Here, we investigate the thermal stability of a critical metabolic enzyme, glyceraldehyde‐3‐phosphate dehydrogenase, from the stony coral Acropora millepora. Binding of the cofactor NAD increases the melting temperature of the enzyme by fully 10 °C. In order to identify the basis for this stabilization, we then determined the crystal structure of the cofactor‐enzyme complex (2.4 Å, PDB ID 6DFZ). This structure is the first of any protein from coral of this genus. The occupancy of NAD across the four monomers of the tetrameric enzyme varies, allowing us to compare the response of the polypeptide structure to the presence of the stabilizing cofactor. We propose that these regions of difference represent areas of the protein important to its thermal stability.

Support or Funding Information

NSF IOS‐1654249

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PDB Structure Data Impacted Discovery and Development of Recently FDA‐Approved Drugs
Authors: Zardecki Christine , Westbrook John , Burley Stephen K.
Published: June 2021

Protein Data Bank (PDB) was established in 1971 as the 1st open access digital data resource in biology and medicine. Today, the PDB currently houses >145,000 atomic level biomolecular structures determined by crystallography, NMR spectroscopy, and 3D electron microscopy. Managed by the Worldwide Protein Data Bank partnership (wwPDB; wwpdb.org), PDB data are used by researchers and educators to understand the role molecules play in health and disease of humans, animals, and plants, food and energy production, and other topics of concern to global prosperity and sustainability.

RCSB PDB operates the US data center for the global PDB archive, and makes PDB data available at no charge to all data consumers without limitations on usage. During calendar 2017, >679 million structure data files were downloaded from the PDB by Data Consumers working worldwide. RCSB PDB served >1 million RCSB.org users worldwide with PDB data integrated with ~40 external data resources providing rich structural views of fundamental biology, biomedicine, and energy sciences, and supported >600,000 PDB101.rcsb.org educational website users around the globe.

RCSB PDB examined the impact of PDB archiving and open access availability on the discovery of 210 new molecular entities (NMEs or drugs) approved by the United States (US) Food and Drug Administration (FDA) between 2010 and 2016. Biological targets for 90% of NMEs are known. Approximately 6,000 PDB structures containing one of these drug targets and/or one approved NME were identified, providing 85% coverage of known targets and 88% coverage of 2010–2016 approved NMEs. Coverage was comparable across all therapeutic areas. Most structures were both published and freely accessible from the PDB long before the NMEs were approved. Additional analyses revealed these PDB structures were cited by more than 200,000 PubMed‐indexed publications reporting pre‐competitive research on the would‐be drug targets. PDB structures and citing publications contributed substantially to understanding of the target biology, and led to biopharmaceutical company investment in successful drug discovery/development programs.

Overall, access to PDB data contribute to patent applications, drug discovery and development, publication of scientific studies, innovations that can lead to new product development and company formation, and STEM education.

Support or Funding Information

RCSB PDB is funded by a grant (DBI‐1338415) from the National Science Foundation, the National Institutes of Health, and the US Department of Energy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Salt Bridge between α4 and α5 Helices Drives Differences in Flexibility and Potency of Inhibition among Regulator of G‐protein Signaling (RGS) Proteins
Authors: Shaw Vincent S , Quinn Josiah A , Mohammadi Mohammadjavad , Vashisth Harish , Neubig Richard R
Published: June 2021

Regulators of G‐protein Signaling (RGS) proteins accelerate the rate of GTP hydrolysis, decreasing G‐protein signaling. Because there are many RGS isoforms with distinct tissue expressions, selective inhibition of RGS action may allow improved tissue specificity over global GPCR agonist application. RGS inhibitors discovered to date act by covalent modification of cysteine residues, and are specific for RGS4. However, single‐cysteine RGS mutants vary widely in their potency of inhibition by thiadiazolidinone (TDZD) inhibitors, with RGS19 being most potently inhibited. We have previously demonstrated a correlation between RGS protein flexibility and potency of inhibition by TDZDs. We hypothesized that differences in a salt bridge‐forming residue between the α4 and α5 helices drives differences in flexibility and potency of inhibition among RGS proteins.

An acidic residue on the α4 helix that takes part in an α4‐α5 salt bride network is shared by RGS4 and RGS8 (D90 and E84 respectively), but not by RGS19 (L118). Upon mutation of RGS19 to induce formation of this salt bridge (L118D), the protein became 7°C more stable than WT, as measured by DSF. A mutation that removed this salt bridge in other isoforms caused an increase in melt temperature of 5°C in RGS4 (D90L) and a decrease in melt temperature of 8°C in RGS8 (E84L). Hydrogen‐deuterium exchange was used to evaluate the flexibility of mutant proteins. The α4 helix, which contains the mutation site, was found to be protected from deuterium exchange on addition of a salt bridge (RGS19 L118D), but have higher exchange compared to WT in both mutants where a salt bridge was removed (RGS4 D90L and RGS8 E84L). Changes in the α4 salt bridge‐forming residue also induced changes in the potency of inhibition of Gα binding by TDZD inhibitor CCG‐50014. RGS19 L118D had a lower potency of inhibition than WT RGS19, while mutations at RGS4 D90L and RGS8 E85L increased potency of inhibition compared to WT proteins. Together, these results show that differences in an α4 salt bridge‐forming residue are responsible for flexibility differences among RGS isoforms, and demonstrate a causal relationship between RGS protein flexibility and potency of TDZD inhibitors.

Support or Funding Information

NSF grant 1507588

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The lower base of corrinoid small molecules regulates reductive dehalogenase enzyme function in Dehalococcoides species
Authors: Jiang Nannan , Yan Jun , Löffler Frank E.
Published: June 2021

Key interactions between proteins and small molecules regulate numerous environmental reactions. Such is the case with reductive dehalogenases that dechlorinate toxic chlorinated solvents only in the presence of corrinoid small molecules. Previous studies have demonstrated that the lower bases attached to these small molecule cofactors affect the activity of reductive dehalogenases differently in corrinoid auxotrophic Dehalococcoides mccartyi strains, which prefer the 5,6‐dimethylbenzimidazole (DMB) lower base. In contrast, Geobacter sulfurreducens strain PCA produces a small molecule corrinoid with 5‐hydroxybenzimidazole (5‐OHBza) as the lower base, which fails to support dechlorination catalyzed by reductive dehalogenases. Here, we cloned and heterologously expressed a unique Geobacter lovleyi O‐methyltransferase gene implicated in the methylation of 5‐OHBza to 5‐methoxybenzimidazole (5‐OMeBza). Unlike 5‐OHBza, 5‐OMeBza does support D. mccartyi reductive dechlorination activity. We demonstrated using HPLC and LC‐MS that the G. sulfurreducens mutant expressed the O‐methyltransferase, as evidenced by the native 5‐OHBza‐cobamide being converted into the 5‐OMeBza‐cobamide. Next, we co‐cultured D. mccartyi and the recombinant G. sulfurreducens in the absence of exogenous vitamin B12, which supported D. mccartyi growth and the complete dechlorination of cis‐1,2‐dichloroethene to ethene. These studies demonstrated that the O‐methyltransferase catalyzes a critical step in lower base biosynthesis and provides a genetic basis for the observation that D. mccartyi reductive dechlorination activity occurs in co‐cultures with G. lovleyi but not with G. sulfurreducens. Our results emphasize the critical role of small molecule interactions directing protein function in the case of dechlorinating toxic chlorinated solvents.

Support or Funding Information

The authors acknowledge funding support from the NIH and SERDP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improving Ligand Geometry in Protein Data Bank Structures Computationally
Authors: Miller Beck , Hudson Brian P , Shao Chenghua , Wang Lu , Zardecki Christine , Burley Stephen K.
Published: June 2021

Information about the three‐dimensional structures of complex biological molecules is an indispensable research tool for many fields of study, including biology, chemistry, pharmacology, and computer science. The Protein Data Bank (PDB) is an public digital archive providing access to structures of nucleic acids, proteins, and large macromolecular machines determined experimentally by X‐ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and 3‐dimensional electron microscopy (3DEM). PDB structures undergo validation for structural plausibility upon their deposition into the PDB using the program Mogul, which cross references each deposited structure against similar data in the Cambridge Structural Database (CSD). Ligands associated with PDB structures are a common source of structural inaccuracy. In this study, a programmatic pipeline was developed to determine if ligand geometric accuracy could be improved using known computational tools. Ligands with dubious geometry were identified through a Python mining code. A sample set of the identified structures was remodeled into minimum energy conformations using the molecular dynamics program AMBER and then fit to the original experimental density maps using the molecular modeling program Coot. The refined models were compared to the original models in the PDB as well as to similar structures in the CSD using the deposition statistical validation server. Preliminary results suggest that the pipeline effectively identifies and improves ligand structures that possess inaccurate geometry. This method can be used to evaluate and improve the accuracy of specific ligands in structures both archived and pending deposition.

Support or Funding Information

This work was supported by an NSF REU and the RCSB PDB [NSF (DBI‐1338415), NIH, and DOE.]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomaterials with Functionally Graded Stiffness
Authors: Siemers Bram , Stempien Alana , Craven Rachel , Napiwocki Brett , Crone Wendy
Published: June 2021

There are many different tissues in the body, each possessing different mechanical properties and varying amounts of stiffness that are needed for cells to function properly and survive. A challenge to recreating in vitro tissues is to accurately recapitulate the mechanical properties of the native tissue. The focus of this research was to produce a substrate with a functionally graded stiffness for use with in vitro culture of cardiomyocytes, the cells of the heart responsible for contraction.

Young's modulus, a measure of stiffness of materials, is a key characteristic parameter for tissues. Currently, there is a lack of substrate systems that span the range of Young's modulus needed to represent both healthy and diseased myocardium. This is problematic when trying to model cardiovascular disease. For example, after myocardial infarction, there are regions of soft and stiff underlying tissue that impact the behavior of cardiomyocytes differently.

The goal of this research was to create a substrate system with a stiffness gradient spanning from 5 to 50 kPa upon which the cardiomyocytes derived from stem cell could be cultured. This in vitro substrate system was designed to mimic the range of physiological environments in which cardiomyocytes exist in vivo. This substrate model is specifically being designed to aid in better understanding and reducing cardiovascular disease.

The method investigated that showed the most promise was UV curing of polyacrylamide hydrogels. A gradient photomask was designed to allow different amounts of UV light to penetrate over the span of the substrate. This mask was then placed over the prepolymer gel during UV exposure. This method produced a stiffness gradient of 55 to 75 kPa over a 1 cm distance. Further work is needed to optimize the photomasks and gel reagents to produce the lower end of modulus representative of healthy myocardium.

Support or Funding Information

Supported with funding from the Karen Thompson Medhi Professor at the University of Wisconsin‐Madison and the National Science Foundation through the University of Wisconsin‐Madison Materials Research Science and Engineering Center (DMR‐0520527) and Nanoscale Science and Engineering Center (DMR‐0425880). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Olfactory Receptor Family 7 Subfamily C Member 1 Expression in T‐cell Acute Lymphoblastic leukemia Identifies a Potential Stem Cell Sub‐population
Authors: Jeon Dayoung , Silva‐Espinoza Juan Carlos , Ahmed Haidar , Vines Charlotte , Bill Colin
Published: June 2021

Cancer stem cells (CSCs) are thought to be a self‐renewing population that is difficult to kill during standard chemotherapy due to the capacity of the cells for self‐renewal and multidrug resistance. Our lab studies T‐cell acute lymphoblastic leukemia (T‐ALL), which is an aggressive form of leukemia. T‐ALL cells, which can hide from chemotherapeutic treatment regimens within the central nervous system ( CNS), use different chemokine and adhesion receptors to invade the CNS; entry into the CNS contributes to the aggressiveness of the disease. Therefore, migration into the CNS necessitates the use of aggressive treatments such as cranial irradiation and intensified intrathecal chemotherapy, which causes neurocognitive deficits, endocrine disorders, and growth impairment. A recent study showed that the ectopic expression of Olfactory Receptor Family 7 Subfamily C Member 1 (OR7C1) correlates with expression of stem cell markers including SOX2, POU5F1, and LGR5. These stem‐cell markers correlate with decreased survival rates, implicating the expression of these stem cells in chemo‐resistant cancers. Therefore, we hypothesized that the expression of these stem cell markers in T‐ALL cell lines would contribute to the aggressiveness of the disease. Using flow cytometry, we found approximately 1% of HuT78, adult human T‐ALL cells, express OR7C1. Furthermore, while we were able to detect OR7C1 protein expression in HuT78 cells through both Western blot and RT‐PCR, we found that peripheral blood mononuclear cells (PBMCs) lack OR7C1. Currently, we are determining if the expression of OR7C1 leads to the expression of stem cell transcription factors in HuT78, CCRF‐CEM pediatric T cell lines, and PBMCs. These findings will allow us to determine the prognostic significance of OR7C1 in the progression of T‐ALL.

Support or Funding Information

NSF award DUE‐1565063

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ferric Reductase Knockdown in Drosophila S2 Cells
Authors: Arismendiz Omar Alexis Rodriguez , Holst Jessica , Ochoa Alma , Gorman Maureen J. , Ragan Emily J.
Published: June 2021

Iron is an essential micronutrient that is potentially harmful due to its redox properties, which is why its transport between cells must be a highly regulated process. As compared to mammals, very little is known about iron uptake in insects, because receptors for ferritin or transferrin have not been identified in insects. The goal of the proposed research is to understand the physiological mechanisms of iron transport in insects. We are testing the hypothesis that the reduction of iron ions in the hemolymph leads to iron transport into cells. Hemolymph contains iron in the ferric form, but iron transporters in animal cells are specific to ferrous iron; therefore iron uptake via an influx transporter is likely to require a reduction step. We silenced a putative ferric reductase CG8399 by RNA interference (RNAi) to determine if it contributes to ferric reductase activity and impacts the iron content of the cells. We analyzed the efficiency of knockdown of CG8399 by real time, quantitative PCR (qPCR).

Support or Funding Information

NSF IOS‐1656407

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pilot Studies of Two Possible Iron Uptake Mechanisms in Insect Cells
Authors: Coca Michelle , Najera Diana G , Gorman Maureen J
Published: June 2021

Iron plays an important role in energy metabolism and other essential physiological processes; however, because iron can also be toxic, its uptake by cells must be strictly regulated. In humans, there is a well‐understood pathway of iron uptake and multiple poorly understood pathways. How iron is transported into insect cells is unknown. The goal of this study was to test two models of iron uptake by cultured insect cells. The first model involves endocytic uptake of protein‐bound iron. The second model involves uptake of iron through a ferrous transporter. For our first set of experiments, we used the well‐studied Drosophila melanogaster S2 cell line. We found that S2 cells express rab5 (an essential component of endosomes), secreted ferritin, membrane‐bound transferrin, two ferric reductases, and a ferrous transporter. Treating cells with endocytosis inhibitors had no effect on iron content; therefore, endocytosis does not seem to be required for iron uptake by S2 cells. In contrast, sequestering iron with a ferrous chelator decreased the iron content of treated cells, suggesting that the ferrous form of iron is involved in the uptake process. We wanted to take advantage of a newly developed chemically defined medium (ZB) to learn more about the mechanism of iron uptake, but we were unable to grow S2 cells in ZB medium. We found that Kc167 and Cl.8 cells, which do grow in ZB medium, express the same set of iron‐related genes as S2 cells. To test the hypothesis that endocytosis is involved in iron uptake by D. melanogaster cells, we are in the process of evaluating the effect of RNAi‐mediated knockdown of rab5 on cellular iron content. Toward this goal, we have synthesized dsRNA that targets rab5 and optimized a method to measure cellular iron content. To test the hypothesis that a ferrous transporter is involved in iron uptake, we will measure the effect of RNAi‐mediated knockdown of divalent metal transporter 1 (DMT1) on cellular iron content. Our tentative conclusion is that insect cells take up iron via a mechanism that involves a ferrous transporter. A better understanding of iron uptake in insects could lead to better insect control strategies and also provide insight into the less understood iron uptake mechanisms of humans.

Support or Funding Information

NSF IOS 1656388

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biological Synthesis of Short Glycogen Polymers
Authors: Beacham Branden , Cuellar Itzayana G. , Snyder Nicole L. , Campbell A. Malcolm
Published: June 2021

Although humans naturally synthesize full‐length glycogen, chemically producing short glycogen polymers in the laboratory is complex and takes months. The goal of our research is to efficiently synthesize short glucose polymersusing the human primer enzyme glycogenin. Glycogen is large polymer of glucose that is produced by many organisms, including humans. Inside cells, glycogen production requires two protein enzymes. The first enzyme, glycogenin, assembles up to 10 glucose molecules to form a glycogen “primer” molecule. Glycogen synthase then uses the short glucose polymer as aprimer to synthesize the much larger glycogen molecule. In our study, we cloned the human glycogenin cDNA and used it to produce the primer enzyme inside E. coli cells. We then assessed the ability of therecombinant human protein to produce short glucose polymers in vitroand analyzed the results to confirm glucose primer production. Here we present our preliminary results demonstrating the potential for our method to produce glycogen primers that can serve as a scaffoldsto produce derivatized glucose‐basedpolymers. The resulting glycopolymers are potential candidates for therapeutic applications.

Support or Funding Information

Funding provided by the Davidson Research Initiative, NSF grants to Davidson College (RUI‐1613203 and RUI‐1708546) and the James G. Martin Genomics Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The resilience of cancer‐specific protein galectin‐3: Implications in biological functions
Authors: Dam Tarun , Kadav Priyanka , Welch Christina , Edwards Jared , Bandyopadhyay Purnima
Published: June 2021

Structural degradation is detrimental for protein activity and functions. We recently found that the tumor‐associated protein galectin‐3 (Gal‐3) is apparently an exception to this rule. Gal‐3 plays important roles in cancer but its functions are not fully understood. We have found that the affinity purified Gal‐3, when stored with lactose at 4°C for more than six weeks, undergoes complete degradation even in the absence of collagenases. Full‐length Gal‐3 after full degradation produced a single protein band of 17 kDa. This molecular weight is similar to that of the CRD (carbohydrate recognition domain) portion of Gal‐3. Conventionally, the CRD is separated from intact Gal‐3 by enzymatic digestion using collagenases. However, in the present case, Gal‐3 apparently self‐degraded to produce the CRD without the catalytic action of collagenases. Our data indicate that degradation of Gal‐3 significantly alters the protein but the CRD remains intact and functionally active. Like the full‐length Gal‐3, the CRDs interacted with thyroglobulin, chondroitin sulfate A and C. However, the CRDs could not cross‐link these multivalent ligands as revealed by our spectroscopic analysis. Our current observations suggest that Gal‐3 retains it ligand recognition properties even when it is substantially degraded in a challenging cellular environment. Thus the degraded Gal‐3 can still carry out some of the functions of intact Gal‐3. Although the CRDs retain the binding property of its intact precursor (Gal‐3), they lose the ability to cross‐link cellular receptors. As a result, the CRDs generated through degradation may influence cell signaling by competing with intact Gal‐3 for the same receptors in cellular environment.

Support or Funding Information

National Science Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of recurrent fusion genes across multiple cancer types
Authors: Luo Jianhua , Michalopoulos George , Yu Yanping
Published: June 2021

Chromosome changes are one of the hallmarks of human malignancies. Chromosomal rearrangement is frequent in human cancers. One of the consequences of chromosomal rearrangement is gene fusions in the cancer genome. We have previously identified a panel of fusion genes in aggressive prostate cancers. In this study, we showed that 6 of these fusion genes are present in 7 different types of human malignancies with variable frequencies. Among them, the CCNH‐C5orf30 and TRMT11‐GRIK2 gene fusions were found in breast cancer, colon cancer, non‐small cell lung cancer, esophageal adenocarcinoma, glioblastoma multiforme, ovarian cancer and liver cancer, with frequencies ranging from 12.9% to 85%. In contrast, four other gene fusions (mTOR‐TP53BP1, TMEM135‐CCDC67, KDM4‐AC011523.2 and LRRC59‐FLJ60017) are less frequent. Both TRMT11‐GRIK2 and CCNH‐C5orf30 are also frequently present in lymph node metastatic cancer samples from the breast, colon and ovary. Thus, detecting these fusion transcripts may have significant biological and clinical implications in cancer patient management.

Support or Funding Information

NCI, DOD and UPCI

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of STAT3 by Human Serotonin 2C Receptor
Authors: Maisha Musarrat , Curtis M.E. , Fears L.S , Lanaghan Zeljka Miletic , Ivy MT , Fentress Hugh
Published: June 2021

Serotonin (5‐HT) is an indolamine neurotransmitter involved in a variety of functions including regulation of mood, appetite, sleep, learning and memory, and neuronal excitability. 5‐HT elicits its effects by binding to at least 14 different receptor subtypes that mediate both excitatory and inhibitory neurotransmission. The many classes of serotonin receptors have been subjected to significant studies in order to discern the functionality and produce effective therapeutic targets. One of the receptors, the 5‐HT2C receptor, has been studied and used as a target for antipsychotic medication. 5‐HT2C subtype receptor exists as a 7‐transmembrane spanning G protein‐coupled receptor (GPCR) found in the central nervous system, and is the only GPCR known to undergo RNA editing, creating 14 known isoforms in the human brain which affect its ability to couple with G proteins and other signaling proteins. The structurally similar 5‐HT2A receptor has been shown to activate the G protein independent JAK/STAT pathway. The purpose of this study was to determine if the human 5‐HT2C receptor also activates the JAK/STAT pathway. Human Embryonic Kidney (HEK) 293 cells were transfected with human 5‐HT2C receptors to produce stable cell lines. Cells were then treated with vehicle (control), 5‐HT, olanzapine (antagonist), and SB206553 (inverse agonist) for 30 minutes or 1 hour. Treated cells were lysed, proteins were separated by SDS‐PAGE, and levels of phosphorylated JAK2 and STAT3 were analyzed using western blots. In cells treated for 30 minutes with vehicle, phosphorylation of STAT3 was observed in cells transfected with the 5‐HT2C receptor but not in untransfected cells indicating constitutive phosphorylation of STAT3 by the 5‐HT2C receptor. Upon treatment with serotonin, phosphorylated levels of STAT3 were increased in cells expressing the 5‐HT2C receptor while STAT3 phosphorylation was completely blocked with cells treated with olanzapine or SB206553. Phosphorylated levels of JAK2 were unaffected across all treatments. Phosphorylated STAT3 levels showed the same patterns in cells expressing the 5‐HT2C receptor when treated for 1 hour. These findings indicate that 2C receptors are activating STAT3 independently of JAK2 and may be involved in cell growth, differentiation, transcription of other STAT genes, and immune response. Future studies will examine different edited isoforms of the 5‐HT2C receptor and whether STAT3 activation is dependent or independent of G‐protein activation to elucidate the phosphorylation mechanism of STAT3 by the human 5‐HT2C receptors.

Support or Funding Information

Supported by NSF Grant HRD‐1238723 to HMF

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinase and Mitochondrial Targets for Reversing the Adverse Effects of Organophosphates on Axonal Transport
Authors: Naughton Sean X , Wei Zhe , Guangyu Wu , Terry Alvin V
Published: June 2021

Organophosphates (OPs) represent a class of chemicals with a wide range of uses in industrial and agricultural settings as pesticides, but they have also been associated with a variety of adverse health effects in humans including deficits in cognition. While the acute toxicity of OPs is associated with the inhibition of cholinesterase enzymes, our laboratory has previously demonstrated that repeated exposures to levels of OPs below the threshold for acute toxicity can negatively affect non‐cholinesterase targets (i.e., effects that might contribute to the cognitive defects). These negative effects of OPs include deficits in the axonal transport of membrane bound organelles (MBOs) as well as alterations in both the transport and morphology of mitochondria. Postmortem analyses of brain tissues of rats treated with the OP diisopropylfluorophosphate (DFP) and in vitro phenotypic screening assays revealed that targeting specific signaling kinases that regulate axonal transport and approaches to improve mitochondrial function might represent rational therapeutic strategies to reverse the negative effects of OPs. These hypotheses were validated in live cell imaging studies in vitro, where DFP‐induced impairments in both anterograde and retrograde AXT were attenuated in a dose‐dependent manner with the glycogen synthase kinase (GSKIIIβ) inhibitor, lithium chloride. Similarly, the mitochondrial‐targeting antioxidant methylene blue also attenuated the negative effects of DFP on anterograde and retrograde axonal transport in a dose dependent manner. Studies are in progress to determine the effects of these potential therapeutic agents on specific components of the axonal transport process (e.g., number of stationary particles, directional changes, pauses, etc.).

Support or Funding Information

Congressionally Directed Medical Research Programs (CDMRP), Medical College of Georgia Foundation, Augusta University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of aging on oxygen permeability of wild type (WT) and AQP1‐RhAG double knockout (dKO) mouse red blood cells
Authors: Zhao Pan , Boron Walter F
Published: June 2021

The dogma that all gases move through red blood cell (RBC) membranes by dissolving in and diffusing through the membrane lipid is no longer correct. Our preliminary data on mouse RBCs genetically deficient in both aquaporin‐1 (AQP1) and Rh complex (mainly RhAG) suggest that these two proteins account for ~60% of membrane O2 permeability. Using stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra, we now examine the effect of aging on the rate constant of Hb deoxygenation (kHbO2) of RBCs from WT mice and mice genetically deficient in both AQP1 and RhAG (double knockout, dKO). Studying mice at five ages (2~3 months, 6 months, 12 months, 18 months, and 24 months), we find that RBCs from WT mice at ages from 2~3 months through 12 months all have similar kHbO2 values (N = 6 mice/group). RBCs from WT mice at 18 (N = 6) and 24 months (N = 2) have mean kHbO2 values that—compared to RBCs from WT mice 2~3 months old—decrease by 8% at 18 months and by 10% at 24 months. These decreases in mean kHbO2 do not yet reach statistical significance, using a Holm‐Bonferroni correction. Regarding RBCs from dKO mice, we observe similar kHbO2 values for all ages from 2~3 months to 18 months (N=5 for 18 months), inclusive. RBCs from dKO mice at 24 months are currently under investigation. We plan to extend this study to RBCs from mice genetically deficient in just AQP1 or RhAG. If the trends continue (i.e., for RBCs from aging WT but not dKO to exhibit a fall in kHbO2), it would be consistent with the hypothesis that the non‐AQP1/RhAG component of RBC membrane O2 permeability is independent of aging, whereas the AQP1 and/or RhAG component declines with aging.

Support or Funding Information

Supported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The right ventricle has more resident immune cells than the left ventricle
Authors: Gorr Matthew W , Sriram Krishna , Muthusamy Abinaya , Chinn Amy M , Insel Paul A
Published: June 2021

The right ventricle (RV) pumps deoxygenated blood from the systemic circulation to the lungs for reoxygenation. Diseases such as pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease are associated with hypoxia that leads to an increase in pulmonary vascular resistance (PVR), resulting in RV hypertrophy and dysfunction. The RV may differ from the left ventricle (LV) in the pathobiology in response to increases in afterload and no specific therapy exists for RV dysfunction. In order to gain insight into the biology of the RV compared to the LV, and perhaps uncover potentially novel therapeutic approaches for RV dysfunction, we performed RNA‐sequencing on RV and LV tissue from rats in normal ambient conditions or subjected to hypoxia (10% O2) for two weeks. Gene ontology and pathway analysis of the RV compared to the LV revealed multiple transcriptomic differences under ambient conditions and in particular, increased expression in the RV of genes related to immune function. Immune cell profiling by flow cytometry of rat cardiac digests, analyzed as the percentage of CD45+/CD11b/c+ cells in the cell population, revealed that in both ambient conditions and hypoxia, the RV had a significantly greater percentage of double‐positive cells compared to the LV (normoxia: average % ± SEM, 8.55 ± 1.71 RV vs. 2.04 ± 0.62 LV and hypoxia: 6.59 ± 2.71 vs. 1.55 ± 0.4; P <0.005, two‐way ANOVA, n=4 rats/group). Hypoxia had no significant effect on these values (P>0.05). These data reveal that the RV has a greater number of resident immune cells compared to the LV, possibly contributing to differences in pathobiology. Further investigation may reveal immune‐related therapeutic strategies for RV disease.

Support or Funding Information

Supported by Department of Defense (W81XWH‐14‐1‐0372)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling the Evolution of Crocodyliform Cranial Shape Through Time
Authors: Wilberg Eric , Turner Alan , Smaers Jeroen
Published: June 2021

Crocodyliforms have a rich fossil record, attained global distribution, and filled a wide variety of ecological niches throughout their history. The group has explored a broad range of cranial morphologies through time, particularly in relation to rostral and supratemporal fossa (STF) form. These character complexes are functionally related, with the snout acting as the prey capture apparatus and the STF as the origin for the pseudotemporalis and/or adductor mandibulae externus profundus musculature. A correlation between STF size and snout shape has been previously suggested (e.g. taxa with long slender snouts tend to have larger STF, possibly related to the importance of the aforementioned muscles in their prey capture strategy). To investigate evolutionary patterns of these cranial modules we quantified snout and supratemporal fossa shape using sliding semilandmark‐based geometric morphometric methods. We then modeled the evolution of these modules across a time‐calibrated phylogeny of Crocodyliformes, testing for differences in allometric patterning (using new functions in the R package bayou and phylogenetic ANCOVA as implemented in evomap). Our results indicate a significant shift in the covariation of STF relative to snout length at the origin of Eusuchia. In comparison with non‐eusuchian crocodyliforms, eusuchians exhibit less change in STF size relative to snout length. This shift in phenotypic covariation is potentially related to a change in pattern (but not strength) of developmental integration between these modules.

As these modules are related to feeding, this shift in phenotypic covariation may indicate an important transition in the evolution of the crocodyliform feeding apparatus at the origin of Eusuchia. That non‐eusuchian crocodyliforms tend to have a larger STF suggests a reliance on muscles occupying the dorsal temporal fenestra, while the relatively smaller STF of eusuchians may mark a shift to an increased role of the pterydoideus musculature responsible for the extreme bite forces of extant crocodylians. If such a model holds true, it may serve to best explain the observed shift in covariation between these modules. The timing of this transition in our analysis matches closely with a previous study modeling the evolution of bite force in the group based on a different osteological proxy. Collectively this suggests that the modern crocodylian feeding apparatus developed around the origin of the crown group, coincident with the establishment of the eusuchian secondary palate.

Support or Funding Information

National Science Foundation grant NSF‐DEB 1754596

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantifying Ubiquitination Signaling with a Chemical Proteomics Strategy
Authors: Li Yunan , Luo Ang , Erber Luke , Chen Yue
Published: June 2021

Stoichiometric analysis of posttranslational modifications is an emerging strategy for absolute quantification of the modification's fractional abundance. In this study, we present a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination, named Isotope‐BAlanced Quantification of Ubiquitination (IBAQ‐Ub). The strategy utilizes a new amine‐reactive chemical tag (AcGG‐NHS) for highly efficient labeling of amine residues. The new chemical tag is structurally homologue to the GG remnant of ubiquitin on modified lysine following trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and the secondary stable isotopic labeling. Quantitative analysis of labeled peptides with HPLC and high resolution mass spectrometry allows unbiased measurements of absolute site‐specific ubiquitination stoichiometry. Using recombinant protein standards and isotopic serial dilution, we showed that the strategy is highly robust, sensitive and accurate. We further designed a SILAC‐based experiment to demonstrate the efficient labeling of lysine with the new AcGG tag in complex cell lysates. Application of the IBAQ‐Ub workflow for the analysis of enriched histone fractions allowed us to measure the stoichiometry of histone H2B C‐terminal ubiquitination. Comparative analysis of stoichiometric dynamics upon proteasome inhibitor treatments showed a dramatic decrease of histone H2B ubiquitination levels and a significant increase of polyubiquitination K48 linkage. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways in cells and diseases.

Support or Funding Information

University of Minnesota start‐up fund and National Science Foundation (CHE‐1753154)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of WWOX and Zfra in limiting neurodegeneration
Authors: Chang Nan‐Shan , Lin Yu‐Hao
Published: June 2021

Human and animal newborns with WWOX/Wwox gene deficiency suffer severe neural diseases, seizure, microcephaly, metabolic disorders, and early death. Neuronal heterotopia, which accounts for seizure, occurs in the newborns due to enhanced migration of cortical neurons to the epicortex. When tumor suppressor WWOX is Tyr33 phosphorylation, it strongly inhibits cancer growth in vitro and in vivo. When Tyr33 is de‐phosphorylated and Ser14 is phosphorylated, endogenous pS14‐WWOX is associated with enhanced growth of cancer stem cell spheres and solid tumors, and progression of neurodegeneration in patients with Alzheimer's disease (AD). We recently reported that a small zinc‐finger like Zfra peptide, e.g. Zfra1‐31 or 4–10, dramatically suppresses Ser14 phosphorylation in WWOX (>90% pS14‐WWOX), which leads to significant suppression of cancer growth and restoration of memory loss by blocking neurodegeneration in triple transgenic mice (3xTg) for AD. Also, when 3xTg mice received Zfra via tail vein injections at month 3, Zfra prevented age‐dependent loss of short‐term and long‐term memory in these mice. Zfra accelerates the degradation of aggregated proteins (e.g. amyloid beta) and activates a novel spleen non‐T/non‐B Hyal‐2+ CD3‐CD19‐lymphocyte, designated Z cells. Intringuingly, activated Z cells have a memory anticancer function, even though these cells have never gotten a chance encountering cancer cells or cancer cell antigens. Whether Z cells block neurodegeneration is unknown. Wwox heterozygous mice have an enhanced memory loss with age, compared to 3xTg mice. This is due, in part, to aggregation of a cascade of proteins, including initiators TRAPPC6AΔ, TIAF1, and SH2GLB2 (designated TTS) for causing amyloid beta and tau aggregation in the brain. In Wwox knockout mice, protein aggregates can be found in the cortex and hippocampus in 3 weeks after birth. In parallel, downregulation of WWOX protein in middle‐aged humans may lead to the TTS cascade of protein aggregation that allows formation of amyloid plaques and tau aggregation in old ages. Our study reveals an inhibitory role of WWOX and Zfra in suppressing neurodegeneration.

Support or Funding Information

Research was supported by the Ministry of Science and Technology, Taiwan (MOST 105‐2320‐B‐006‐046, 105‐2320‐B‐006‐036,106‐2320‐B‐006‐017, and 106‐2320‐B‐006‐061), the Department of Defense, USA (W81XWH‐08–1‐0682), the National Health Research Institute, Taiwan (NHRI‐EX107‐10734NI) to NS Chang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2
Authors: Tong Ashley L , Schlau‐Cohen Gabriela , Ogren John , Cao Jianshu , Chenu Aurelia , Gordon Sam , Blankenship Robert , Lu Yue
Published: June 2021

Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light‐harvesting process begins with absorption of solar energy by an antenna protein called Light‐Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light‐harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non‐native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein‐protein interactions and lipid‐protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light‐harvesting complexes in a near‐native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid‐protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent‐solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to tilting of the peripheral bacteriochlorophyll in the B800 band. These results highlight the importance of well‐defined systems with near‐native membrane conditions for physiologically‐relevant measurements.

Support or Funding Information

National Science Foundation Beckman Young Investigator Department of Energy

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isolation and Identification of C‐Mannosylated Hsc70 Conjugates in RAW 264.7 Macrophage‐Like Cells
Authors: Parsons Michael Arland
Published: June 2021

Post‐translational addition of carbohydrate molecules is often necessary for proteins to be fully active. This process, known as glycosylation, usually involves a carbon‐nitrogen or carbon‐oxygen bond. There is, however, a form of glycosylation that involves formation of a carbon‐carbon bond. C‐linked glycosylation in higher organisms consists of the addition of α‐mannopyranoside from dolichyl‐phosphate mannose onto the C‐2 carbon of tryptophan's indole functional group, generating a residue known as C‐mannosyltryptophan. Though the function of this modification is not well characterized, the addition of α‐mannopyranoside to tryptophan residues is thought to play an important role in the innate immune response. (Muroi et al.; Hartmann and Hofsteenge; Hofsteenge et al.)

It is known that the consensus sequence for C‐mannosylation is Trp‐x‐x‐Trp/Cys (x could be any amino acid), in which the first tryptophan is modified. (Furmanek and Hosteenge) This canonical sequence has been found in several signaling molecules such as cytokines and cytokine receptors.

It has been shown that Hsc70 interacts with proteins containing the C‐mannosyltryptophan residue. With this in mind, Hsc70 pulldowns were done by the Rakus Lab in an attempt to capture some of these conjugates. Several proteins were identified using this procedure, and mass spectrometry showed a mass shift of 162 Da on tryptophan containing peptides – indicative of a C‐mannosyltryptophan residue.

One of these modified peptides was identified as belonging to a Type I cytokine receptor – Interleukin 12 Receptor β2 (IL12Rβ2). Notably, one of its target molecules, interleukin‐12 (IL12), also contains the canonical sequence and has been shown to contain a C‐mannosyltryptophan residue. (Doucey et al.)

Anti‐ IL12Rβ2 antibodies were used in an attempt to isolate IL12Rβ2 directly. The same was done for a similar protein IL12Rβ1. While IL12Rβ2 appeared to be present in western blots, mass spec data failed to confirm this. IL12Rβ1 was not seen in western blots or mass spec. Upon successful isolation, a more thorough mass spec analysis can confirm or deny the existence of C‐mannosyltryptophan residues in these receptors.

Support or Funding Information

Marshall University Chemistry Department; NSF (OIA‐1738707)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.