bluuebunny/crossref_metadata_2025 huggingface dataset:
High Fat Diet Blunts the Activation of Canonical Wnt Signaling Pathway Induced by Radiation Therapy in Mouse Colon Epithelia
Authors: Xu Guanying , Emmons Russel , De Lisio Michael , Pan Yuan‐Xiang , Chen Hong
Published: June 2021

Background

Radiation stimulates multiple metabolic pathways in colon epithelial cells in mice, including the Wnt signaling pathway. Activation of Wnt/β‐catenin pathway was indicated to increase resistance to radiation therapy. On the other hand, we have shown that long‐term consumption of high fat diet inhibits Wnt signaling through the down‐regulation of β‐catenin expression in colon.

Objectives

The main goal of this present study is to examine the impact from high fat diet on the expression of genes involved in the Wnt/β‐catenin pathway on colon epithelial in mice exposed to radiation.

Methods

In this study, 5‐week‐old male mice were fed either a control diet (n=22, 10% kcal fat) or a high‐fat diet (n=24, 45% kcal fat) for 11 weeks. Half of the mice were sampled as pre‐irradiation (Pre‐CON and Pre‐HF). The rest of the mice were treated with a single dose of radiation at 3 Gy (IR‐CON and IR‐HF) and tissue collection was conducted after 4 more weeks of respective dietary treatments. mRNA expression of Wnt pathway genes were analyzed in the colon tissue.

Results

Radiation significantly increased mRNA expression of canonical Wnt signaling related genes including APC, AXIN1, CTNNB1 (β‐catenin) and cMyc in IR‐CON comparing to Pre‐CON (p<0.05). On the other hand, APC, AXIN1 and cMyc remain unchanged in IR‐HF comparing to Pre‐HF, only CTNNB1 increased. Sfrp2, a Wnt signaling antagonist, was significantly increased in IR‐HF comparing to Pre‐HF (p<0.05), while remained unchanged in IR‐CON comparing to Pre‐CON. Furthermore, mRNA expression of a cell cycle control gene, p21, was significantly increased in IR‐HF. This is accompanied with a significant decrease of CyclinD1 expression (p<0.05, when comparing to Pre‐HF).

Conclusion

Overall, analysis of mRNA expression suggests that high fat diet blunts the effects of radiation on inducing the Wnt signaling pathway, which is associated with cell cycle pathway in mouse colon epithelia. Our study shows that high fat diet may interfere with radiation treatment in colon.

Support or Funding Information

This project was supported in part by the Campus Research Board of University of Illinois at Urbana‐Champaign and the USDA Cooperative State Research, Education and Extension Service, Hatch project numbers # ILLU‐698‐923 and ILLU‐698‐379 to H.C.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of eukaryotic initiation factor 3 in the non‐canonical mechanism of Barley Yellow Dwarf Virus translation initiation
Authors: Powell Paul , Bhardwaj Usha , Mitra Sondeb , Gonzalez Ruben , Goss Dixie
Published: June 2021

Plant viruses are a world‐wide threat both economically and to the food supply. Many of these are positive‐strand RNA viruses that have developed a myriad of non‐canonical translation initiation mechanisms. These unique mechanisms circumvent cap‐dependent translational controls and enable the viruses to outcompete host cell mRNA for the translation machinery. Many RNA viruses lack the canonical 5′ 7‐methylguanosine cap in their mRNAs, thereby allowing them to bypass canonical translation initiation. Instead, these mRNAs utilize structures in the 3′ untranslated region (UTR) of their mRNA known as a 3′ CITE (cap‐independent translation element) to drive non‐canonical translation initiation. Recently, we have shown that a 3′ CITE in Barley Yellow Dwarf Virus (BYDV), termed the 3′ BTE (BYDV 3′ CITE), can bind eukaryotic translation initiation factor (eIF) 4G, eIF4A, and ATP‐bound eIF4B in order to recruit the 40S ribosomal subunit and form a pre‐initiation complex (PIC) at the 3′ BTE. These eIFs cannot directly recruit the 40S subunit to the 5′ UTR of BYDV, but must instead transfer the 40S subunits to the 5′ end of the mRNA scanning, AUG detection, and translation elongation to proceed. This transfer process is aided by a long‐distance, ‘kissing‐loop’, base pairing interaction between stem‐loops in the 5′ and 3′ ends of BYDV. This kissing loop interaction is relatively weak, leading to the hypothesis that additional factors are required to create a stable 5′–3′ interaction capable of efficiently transferring the PIC. Data from pulldown experiments have shown that, in addition to eIF4G, almost every subunit of eIF3 is bound to the BTE. Single‐molecule fluorescence co‐localization experiments have shown that eIF3 specifically and significantly enhanced binding of 40S subunits to the BTE. Moreover, fluorescence anisotropy‐based binding studies have shown that eIF3 binds directly to both the BTE and the 5′ UTR of BYDV, that the BTE and 5′ UTR do not compete for binding to eIF3, and that the presence of the 40S subunit increases the affinity with which eIF3 binds to the BTE and 5′ UTR. Collectively, these data suggest that eIF3, the 40S subunit, the 3′ BTE, and the 5′ UTR are components of a single macromolecular complex. Based on this interpretation of our data, we propose that eIF3 bridges the 3′ and 5′ ends of BYDV and aids the kissing loop interaction in transferring the PIC from the 3′ to the 5′ end of BYDV.

Support or Funding Information

Grant Support: NSF MCB‐1513737 (to DJG), NSF‐IGERT Fellowship 0965983 (to PP), and NIH GM 084288 (to RLG).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pervasive, coordinated protein level changes driven by transcript isoform switching
Authors: Brar Gloria , Otto George , Cheng Ze , Powers Emily , Keskin Abdurrahman , Jovanovic Marko
Published: June 2021

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous, genome‐wide measurements of mRNA, translation and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti‐correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases—or over 8% of all measured genes—involves temporally regulated switching between production of a canonical, translatable transcript and a 5′ extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether an mRNA is induced or not, but rather based on the type of transcript produced.

Support or Funding Information

This work was supported by NIH funding to G.A.B. (DP2‐GM‐119138), investigator awards from the Alfred P. Sloan Foundation and Pew Charitable Trusts to G.A.B., UC‐Berkeley start‐up funding to G.A.B. G.M.O. is supported by an NIH training grant (GM0007232) to UCB MCB. E.P. is supported by an NSF predoctoral fellowship. M.J. was supported by the Marie Sklodowska‐Curie IOF and he and A.K. are supported by Columbia start‐up funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetics and Binding Studies of the (NRPS)‐Independent Siderophore (NIS) Synthetase DesD
Authors: Lathan Kiera , Hoffmann Katherine
Published: June 2021

Pathogenic bacteria have always been a problem in human health. They do not discriminate when it comes to finding a human host for them to survive in, so it effects all people regardless of ethnicity and socioeconomic background. Pathogenic bacteria use their host to supply them with something that they lack. In the case of iron, bacteria synthesize, secrete, and reacquire small molecule chelators called siderophores. Our lab is specifically focusing on siderophores made with the non‐ribosomal peptide synthetase independent siderophore (NIS) pathways, which always contain at least one NIS synthetase family protein. These proteins are excellent drug targets because of their association with virulence, their unique chemistry, and their unique fold. A drug that successfully targeted and inhibited this family of proteins might represent a new class of antibiotics.

The best studied NIS synthetase is Desferrioxamine synthetase D (DesD) from Streptomyces coelicolor, which makes the group of siderophores called desferrioxamines. Desferrioxamine E is the biggest siderophore made of 3 N‐hydoxysuccinylcadaverine units repeatedly added by DesD and then cyclized. This broad substrate specificity (the variation in size recognized by the same protein), is the reason why we are interested in mapping the thermodynamics of binding to different substrates to see if there is a variation in association constants. We are using Isothermal Titration Calorimetry (ITC) because it is label‐free and have successfully piloted the conditions for the assay. If we are able to delineate the priority requirements for binding, we will progress to designing an inhibitor to maximize the binding affinity. ITC methods will also be used to characterize the kinetics DesD so that a better overall picture of the proteins' biochemistry may be understood.

Support or Funding Information

This work was supported by the National Science Foundation grant to K.M.H. (MSP ‐ 1716986) and by California Lutheran 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.
Characterization of the 3rd and 4th EF‐hand Motifs of NADPH Oxidase 5 by Spectroscopy and Calorimetry
Authors: Wei Chin‐Chuan , Fabry Emily
Published: June 2021

Reactive Oxygen Species generated by NADPH oxidases (Nox's) are implicated in intracellular signaling leading to altered gene expression, protein biosynthesis, apoptosis, kinase activation, and oxygen sensing. Among the Nox family, Nox5 contains an additional EF‐hand domain (EFD) along with transmembrane domain and dehydrogenase domain. It is believed that Ca2+ binding to EFD triggers a conformational change, leading to interaction with its dehydrogenase domain and triggers ROS‐production. However, the Ca2+ binding to Nox5‐EFD and its domain‐domain interaction is not completely understood. Previously, we have characterized the Ca2+ binding to EF1 and EF2 located in the N‐terminus of Nox5 (N‐EFD) using isothermal titration calorimetry (ITC). Here we extended to our study to EF3 and EF4 of C‐EFD. Our preliminary data indicates that EF3 is specific to the Ca2+/Mg2+ ions with an unusually high Ca2+ binding affinity (Ka = ~108 M−1) while EF4 is specific to Ca2+ only. The results were supported by mutation studies, in which Glu at the 12th position of each EF loop were replaced with Gln. The mutants lose their Ca2+ binding ability significantly, which allows us to determine microscopic Ca2+ binding affinity for each EF‐hand. Currently we are measuring the Ca2+ binding using different buffers and at different temperatures, which will reveal the heat capacity change upon Ca2+ binding. The heat capacity along with melting measured with differential scanning calorimetry will provide valuable information about the conformational change of EFD.

Support or Funding Information

National Science Foundation CHE‐1608484

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Biochemical and Structural Look into the Functional Role of Transferrin in D. melanogaster
Authors: Weber Jacob , Brummett Lisa , Geisbrecht Brian , Kanost Michael , Gorman Maureen
Published: June 2021

Transferrins (TFs) are a family of vertebrate and invertebrate proteins that have high affinity for ferric iron. The extensively studied mammalian TFs are typically bilobal in structure and have iron binding sites in both their lobes—critical to their essential role in proper iron sequestration and transport in blood. These sites coordinate iron binding through four conserved residues—aspartate, tyrosine, tyrosine and histidine—and a carbonate ion cofactor. Aside from their function of transporting iron to cells, their ability to sequester and tightly bind iron is a crucial defense against invading pathogens. Many insect TFs show less sequence conservation and have only one iron binding site. They have not been adequately studied for their roles in iron metabolism and immunity. Our research aims to determine the function(s) of insect TF via biochemical and structural analysis using Drosophila melanogaster transferrin (DmTsf1) as a model. We purified DmTsf1 from a baculovirus expression system using ammonium sulfate precipitation, and ion exchange and gel filtration chromatography, and generated apo‐ and holo‐forms. Apo‐DmTsf1 but not holo‐DmTsf1 inhibited Escherichia coli growth, suggesting an immune function that relies on iron sequestration. A homology model of DmTsf1 showed a similar bilobal structure but iron binding in only one lobe. The putative binding residues are glutamate, tyrosine, and tyrosine. If the model is correct, a loss of the histidine residue in DmTsf1 should result in a lower binding affinity for iron. We are using crystallography to identify the actual iron binding residues. Initial protein‐ligand binding kinetics using equilibrium dialysis have shown Apo‐DmTsf1 to indeed having one binding site for ferric iron and a lower Kd of 10−18. Further biochemical pursuits into the role of insect TFs will include examining DmTsf1's ability to bind other metals.

Support or Funding Information

NIH NGMS R37 GM041247, NSF IOS 1656388

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of the Hsp70 Protein Quality Control System by Phosphorylation of Chaperones and Co‐chaperones
Authors: Page Richard
Published: June 2021

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of the co‐chaperones CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Hsp70 can only interact with one of the co‐chaperones at a time, thus modulating interactions of Hsp70 with either CHIP or HOP controls the Hsp70 protein quality control axis by shunting misfolded client proteins to either the degradative pathway or the refolding pathway. Interactions of Hsp70 with CHIP promote CHIP‐mediated degradation via the ubiquitin proteasome pathway. Interactions of Hsp70 with HOP promote protein refolding since HOP connect Hsp70 with the 90 kilodalton heat shock protein Hsp90. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Thus, phosphorylation of Hsp70 Thr636 acts as a switch that directs the output and function of cellular protein quality control. Phosphorylation of CHIP Ser20 or CHIP Ser24 also modulates the interactions between CHIP and Hsp70, providing yet another level of regulation for the Hsp70 protein quality control system. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and HOP‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex.

Support or Funding Information

NSF 15‐52113

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 Misfolded Protein Client as a Determinant of Substrate Ubiquitination by the Hsp70/CHIP Complex
Authors: Paththamperuma Chathura , Zhang Huaqun , Page Richard
Published: June 2021

The E3 ubiquitin ligase CHIP (carboxy terminus of Hsc70 interacting protein) targets misfolded proteins chaperoned by heat shock protein 70 (Hsp70) for ubiquitination and subsequent proteasomal degradation. Hsp70 recruits and attempts to refold misfolded proteins, or “clients”, in iterative, ATP‐dependent cycles. In contrast, binding of Hsp70 to the tetratricopeptide repeat (TPR) domain of CHIP, allows for client proteins to be recruited for ubiquitination by CHIP. Ubiquitination of lysine residues on a client protein allows CHIP/Hsp70 complexes to promote the proteasomal degradation of the client. Thus, the CHIP/Hsp70 complex stands at the crossroads of degradative and refolding pathways of client proteins which is crucial for cellular protein quality control. The current knowledge in the field lacks a clear understanding of how the CHIP/Hsp70 complex executes its triage mechanism that directs misfolded proteins for either refolding or degradation. Recent models of ATP and ADP bound Hsp70 revealed the distances between the substrate binding domain (SBD) and the E2~ubiquitin conjugate active site as ~120 Å and ~60 Å respectively. This implies, with the dynamic conformational changes of Hsp70 complex, the ADP‐bound state of Hsp70 organizes the chaperoned ubiquitination complex by properly orienting the client with respect to CHIP which allows the client protein to span this 60 Å gap and accept the ubiquitin from the bound E2‐ubiquitin conjugate. Therefore, the distance between Hsp70 binding site and the active site of the E2~ubiquitin conjugate bound to CHIP plays a significant role as a determinant in the triage mechanism of CHIP/Hsp70. In this study, we focus on investigating the relationship between key distances within the CHIP/Hsp70/client complex and the ubiquitination activity of the CHIP/Hsp70 complex by utilizing in‐vitro ubiquitination assays of modified substrates.

Support or Funding Information

NSF 15‐52113

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 relationship of structural stability to temperature‐dependent activity in a family of thermophilic inteins”
Authors: Exconde Patrick M. , Gomez Alvin V. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational process by which a self‐catalyzing intervening sequence called an intein removes itself from a precursor protein and concomitantly ligates the flanking sequences, known as exteins. Our inteins of interest interrupt the DNA Polymerase II from the extreme thermophiles Thermococcus sibiricus (Tsi) and Thermococcus gammatolerans (Tga). We are interested in these inteins because of their sequence similarity to those from the extreme thermophiles Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), all of which are mini‐inteins that lack a homing endonuclease domain. The Pho intein has been found to require higher temperature for splicing than the Pab intein, perhaps due to a shorter, more conformationally‐restricted region that replaces the missing homing endonuclease domain. The Tga and Tsi inteins have loop lengths more similar to the Pho intein. A splicing assay under increasing pressure will determine if there is a difference in activity in the two inteins that vary in the depths at which the parent organisms dwell. A preliminary assay at atmospheric pressure with an optimal isothermal temperature of 52°C showed that the Tga intein splices more efficiently than the Tsi intein. We will measure the temperature dependence of the intrinsic tryptophan fluorescence to link the overall stability of the fold to the difference in activity.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Camille and Henry Dreyfus Foundation (KVM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Salt‐Dependent Protein Splicing: In Vitro Enzymology and In vivo Physiological Relevance
Authors: Janton Christopher J. , Gomez Alvin V. , Makkay Andrea M. , Papke R. Thane , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational process in which two separate polypeptides are produced from a single gene. Splicing involves the excision of an intervening protein sequence, called the intein, and ligation of the flanking exteins. Inteins have been found in halophilic archaea, including Halobacterium salinarum (Hsa), and previous work has demonstrated that in vitro splicing of the Hsa DNA Polymerase II (Polll) intein only occurs under reducing conditions with high salt concentrations. We compared the in vitro activity of the Hsa Polll intein to that of the only other Hsa intein, which interrupts a Cell Division Control protein (Cdc21a). The Hsa Cdc21a intein does not splice in E. coli, but can be induced to splice at similar conditions to the Hsa Polll intein in vitro. Additionally, we used a Cys1Ala mutation to study the extent of the third step of splicing. We are creating intein‐less strains of H. salinarum, and will use these to study the fitness cost of maintaining inteins under varying growth conditions.

Support or Funding Information

This work was supported by NASA (grant NNX15AM09G to RTP), the National Science Foundation (grants MCB1244089 and MCB1517138 to KVM) and the Dreyfus Foundation (KVM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endonuclease and splicing activity of a Haloferax volcanii intein
Authors: Cawood Alexandra
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro at high salt. Collaborators have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Variable Salt Dependence of Mini‐Inteins from Haloquadratum walsbyi
Authors: Conroy Owen R. , Gomez Alvin V. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the protein (exteins) while simultaneously ligating the two flanking exteins. Inteins can be found in many types of archaebacteria, including halophiles, which thrive in environments with high salt concentrations. Previous results have shown that mini inteins of Haloquadratum walsbyi, such as those interrupting the cdc21 protein, require high concentrations of salt to splice. One intein that interrupts the DNA Gyrase B splices in E. coli, whereas similar inteins have required high salt incubation in vitro for splicing. The Hwa cdc21a, cdc21c, and PolIIb inteins were likely present in the most recent halophile ancestor as they are monophyletic within the halobacteria, the PolIIa and cdc21b inteins are polyphyletic, and the GyrB intein is likely related to inteins from bacteria. It is hypothesized that the GyrB intein has more recently invaded the halobacteria, therefore making the intein less stable to unfolding in the presence of salt when compared to earlier invading inteins, which many have adapted to high salt environments and are dependent on high salt concentrations for activity.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A bacterial flavin‐dependent oxidoreductase that captures carbon dioxide into biomass
Authors: Mattice Jenna , Streit Bennett , Prussia Greg , Peters John , DuBois Jennifer
Published: June 2021

Atmospheric carbon dioxide is used as a carbon source for building biomass in plants and photosynthetic microbes. Non‐photosynthetic processes that also fix carbon dioxide have more recently been discovered. This research focuses on a microbial mechanism for coupling acetone to CO2 to make a central metabolite, acetoacetate. The key reaction is catalyzed by NADPH‐2‐ketopropyl‐coenzyme M oxidoreductase/carboxylase (2‐KPCC), a bacterial enzyme that is part of the flavin and cysteine‐disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. Our research asks: how has nature repurposed a DSOR to, uniquely, break a C‐S bond and then trap and fix CO2? 2‐KPCC lacks a conserved, catalytically essential acid‐base histidine possessed by all other DSOR enzymes, having instead a phenylalanine (F501) at the same position. Mutagenesis showed that a F501H mutant has a similar rate of catalytic turnover; however, the product is acetone instead of acetoacetate (Figure 1). We hypothesized that F501 is important for both the reductive half reaction – which generates the reactive, C‐S bond breaking form of the active site – and for the oxidative half, in which an enolacetone intermediate reacts with either the correct (CO2) or incorrect (H+) electrophile. In this study, we used real‐time and spectroscopic methods to examine the reductive half reaction. In typical DSORs, this reaction generates a Cys/FAD charge transfer species. However, we showed that 2‐KPCC generates an electronically unique form of the active site, in which the flavin is oxidized and a pair of active site histidines are reduced and protonated. We hypothesize that this form of the active site generates the substrate‐reactive Cys in a more nucleophilic form, where it is capable of cleaving relatively strong C‐S bonds. The resulting enolacetone carbanion is an extremely potent nucleophile that is capable of directly attacking CO2. Research on 2‐KPCC and other biological CO2 fixation methods adds to our arsenal of strategies for carbon dioxide capture and use.

Support or Funding Information

DOE:DE‐FG02‐04ER15563

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrostatic interactions in natural enzymes: What can we learn for enzyme design?
Authors: Ondrechen Mary Jo , Coulther Timothy A. , Ngu Lisa , Beuning Penny J.
Published: June 2021

The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic power and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, and E. coli ornithine transcarbamoylase. In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. An approach to build these properties into the initial designs is proposed.

Support or Funding Information

NSF MCB‐1517290 (MJO & PJB) and a National Institute of Justice Fellowship (TAC)

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 Influence of Pressure on the Activity of Enzymes from Deep‐sea Extremophiles
Authors: Ramsoomair Christian Kieran , Chavez Angel , Ruiz Mercedes Anessa , Gomez Alvin V. , Mills Kenneth Vincent
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide, or intein. We studied inteins that interrupt the DNA Polymerase II from three different extremophiles, H. salinarum (Hsa), Pyrococcus abyssi (Pab), and Pyrococcus horikoshii (Pho). Intriguingly, all three of these inteins splice conditionally based on environmental cues. We hypothesized that since deep‐sea extremophiles such as P. abyssi and P. horikoshii live under moderately high pressures (150–200 atm), splicing will be unaffected or perhaps even enhanced under pressure. We also hypothesized that inteins from surface dwelling extremophiles like Hsa will not function as efficiently at moderate pressure. Lastly, we hypothesized high pressure (2000 atm and above), regardless of the native environment, will destabilize, unfold, and inactivate the activity. Results suggest that moderate pressures (200 atm and below) do not affect the splicing efficiency of inteins from either deep‐sea extremophile. While similarly unaffected at moderate pressure (100 atm), the function of Hsa inteins appear to be significantly diminished by higher pressures (1000 atm and above). In contrast, higher pressures appear to have no negative effect on the splicing of inteins from the deep‐seas extremophiles, and in some cases can even increase the splicing efficiency.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) (KVM), the Camille & Henry Dreyfus Foundation, and by Kim and Wendell P. Weeks P15 to the Holy Cross Alumni/Parent Summer Research Scholarship Fund (CKR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid abnormalities and hepatic dysfunction associated with Yersinia pestis infection in non‐human primate model
Authors: Gautam Aarti , Muhie Seid , Chakraborty Nabarun , Hoke Allison , Donohue Duncan , Miller Stacy Ann , Hammamieh Rasha , Jett Marti
Published: June 2021

Aerosolized Yersinia pestis causes pneumonic plague and is a highly virulent form of the infection. When left untreated, the fatality rate is very high, and survivors can suffer from complex clinical consequences. The exact mechanisms of disease progression are presently unclear. In this study, we used metabolite profiling to gain mechanistic insight into the progression of the disease. African green monkeys (Chlorocebus sabaeus) were challenged with the highly virulent Y. pestis strain CO92 by the aerosol route, plasma samples were obtained, and untargeted metabolomics profiling of plasma was performed using both liquid and gas chromatography with mass spectrometry. At early time points post‐exposure (6–18 h), we found increases in the abundance of certain polyunsaturated fatty acids, long chain fatty acids, carnitines, and dicarboxylate fatty acids in exposed animals when compared to their time‐matched control samples. The metabolites from lysolipids and bile acid metabolism pathways were lower in exposed animals when compared to their time‐matched controls during the entire course of infection. Many of the metabolites from amino acid pathways were perturbed, and a few from the leucine, isoleucine, and valine pathways were elevated during the course of infection, whereas metabolites from the urea cycle, arginine, and proline pathways were lower. The presence of branched chain amino acids in plasma, in addition to mobilization of fatty acids for subsequent β‐oxidation, indicated an increased energy demand. The involvement of several lipid pathways post‐infection suggested that there was activation of biological pathways linked to inflammation and oxidative stress. Enrichment analysis indicated perturbations in the bile acid pathway during the entire course of infection along with multiple metabolites that may have involvement in liver function indicating potential hepatic dysfunction. Integration of metabolomics and transcriptomics data from blood identified linoleate as a core metabolite with cross‐talk with multiple genes from various time points. Collectively, the data from this study provided new insights into the mechanisms of Y. pestis pathogenesis and into the host‐pathogen interactions that shape the outcome of the infection.

Disclaimers: Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

Support or Funding Information

Funding Support from DTRA is highly acknowledged

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atypical Protein Kinase C‐specific Activity Reporter Reveals Novel Activation Mechanism of Atypical Protein Kinase C by Sphingosine 1‐phosphate
Authors: Kajimoto Taketoshi , Caliman Alisha D. , Tobias Irene S. , Okada Taro , McCammon J. Andrew , Nakamura Shun‐ichi , Newton Alexandra C.
Published: June 2021

The protein kinase C (PKC) family comprises 9 members; conventional PKC isozymes (α, β γ), novel PKC isozymes (δ, ɛ, θ, η), and atypical PKC isozymes (ζ, ι/λ). It is well established that conventional and novel PKC isozymes are activated by second messengers, diacylglycerol and, for the conventional PKC isozymes, calcium ion. However the activation mechanism and physiological function of atypical PKC isozymes are less well understood. Here we show that a lipid mediator, sphingosine 1‐phosphate (S1P) controls the cellular activity of atypical PKC isozymes. First, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies reveal that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated biochemically. Our results are consistent with a model in which S1P binds the kinase domain of atypical PKC isozymes, an event that releases autoinhibitory constraints to lock the PKC in an active conformation. Our results provide a new molecular mechanism for controlling the cellular activity of atypical PKC.

Support or Funding Information

This study was supported by NIH to A.C.N., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., JSPS KAKENHI to S.N. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of GPCR Kinase 2 in Intact Cells: A Proteomic Approach
Authors: Sterne‐Marr Rachel , Vijay Jesus , Keyoskey Joshua , Shareef Afzaal
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) were first discovered by their ability to phosphorylate GPCRs in an agonist‐dependent manner. GRKs belong to the AGC kinase family of kinases. Activation of AGC kinases is often accompanied by phosphorylation of activation loop residues as well as other conformational changes in catalytically important residues. Crystal structures of GRK2 suggest that the activation segment region is in a relatively “active” conformation in the absence of phosphorylation. Nonetheless, a phosphoproteomic study (Oppermann et al., 2009) indicated that GRK2 Ser350 and Thr353 can be phosphorylated in intact cancer cells. Ser350 and Thr353 reside in the activation loop and P+1 site of the peptide‐binding site, respectively. To test the role of Ser350 and Thr353 phosphorylation in GRK2 function, we substituted alanine and aspartate at these two positions and tested the ability to phosphorylate the beta‐adrenergic receptor. We found that Thr353 is particularly important for phosphorylation of beta‐adrenergic receptor by GRK2 in COS‐7 cells, since conversion to Ala or Asp prevented substrate phosphorylation. In cells transfected with both beta‐adrenergic receptor and GRK2, multiple forms of GRK2 were detected in cells treated with agonist. The multiple forms are consistent with various phosphorylation states. Our goal is to assess Ser350/Thr353 phosphorylation in intact cells using phosphoproteomic approaches. To this end, we engineered a hexahistidine tag on GRK2 expressed from a mammalian cell vector to facilitate its purification and have demonstrated that the tagged kinase is fully functional in agonist‐dependent receptor phosphorylation. We are further developing methods to assess GRK2 phosphorylation by isoelectric focusing and mass spectrometry.

Support or Funding Information

This work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering DYRK1A Signaling using Proteomics and Transcriptomics
Authors: Poss Zachary , Ebmeier Christopher , Simpson Helen , Pagratis Maria , McClure‐Begley Tristan , Old William
Published: June 2021

Trisomy 21 is the most common genetic manifestation of intellectual disability, with deleterious brain phenotypes thought to be caused by increased dosage of critical genes on chromosome 21, including the kinase DYRK1A. Additionally, individuals with Down syndrome are at an increased risk of developing Alzheimer's disease, immune dysfunction and certain cancers, and DYRK1A has been implicated in each of these conditions.

Although it is clear that DYRK1A is critical for neural development, little is known about the substrates it modifies and how its dosage and activity contribute to trisomy 21 phenotypes. We employed phosphoproteomic and transcriptomic approaches in a 3D neuronal model of the developing trisomy 21 brain and found that DYRK1A phosphorylates and regulates key factors involved in splicing and transcription regulation. As both of these processes are central to neural differentiation, these data suggest that DYRK1A activity may be critical for the transition from progenitor to differentiated neurons. Additionally, dysregulation of this transition may contribute to phenotypic brain hallmarks exhibited within the trisomy 21 population, which express an extra copy of DYRK1A.

Support or Funding Information

Linda Crnic Institute for Down Syndrome, Anna and John J. Sie Foundation, DARPA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of a research‐based framework to guide instructional design and assessment related to noncovalent interactions in a biochemical context
Authors: Loertscher Jennifer , Feola Stephanie , Lemons Paula , Lewis Jennifer , Mercer Allison , Minderhout Vicky
Published: June 2021

Noncovalent interactions are difficult for students to conceptualize throughout the undergraduate chemistry sequence, which results in misconceptions that can make mastering complex biochemistry concepts difficult. In an effort to elevate the quality of teaching and learning in this area, a construct map was developed that outlines a common learning trajectory of the physical basis of interactions. Utilizing this guideline, a series of activities and assessments were created and implemented by a biochemistry educator who participated in the iterative workshops that developed the construct map. We have analyzed students' responses to newly‐developed instructional materials and exam questions and have characterized themes related to students' understanding of noncovalent interactions. We ultimately hope to gain insights that will result in further changes in teaching practice to better support student learning. This work highlights the interplay of research and practice in undergraduate level biochemistry curriculum.

Support or Funding Information

National Science Foundation DUE‐1224868

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and construction of a structural model of the bifunctional GlmU protein in complex with N‐acetyl‐D‐glucosamine‐1‐phosphate and uridine‐diphosphate‐N‐acetylglucosamine
Authors: Tollar M. Roarke , Edwards Titus D. , Nguyen Nhan , Novak Walter R.P.
Published: June 2021

The focus of this project was to explore the structure of the GlmU protein from E. coli (PDB ID: 2OI7) and to design and build a physical model that illustrates the key functional features of the protein. GlmU is a bifunctional bacterial protein that synthesizes uridine‐diphosphate‐N‐acetylglucosamine (UDP‐GlcNAc) from D‐glucosamine 1‐phosphate, acetyl‐CoA, and UTP. The structure described here is in complex with the products desulfo‐coenzyme A, N‐acetyl‐D‐glucosamine‐1‐phosphate and UDP‐GlcNAc. The N‐terminal uridyltransferase domain is composed of two beta‐hairpins and a seven‐stranded beta‐sheet surrounded by alpha‐helices. The C‐terminal acetyltransferase domain is a left‐handed parallel beta‐helix. GlmU functions biologically as a homotrimer. The three N‐terminal uridyltransferase active sites are independently formed by each monomer; however, each of the three acetyltransferase active sites is comprised of residues from the beta‐helix domain of all three monomers. This site is formed by one face of the beta‐helix and a loop region of the first monomer, a different face of the second monomer beta‐helix, and the C‐terminal tail of the third monomer.

Support or Funding Information

This work was funded in part by NSF‐DUE 1725940 for the CREST Project and the Wabash College Haines Biochemistry fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutant huntingtin affects p53 function in a context‐dependent manner in a cell model of Huntington's disease
Authors: Ellison Genevieve A. , Garcia Loren S. , Cornett Jonathan C.
Published: June 2021

Huntington's disease (HD) is an adult‐onset neurodegenerative disease caused by expansion of a polyglutamine (polyQ) repeat in the N‐terminal region of the huntingtin (htt) protein. In HD, multiple N‐terminal mutant htt fragments, smaller than the first 500 amino acids, have been found to accumulate in disease tissue. These N‐terminal htt fragments associate abnormally with many cellular proteins, disturbing their function. The p53 tumor suppressor protein has been shown to abnormally interact with mutant htt and is associated with cellular dysfunction in HD. However, it is unknown whether different htt fragments affect p53 function differentially. Here, we investigated the affect of different length N‐terminal mutant htt fragments on p53 localization and function in a human cell model of HD. We found that shorter htt fragments cause more accumulation of p53 protein in the nucleus which is associated with decreased cell viability. Our results demonstrate the importance of mutant htt protein context in HD. While the p53 pathway is implicated as a potential target to ameliorate Huntington disease progression, the therapeutic benefit may be limited by the relative abundance of shorter N‐terminal htt fragments in target cells.

Support or Funding Information

This work was supported by the National Science Foundation under Grant No. 1458212. JC was supported in part by a faculty fellowship from the Appalachian College Association.

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