bluuebunny/crossref_metadata_2025_split huggingface dataset:
CAMKK2‐dependent AMPK activation is required to drive cell cycle progression and hepatocyte proliferation after partial hepatectomy in the rat
Authors: Juskeviciute Egle , Antony Anil Noronha , Crumm Sara , Hoek Jan 
Published: June 2021

Liver regeneration is a unique repair response to tissue damage where normally quiescent liver cells reenter the cell cycle and undergo cell division, regaining the damaged or lost tissue, while maintaining the differentiated function of the tissue. In previous studies, we and others demonstrated that 70% partial hepatectomy (PHx) in the rat leads to an instantaneous (within 30 sec) release of up to 25% of ATP from the remnant liver. Released ATP served to activate purinergic receptors that enhance cellular stress responses and immediate‐early gene expression. ATP loss was mediated at least in part by a transient opening of connexin hemichannels and activation of stress signals and cell cycle progression was inhibited by hemichannel blocking peptides. The low ATP levels persist through the cell cycle progression and tissue ATP levels did not recover until 24–48 hrs post‐PHx. In the current study, we assessed the intracellular signaling events initiated by ATP loss. The decline in tissue ATP levels was accompanied by a persistent increase in ATP/AMP ratio and a transient activation of AMPK in the remnant liver, which peaked within 1 min after PHx, but then declined to basal levels within 15 min, despite the persistence of a low ATP/AMP. A second, extended phase of AMPK activation occurred, which peaked at 12–24 hrs after 70% PHx without being accompanied by major changes in tissue ATP/AMP ratios. This secondary AMPK activation in the regenerating rat liver accompanied cell cycle progression and DNA synthesis through G1‐S phase. After 30% PHx, a model that exhibits the hepatocyte priming events, but does not display the synchronized cell cycle progression and proliferation response, the initial ATP release and signaling events were comparable, but the secondary peak of AMPK activation is absent in the 30% PHx. Treatment with STO‐609, a selective inhibitor of CAMKK2, suppressed the second AMPK activation peak and inhibited hepatocyte proliferation after 70% PHx, suggesting the importance of calcium signaling in the regenerating liver. We conclude that an AMP‐independent CAMKK2‐mediated activation of AMPK is essential for effective cell cycle progression after PHx.Support or Funding InformationSupported by AA018873This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Dynamics of D‐Amino Acid Oxidase Using Molecular Dynamics Simulations
Authors: Jonsson Amanda L , Kueffer Lauren , Birdsong Garrick 
Published: June 2021

The D‐amino acid oxidase (DAAO) enzyme catalyzes the oxidative deamination of D‐amino acids to give ammonia and an α‐keto acid, coupled to the reduction of the cofactor FAD. In humans DAAO plays a key role in modulating the degradation pathway of the important signaling molecule D‐serine in the brain. We ran all‐atom, explicit‐solvent molecular dynamics simulations of the wild‐type human DAAO, several mutations known to increase DAAO activity or FAD binding (D31H, R279A, and W209R), and mutations known to decrease activity or binding (R199Q and R199W). Theses mutations are spread throughout the protein structure, some far from the FAD‐binding site and active site. We aim to better understand how small changes in the sequence can lead to changes in overall activity and binding affinity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An N‐terminal truncated isoform of the ER‐phagy receptor FAM134B (FAM134B‐2) regulates starvation‐induced ER‐to‐lysosome protein degradation
Authors: Miyazaki Shinobu , Kohno Shohei , Shiozaki Yuji , Keenan Audrey , Miyazaki Makoto 
Published: June 2021

Autophagy is a conserved system that adapts to nutrient starvation, after which proteins and organelles are degraded to recycle amino acids in response to starvation. Recently, the endoplasmic reticulum (ER) was added to the list of targets of autophagic degradation. Autophagic degradation of the ER (ER‐phagy) is considered a key mechanism in maintaining ER homeostasis. Four ER‐resident proteins (FAM134B, CCPG1, SEC62, and RTN3) have been identified as ER‐phagy cargo receptors, which contain LC3‐interacting regions. In this study we identified an N‐terminal truncated isoform of FAM134B (FAM134B‐2) that contributes to starvation‐induced ER‐phagy. FAM134B‐2 but not full length FAM134B (FAM134B‐1) is expressed in a fed state. Starvation drastically induces FAM134B‐2 but no other ER‐phagy cargo receptors through transcriptional activation by C/EBPβ. C/EBPβ overexpression increases FAM134B‐2 recruitment into autophagosomes and lysosomal degradation. In addition, FAM134B‐2 is preferentially localized in the smooth ER. This study demonstrates that the C/EBPβ‐FAM134B‐2 axis regulates starvation‐induced smooth ER‐phagy.Support or Funding InformationR01DK096030, R01HL117062, R01HL133545 and R01 HL132318.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nonspecific Electrostatic Interactions between Tau and 14‐3‐3ζ Promote Tau Aggregating
Authors: Xiong Junwen , Gao Meng , Huang Yongqi 
Published: June 2021

Aberrant accumulation of microtubule associated protein tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies. Previous studies have suggested that the scaffolding protein 14‐3‐3ζ promotes tau aggregating by interacting specifically with the pSer324 14‐3‐3ζ‐binding motif within the third microtubule binding repeat. Therefore, the tau/14‐3‐3ζ interaction is a promising drug target for Alzheimer's disease treatment. Indeed, inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. However, 14‐3‐3ζ has also been shown to promote aggregation of unphosphorylated tau protein, suggesting that nonspecific interactions between tau and 14‐3‐3ζ may be also involved in tau aggregating. In this work, the interactions between tau K18 construct and 14‐3‐3ζ were investigated. We compared the aggregating propensity for three tau species (i.e. WT, pS324, and S324A) in the presence or absence of 14‐3‐3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14‐3‐3ζ, phosphorylation only showed marginal effect on 14‐3‐3ζ mediated tau aggregating. Furthermore, WT and S324A tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Although 14‐3‐3ζ specific inhibitors reduced the binding affinity of pS324 tau with 14‐3‐3ζ, NMR spectroscopy showed that nonspecific interactions between pS324 tau and 14‐3‐3ζ remained when 14‐3‐3ζ specific inhibitors were present. Our results suggest that nonspecific interactions between tau and 14‐3‐3ζ play an important role in the 14‐3‐3ζ mediated tau aggregating process.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Basis of ClpP Protease Activation by Small Molecules
Authors: Houry Walid A 
Published: June 2021

Bacterial ClpP is a highly conserved, self‐compartmentalizing, cylindrical serine protease required for maintaining cellular proteostasis. Acyldepsipeptides (ADEPs) and activators of self‐compartmentalized proteases (ACPs) are small molecules that cause the dysregulation and activation of ClpP, which leads to bacterial cell death, highlighting their potential use as novel antibiotics. However, the molecular basis of ClpP activation by small molecules remains undetermined. Structural changes in ClpP from Neisseria meningitidis and Escherichia coli upon binding to novel ACP and ADEP analogs were probed by X‐ray crystallography, methyl‐TROSY NMR, hydrogen‐deuterium exchange, and small angle X‐ray scattering. ACP and ADEP were found to induce distinct conformational changes in the tertiary structure of ClpP. However, ordered N‐terminal axial loops, reorganization of the hydrogen bonding network at the entrance pore of ClpP, and reduced structural heterogeneity of the ClpP cylinder are common features of activator‐bound complexes. Activated ClpP mutants were obtained that recapitulate these effects. Based on our data, we propose a unified mechanism for compound‐based ClpP activation.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular regulation of lysine trimethylation by the P. aeruginosa methyltransferase EftM
Authors: Dey Debayan , Kuiper Emily G , Goldberg Joanna B , Conn Graeme L 
Published: June 2021

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe life‐threatening lung and other infections in individuals with cystic fibrosis, compromised immune systems or severe burns. P. aeruginosa is also intrinsically resistant to many antibiotics making infections difficult to treat once established. We previously showed that the S‐adenosyl‐L‐methionine‐dependent methyltransferase EftM is responsible for trimethylation of lysine 5 of the translation factor EF‐Tu and that this modification enhances P. aeruginosa adhesion to host epithelial cells. Additionally, methyltransferase activity, and thus EF‐Tu modification, is temperature regulated via the intrinsic thermolability of EftM. Understanding EftM's complete mechanism of action may reveal new strategies for developing effective therapeutics targeting this pathogen. As the next step in this process, we now aim to define the substrate recognition and catalytic mechanisms of EftM. A phylogenetic reconstruction of the larger EftM family was generated to identify conservation patterns of putative key residues involved in interaction with EF‐Tu and facilitating catalysis. This analysis was supported by generation of an EftM homology model and protein‐protein docking studies to propose a EftM:EF‐Tu binding model. This model and phylogentic insights were used to guide site‐directed mutagenesis of residues in EftM and EF‐Tu to test their role in protein‐protein recognition using methyltransferase activity and binding assays. Finally, to begin investigating the role of protein dynamics and potential allosteric effects of SAM cosubstrate binding, other computational methods such as normal mode analysis were used to propose a coarse‐grain model of dynamics of EftM in parallel with experimental hydrogen‐deuterium exchange coupled with mass spectrometry (HDX‐MS) analysis of EftM and its complexes with SAM and EF‐Tu. In conclusion, our results provide the first structural insight into the EftM:EF‐Tu recognition, which paves the way for further understanding EftM's substrate recognition and catalytic mechanisms.Support or Funding InformationCystic Fibrosis Foundation Postdoctoral Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Dynamics Couple Substrate Recognition with Allosteric Domain Communication in Nonribosomal Peptide Synthetases
Authors: Frueh Dominique P , Mishra Subrata H , Kancherla Aswani K , Marincin Kenneth A , Nerli Santrupti , Sgourakis Nikolaos G , Dowling Daniel 
Published: June 2021

Timely control of protein interactions is central to modulate biological activity such as gene activation, cell signaling or metabolism. Here, events such as post‐translational modifications and substrate or protein binding affect proteins so as to govern their interactions with partner proteins and repress or promote activity. In spite of progress in identifying protagonists of molecular communication and predicting the outcome of molecular events, it has proven more challenging to determine how molecular responses within proteins translate into responses between proteins, particularly when remote sites are involved within proteins. Such challenges are inherent to understanding microbial, multi‐domain enzymatic systems called nonribosomal peptide synthetases (NRPSs). NRPSs employ contiguous repeats of domains to covalently tether substrates onto carrier proteins (CP) and assemble them into complex products through intervening condensation or cyclization domains. The pharmaceutical properties of NRPS products (e.g. antibiotics) have spurred attempts at engineering NRPSs, but dynamics between domains have hampered progress, as it remains unclear whether and how the many catalytic steps required for synthesis relate with transient domain communication. Using NMR, we demonstrate that large scale structural dynamics within a 52 kDa cyclization domain sense substrates tethered to partner CPs to not only promote binding between both partner domains but also to remodel both the active site and a remote binding site for downstream partners, thus revealing a path for allosteric communication involving three partner domains. We provide a 3D structural description of the enzyme dynamics and demonstrate that mutations that impede catalysis injure the same dynamic allosteric network, suggesting that catalysis efficiency may be streamlined with productive domain communication and substrate recognition through dynamic responses. Our results reveal and describe at the molecular level a substrate driven allosteric response in NRPSs, and they exemplify how structural dynamics within proteins can couple substrate recognition with active site remodeling and promotion of both local and remote protein communication.Support or Funding InformationNIH‐R01GM104257This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐activity relationships of fragment‐based inhibitors of Trichomonas vaginalis adenosine/guanosine preferring nucleoside ribohydrolase
Authors: Emilcar Melissa , Leonardo Angelica , Li Tian , Dulloo Zaafir , Kabir Nafeesathul Hanan , Gil Ari , VanAlstine‐Parris Melissa A. , Stockman Brian J. 
Published: June 2021

Trichomoniasis is a sexually transmitted infection caused by the parasite, Trichomonas vaginalis. Infections are currently treated with 5‐nitroimidazole drugs, such as metronidazole and tinidazole. However, strains of the parasite with resistance to these drugs have emerged, indicating the need for new treatments with novel mechanisms. Since the parasite is unable to perform de novo synthesis of nucleobases, it must obtain them from its host using salvage pathway enzymes including adenosine/guanosine preferring nucleoside ribohydrolase (AGNH), an essential enzyme involved in the pyrimidine salvage pathway. We previously used fragment screening to identify ligand‐efficient fragment inhibitors of AGNH. Medicinal chemistry efforts were then focused on several fragment scaffolds including benzimidazoles and phenyl pyridines. IC50 values were determined using the same 1H NMR‐based activity assays as the fragment screens. The resulting structure‐activity relationships suggest that the fragment scaffolds interact primarily with the nucleobase regions of the active site rather than the ribose pocket. Collectively, the data define emerging structure‐activity relationships that suggest likely vectors and chemical modifications for improving inhibition potency while maintaining ligand efficiency. The data establishes a platform for ongoing medicinal chemistry development of compounds with nM potency that will provide the tools for in vitro target validation against both 5‐nitroimidazole‐sensitive and 5‐nitroimidazole‐resistant T. vaginalis strains.Support or Funding InformationResearch was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI128585 to BJS and MAVP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Translation of 1H and 19F NMR‐based activity assays to in vitro characterization of nucleoside hydrolase activity in cell extracts and whole cells
Authors: Mahmood Maham , Canestrari Madison , Thuilot Samantha , Stockman Brian J. 
Published: June 2021

Trichomoniasis, the most prevalent non‐viral sexually transmitted infection in the world, is caused by the parasitic protozoan Trichomonas vaginalis. Studies have indicated an association between T. vaginalis and a higher susceptibility to various other infections including chlamydia, HIV, and syphilis. The parasite has shown increasing resistance to the current treatment of 5‐nitroimidazole drugs such as metronidazole. T. vaginalis is incapable of de novo synthesis of purine and pyrimidine rings, so it must rely on salvage pathway enzymes such as adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH) to scavenge nucleobases. Both enzymes have been screened to identify fragment inhibitors with high ligand efficiencies to use as starting points for drug design. AGNH and UNH can be validated as antitrichomonal targets by demonstrating a correlation between enzyme inhibition and antitrichomonal activity. Escherichia coli cells with endogenous nucleoside ribohydrolase gene expression of rihA (ybeK), rihB (yieK) and rihC (yaaF) were used as a surrogate to develop protocols for observing in vitro enzyme activity. A 1H NMR‐based activity assay for AGNH using adenosine as the substrate and an 19F NMR‐based activity assay for UNH using 5‐fluorouridine as the substrate were previously developed for compound screening. These assays proved remarkably robust for observing nucleoside hydrolase activity in cell extracts and in whole cells. Signals for substrate and product are clearly distinguishable from background signals arising from the cell contents. Reactions have been shown to be cell‐dependent, indicating that both enzymes are intracellular and that substrate can rapidly enter the cells. Similar experiments are now in progress using T. vaginalis strains to validate the molecular mechanisms of inhibition.Support or Funding InformationResearch was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI128585 to BJS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NITROGEN STARVATION‐INDUCED PHOSPHORYLATION OF GPA2 ACTS AS A MOLECULAR SWITCH IN PROMOTING SPORULATION
Authors: Huang Shan , Benben Alex , Green Robert , Keaveney Shannon , Wang Yuqi 
Published: June 2021

G proteins are important molecular switches that facilitate the transmission of a variety of signals from outside to the inside of the cells. G proteins are highly conserved, which allows studying their mechanisms of regulation in model organisms such as the budding yeast Saccharomyces cerevisiae. Gpa2 is a yeast G alpha protein that functions in the nutrient signaling pathway. Through the usage of phos‐tag, we found that Gpa2 protein undergoes phosphorylation and its level of phosphorylation is markedly increased upon nitrogen starvation. Gpa2 interacts with glycogen synthase kinase (GSK), and disrupting GSK activity diminishes the level of Gpa2 phosphorylation. Functionally, phosphorylation enhances plasma membrane localization of Gpa2 and promotes sporulation in response to nutrient deprivation condition. Together, our study reveals a mechanism by which GSK‐and nutrient‐dependent phosphorylation of Gpa2 serves as a molecular switch that regulates sporulation in yeast.Support or Funding InformationWe thank Drs. Claudina Rodrigues‐Pousada, Henrik G. Dohlman, and Jeremy Thorner for generously providing strains, plasmids and antibodies.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 Ellis van Creveld protein 1 binds and hydrolyzes GTP to a mixture of GMP and GDP and possesses the characteristics of large GTPases
Authors: ODUNUGA ODUTAYO O , NGUYEN GIANG T 
Published: June 2021

Mutations in the genes that code for EVC1 and EVC2 proteins cause Ellis van Creveld (EvC) syndrome and its milder form, Weyers acrofacial dysostosis (WAD). EVC1 physically binds to EVC2 and together, they are tethered to the base of the primary cilium by the EFCAB7/IQCE protein complex. This multi‐protein assembly positively regulates the Sonic and Indian hedgehog (Hh) signaling pathways by promoting downstream processes after activation of the receptor, smoothened (smo), by the hedgehog proteins. Bioinformatic analysis revealed the presence of several structural motifs in EVC1 sequences including P‐loop, leucine zipper, transmembrane and nuclear localization signal. Using a fragment of EVC1 from mice and humans that contains P‐loop and leucine zipper regions, we present experimental evidence to show that EVC1 protein possesses a high intrinsic GTPase activity with a kcat of ~10 min−1. In addition, dimerization of EVC1 via its leucine zipper causes the protein to hydrolyze GTP to a mixture of GDP and GMP at unequal ratios. The protein binds GTP at milli to micro molar concentrations, indicating low substrate affinity. These kinetic parameters are similar to those displayed by large GTPases such as Dynamin. We propose that the GTP‐hydrolyzing activity of EVC1 protein is an important mechanistic step in the regulation of the hedgehog signaling pathways.Support or Funding InformationWelch Foundation Grant # AN‐0008This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.