bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purinergic Signaling Controls Fibroblast Growth Factor‐21 Expression in Skeletal Muscle through Akt/mTOR Pathway.
Authors: Arias‐Calderón Manuel , Morales Camilo , Vasquez Walter , Hernández Nadia , Jaimovich Enrique , Buvinic Sonja 
Published: June 2021

Fibroblast Growth Factor‐21 (FGF‐21) is a myokine which induce fiber type transformation and metabolic adaptations in muscle. However, little is known about the molecular mechanisms involved in the regulation of FGF‐21 expression in skeletal muscle in non‐pathological conditions. We have previously described that purinergic signaling is related with muscle adaptations and IL6‐myokine production through the “excitation‐transcription coupling” (ETC) mechanism, in which extracellular ATP (eATP) promotes gene expression in skeletal muscle. We have also demonstrated that eATP activates the PI3K/Akt signaling pathway in myotubes. Considering that Akt has been proposed as a master regulator for FGF‐21 expression control, the aim of this work is to determine the role of extracellular ATP over the FGF‐21 production in adult mouse skeletal muscle, and their dependence on the Akt/mTOR signaling pathway. Mice FDB‐isolated fibers or whole‐muscle were used in this study. The mRNA levels of FGF‐21 after electrical (20 Hz, 270 pulses, 0.3 msec each) or exogenous ATP (100 μM) stimulation were assessed by qPCR. FGF‐21 protein levels were assessed by immunoblot. We used pharmacological inhibitors of purinergic receptors (100 μM Suramin) and Akt/mTOR pathway inhibitors (50 μM LY294002; 1 μM Akt inhibitor VIII; 100 nM rapamycin), to turn off different interactors of the putative signaling pathway. ATP stimulation (100 μM) evoked an 8‐fold increase in FGF‐21 mRNA levels in isolated FDB fibers at 30 min post stimulation. The increase in FGF‐21 mRNA levels evoked by eATP was inhibited by suramin, a general antagonist of P2Y/P2X nucleotide receptors. Electrical stimulation also evokes an increase in FGF‐21 mRNA levels, which was inhibited by both nifedipine (a DHPR blocker) and suramin. Exogenous ATP increased protein levels of FGF‐21 in whole FDB‐muscles at 120 min, in a concentration‐dependent manner, with a maximal increase of 3.5 folds with 3 μM ATP. The increase in FGF‐21 protein level evoked by ATP, was abolished by LY294002 (a PI3K inhibitor), Akt inhibitor VIII, and rapamycin (mTOR inhibitor). In this work, we demonstrated for the first time that extracellular ATP evokes an increase in the mRNA and protein levels of FGF‐21 in skeletal muscle. The increase on FGF‐21 levels evoked by ATP is dependent on Akt/mTOR pathway activation. These results suggest that ETC mechanism could be involved in regulation of FGF‐21 expression in skeletal muscle.Support or Funding InformationFunded by CONICYT PCHA 21151035(MA‐C), 63140009(CM) and Fondecyt 1151353(SB), 1151293(EJ)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conserved salt‐bridge competition triggered by phosphorylation regulates the protein interactome
Authors: Skinner John James , Wang Sheng , Lee Jiyoung , Ong Colin , Sommese Ruth , Sivaramakrishnan Sivaraj , Koelmel Wolfgang , Hirschbeck Maria , Schindelin Hermann , Kisker Caroline , Lorenz Kristina , Sosnick Tobin , Rosner Marsha Rich 
Published: June 2021

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt‐bridge competition or “theft” mechanism that enables a phospho‐triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf‐1 to inhibiting G‐protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G‐Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo‐oligomers and 30% of hetero‐oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt‐bridge theft mechanism represents a facile strategy for promoting or disrupting peptide interactions using solvent‐accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.Support or Funding InformationGM087630 (to M.R.R.), GM55694 (to T.S.), Deutsche Forschungsgemeinschaft (FZ82: K.L., C.K., and H.S.; SFB688, TPA17: K.L.) and the Ministry for Innovation, Science and Research of the Federal State of North Rhine‐Westphalia (K.L.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allosteric Regulation of Protein Kinases Using Optogenetics
Authors: Shaaya Mark , Huyot Vincent , Zhurikhina Anastasya , Tsygankov Denis , Natarajan Viswanathan , Karginov Andrei 
Published: June 2021

Activation of single protein kinase can result in a very distinct signaling output depending on the duration, location, and frequency of its activation in the cell. However, interrogation of kinase‐mediated processes remains challenging due to the paucity of tools that precisely regulate kinases in space and time. To overcome existing limitations, we developed a novel approach for light‐mediated regulation of kinases. We engineered a light‐regulated allosteric switch (LightR), a small domain based on Vivid (VVD) photoreceptor, that undergoes conformational changes upon illumination with blue light. Insertion of LightR at a specific location in the catalytic domain of Src kinase enables light‐mediated control of its activity. In the dark, engineered LightR‐Src is inactive. Illumination of cells with blue light leads to robust activation of the kinase. Activation of LightR‐Src is specific, fast, and reversible. This allows us to control duration of kinase activation as well as induce repeated cycles of kinase activation/inactivation in living cells. Moreover, the level of activation can be controlled by changing light intensity. Specific modifications of the LightR domain allow us to modulate the activation/inactivation kinetics and the level of activity of LightR‐Src, enabling additional “tuning” of the engineered kinase and its optimization for different applications. Activation of LightR‐Src in living cells induces robust cell spreading and protrusive activity, which are known Src‐induced phenotypes described in previous studies. Importantly, the morphological changes stop as soon as the kinase is inactivated, demonstrating reversibility of the manipulated signaling. Repeated activation/inactivation of LightR‐Src induces cycles of morphodynamic activity of the cells, showing the capability of this tool to mimic oscillation of kinase signaling in living cells. Localized illumination of cells induces local formation of protrusions and cell polarization towards the light, demonstrating spatial regulation of LightR‐Src. Our study also reveals that continuous local activation of LightR‐Src at the cell periphery induced local oscillations of protrusive activity with defined periodicity. Insertion of LightR domain at a homologous position in Abl kinase also enabled light‐mediated regulation of its activity. These results, and the high structural similarity among catalytic domains of kinases, suggest broad applicability of this tool for regulation of different kinases. Furthermore, this approach could be potentially employed to control the activity of other enzymes in time and space.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing an Optogenetic System for Manipulating Protein Localization in Fission Yeast
Authors: Mehdi Maahum , Goss John Woodward 
Published: June 2021

The cellular function of a protein is largely dependent upon proper localization, particularly in complex protein networks that regulate stages of cell division. Many of these regulatory proteins are highly conserved and essential for cell viability. As a result, manipulating protein function in vivo often involves controlling expression levels using inducible promoters or generating point mutations to disrupt function, both of which have limited temporal resolution for observing phenotypes. An emerging technology to manipulate the function of essential proteins involves the field of optogenetics. This approach utilizes light‐inducible dimerization modules to enable rapid, reversible spatial and temporal manipulation of in vivo protein localization. Transiently altering protein localization to temporarily disrupt protein function can provide insight into how that protein contributes to a cellular pathway.In this study, we utilize molecular cloning to adapt a CRY2‐CIBN optogenetic light‐inducible dimerization system into common fission yeast expression vectors. These vectors enable us to incorporate genetically encoded cryptochrome fusion tags onto genes of interest in their native locus of the fission yeast genome using homologous recombination. We can then induce dimerization of the CRY2‐CIBN domains in the resulting tagged‐proteins in a rapid and reversible manner using 488nm light, which alters their spatio‐temporal localization in vivo allowing for direct observation of the downstream effects on cellular processes in real time. We utilize this system to manipulate localization of an essential regulatory kinase involved in cytokinesis as a method to further characterize its contribution to successful cell division. This optogenetic system is a valuable tool for studying the fission yeast cell cycle that enables researchers to better understand the role proteins play in regulation of this essential, highly conserved process.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Convergence of Wnt, Growth Factor and Trimeric G‐protein Signals on the Signaling Scaffold Daple
Authors: Ear Jason , Aznar Nicolas , Dunkel Ying , Sun Nina , Satterfield Kendall , He Fang , Lopez‐Sanchez Inmaculada , Ghassemian Majid , Sahoo Debashis , Kufareva Irina , Ghosh Pradipta 
Published: June 2021

Aberrant signal transduction is a hallmark of cancer biology. Pathways commonly deregulated include G‐protein signaling, Wnt signaling, and Receptor Tyrosine kinase (RTK) signaling. While typically studied individually, a growing amount of evidence indicate that these pathways are intertwined. Recent studies have demonstrated that Daple (CCDC88A) is multimodular domain containing protein that can tether various pathways together, and plays a major role in colorectal cancer (CRCs) progression. Daple is a Dishevelled (Dvl)‐ and Frizzled (FZD) ‐binding protein and a guanine‐nucleotide exchange factor (GEF) for the trimeric G protein, Gαi. Daple enhances β‐catenin‐independent (non‐canonical) Wnt signaling through its ability to activate the pathway downstream of FZD7 upon Wnt5a stimulation. Here we identified that Daple is a substrate of multiple RTKs and non‐RTKs, and hence, can serve as a point of convergence for all three signaling cascades. We show that phosphorylation by both RTKs and non‐RTKs near the atypical PDZ‐binding motif (PBM) on Daple dissociated the Daple:Dvl complexes and augmented the ability of Daple to bind and activate Gαi; which potentiated β‐catenin‐independent Wnt signals and triggered epithelial‐mesenchymal transition (EMT) in a manner similar to that triggered by Wnt5a/FZD7. Although Daple acts as a tumor suppressor in the healthy colon, concurrent upregulation of Daple and the prototype RTK, epidermal growth factor receptor (EGFR), in colorectal tumors from patients was associated with poor prognosis. We conclude that Daple‐dependent activation of Gαi and enhancement of β‐catenin‐independent Wnt signals is not just triggered by Wnt5a/FZD7 to suppress tumorigenesis, but also is hijacked by growth factor‐RTKs to stoke tumor progression. Thus, this work defines a previously unknown crosstalk paradigm amongst growth factor RTKs, trimeric G‐proteins, and Wnt/FZD in cancer biology.Support or Funding InformationThis work was supported by NIH grants CA100768, CA160911 and DK099226 (to P.G). P.G. was also supported by the American Cancer Society (ACS‐IRG 70‐002) and by the UC San Diego Moores Cancer Center. J.E is supported by an NIH‐institutional training grant (2T32CA067754‐21A1). I.K. was supported by NIH grants GM071872, AI118985, and GM117424. I.L‐S was supported by a fellowship from the American Heart Association (AHA #14POST20050025). F.H was supported by the State Scholarship Fund of China Scholarship Council (No.201208510048) and fund from West China Hospital, Sichuan University, PR China, during her tenure as a visiting professor to UCSD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Beta2‐Adrenergic Receptor Stimulation Improves Whole‐Body Glucose Homeostasis through Skeletal Muscle Metabolic Reprogramming
Authors: Meister Jaroslawna , Bone Derek , Knudsen Jonas Roland , Lee Regina , Cohen Amanda , Kleinert Maximilian , Berdeaux Rebecca , Richter Erik , Wess Jürgen 
Published: June 2021

Type 2 diabetes (T2D) has emerged as a major health problem worldwide. Since skeletal muscle (SKM) is the major site of insulin‐stimulated glucose disposal in the body, SKM insulin resistance plays a crucial role in the pathogenesis of this disorder. Drugs that could enhance or mimic insulin action in SKM hold great therapeutic potential for the treatment of obesity and T2D.Previous work carried out with cultured SKM cells indicates that activation of SKM β2‐adrenergic receptors (β2‐ARs) can promote glucose uptake. In rodent models of T2D, chronic administration of clenbuterol, a selective β2‐AR agonist, greatly improves glucose tolerance. However, the mechanism and in vivo tissue(s) responsible for the actions of clenbuterol are still unclear. This study was designed to test the hypothesis that the beneficial metabolic effects of clenbuterol observed in vivo are mediated by SKM β2‐ARs. For this purpose, we generated mice that lack β2‐ARs or the Gαs subunit selectively in SKM (SKM‐β2‐AR‐KO and SKM‐Gs‐KO mice, respectively). We also generated transgenic mice expressing a Gs‐DREADD selectively in SKM (SKM‐Gs‐DREADD mice).As expected, chronic administration of clenbuterol via drinking water improved glucose tolerance in wild‐type (WT) mice without affecting glucose‐stimulated insulin secretion or insulin tolerance. The SKM selectivity of this chronic clenbuterol effect was confirmed using SKM‐β2‐KO, SKM‐Gs‐KO, and SKM‐Gs‐DREADD mice. RNA‐seq and functional data indicated increases in SKM glucose accumulation, glycolysis, and glycogen synthesis following chronic treatment, suggesting a switch of SKM metabolism towards enhanced glucose utilization.Multiple components of known, but also less studied metabolic pathways seem to be simultaneously involved in mediating the beneficial metabolic effects of chronic β2‐AR stimulation.Our study represents a striking example of the complexity of β2‐AR signaling in vivo. Further analysis of β2‐AR signaling pathways in SKM could help identify potential drug targets for new T2D treatments.Support or Funding InformationThis research was funded by the Intramural Research Program of the NIDDK.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 a phospholipase C‐like protein (TbPI‐PLC2) from Trypanosoma brucei
Authors: Negrao Nuria Waddington , King‐Keller Sharon , Huang Guozhong , Docampo Roberto 
Published: June 2021

Trypanosoma brucei is the causative agent of African Trypanosomiasis, a deadly disease affecting humans and cattle. There are very few drugs to treat the disease and evidence of mounting resistance raises the need for new drug development. The inositol 1,4,5 triphosphate/diacylglycerol (IP3/DAG) pathway regulates important processes in many organisms. T. brucei has an active IP3 receptor, localized to the acidocalcisome, that is essential for infection in mice. In previous work (King‐Keller et al., Eukaryot. Cell 14:486–494, 2015) we characterized a phosphoinositide phospholipase C (TbPI‐PLC1, Tb927.11.5970) from T. brucei that contains a domain organization characteristic of PI‐PLCs, such as X and Y catalytic domains, an EF‐hand calcium‐binding motif, and a C2 domain, but lacks a pleckstrin homology (PH) domain. In addition, TbPI‐PLC1 contains an N‐terminal myristoylation consensus sequence only found in trypanosomatid PI‐PLCs. Here we report the presence of a second PI‐PLC‐like protein (TbPI‐PLC2, Tb927.6.2090) that is very similar to TbPI‐PLC1 but lacks the Y catalytic domain and the C2 domain and possesses instead a PDZ domain. Recombinant TbPI‐PLC2 hydrolyzes neither phosphatidylinositol (PI) nor phosphatidylinositol 4,5‐bisphosphate (PIP2), and does not modulate TbPI‐PLC1 activity. However, knockdown of TbPI‐PLC2 expression alone or together with downregulation of TbPI‐PLC1 expression by RNAi resulted in growth inhibition. This is in contrast with the lack of effect of downregulation of expression of TbPI‐PLC1 alone. TbPI‐PLC2 has a plasma membrane and intracellular localization and it might be involved in IP3 binding as has been reported for the phospholipase C‐related catalytically inactive protein 1 (PRIP‐1) of mammalian cells (Uji et al., Life Sci 72:443–453, 2002). The PDZ domain could be involved in this binding and this is being investigated.Support or Funding InformationNIH grant # AI077538This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial adenylate kinase 4 regulates dual AMPK and mTORC1 activation
Authors: Gonzales Carlos , Nguyen Anh Phuong , Abdullah Amelia , Meraz Ryan , Castellanos Ernesto , Nittayo Benjamin , Lanning Nathan 
Published: June 2021

In order to maintain cellular homeostasis, mitochondria integrate bioenergetic, metabolic, and redox cues. Signals both converge on and emanate from mitochondria to respond to varied cellular stressors. Therefore, mitochondrial proteins must play critical roles in maintaining cellular homeostasis. In a search for mitochondrial proteins that impact cellular bioenergetics, we identified the adenylate kinase, AK4, as the most potent regulator of ATP levels out of >1,000 mitochondrial proteins. We also found AK4 expression to significantly impact AMKP and mTORC1 activation status as well as cells' ability to respond to energetic stressors. Suppressing AK4 expression increases steady‐state cellular ATP levels by up to 30%, alters cellular respiration metabolite pools, and results in concomitant activation of both AMPK and mTORC1 signaling. Together, these effects of suppressed AK4 expression allow cells to thrive in the context of nutrient deprivation‐induced stress. The profound effects of AK4 expression on bioenergetics, survival under stress, and dual activation of the AMPK and mTORC1 energy sensing pathways position AK4 as a potential master regulator of mitochondria‐dependent cellular homeostasis.Support or Funding InformationNIH 1R15GM123382 CSUPERB New Investigator GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using embryonic zebrafish to evaluate the effects of exposure to novel bisphenol analogues
Authors: Bruton Laura N. , Zhou Xinrui , Timmons Shannon C. , Morrissette Jeffrey M. 
Published: June 2021

Bisphenol A (BPA) is a well‐established endocrine disruptor that has been shown to induce developmental and behavioral changes in both human and animal models. Gestational exposure of embryonic zebrafish to low nanomolar doses of bisphenol A (or S) altered hypothalamic neurogenesis during development and induced hyperactive swimming behaviors. There are many potential bisphenol analogues, but little is known about their toxicity or ability to act as endocrine disruptors. We have synthesized a small library of novel bisphenol analogues and are testing their toxicity in a zebrafish larval toxicity assay. We are also using an ELISA‐based estrogen binding assay to assess the analogues' affinity for estrogen receptors. We will be exposing zebrafish embryos to ranging concentrations of bisphenol analogues during time periods of 10–16 hpf (hours post‐fertilization), 16–24 hpf, and 24–36 hpf. At 5 dpf (days post‐fertilization) zebrafish locomotor activity in control and analogue exposed fish will be assessed using video‐tracking software quantifying bursts of hyperactivity. Results from these experiments will be discussed.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 and expression of Rhizophora mangle RmMT2, a type 2 metallothionein gene, in response to heavy metal exposure
Authors: Snyder Ian D. , Acacio Samantha , Trabing Rebecca M. , Shor Audrey C. 
Published: June 2021

Mangrove forests are important to the coastal ecosystems for their preservation of coastline landmass and offering habitats for marine organisms. Mangrove ecosystems also play a significant role in phytoremediation, or the uptake of toxic metals from the surrounding environment. This process is likely due to the expression of proteins known as metallothionines, a class of cysteine rich proteins whose sulfhydryl groups chelate and deactivate toxic metal ions. This study sought to better understand the expression and regulation of RmMT2A as a function of exposure to heavy metals. It was hypothesized that Rhizophora mangle, the red mangrove, possess the MT2A protein to combat abiotic stress, and its regulation is directly correlated with increasing concentrations of toxic metals in the surrounding environment. To investigate these hypotheses, R. mangle seedlings were exposed to increasing concentrations of toxic metals (CdCl2 and Pb(C2H3O2)2) for 3, 7, and 12 days, at concentrations 0 μM, 2 μM, 10 μM, 20 μM and 40 μM. Leaf samples were harvested, and total mRNA was extracted. Qualitative PCR was performed confirming the presence and expression of the RmMT2A gene and real‐time quantitative PCR assessed expression as a function of increased heavy metal exposure. Subsequent analysis will determine the metal‐binding properties of the GST‐RmMT2A fusion protein as well as the stress resistance properties of the gene. Metal tolerance was measured in Escherichia coli BL21 (DE3) transformed with RmMT2A. These data may suggest that the RmMT2A gene plays an important role in the efficiency by which R. mangle play as biogeochemical barriers to heavy metals.Support or Funding InformationThis research was funded by the Department of Mathematics and Science, Saint Leo 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.