bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an agonist binding site motif that regulates biased signaling of GPCRs through altered conformations of intracellular loop 2
Authors: Soto Marta Sanchez , Verma Ravi Kumar , Willette Blair K.A. , Gonye Elizabeth C , Moore Annah M , Chun Lani S , Moritz Amy E , Yano Hideaki , Free R Benjamin , Shi Lei , Sibley David R 
Published: June 2021

We recently identified a signaling‐biased agonist of the D2 dopamine receptor (D2R), MLS1547, that stimulates G protein‐mediated signaling, but is ineffective in recruiting β‐arrestin. MLS1547 was found to uniquely interact with a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop (EL2) of the D2R. Detailed investigations on the role of these specific residues lead to the identification of F189 (residue 5.38 using Ballesteros‐Weinstein numbering) as a micro‐switch for regulating D2R interactions with β‐arrestin. As position 5.38 is relatively conserved (frequently Phe or Tyr) in class A GPCRs, we constructed alanine mutations (Phe/Tyr to Ala) at this position within all D2‐like receptors (D2R, D3R, and D4R), the β2‐adrenergic receptor (β2R) and the V2 vasopressin receptor (V2R). Strikingly, we found that the alanine 5.38 mutations negated the receptors' ability to recruit β‐arrestin in response to agonists, while G protein‐signaling efficacy was maintained. These data suggest that the presence of a Phe or Tyr residue at position 5.38 in these GPCRs is critical for stabilizing an activate state for recruiting β‐arrestin. To investigate how alterations at this position produce conformational rearrangements resulting in signaling bias, we used the β2R, for which active state crystal structures are available, to build both β2R‐WT and β2R‐Y199A models in complex with the full agonist BI‐167107, and performed extensive molecular dynamics simulations. Using this approach, we identified residues that differentially interact with BI‐167107 in the β2R‐WT vs. β2R‐Y199A leading to conformational rearrangements that propagate through the TM3‐TM4‐TM5 interface to the intracellular side of the receptor. These coordinated changes result in a different tilt of TM4, face shift of TM4 and TM5 on their extracellular sides, and an altered orientation of IL2 in the β2R‐Y199A compared to the β2R‐WT. Strikingly, such coordinated changes and altered IL2 conformations are reminiscent of the differences between the recently solved cryo‐EM structure of the rhodopsin‐Gi complex and the crystal structure of the rhodopsin‐β‐arrestin complex. These results describe a structural basis for how ligand binding site alterations can modulate GPCR coupling to different transducers resulting in biased signaling.Support or Funding InformationNINDS Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of Mg2+ and Solute Carrier Family 41 to Pulmonary Hypertension in Rat Models
Authors: Lin Mo‐Jun , Wang Dan , Lin Da‐Cen , Jiao Hai‐Xia , Sham James SK 
Published: June 2021

Pulmonary hypertension (PH) is a debilitating disease and characterized by profound vascular remodeling. Many studies indicate that it is related in part to the alterations of Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Mg2+ ion is closely related to Ca2+ ion and is the second‐most abundant divalent cation in animal cells. It is required for the regulation of diverse cellular functions, including cell growth, proliferation, and migration. The Solute Carrier 41 family (SLC41) members including A1, A2 and A3 are Mg2+ transporters, and they have been identified in lung vascular tissue. However, the changes in their expression and functions of SLC41 transporters in PH have not been examined. The present study was designed to investigate whether Mg2+ and its transporters are involved in PH development. PH rats were established by a single injection of MCT or exposed to normobaric hypoxia (CH, 10% O2) for 21 days. Another set of animals received the same treatment were fed with Mg2+ diet supplement (10 mL/kg/d) for 21 days. The expression of SLC41A1/2/3 mRNA and protein in PAs of control and PH rats with or without Mg2+ supplement were determined by Q‐PCR and Western blot. SLC41A1‐2 expressions were significantly increased in PAs of MCT or CH‐treated rats, while mRNA and protein of SLC41A3 were decreased in PH rats. Moreover, PH was attenuated and the changes in SLC41A1‐3 expression were reversed in rats treated with Mg2+ diet supplement. The roles of Mg2+ and SLC41 proteins in PASMCs proliferation and migration were further examined in PASMCs isolated from control rat. They were treated with a control sequence or a SLC41A1/2 targeting siRNA and cultured under low (0.15 mM), normal (1.2 mM) and high (4.8 mM) level of Mg2+. PASMC proliferation monitored using MTS reagent or EdU staining and, migration assessed with Transwell or wound healing assay were markedly inhibited after incubation with high Mg2+ for 48h. Furthermore, siRNA targeting SLC41A1/2 attenuated PASMC proliferation. Our results, hence, clearly show that the SLC41 transporters contribute to PH, and Mg2+ diet supplement could effectively alleviate PH; In addition, Mg2+ and SLC41A1/2 actively participate in PASMC proliferation and migration. These results provide the basis for future study on the roles of Mg2+ and its transporters in PH development.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China NSFC31571179 and NSFC31371165This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐Quadruplex Loop Length Regulates PARP‐1 Enzymatic Activation
Authors: Edwards Andrea , Marecki John , Byrd Alicia , Raney Kevin 
Published: June 2021

Guanine‐rich sequences can interact by Hoogsteen‐hydrogen bonding to form planar quartets that stack into four‐stranded structures known as G‐Quadruplexes (G4DNA). G4DNA is non‐random, overrepresented at key regulatory regions of the cell such as promoters of proto‐oncogenes and telomeres. The overrepresentation in such sites suggests G4DNA serves a significant role in the cell. Poly(ADP‐ribose) polymerase 1 (PARP‐1) is an enzyme that mediates production of polymers of ADP‐ribose units (PAR) that serve as a signal for important cellular processes. PARP‐1 plays a role in the regulation of gene expression, transcription, chromatin remodeling, and DNA repair. Interestingly, PARP‐1 interacts with G4DNA. We show that PARP‐1 binds several G4DNA structures with tight affinity. We also confirm previous evidence revealing that PARP‐1 is enzymatically activated by c‐KIT G4DNA in a DNA structure‐specific manner. In addition, our activity assays reveal that while PARP‐1 binds various G4DNA structures only a subset promote PARP‐1‐mediated PARylation. We show that PARP‐1 enzymatic activation is regulated by G4DNA loop length where longer loops, at least >1 nucleotide, confer higher PARP‐1 activation while shorter loops reduce PARP‐1 activation. Our approach supports a PARP‐1 and G4DNA interaction but emphasizes PARP‐1's catalytic activity is G4DNA loop length‐dependent.Support or Funding InformationNational Institutes of Health Grant R35 GM122601 (to K.D.R.) UAMS Initiative for Maximizing Student Development Program (IMSD) Grant R25 GM083247This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the impact of splicing protein Dib1 on the pre‐messenger RNA interactions with the spliceosome
Authors: Potts Camille , Goldstein Rachel , Pittner Nicholas , Widom Julia , Duran Elizabeth , Walter Nils , Maeder Corina 
Published: June 2021

In eukaryotic organisms, pre‐messenger RNA splicing facilitates the removal of non‐coding regions of RNA to create a functional mRNA. Pre‐mRNA splicing is catalyzed by the spliceosome, a large, dynamic, molecular machine composed of five small nuclear ribonucleoproteins (snRNPs). Dib1, a key protein of the spliceosome, resides in the U4/U6.U5 triple‐snRNP. Dib1 is essential for cell viability and is conserved from yeast to humans. Dib1 must depart the splicing complex before the catalytic steps of splicing can occur; however, how Dib1's departure is facilitated is unknown. Dib1 and pre‐mRNA are in close proximity in the pre‐catalytic spliceosome (Plaschka et al. Nature 2017); characterizing the interactions between Dib1 and pre‐mRNA may be necessary to understand Dib1's departure. We are characterizing potential interactions between Dib1 and pre‐mRNA using single molecule fluorescence colocalization experiments in collaboration with Dr. Nils Walter at the University of Michigan. An open question is how and whether Dib1 and pre‐mRNA are co‐localized in the splicing machinery. We are addressing this question using Cy5‐labeled Dib1 and Cy3‐labeled pre‐mRNA. We have labeled purified Dib1 with a Cy5 fluorophore and have been able to splice Cy3‐labeled pre‐mRNA. Progress and results of the single molecule fluorescence colocalization between Dib1 and pre‐mRNA will be discussed. Another area of interest is how temperature‐sensitive mutant Dib1, which causes a loss of splicing function in Saccharomyces cerevisiae, may affect the pre‐mRNA throughout the splicing cycle. To this end, we are characterizing pre‐mRNA conformation in S. cerevisiae extracts containing wild type or mutant Dib1 protein. We are using single molecule fluorescence resonance energy transfer (smFRET) to analyze the conformation of pre‐mRNA labeled with a Cy3 fluorophore at the 5′ splice site and a Cy5 fluorophore at the branch point. Comparison of the smFRET analysis will show whether mutant Dib1 causing loss of function is associated with a change of RNA conformation. Progress and results of the smFRET analysis of pre‐mRNA will be discussed.Support or Funding InformationNIH R15GM120720; Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding to the ribosome by eIF4B drives yeast translational control in response to membrane stressors
Authors: Walker Sarah E , Liu Xiaozhuo E , Moshiri Houtan E 
Published: June 2021

The eIF4 group of eukaryotic translation initiation factors promotes mRNA binding to the ribosome. Increased levels or activities of these factors have been associated with a number of human diseases, highlighting the need for additional mechanistic information for these proteins. Our previous work indicated that ribosome binding by the N‐terminal domain of yeast eIF4B is critical for robust initiation in vitro and in vivo. In contrast, RNA‐binding activities by the RRM domain of eIF4B were dispensable. Our work left open the possibility that the RRM of eIF4B could allow for enhanced translation under stress conditions. Here we have compared the effects of disrupting RNA and ribosome binding of yeast eIF4B under ~1400 different growth conditions. The RRM was dispensable for vegetative growth in all conditions tested, but we found that ribosome binding promotes growth in response to a number of stressors through changes in translation. In particular, the NTD confers a strong growth advantage in the presence of the denaturing reagent, Urea and a number of other osmolytes that require robust cellular integrity for survival. Ribosome profiling of cells with and without the ribosome‐binding NTD of eIF4B reveals gross changes in translation of mRNAs containing longer than average and highly structured 5‐prime untranslated regions in response to Urea stress. These changes are dependent on the ribosome‐binding activity of eIF4B, which suggests eIF4B regulates mRNA recruitment as a part of the ribosomal preinitiation complex rather than by activating isolated mRNPs prior to ribosome binding. This analysis indicates the cellular response to urea in yeast includes a translational component, driven by increased translation of proteins associated with the cellular periphery. The results of these studies highlight the importance of the general translation factor eIF4B in adapting to external conditions, and allow us to tie the mechanical functions of this translation factor to specific cellular phenotypes.Support or Funding InformationThis work is supported by NIH grant R00GM119173 and startup funds from SUNY at Buffalo‐College of Arts and Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monofunctionalization with dextrans endows cell‐penetrative properties to proteins
Authors: Kilgore Henry Ralph , Ressler Valerie T , Raines Ronald T 
Published: June 2021

Dextrans have proven to be a widely useful class of polysaccharides, with applications ranging from antithrombotic agents to enhancers of vaccine stability. We have examined an unprecedented property of dextrans: the capacity to convey cargo into the cytosol. Mono‐functionalized dextran–flourogenic probe and dextran–protein conjugates were prepared to assess the ability of dextran to mediate cellular delivery. We show that dextran conjugates are taken up in an energy‐dependent process culminating in cytosolic access. Several cell types were examined, indicating the generality of this uptake pathway. These observations motivated the preparation of dextran–cytochrome C conjugates. Release of cytochrome C from the intermembrane space activates the intrinsic apoptosis pathway. Cytotoxicity measurements of dextran–cytochrome C conjugates thus provide a straightforward metric for cytosolic access of dextran–protein conjugates. We verify that cytochrome C mediates cell death by measuring activation of downstream caspases 3 and 7, indicating that dextran–cytochrome C conjugates promote formation of the apoptosome. These experiments document a new and exciting frontier for polysaccharide bioconjugates and support the existence of an uptake pathway for 1,6‐linked polysaccharides.Support or Funding Information[This work was supported by Grant R01 GM044783 (NIH).]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimizing Lipid Extraction in Anabaena
Authors: Menning Hope Deann , Mazzer Paula Deann 
Published: June 2021

Lipidomics provides a great snapshot technique to determine how the lipids work in a cell. We do not know a lot about the lipids of cyanobacteria. We set out to optimize an extraction protocol, to figure out the different lipids in a type of blue green algae, Anabaena. Our best extraction by mass is with a 10‐minute sonication step with a modified Bligh and Dyer technique. After extraction, analysis was conducted using an FTICR‐MS and derivatization of fatty acid methyl esters. We have developed the baseline lipidome for Anabaena as a starting point for future research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MK‐STYX Impacts Autophagy and Microtubule Dynamics
Authors: Mattei Andrew M. , Christian Patrick S. , Hinton Shantá D. 
Published: June 2021

Macroautophagy (autophagy) is a critical process that degrades cytoplasmic components to maintain homeostasis and which requires microtubule interactions to function. The autophagosomes (double membrane vesicles) envelop substrates and then are transported along microtubules through motor proteins to fuse with degradative lysosomes. The formation of the autolysosome allows the breakdown and degradation of various cellular targets such as stress granules (SG; stalled mRNA). We reported that the pseudophosphatase MK‐STYX [MAPK (mitogen‐activated protein kinase) phosphoserine/threonine/tyrosine‐binding protein] inhibits SG. Since autophagy and MK‐STYX each negatively affect stress granule assembly, we sought to determine whether MK‐STYX has a role in autophagy and microtubule dynamics. Our studies show that MK‐STYX causes cytosolic TFEB (Transcription factor E‐Box; the autophagy “master switch”) to become nuclear and form peripheral nuclear aggregates independently of nutrient status. Whereas, MK‐STYXactive (active mutant in which catalytic activity has been “restored”), only increased TFEB cytosolic aggregates in serum starved cells. TFEB also localizes to the lysosomal surface, acting as negative regulator of lysosomal and autophagosomal biogenesis. MK‐STYX alters lysosomal and autophagosomal dynamics; we observed oversized lysosomes in the presence of MK‐STYX. Furthermore, autophagosomes localized to the distal ends of HEK‐293 cellular extensions in the presence of MK‐STYX. In addition, MK‐STYX increases the phosphorylation of the essential autophagosome constituent LC3 (microtubule‐associated proteins 1A/1B light chain 3B). To determine MK‐STYX's effects on microtubule function, we over‐expressed MK‐STYX in HEK‐293 cells in the presence or absence of nocodazole, which disrupts microtubules. MK‐STYX did not affect microtubules dynamics directly, suggesting that further analysis of the motor proteins involved in stress granule regulation and/or autophagy is needed. Investigating MK‐STYX as a player in autophagic mechanisms is an intriguing scientific trajectory. Autophagy plays a major role in prominent cellular processes ranging from initial developmental stages to the onset of progressive human pathologies such as neurodegenerative diseases and cancer. These studies suggest that MK‐STYX may have an important role in autophagy, illuminating the importance of pseudoenzymes in regulating critical cellular pathways.Support or Funding InformationSupport for this research was provided by two Howard Hughes Medical Institute Research Fellowships and a National Science Foundation Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Live‐Cell Fluorescent Visualization of T3SS Needle and Its Dynamics
Authors: Cheng Dorothy , Wimmi Stephan , Diepold Andreas 
Published: June 2021

The Type III Secretion System (T3SS) is a complex transmembrane apparatus used by many Gram‐negative pathogens for protein translocation and host invasion. An essential T3SS component is the needle, a hollow substructure extending from the bacterial membrane, spanning the space from the bacterium to the host cytosol. The needle is formed by helical polymerization of a single small protein, which is secreted by the T3SS and assembled outside the bacterium. Visualization of the needle previously depended on immunofluorescence, which requires cell fixation. We hereby describe a simple methodology for T3SS needle fluorescent tagging and visualization, which enables live‐cell microscopy and investigation of needle kinetics and dynamics.Support or Funding InformationThis study was supported in part by the DAAD RISE Scholarship, Reed College Biology Undergraduate Student Travel Award, Reed College Summer Opportunity Fellowship, Reed College Undergraduate Research Opportunity Grant and Galakatos funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skin Lipid Abnormalities in Patients with a History of Eczema Herpeticum Indicate the Involvement of the Sphingosine‐1‐Phosphate Signaling System in HSV Viral Replication
Authors: Berdyshev Evgeny , Goleva Elena , Bronova Irina , Jung John , Bronoff Anna S. , Norquest Kathryn , Richers Brittany , Leung Donald YM 
Published: June 2021

RationaleAtopic dermatitis (AD) with eczema herpeticum (EH) can result in systemic infections and can be life‐threatening. The purpose of this study was to identify differences in non‐lesional skin between AD with a history of recurrent EH (ADEH+), AD without EH (ADEH−), and non‐atopic (NA) controls to gain insight into mechanisms involved in EH pathogenesis using a novel minimally invasive skin tape strip (STS) methodology and mass spectrometric approaches.MethodsSTS layers 5–6 from 13 NA, 13 ADEH−, and 15 ADEH+ adult subjects were collected from non‐lesional skin. AD subjects were balanced by gender, age and disease severity; STS were subjected to lipidomic and filaggrin breakdown products analyses by liquid chromatography tandem mass spectrometry (LC‐MS/MS). For the in vitro experiments, differentiated primary human keratinocytes were transfected with control or sphingosine‐1‐phosphate (S1P) lyase (SGPL), sphingosine kinase‐1 (SPHK1), or sphingosine kinase‐2 (SPHK2) siRNA for 48h and infected with HSV‐1 (0.1 MOI). The effect of enzyme silencing on sphingolipidome was evaluated via the LC‐MS/MS. Viral replication was assessed in 24h with qPCR and plaque assay.ResultsADEH+, as compared to ADEH− and NA, subjects had a significantly increased proportion of short‐chain over long‐chain NS‐ceramides (sphingosine N‐acylated with non‐hydroxy fatty acids) (p=0.002), and increased levels of sphingomyelins (p=0.006). In contrast to ADEH−, ADEH+ subjects demonstrated increased level of free C18‐ and C17‐sphingosines (p=0.007 and 0.052, correspondingly) that suggests upregulated skin sphingosine‐1‐phosphate (S1P) metabolism and signaling. A comparison of the effect of silencing of major enzymes that directly regulate S1P levels (SPHK1, SPHK2, SGPL) in primary human keratinocytes in vitro revealed that only the silencing of SGPL resulted in increased viral replication after keratinocyte inoculation with HSV‐1 (Mean±SE, 0.55±0.09 vs. 0.25±0.05 for S1PL vs. control siRNA transfected keratinocytes, p<0.01, as detected by qPCR).ConclusionsSTS from ADEH+ subjects and in vitro studies indicate involvement of the S1P signaling system in EH and suggest novel potential therapeutic targets to treat EH.Support or Funding InformationThis study was funded by the Atopic Dermatitis Research Network supported by NIH/NIAID grant U19 AI117673.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.