bluuebunny/crossref_metadata_2025_split huggingface dataset:
Error‐Prone DNA Polymerase IV preserves the memory of its interaction with RecA
Authors: Tashjian Tommy F. , Lin Ida , Nguyen Brian H. , Godoy Veronica G. 
Published: June 2021

DNA Polymerase IV (DinB) is a highly conserved, error‐prone DNA polymerase (DNAP) expressed as part of Escherichia coli's DNA damage response. This polymerase is well known for its ability to perform translesion synthesis, or to bypass DNA damage on the template strand that would otherwise cause lethal replication fork stalling. As a consequence of this ability to accommodate DNA lesions, DinB is highly mutagenic compared to high fidelity DNAPs (100‐fold less accurate than replicative DNAP III). Despite its potential for mutagenesis, this enzyme is present at high concentrations (~250 nM) even in the absence of DNA damage; approximately 8‐fold higher than the replicative DNAP III. Upon induction of the DNA damage response, DinB is the most abundant DNAP in the cell (~2500 nM). DinB mutagenic potential and high intracellular concentration suggests that its activity must be strictly regulated. Indeed, it has been shown that DinB's activity is regulated by protein‐protein interactions.In E. coli DinB is known to interact with the cells' highly conserved main recombinase, RecA. It has been shown that this interaction increases DinB's fidelity on damaged templates in vitro. In addition, our previous work identified a variant of DinB, DinB(C66A), which contains a single amino acid substitution in the DinB family highly conserved C66 residue. We previously showed that DinB(C66A) has altered binding to RecA and does not benefit from an increase in fidelity upon interaction with RecA.We find that DinB undergoes a detectable conformational change upon interaction with RecA in vitro. Our evidence also suggests that the DinB interaction with RecA is short lived. Remarkably the conformational change in DinB, which is effected by RecA, persists after RecA protein is removed by chromatography. In addition, DinB becomes more susceptible to proteolysis. We also find that the variant DinB(C66A), which does not show increased fidelity upon RecA‐interaction, either does not undergo the conformational change, or it is unable to stably maintain said conformational change. These data suggest that DinB recalls previous interaction with RecA, which in turn affect the fidelity of DinB beyond the time of interaction with RecA. The interplay between DinB and RecA represents a clever mechanism by which RecA is recycled and available for its many other cellular functions while at the same time DinB fidelity is elevated preventing high mutagenic burden despite DinB's high intracellular concentrations.Support or Funding InformationResearch funded by National Institute of General Medical Sciences (RO1GM088230 to VG) and Northeastern University Office of the Provost (Graduate Dissertation Research Grant to TT).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure of Eukaryotic CMG Helicase at a Replication Fork and Implications for Replisome Architecture and Origin Initiation
Authors: Yuan Zuanning , Georgescu Roxana , Lin Bai , de Luna Almeida Santos Ruda , Zhang Dan , Yurieva Olga , O'Donnell Michael , Li Huilin 
Published: June 2021

Helicases are a class of motor proteins that move directionally along a nucleic acid phosphodiester backbone and separate two annealed nucleic acid strands by using ATP hydrolysis. Helicases exist in all living organisms. Based on their shared sequence motifs, they are classified into 6 classes in which superfamilies 1 and 2 are not ring‐shaped structure and superfamilies 3–6 are ring‐forming helicases. In eukaryotes, DNA helicase CMG consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like all hexameric helicases, is shaped like a ring with two tiers referred to as an NTD tier (N‐terminal domain) and a CTD tier (C‐terminal domain); the CTD tier contains the ATP motors. In principle, either tier could translocate ahead of the other during movement on DNA. By using single‐particle cryo‐EM of an active CMG on the forked DNA with a dual biotin‐streptavidin block, we solved the first structure of helicase in complex with a DNA fork. The DNA duplex stem penetrates into the central channel of the NTD tier and the unwound leading ssDNA traverses the channel through the NTD tier into the CTD motor tier, 5′‐3′ through CMG. Therefore, during translocation the CTD motor is behind the NTD ring that is pushed ahead to face the fork, opposite the currently accepted polarity. The polarity of the NTD ahead of the CTD places the leading Polɛ below CMG and Polα‐primase at the top of CMG, above the unwinding point of the replication fork. The DNA direction requires that the Mcm rings must first pass one another when the Mcm2–7 double hexamer come apart. Interestingly, the new NTD‐to‐CTD polarity of translocation reveals an unforeseen quality control mechanism that each Mcm2–7 is fully converted from encircling dsDNA to ssDNA before they can depart from the origin. We propose this architecture of two motors head to head may generate energy that underlies initial melting at the origin.Support or Funding InformationThis work was funded by NIH Grants GM111472 (to H.L.) and GM115809 (to M.E.O.); the Van Andel Research Institute (H.L); and the Howard Hughes Medical Institute (M.E.O.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXC1 is Over‐expressed and is More Stable in Triple Negative/Basal‐Like Breast Cancer
Authors: Elian Fahed A. , McMullen Todd PW. , Brindley David N. , Walter Michael A. 
Published: June 2021

Rapidly accumulating evidence implicates forkhead box C1 (FOXC1) in Triple Negative Breast Cancer (TNBC), particularly in Basal‐Like Breast cancer (BLBC). Recently additional studies have demonstrating that FOXC1 is also a major player in hepatocellular carcinoma (HCC), endometrial cancer, Hodgkin's lymphoma (HL), non‐Hodgkin's lymphoma (NHL), and others. The FOXC1 gene encodes a transcription factor that is crucial to mesodermal, neural crest, and ocular development. Loss of function mutations in FOXC1 have been shown to cause autosomal dominantly inherited Axenfeld‐Rieger's Syndrome (ARS), a developmental disorder associated in eye anomalies and glaucoma. Interestingly, while FOXC1 missense mutations that cause ARS reduce FOXC1 activity, increased FOXC1 function now appears to be often linked to more aggressive cancer phenotypes in TN/BL‐BC, HCC, HL, and NHL.We have investigated the mechanism(s) by which FOXC1 activity is increased in BLBC. Samples were obtained from TNBC tumors, Triple Positive Breast Cancer (TPBC) tumors or from breast tissue from normal patients. Using quantitative PCR, we found that FOXC1 was significantly over‐expressed in TNBC patients as compared to controls. In contrast, FOXC1 mRNA was significantly less expressed in TPBC samples as compared to either control or TNBC samples. FOXC1 protein half‐life was significantly longer in the TNBC/BLBC cell lines (HS 587T and BT 549) compared to FOXC1 protein's half‐life in HeLa cells. Studies of FOXC1 Copy‐number variation (CNV) in TNBC/BLBC cell lines reveals that cell lines that have higher levels of FOXC1 protein (HS 587T, BT‐549) have extra copies of FOXC1. This contrasts with a cell line with lower expression of FOXC1 protein, MDA‐MB‐231, which may be explained by a deletion of FOXC1 in this TNBC cell line. Our results suggest that increased FOXC1 function in TNBC/BLBC results from over‐expression of FOXC1 in tumors of TNBC patients that appears to be the result of changes to FOXC1 stability and amplification of FOXC1 copy number. We predict that understanding the mechanism(s) underlying FOXC1's activation in cancer could aid in designing improved therapies for TNBC patients.Support or Funding InformationVerag Donation to MAWThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Receptor‐Mediated Delivery of CRISPR‐Cas9 Endonuclease for Cell Type Specific Gene Editing
Authors: Roy Marc , Rouet Romain , Thuma Benjamin , Lintner Nathanael , Rubitski David , Finley James , Wisniewska Hanna , Mendonsa Rima , de Oñate Lorena , Hirsh Ariana , Barrón Joan Compte , McLellan Thomas , Bellenger Justin , Feng Xidong , Varghese Alison , Chrunyk Boris , Borzilleri Kris , Hesp Kevin , Zhou Kaihong , Ma Nannan , Tu Meihua , Wilson Ross , Dullea Robert , McClure Kim , Liras Spiros , Mascitti Vincent , Doudna Jennifer 
Published: June 2021

CRISPR‐Cas RNA‐guided endonucleases hold great promise for disrupting or correcting genomic sequences through site‐specific DNA cleavage and repair. However, the lack of methods for cell‐ and tissue‐selective delivery currently limits both research and clinical uses of these enzymes. Here we report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9‐ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Live cell imaging studies are consistent with a receptor‐mediated endocytosis mechanism of active uptake of the RNP. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor‐facilitated and cell‐type specific gene editing that did not rely on electroporation or the use of transfection reagents. This approach provides a framework for applications to selective gene editing in the liver and offers a potentially general mechanism for cell‐, tissue‐, or organ‐targeted gene editing by receptor‐mediated delivery of genome‐editing enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
JNK‐mediated eIF4E phosphorylation and signaling in fibrotic functions of lung‐resident mesenchymal cells (MCs)
Authors: Walker Natalie Maureen , Lama Vibha N. 
Published: June 2021

INTROWe have recently demonstrated that cap‐dependent translation regulated through 4E‐BP1/eIF4E signaling plays a critical role in regulating fibrotic functions of activated mesenchymal cells, specifically collagen I expression. Fibrotic MC signaling to 4EBP1 is resistant to rapamycin, and resistance to TOR inhibitors has also been described in cancer. This underscores the need for better therapeutic options targeting aberrant translation, and further understanding of upstream signaling mechanisms regulating translational control will be key. Here we investigate mechanisms driving cap‐dependent translation upstream of eIF4E Serine 209 phosphorylation in lung MCs.Materials/methodsLung‐resident mesenchymal cells were isolated from Bronchoalveolar lavage fluid of lung transplant recipients with and without fibrosis by plastic adherence. MCs were treated for 2 hours with 10uM SP600125 (JNK inhibitor), U0126 (MEK inhibitor), SB202190 (p38inhibitor), or the MNK kinase inhibitors CGP57380 and eFT‐508. Constitutively active JNK1 (MKK7‐JNK1 fusion) was shuttled into plentiLOX‐IRES‐puro and packaged into lenti‐viral particles. Non‐fibrotic MCs were infected for 48 hours, lysed and protein expression was analyzed by western blot.RESULTSFibrotic MCs isolated from patients with Bronchiolitis obliterans had significantly higher cap‐dependent translation compared to non‐fibrotic MCs when measured utilizing luciferase reporter assay (n=7; p<0.01). Fibrotic MCs had significantly higher phosphorylation of eIF4E at serine 209 compared to non‐fibrotic MCs when baseline expression was measured by western blotting (n=8; p<0.01), suggesting elevated translational activity. MCs treated with rapamycin (250 nM) for 2 hours demonstrated increased phosphorylation of eIF4E (S209), which was sensitive to MNK kinase inhibition. Treatment of fibrotic MCs with JNK MAPkinase inhibitor SP600125 or the MNK inhibitors eFT‐508 and CGP57380 led to a significant decrease in collagen I expression and eIF4E (S209) phosphorylation, while MEK and p38 inhibitors did not. Expression of constitutively active JNK led to a significant increase in phosphorylated eIF4E (S209) in non‐fibrotic MCs, implicating JNK activation as key to causing eIF4E phosphorylation and potentially increased cap‐dependent translation.ConclusionThese data demonstrate that cap‐dependent translation downstream of 4E‐BP1/eIF4E is elevated in fibrotic MCs and that eIF4E phosphorylation at serine 209 is sensitive to JNK/MNK inhibition. This suggests that the JNK/MNK signaling axis upstream of 4E‐BP1/eIF4E and hence translation is key to fibrotic functions of MCs during pathogenesis of fibrotic lung diseases, and a viable target for treating fibrosis.Support or Funding InformationThis abstract is funded by NIH grants: RO1F035722 and RO1F037975.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RpoS recovery from phosphate starvation
Authors: Hamdallah Issam Nihad , Majdalani Nadim , Tripathi Arti , Gottesman Susan 
Published: June 2021

Acclimation of E. coli to environmental stress is dependent on changes in gene expression. RpoS is a sigma factor that helps the cell deal with a range of stresses. When cells are not under stress, RpoS is rapidly degraded; however, during stationary phase or under other stresses during growth, RpoS accumulates due to increased translation or decreased proteolysis. Under phosphate starvation, RpoS degradation is inhibited by a small antiadaptor protein, IraP. IraP achieves RpoS stabilization by blocking adaptor protein RssB from delivering RpoS to the ClpXP protease for degradation. While the pathway for RpoS stabilization under phosphate starvation is well defined, recovery from stress is less well understood. Western blot analysis indicates that when phosphate is introduced into phosphate‐deprived cultures, RpoS levels sharply decrease to barely detectable levels within five minutes of exposure, which strongly suggests that rapid degradation is restored when phosphate is restored to cultures. We are examining the mechanism behind this recovery process by monitoring the fate of IraP and control of the adaptor protein RssB in the presence of phosphate. We hope this study will help us better understand how the interplay of stress induction and recovery govern adaptations under nutrient starvation. The lessons learned from the recovery of IraP may also provide insight into the mechanism of recovery of the two other known antiadaptors, IraD and IraM.Support or Funding InformationNIH Intramural FundingThis 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 Protein‐Protein Interaction Between NIL‐16 and HDAC3
Authors: Dailey Leandrew A. , Baugh Keith Daniel , Fenster Steven Daniel 
Published: June 2021

Neuronal Interleukin‐16 (NIL‐16) is the neuronal‐specific splice isoform of the interleukin‐16 (IL16) gene. NIL‐16 shows restricted expression to the hippocampus and cerebellum: brain regions involved in learning and memory. NIL‐16 shares an identical C‐terminal protein‐coding sequence with pro‐IL‐16; the shorter, immune‐specific splice isoform that is the pro‐inflammatory precursor to the cytokine IL‐16. The mutual amino acid sequence between pro‐IL‐16 and NIL‐16 contains three PDZ protein interaction domains (PDZ‐1, PDZ‐2 and PDZ‐2). In addition to serving as the precursor to IL‐16, pro‐IL‐16 regulates immune cell proliferation through a PDZ‐2 mediated‐interaction with the nuclear‐associated protein HDAC3. Emerging evidence indicates that HDAC3 plays a critical role in regulating fundamental mechanisms involved in brain development and mechanisms of learning and memory. Here, we hypothesize that like pro‐IL‐16, a similar protein‐protein interaction exists between NIL‐16 and HDAC3 suggesting a possible role in mediating important processes in neurons. To test this hypothesis, we conducted pulldown assays with mouse cerebellar brain extract using fusion proteins coding for the PDZ‐2 domain of NIL‐16 and the PDZ‐interacting motif of HDAC3. Our results indicate that we can affinity purify HDAC3 from cerebellar extract with the PDZ‐2 domain of NIL‐16. Likewise, we also affinity purified NIL‐16 with the PDZ‐interacting motif of HDAC3. Additionally, using immunofluorescent microscopy, we show overlapping distribution of NIL‐16 and HDAC3 expressed in COS‐7 cells and in cultured cerebellar granule neurons. Finally, we performed co‐immunoprecipitation assays with cerebellar extract demonstrating preliminary evidence that NIL‐16 and HDAC3 form an in vivo protein complex in neurons.Support or Funding InformationResearch in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping the interface of alpha globin and eNOS: implications for increasing endogenous NO therapeutically
Authors: Keller TC Stevenson , Swope Nicole K. , Columbus Linda , Isakson Brant E. 
Published: June 2021

Nitric oxide signaling is a powerful mechanism for the regulation of blood pressure homeostasis, inducing vasodilation and reducing peripheral resistance. As such, disruption of NO‐inhibitory protein interactions may prove to be a novel therapeutic for the treatment of hypertensive disease states. Focusing on the recent discovery of the alpha globin/eNOS complex in vascular endothelium, we have designed an alpha globin mimetic peptide that binds to eNOS and increases NO signaling and dilation. A key step in the development of therapeutic agents targeting this complex is a high‐resolution structural model of the binding interface of alpha globin and eNOS. Thus, we have used solution NMR to determine the relevant structural details of the alpha globin mimetic peptide known to bind to eNOS. Interestingly, this twenty residue peptide has a stable population of both proline cis and trans isomers, potentially influencing the affinity of the peptide/eNOS interaction. Crosslinking mass spectrometry allows for the mapping of interacting residues between the peptide and eNOS after enzymatic digestion. Using cis and trans proline peptide ensembles, we have generated models of the peptide/eNOS complex through docking simulations which can be verified with full‐length alpha globin. The different conformations adopted by the peptide allows for potential new interaction modes that may not be possible with full alpha globin protein, thereby possibly increasing peptide/protein binding affinity. Determining the binding interface of the alpha globin/eNOS complex allows for targeted searches for molecular inhibitors of this alpha globin/eNOS interaction, in efforts for novel therapeutics to combat vasoconstrictive disease.Support or Funding InformationAmerican Heart Association Grant 16PRE31180040 (TCSK)NIH 088554 (BEI)NIH 087828 (LC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatic Endoplasmic Reticulum Associated Degradation (ERAD) manages FGF21 levels and metabolism via CREBH during fasting‐feeding and growth
Authors: Bhattacharya Asmita , Zhang Kezhong , Qi Ling 
Published: June 2021

The liver regulates growth and systemic energy homeostasis through inter‐organ communication via the secretion of growth factors, hormones and peptides. Fibroblast growth factor 21 (FGF21) is a liver‐derived, fasting‐induced hormone with broad effects on growth, nutrient metabolism and insulin sensitivity, and huge potential as an anti‐diabetes/obesity therapeutic. Here, we report the discovery of a novel regulatory mechanism repressing FGF21 expression under growth and fasting‐feeding. The SEL1L‐HRD1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)‐associated degradation (ERAD) machinery, responsible for the recruitment, ubiquitination and retro‐translocation of ER proteins for cytosolic proteasomal degradation. Mice with liver‐specific deletion of SEL1L exhibit growth retardation and improved metabolism with markedly elevated circulating FGF21, phenocopying FGF21 transgenic mice or pharmacological models. Mechanistically, we show that the SEL1L‐HRD1 ERAD complex controls FGF21 transcription by regulating the turnover (and thus nuclear abundance) of ER‐resident transcription factor CREBH, while having no effect on the other well‐known FGF21 transcription factor PPARα. We further show a physiologically‐regulated, inverse correlation between SEL1L‐HRD1 ERAD and CREBH‐FGF21 levels under fasting‐feeding and growth. This study not only establishes the importance of ERAD in liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic “ERAD‐CREBH‐FGF21” axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.Support or Funding InformationAHA predoctoral fellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Mitophagy by the Parkin Ubiquitin Ligase and PINK1 Ubiquitin Kinase
Authors: Gehring Kalle 
Published: June 2021

Familial forms of Parkinson's disease arise from loss of the mitochondrial quality control pathway mediated by PINK1, a protein kinase, and Parkin, an E3 ubiquitin ligase. Together, they regulate the disposal of damaged mitochondria by autophagy (mitophagy). When damaged, mitochondria accumulate PINK1, which phosphorylates ubiquitin to recruit cytosolic Parkin. Normally quiescent due to autoinhibitory interactions, Parkin is activated by both phosphorylation by PINK1 and binding phosphorylated ubiquitin. The molecular mechanisms that activate activate Parkin have been the focus of much speculation. Here, I describe the crystal structure of the active conformation of Parkin that shows phosphorylation of parkin by PINK1 leads to a dramatic conformational change to derepress its E2‐binding and catalytic sites. Using a variety of experimental approaches, the activation pathways mediated by phospho‐ubiquitin binding and Parkin phosphorylation are compared. The study deepens our understanding of the mechanisms of Parkin activation and suggests new approaches for the treatment of Parkinson's disease by activating Parkin with small molecules.Support or Funding InformationCanadian Institutes of Health Research and the Michael J Fox FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.