bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epidermal Growth Factor‐Induced Phosphorylation Changes in Rat Inner Medullary Collecting Duct
Authors: Chou Chung‐Lin , Knepper Mark A 
Published: June 2021

Epidermal growth factor (EGF) is a potent mitogenic agent promoting cell proliferation, differentiation and survival. The kidney thick ascending limb of Henle's loop and distal convoluted tubule were identified as major sites of EGF synthesis. Downstream from these sites, EGF has been shown to modulate the osmotic water permeability of the collecting duct. The present study sought to determine the EGF‐elicited phosphorylation changes in inner medullary collecting duct (IMCD). Rat IMCD suspensions were treated with 1 μM EGF or vehicle for 30 min. Samples from three replicates were analyzed by TMT isobaric labeling quantitative protein mass spectrometry after phosphopeptide enrichment. A total of 12260 phosphopeptides with unique phosphorylation sites were quantified in EGF‐treated IMCD, with 78 peptides increased and 100 peptides decreased in phosphorylation based on dual statistical criteria. The enriched motif of upregulated phosphopeptides is consistent with activation of proline‐directed kinases, with a unique motif of [S/T]‐P‐K. DAVID analysis of 178 peptides (in 142 proteins) that underwent significant phosphorylation change shows ErbB as the prominent KEGG signaling pathway, confirming that EGF activates ERK mitogen‐activated protein kinase (MAPK), Mapk1 at T183/Y185 and Mapk3 at T203/Y205, via the canonical Ras‐Raf‐MEK pathway. There are five proteins (Atf2, eIF4ebp1, Raf1, Slc9a1, SP1) with phosphorylation sites that are known ERK targets. Among cytosolic docking proteins that can direct the intracellular effects of EGF, several proteins that underwent phosphorylation changes are associated with the PI3k‐Akt‐Mtor pathway, which regulates translation initiation complex eIF4F formation and the translation elongation processes (Ago2, eIF2ak3, eIF2ak4, eIF4ebp1, Eef2, Eef2k, Fbxo6, Pdcd4). In addition, several proteins associated with the Plcγ calcium signaling pathway were significantly altered in phosphorylation. These include Plcg1, Plcb4, Itpr2, Camk2g, Ppp3cb (calcineurin B) and Nos1. EGF had no significant effect on known vasopressin‐regulated phosphorylation sites of the aquaporin‐2 water channel (AQP2) (S256, S261, S264, S269) or the vasopressin‐regulated urea channel (UT‐A1) (S84, S486, S499). However, several modulators of cytoskeleton rearrangement (Cfl1, Fnbp1, Macf1, Parva, Plec, Sipa1l1, Specc1l, Trip10) and trafficking proteins (Dnajc5, Epn3, Mvb12a, Stx6, Ston2, Sytl5, Trak2) underwent phosphorylation changes. EGF also induced phosphorylation changes in several RNA polymerase II‐associated nuclear proteins (Cdk12, Gtf2f1, Leo1, Iws1, Rbm5, Rpap3, SP1). This data can be useful in further investigation of the EGF's action in renal collecting duct cells.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Protein Phosphatase 1 prevents high potassium mediated downregulation of the thiazide‐sensitive sodium chloride cotransporter NCC
Authors: Kortenoeven Marleen L.A. , Wu Qi , Cheng Lei , Fenton Robert A. 
Published: June 2021

The “aldosterone paradox” is a poorly understood physiological mechanism in which increases in the hormone aldosterone either serve to stabilize blood pressure by increasing sodium‐chloride (NaCl) reabsorption or to excrete potassium (K+). Altered NaCl transport via the NaCl cotransporter NCC in the renal distal convoluted tubule (DCT) has been suggested to play a central role in switching from one response to the other. Under low NaCl intake, NCC abundance and activity is high, whereas it is low during high K+ intake ‐ leading to increased delivery of NaCl to downstream segments and increased electrogenic K+ secretion. Despite its essential role, little is known about the molecular alterations in the DCT during the aldosterone paradox, and the proteins involved in the regulation of NCC during this response.To define the proteome of the DCT and how it is modified by increased circulating aldosterone levels subsequent to long term low dietary NaCl or high K+ intake, transgenic mice with expression of enhanced green fluorescent protein (eGFP) under the control of the parvalbumin promoter were used. These mice have specific eGFP‐labeling of DCT cells, allowing high purity fluorescence‐activated cell sorting (FACS) of DCT cells. Mice were maintained on low NaCl or high K+ diets for 4 days, which resulted in increased aldosterone levels and a change in NCC abundance. Following FACS, the DCT proteome was determined by liquid chromatography‐coupled tandem mass spectrometry. 3078 unique proteins were identified in the DCT. 210 proteins were significantly altered in abundance following a low NaCl diet. 625 were significantly altered in abundance following a high K+ diet. Protein Phosphatase 1α (Pp1α) abundance was only increased by high K+ diet. In freshly isolated mouse tubules from the renal cortex, a higher K+ concentration in the medium for 1–4 days resulted in a significant decrease in NCC expression and increased expression of PP1α, mimicking the in vivo situation. The K+‐induced decrease of total NCC levels could be inhibited by treating the cells with the PP1‐blocker tautomycetin, suggesting that PP1 is involved in the K+‐induced downregulation of NCC. In summary, these results suggest that PP1α is involved in the decreased NCC abundance seen during long term high potassium intake.Support or Funding InformationDanish Medical Research Council, Fondation LeDuq, Novo Nordisk Foundation, Aarhus University Research Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Apolipoprotein A‐IV Receptor: a Possibility of an Orphan GPCR?
Authors: Mahmood Ammar , Stein Lauren , Tso Patrick , Samson Willis , Yosten Gina 
Published: June 2021

Apolipoprotein‐A4 (apoA‐IV) is a 46 kDa protein that participates in many physiological functions including glucose homeostasis, lipid metabolism, and satiation. Thus, apoA‐IV could serve as a potential target candidate for the treatment of obesity, diabetes, and cardiovascular diseases. A previous study showed that apoA‐IV binds to the plasma membrane of human hepatic cells, but the membrane receptor responsible for apoA‐IV binding remains unknown. In addition, apoA‐IV exerts its physiological roles through PI3K/Akt signaling pathway in rat hypothalamus and mouse adipocytes. We therefore hypothesize that apoA‐IV interacts with a G‐protein‐coupled receptor, more specifically an orphan GPCR. To test this hypothesis, we are employing our Deductive Reasoning Strategy to match apoA‐IV with its potential receptors. Human gastric carcinoma cells (KATO III) and human liver hepatocellular carcinoma cells (HEPG2) treated with apoA‐IV exhibited significant increases in c‐FOS mRNA expression as measured by qPCR. In addition, KATO III and HEPG2 cells expressed many of the overlapping orphan GPCRs. These results suggest that an orphan GPCR for apoA‐IV ligand may potentially exist. Testing other cell lines for their responsiveness to apoA‐IV treatment and expression for orphan GPCRs as well as employing siRNA‐mediated knockdown of orphan GPCRs that are expressed by all tested cell lines is being performed to identify a potentially specific orphan GPCR that interacts with apoA‐IV. Identification of apoA‐IV receptor would provide a novel platform for apoA‐IV‐based therapeutics.Support or Funding InformationNational Institute of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Varying Cation Types Differentially Stabilizes Poly(dA:dT) DNA
Authors: Schultz Thomas , Grilley Dan 
Published: June 2021

The two meters of DNA in your cells is packaged by eight histone proteins into a complex called a nucleosome, which allows it to fit into a space one millionth of a meter in diameter. Storage in nucleosomes makes DNA inaccessible to the cellular machinery that is responsible for reading the information contained within the DNA and expressing the genetic code. Nucleosomes form on only approximately 70% of the DNA, and are excluded from forming on poly(dA:dT) tracts (A‐tracts). In nucleosomes, AA base steps are preferentially found at positions of high bending towards the minor groove such that a pattern of AA base steps spaced 10 base pairs apart leads to a highly stable nucleosome. Conversely, many AA base steps linked together to form an A‐tract will exclude nucleosomes. This A‐tract nucleosome exclusion is biologically important as it allows A‐tracts to act as regulatory elements and provide access to DNA binding proteins such as transcription and replication factors. In the context of a nucleosome and in naked DNA, AA base steps are known to form a narrowed minor groove. This narrowed minor groove brings the negative charges of the phosphodiester backbone into closer proximity which is normally not favored due to like‐charges repulsing. Our research is focused on the energetics of forming the narrowed minor groove that occurs in A‐tracts with a specific focus on the stabilization caused by cations. Using absorbance and circular dichroism spectroscopy, we show that the structure and stability of A‐tract DNA is dependent on the size and hydrogen bonding capacity of the cations that are in solution. This cation dependence tracks with the size of the A‐tract DNA, supporting the idea that there is cooperativity between AA base steps. To test the biological importance of these findings, we performed in vitro nucleosome reconstitutions in which generic sequence DNA and A‐tract DNA compete for limited quantities of histone proteins. Using these competitive reconstitutions, we show that A‐tracts are excluded from nucleosomes in a manner that depends on the cation in solution. Our results show that A‐tract structure, unlike generic sequence DNA, is sensitive to the type of cation that is present inside of cells, and that this sensitivity is important for understanding the packaging of DNA into nucleosomes.Support or Funding InformationThis research was supported by an Undergraduate Research and Creativity grant to TS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Lysine Acetylation Regulates the Choice of the Okazaki Fragment Maturation Pathway
Authors: Balakrishnan Lata , Njeri Catherine , Ononye Onyekachi , Surendran Sneha , Howald Olivia , Kaur Jasmeet 
Published: June 2021

Eukaryotic DNA replication machinery has evolved to accurately maintain the human genome through decades of life and across generational time. Mutations in the genome can initiate and promote human diseases, such as cancer and neurodegenerative disorders. During replication, lagging strand synthesis initiated by the error‐prone DNA polymerase alpha primes template DNA with a RNA/DNA initiator primer. This is further elongated by high‐fidelity DNA polymerase delta, creating short stretches of DNA, known as Okazaki fragments. Because millions of these fragments need to be created and accurately ligated per cell division, the risk of mutagenesis is higher in the lagging strand compared to the leading strand. Maturation of the Okazaki fragment necessitates the displacement of the initiator primer, to remove the RNA segment prior to ligation. Two redundant pathways have been proposed in this process, one, known as the short flap pathway, rapidly removes the RNA primer and allows ligation of the polymerase alpha synthesized DNA primer. The other pathway, known as the long flap pathway, promotes displacement of the entire initiator primer and the region is resynthesized by the high‐fidelity DNA polymerase delta. Many of the proteins involved in Okazaki fragment maturation pathway are post‐translationally modified by lysine acetylation. Biochemical studies analyzing enzymatic properties of in vitro acetylated Okazaki fragment proteins show alterations in protein‐DNA, protein‐protein interactions and enzyme function. Our preliminary analysis of in vitro acetylated recombinant Okazaki fragment processing (OFP) proteins show that lysine acetylation regulates enzyme activity in a manner consistent with promoting the long flap pathway. Lysine acetylation inhibits the function of the short flap pathway nuclease, flap endonuclease 1 (FEN1) and stimulates the functions of nearly all the proteins in the long flap pathway. Further, using reporter constructs, we showed that in mammalian cells, increasing cellular lysine acetylation correlated with use of the long flap pathway for repair processing. Our current model proposes that lysine acetylation of Okazaki fragment proteins regulates the choice of the long flap pathway, resulting in higher‐fidelity lagging strand synthesis.Support or Funding InformationThis work was supported by funding from National Institute of Health (GM098328) and IUPUI New Faculty Startup Award.This abstract is from the Experimental Biology 2019 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 , Bai Lin , Santos Ruda , Zhang Dan , Yurieva Olga , O'Donnell Michael , Li Huilin 
Published: June 2021

In eukaryotes, DNA replisome is a large protein complex that carries out DNA replication, which contains several enzymatic protein subunits, such as helicase, RNA primase and DNA polymerase. Replisome generates a DNA replication fork and duplicates both the leading and lagging strand of duplex DNA. Previous studies showed the Ctf4 trimer is proposed to be a hub to coordinate the subunits in replisome, Ctf4 trimer may link CMG and Pol a through binding CIP motif of Sld5 and Pol a. 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.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural basis for teneurin function in circuit‐wiring: A toxin motif at the synapse
Authors: Arac Demet 
Published: June 2021

Teneurins (TENs) are cell‐surface adhesion proteins with critical roles in tissue development and axon guidance. Here we report the 3.1‐Å electron cryo‐microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc‐toxins. The ECR includes a large β‐barrel that partially encapsulates a C‐terminal domain, which emerges to the solvent through an opening in the mid‐barrel region. An immunoglobulin (Ig)‐like domain seals the bottom of the barrel while a β‐propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the β‐propeller acts as a switch to regulate trans‐cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans‐cellular signaling in a LPHN‐dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.Support or Funding InformationNIGMSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functionally Important Conformational Sub‐States in Human Ribonuclease Family
Authors: Bafna Khushboo , Narayanan Chitra , Bhojane Purva P , Bernard David , Howell Elizabeth E , Doucet Nicolas , Agarwal Pratul K 
Published: June 2021

Eight members of the human pancreatic ribonuclease (RNase) family have diverse biological functions including angiogenesis and host defense (cytotoxic and anti‐pathogen activities) but share common structural scaffold and catalyze the hydrolysis of ribonucleic acid (RNA). However, the catalytic efficiency and rate of dynamics among these enzymes differ by more than a million (106) folds. Emerging evidence from other enzyme systems propose that internal motions drive the sampling of short‐lived minor population of conformations (called as sub‐states) that can contain features to promote various steps during the function of an enzyme. Therefore, quantitatively characterizing the conformational sub‐states in the catalytic cycle of enzymes, including substrate binding, structural rearrangements leading to the transition state, product formation and product release will enable us to obtain detailed insights in the role of enzyme dynamics in catalysis by RNases. We have used a combination of theoretical modeling, computer simulations, steady state kinetics and NMR experiments. Our studies indicate that RNases superfamily can be classified into sub‐families with designated biological functions and conserved dynamical behavior. Further, computer simulations show diverse binding preferences across the human RNase family and the conformational sub‐states in various steps of the catalytic cycle differ among the members belonging to each of the sub‐family. Moreover, we observe a ~106 fold difference in the catalytic rates of the human RNases. Additionally, we are using hybrid QM/MM method to model the catalysis of the single stranded RNA that would enable us to identify the distinct conformational sub‐states sampled by each of the members of human RNase family in their respective minimum energy reaction pathway. Results show interesting differences in the conformational sub‐states in the members of human RNases family.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palladin is needed for proper actin‐rich comet tail formation and can functionally replace the Arp2/3 complex during intracellular motility of Listeria bacteria
Authors: Dhanda Arandeep Singh , Vogl A Wayne , Albraiki Sharifah E , Otey Carol A , Beck Moriah R , Guttman Julian A 
Published: June 2021

Listeria monocytogenes (Listeria) bacteria are well‐known for their ability to hijack the eukaryotic actin cytoskeleton during infections. To propagate disease, Listeria generate Arp2/3‐based actin‐rich structures called comet tails that move the bacteria within and amongst host cells. The host protein palladin is a key component of actin‐rich structures normally generated during eukaryotic cell motility, however the precise function(s) of palladin during motility remains unclear. Here we tested the hypothesis that palladin is a crucial factor for Listeria motility and simultaneously used Listeria as a model to determine if palladin itself could functionally replace the Arp2/3 complex. Using palladin‐targeting antibodies, we identified palladin at Listeria invasion sites and comet tails. Strikingly, when we depleted cells of palladin, comet tails became shorter and severely misshapen. In cells expressing palladin mutants defective for actin or VASP binding, comet tails began to disintegrate or became progressively thinner as they moved. Ultrastructural examination of these thin comet tails revealed a switch in the comet tail actin network from highly branched arrays to parallel bundles. To test whether palladin could compensate for the Arp2/3 complex during bacterial motility, we overexpressed palladin in cells treated with the potent Arp2/3 inhibitor CK‐666. In these cells Listeria motility was unperturbed. Next we used a cell line depleted of several Arp2/3 complex subunits. As expected, Listeria were non‐motile during infections of these cells, however palladin overexpression in this Arp2/3 functionally null cell line restored the ability of Listeria to generate the actin‐rich structures formed by the bacteria. To definitively demonstrate palladin's ability to compensate for a lack of functional Arp2/3, we used purified protein components in conjunction with Listeria bacteria to show that actin‐rich structures formed by Listeria are generated in a cell‐free system containing palladin in place of the Arp2/3 complex. In conclusion, we show that palladin structurally organizes bacterial actin‐rich structures and importantly, compensates for the Arp2/3 complex without hindrance during bacterial actin‐based motility.Support or Funding InformationGrant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397 to A.W.V), NIH (grant no. R15 GM120670 to M.R.B), SFU departmental funds and SFU Multi‐Year Funding (A.S.D)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 Imaging of Endothelial Cell Tube Formation: Inhibition by Chondrostatin
Authors: Sandell Linda J , Wang Zhepeng , Franz Carl , Bryan Jennifer , Siegel Adam , Mecham Robert , Wagensail Jessica , Ell Brian , Rapraeger Alan 
Published: June 2021

Cartilage tissue is avascular and resistant to tumor invasion. The basis for these properties has been actively sought for the last 35 years. Based on the assumption that cartilage contains inhibitors of angiogenesis, previous studies have focused on isolating potential vascular inhibitors from cartilage. Here we report that the NH2‐propeptide of type IIB procollagen (chondrostatin), a product of collagen biosynthesis, is capable of inhibiting angiogenesis mediated by the integrins, αVß3 or αVß5 present on endothelial cells. Chondrostatin inhibits tube formation by human umbilical vein cells (HUVEC) and inhibits endogenous endothelial cell outgrowth in a rat aortic ring angiogenesis bioassay. The kinetics of formation of vessel tubes by HUVEC cells was investigated by imaging cells as they formed tubes in a collagen gel. As αVß3 and αVß5 integrins are expressed primarily on endothelial cells, cancer cells and osteoclasts, but not on normal chondrocytes, we propose that cartilage chondrostatin protects cartilage by targeting endothelial cells during chondrogenesis, thus inhibiting angiogenesis and rendering the tissue avascular. We show that chondrostatin also binds to chondrosarcoma, breast and cervical cancer cells via the integrins αVß3 and αVß5 and inhibits migration, eventually inducing cell death.Supported by NIH R0136994