bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular Identification of Doublecortin Positive Immature Neurons in Monkey Cortex
Authors: Heyworth Nadine , Calderazzo Samantha , Kyada Margee , Rosene Douglas 
Published: June 2021

Doublecortin (DCX) is a microtubule‐associated protein expressed transiently during the maturation and migration of newborn neurons. In the adult brain, two neurogenic regions including the Subventricular Zone (SVZ) and the Subgranular Zone (SGZ) continue to produce adult generated neurons (aGN) throughout adulthood. The transient expression of DCX has also been found in various neocortical and allocortical regions in the adult brain of the rodent, rabbit, cat, monkey and human. These regions include the piriform cortex, medial prefrontal cortex, entorhinal cortex, as well as neocortical areas of the temporal lobe and insula. DCX+ cells are reported to localize mainly in superficial layers of cortex (II/III) with scattered DCX cells in deeper cortical layers. Some studies have reported these cells differentiate into excitatory neurons while others report a GABAergic phenotype. In addition to cellular identity, the origin of these neurons remains unresolved with one possibility that these cells are derived postnatally from the SVZ while others contend that they are generated prenatally.To address these issues, tissue was obtained from rhesus monkeys injected with BrdU to label aGN. Sections were processed with immunohistochemistry and confocal microscopy to determine if DCX cells in the temporal lobe co‐localize with BrdU and/or with markers of inhibitory interneurons including Calbindin, Parvalbumin, and Calretinin, or with the excitatory neuron marker CaMKII. Results show no DCX+ cells co‐localized with BrdU, indicating that DCX+ immature neurons are not adult generated and hence are leftover from development. Preliminary analysis of co‐localization with DCX shows that at least some co‐localize with GABergic markers of interneurons.
Investigating the Role of Threonylcarbamoyl Adenosine in HIV‐1 Replication in vivo
Authors: Kirkland Marina , Venketaraman Vishwanath , Swairjo Manal 
Published: June 2021

In all retroviruses, reverse transcription of viral genes is primed by a host‐cell tRNA whose 3′‐end 18 nucleotides are complementary to the viral primer binding site. For lentiviruses, including HIV, the natural primer is tRNALys3 which harbors several modified bases in its anticodon stem‐loop (ASL). Interaction of the fully modified ASL with the HIV RNA template is required for efficient reverse transcription of viral RNA, and continuation of viral life cycle. One of the modified nucleosides of the ASL of tRNALys3 is N6‐threonylcarbamoyl adenosine (t6A37), a universally conserved modification found at position 37 in all tRNAs decoding ANN codons. t6A37 stabilizes the ASL structure and prevents ribosomal frameshifting in the course of translation. Eukaryotic t6A37 biosynthesis is catalyzed by the enzyme Sua5 and the KEOPS complex, comprised of the four subunits Kae1, Pcc1, Bud32 and Cgi121.We hypothesize that t6A37 plays an important role in viral replication in vivo, by stabilizing ASL structure or by direct interaction with viral RNA. To test this hypothesis, we set out to suppress the biosynthesis of t6A37 in CD4+ cells by targeting its biosynthesis genes with siRNA and testing viral load upon HIV‐1 infection. Background expression levels of the core human t6A37 biosynthesis gene Osgep (Kae1 ortholog) were determined by qRT‐PCR. Treatment with anti‐OSGEP siRNA resulted in knockdown of Osgep expression by 60% 48 hrs post‐transfection and sustained for 72 hrs, 4 times longer than the reported half‐life of OSGEP protein in human cells.
Structural Characterization of nc886, a Cellular Non‐coding RNA Regulator of the Antiviral Kinase PKR
Authors: Calderon Brenda , Conn Graeme 
Published: June 2021

The double‐stranded (ds)RNA‐activated protein kinase (PKR) senses dsRNA produced during viral replication and acts as the first line of defense in the innate immune response to viral infection. Activated PKR phosphorylates the eukaryotic initiation factor 2 (eIF2), halting protein synthesis and viral replication. How PKR basal activity is regulated in the absence of viral infection is unknown. Recently, the cellular non‐coding RNA 886 (nc886) was discovered and proposed to act as a novel, endogenous regulator of PKR. We hypothesize that nc886 regulates PKR by binding to the monomeric form and preventing the dimerization step of activation. This maintains PKR in an inactive state that is poised for a rapid response to viral infection. Our goal is to define the molecular mechanism of nc886‐mediated PKR inhibition using RNA structure probing and mutational analyses combined with binding and activity assays. We show that nc886 RNA exists as two stable, structurally and functionally distinct conformations. Ribonuclease digestion reveals that the two conformers differ in their apical region, while the terminal stem portion of the RNA structure is conserved. The two conformers have strikingly different binding affinities for PKR, but both are functional inhibitors of PKR in in vitro kinase assays. By delineating the structural basis for PKR interaction with the different nc886 conformers, we will uncover the structural motifs that control PKR inhibition and determine how PKR is poised to act in the antiviral response.Source of Research Support: NIH R21‐AI097803 and 5T32GM008367
Diversification of a Bacterial ADP‐Ribosyltransferase
Authors: Sung Vicky , Kallert Stephanie 
Published: June 2021

OBJECTIVETo accelerate the enzyme evolution, we diversified a bacterial ADP‐ribosyltransferase, cholix toxin catalytic fragment, to modify different substrates.METHODSPreviously, we identified a diffusible intermediate that could modify target residues in a proximity dependent manner (submitted in the same meeting, Topic #2278‐ASBMB‐Enzyme Mechanism, Number 540). We utilized a phage display library screening strategy to select for variants to bind to different substrates of interests, followed by enzymatic screening for the selected variants retaining enzymatic activity. Site‐directed mutagenesis, enzymatic assays and 2‐D‐electrophoresis analysis were used to detect the in vitro modification of the substrates by the engineered enzymes.RESULTSWe showed several examples of engineered cholix toxin catalytic fragment variants that can modify various substrates, including enhanced green fluorescent protein (EGFP) and mouse immunoglobulins.CONCLUSIONWe anticipate our findings to be a starting point for more sophisticated engineering. Such enzymes can be useful tools for basic research or therapeutic applications.
Investigating the Structural Basis of the CTD Code
Authors: Gibbs Eric , Showalter Scott 
Published: June 2021

The goal of this project is to study the structure and function of the C‐terminal domain of RNA Polymerase II (CTD). The CTD, which contains tandem repeats of the consensus heptad sequence YSPTSPS, orchestrates transcription by recruiting trans‐acting regulatory factors, directed by a set of post‐translational modifications known as the CTD code. Since the discovery of the CTD code, much effort has gone into the identification of novel modifications, the determination of their effects on specific CTD interactions, and the structural characterization of transcription factors in complex with the CTD. Much less is known about the CTD in its unbound state and of the possible structural effects of the post‐translational modifications that constitute the code. Recently, we have begun biophysical characterization of the unmodified and hyperphosphorylated CTD using a combination of techniques, including 13C Direct‐Detect Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry. Preliminary results suggest that the CTD exists in an extended conformation that cannot be adequately described by the previously proposed beta–spiral model. The models we have developed serve as novel structural hypothesis, relevant for determination of the structural basis for the CTD code and the elucidation of the molecular mechanisms of eukaryotic transcription.This work is funded by NSF CAREER award (MCB‐0953918) to Scott A. Showalter.
Unique Sensitivity to Extracellular Proteases and Peptide Protection of the HERG Potassium Channel
Authors: Zhang Shetuan , Guo Jun , Lamothe Shawn , Hogan‐Cann Andrew , Kang Yudi , Yang Tonghua 
Published: June 2021

The human ether‐a‐go‐go–related gene (hERG) encodes the pore‐forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome, arrhythmias and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation of various proteases. We characterized the effects of proteases on hERG/IKr with the aim to develop novel strategies to prevent protease‐mediated damage. Using whole‐cell patch clamp and Western blot analysis we demonstrated that the hERG/IKr channel is selectively cleaved by the serine proteases, proteinase K and protease XXIV. Using molecular biology including making chimerical channels between protease K‐sensitive hERG and insensitive hEAG, we identified that the S5‐pore linker of hERG is the target domain for proteinase K. We discovered that the scorpion toxin BeKm‐1, which binds to the S5‐pore linker of hERG, can effectively protect hERG from proteinase K‐mediated damage. The reduction in mature ERG expression was observed in a rabbit cardiac ischemia model and in hERG‐HEK cells cultured in hypoxic conditions. BeKm‐1 effectively prevented the hypoxia‐induced reduction of the mature hERG expression. In conclusion, hERG is uniquely susceptible to proteases, which may contribute to arrhythmias under conditions such as cardiac ischemia. Protecting hERG channels from proteolytic damage using specific peptides may represent a novel strategy to prevent and treat arrhythmias associated with ischemic heart disease.(Supported by the Canadian Institutes of Health Research & the Heart and Stroke Foundation of Ontario)
Targeting Citrullination in the Injured Retina
Authors: Wizeman John , Nicholas Anthony , Mohan Royce 
Published: June 2021

ObjectiveTo define the contribution of citrullination on GFAP and vimentin in a traumatic eye injury model.Methods: An ocular alkali injury model of retinal gliosis was employed in mice. Retinas from injured mice were examined at different times by western blot or immunochemistry for expression of GFAP, vimentin and citrullinated proteins. Posterior eye‐cups from 7‐day injured mice were placed into explant culture to investigate effects of peptidyl arginine deiminase (PAD) inhibitor Cl‐amidine.Results: GFAP expression increases initially in the soluble extracts, starting as early as 1 hour after injury and continues over the 7‐day post‐injury period, followed by increased abundance of polymeric filaments in insoluble extracts. GFAP filament growth begins at the Muller glial end‐feet and advances towards the outer retina, showing punctate co‐staining for citrullinated precursors that become incorporated into vimentin and GFAP filaments from soluble pools. At later time points, these citrullinated protein species also become abundant in insoluble extracts. In posterior eye explant culture model from 7‐day injured eyes showing reactive gliosis, global increases in high molecular weight citrullinated proteins were detected. Cl‐amidine treatment strongly downregulates these hypercitrullinated proteins and high‐molecular weight citrullinated GFAP species.ConclusionsGFAP appears to be a major target of hypercitrullination in this traumatic injury model of irreversible blindness because of its abundant overexpression. Our findings suggest that PADs are important targets for reversing pathological reactive gliosis.Supported by EY016782 and Solomon Endowed Chair Fund.
N‐chlorination, a Reversible Post‐translational Modification That Activates Chaperone Function in RidA
Authors: Müller Alexandra , Langklotz Sina , Lupilova Nataliya , Kuhlmann Katja , Bandow Julia , Leichert Lars 
Published: June 2021

RidA from Escherichia coli is a member of the highly conserved YjgF/YER057p/UK114 protein family. Members of this family have a multitude of assigned functions, which include translational inhibition (human), Hsp90‐like chaperone activity (fruit fly), and enamine/imine deaminase activity in the branched‐chain amino acid synthesis (Salmonella enterica).In the present study, we describe a novel function of RidA from E. coli. When treated with HOCl (household bleach), RidA is converted into a highly effective chaperone holdase preventing aggregation of the model substrate citrate synthase. Activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system, and glutathione.However, in contrast to Hsp33, another well‐described HOCl‐activated chaperone in E. coli, activation of RidA does not depend on cysteine modification. Instead, upon treatment with HOCl or chloramines, the amino group content of RidA decreases as a result of reversible N‐chlorination of positively charged residues. N‐chlorination increases surface hydrophobicity, which enables RidA to bind its unfolding client proteins. Deletion of ridA results in an HOCl‐sensitive phenotype of E. coli demonstrating its crucial role during HOCl stress. A proteomic analysis of RidA's client proteins revealed that N‐chlorinated RidA binds highly abundant cytosolic proteins to protect them from HOCl‐mediated precipitation. N‐chlorination of positively charged residues thus is a novel post‐translational modification that reversibly turns RidA into a chaperone holdase.
Protein Polyphosphorylation of Lysine Residues by Inorganic Polyphosphate
Authors: Azevedo Cristina , Saiardi Adolfo 
Published: June 2021

Whole genome sequencing revealed that the complexity of higher organisms is not only linked to the number of genes but also to the evolution of a wide range of additional regulatory features such as post‐translational modification of proteins (PTMs). Almost all proteins can be modulated in this way, adding a level of complexity much greater than each gene product. PTMs can be very complex in nature and occur enzymatically and non‐enzymatically. We identified and characterized a new PTM mediated by inorganic polyphosphate (polyP). This linear polymer of orthophosphate linked by ‘high‐energy’ phosphoanhydride bonds, analogous to those found in ATP, is ubiquitous in nature. We named this modification “polyphosphorylation” as it represents the covalent attachment of polyP to target proteins. We found that Nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1) are polyphosphorylated at lysine residues within a conserved N‐terminal PolyAcidic serine (S) and lysine (K) rich (PASK) cluster. Polyphosphorylation negatively regulates Nsr1/Top1 interaction and impairs Top1 enzymatic activity of relaxing super coiled DNA. We demonstrate that modulation of cellular levels of polyP regulates Top1 activity by modifying its polyphosphorylation status. We propose that polyphosphorylation represents a new regulatory mechanism of acidic proteins known to be enriched in the nuclei of both yeast and mammalian cells. Polyphosphorylation of these proteins may add an additional layer of regulation to nuclear signaling, in particular to one of the most energy‐consuming and tightly regulated process in eukaryotic cells, ribosomal RNA biogenesis, where Nsr1 and Top1 play roles.
Arkadia Mediates Sumoylation‐dependent Stabilization of Nrf2
Authors: McIntosh Deneshia , Walters Treniqka , Arinze Ifeanyi 
Published: June 2021

We have previously demonstrated that ubiquitylation of polysumoylated Nrf2 by RING finger protein 4 (RNF4), a SUMO‐targeted ubiquitin ligase (STUbL), targets Nrf2 for degradation in promyelocytic leukemia‐nuclear bodies (PML‐NBs) in HepG2 cells. Here, we show that Arkadia (also called RNF111), another SUMO‐targeted ubiquitin ligase, also ubiquitylated polysumoylated Nrf2 in these cells. The action of Arkadia stabilized Nrf2,measured in whole‐cell lysates or in PML‐NB‐enriched fractions. Using linkage (Lys 48‐ or Lys 63‐) specific ubiquitin antibodies, we show in co‐immunoprecipitation assays that Lys‐63 but not Lys‐48 is involved in Arkadia‐mediated ubiquitylation of polysumoylated Nrf2. Thus, Arkadia‐induced stabilization of Nrf2 is consistent with the notion that polyubiquitin chains linked through Lys‐63 residues do not target proteins for proteolytic destruction. Given that Arkadia has also been reported by others to ubiquitylate polysumoylated PML, thereby targeting it for proteolytic degradation, our study suggests that Arkadia functions as a dual specificity E3 ubiquitin ligase. Arkadia was able to enhance Nrf2‐mediated gene transcription from the heme‐oxygenase (HO‐1) luciferase gene construct whereas RNF4 decreased Nrf2‐mediated transcription from this reporter. RNF4 and Arkadia did not act synergistically suggesting that these two STUbLs affect Nrf2‐mediated gene transcription by two different mehanisms. Arkadia‐mediated ubiquitylation of polysumoylated Nrf2, resulting in stabilization of Nrf2, may explain the Arkadia‐induced increase in Nrf2‐dependent gene transcription.Supported by NIH grants # SC1CA143985, 5T32HL007737, 2 SD1MD000104, and ULITR000445
cAMP Signaling: An Important Event Facilitating Survival and Infectivity of Leishmania.
Authors: Biswas Arunima , Bhattacharya Arijit , Vij Amit , Das pijush 
Published: June 2021

IntroductionLeishmania donovani encounters striking shift in temperature and pH and these act as the key environmental trigger for differentiation. Moreover, the parasite also faces a huge oxidative stress inside macrophages. We showed that differentiation condition‐exposed parasites become resistant to oxidative damage due to increased intracellular cAMP‐mediated responses.ObjectiveFor comprehensive understanding of cAMP signaling, we studied all the enzymes associated with cAMP metabolism, viz. the synthesizing enzyme adenylate cyclase, the degrading enzyme, phosphodiesterase (PDE), the regulatory enzyme pyrophosphatase and the functional enzyme, cAMP‐dependent protein kinase (PKA).ResultsA stage‐specific isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation‐coupled induction of cAMP. The membrane bound acidocalcisomal pyrophosphatase, LdV‐H+PPase, was the major isoform regulating cAMP level in association with LdRACA by modulating the total pyrophosphate pool. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defence. Another cytosolic phosphodiesterase, LdPDED though not differentially expressed, was found to down‐regulate cAMP‐mediated responses by inhibiting catalytic activity of PKA and by increasing its own PDEase activity through phosphorylation. Also, a functional cAMP‐binding effector molecule (a regulatory subunit of PKA, LdPKAR) seemed to modulate metacyclogenesis through induction of autophagy.ConclusionThis study reveals the significance of cAMP signaling in parasite survival and infectivity.
M1 Protein from GAS Protects against Histone Killing
Authors: Döhrmann Simon , LaRock Chris , Anderson Ericka , Ryali Brinda , Ghosh Partho , Nizet Victor 
Published: June 2021

Histones are an essential element of chromatin structure and necessary for eukaryotic survival. Highly positively charged, histones resemble small, cationicantimicrobial peptides (AMPs) of the mammalian innate immune system. The broad‐spectrum bactericidal action of histones against a variety of bacteria was first described in 1958. More recently, a framework for histone function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps (NETs), DNA and histone‐based structures extruded by neutrophils to entrap and kill bacteria. Here, we explore histone resistance of several Gram‐positive bacterial pathogens and observe that one important human pathogen, group A Streptococcus (GAS), is highly resistant to histone killing. A screen of isogenic GAS mutants lacking various virulence factor genes revealed that the surface‐anchored M1 protein is associated with high‐level histone resistance. Studies utilizing purified protein and heterologous gene expression showed M1 to be both necessary and sufficient to confer histone resistance. The capacity of M1 to sequester histones and promote accumulatation of host fibrinogen as a further steric hindrance factor, blocked histone access to the bacterial cell wall. M1‐mediated histone resistance was correlated with enhanced GAS survival in NETs. This is the first description of a Gram‐positive bacterial resistance mechanism to histone killing, which in turn contributes to NET resistance. Histone resistance is likely a contributing factor to the well‐established virulence role of M1 in invasive GAS infection.This research was supported by NIH grant (AI096837) to PG and VN.
From the ECM to Thrombosis: a New Role for the Matrix Enzyme Lysyl Oxidase
Authors: Matsuura Shinobu , Mi Rongjuan , Eliades Alexia , Toselli Paul , Thon Jonathan , Italiano Joseph , Trackman Philip , Papadantonakis Nikolaos , Ravid Katya 
Published: June 2021

IntroductionLysyl oxidase (LOX) catalyzes the oxidation of lysines to aldehydes, providing stability in connective tissues through spontaneous cross‐linking of collagen and elastin. LOX expression is elevated in sera of patients with myeloproliferative neoplasms (MPN), a group of disorders that have in common the proliferation of megakaryocytes and myelofibrosis in bone marrow. Thrombosis is a leading cause of morbidity and mortality in MPN, yet there is still controversy over contributors to the thrombotic events in MPN.Objectiveto test whether Lysyl Oxidase (LOX) is involved in the pathogenesis of thrombosis.MethodsA transgenic mouse model expressing LOX in platelets (Pf4‐Loxtg/tg) was used to analyze platelet activation and thrombus formation.ResultsVessel occlusion after endothelial injury was significantly shorter in Pf4‐Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. While exploring mechanisms involved, we found that Pf4‐Loxtg/tg platelets adhere better to collagen. The higher affinity of Pf4‐Loxtg/tg platelets to monomeric compared to fibrillar collagen, as well as affinity to the GFOGER peptide implies integrin α2β1 as the collagen receptor targeted by LOX. Taken together, our study identified LOX as a new regulator of collagen receptor and thus platelet activation and thrombosis.