scholarly journals R-loop homeostasis and cancer mutagenesis promoted by the DNA cytosine deaminase APOBEC3B

2021 ◽  
Author(s):  
Jennifer L. McCann ◽  
Agnese Cristini ◽  
Emily K. Law ◽  
Seo Yun Lee ◽  
Michael Tellier ◽  
...  
Keyword(s):  
Cytosine Deaminase ◽  
List Type ◽  
Antiviral Protein ◽  
Bioinformatics Analyses ◽  
Single Stranded Dna ◽  
Dna And Rna ◽  
Mutation Model ◽  
Genome Wide ◽  
In Cells

AbstractThe single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here, APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in human cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts overexpressed genes and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B mediates R-loop homeostasis and contributes to R-loop mutagenesis in cancer.HighlightsUnbiased proteomics link antiviral APOBEC3B to R-loop regulationSystematic alterations of APOBEC3B levels trigger corresponding changes in R-loopsAPOBEC3B binds R-loops in living cells and in vitroBioinformatics analyses support an R-loop deamination and mutation model

scholarly journals An Improved 4’-Aminomethyltroxsalen-Based DNA Crosslinker for Biotinylation of DNA

2020 ◽  
Author(s):  
Kevin Wielenberg ◽  
Miao Wang ◽  
Min Yang ◽  
Abdullah Ozer ◽  
John T. Lis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Uv Light ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Long Wave ◽  
Genome Wide ◽  
In Cells

AbstractNucleic acid crosslinkers that covalently join commentary strands have applications as both pharmaceuticals and biochemical probes. Psoralen is a popular crosslinker moiety that reacts with double stranded DNA and RNA upon irradiation with long wave UV light. A commercially available compound EZ-Link Psoralen-PEG3-Biotin has been used in many studies to crosslink DNA and double strand RNA for genome-wide investigations. Here we present a novel probe, AP3B, which uses a psoralen derivative, 4’-aminomethyltrioxsalen, to biotinylate nucleic acids. We show that this compound is 8-fold more effective at labeling DNA in cells and several hundred-fold more effective at crosslinking two strands of DNA in vitro than the commercially available compound EZ-Link Psoralen-PEG3-Biotin.

scholarly journals Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Spencer Hill ◽  
Kurt Reichermeier ◽  
Daniel C Scott ◽  
Lorena Samentar ◽  
Jasmin Coulombe-Huntington ◽  
...  
Keyword(s):  
Chain Extension ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Extension Activity ◽  
The Stability ◽  
Genetic Buffering ◽  
Ligase Function ◽  
In Cells

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

scholarly journals Genetic Characterization of the Bile Salt Response in Lactobacillus plantarum and Analysis of Responsive Promoters In Vitro and In Situ in the Gastrointestinal Tract

2004 ◽  
Vol 186 (23) ◽  
pp. 7829-7835 ◽  
Author(s):  
Peter A. Bron ◽  
Maria Marco ◽  
Sally M. Hoffer ◽  
Esther Van Mullekom ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Lactobacillus Plantarum ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
Associated Functions ◽  
Maximal Growth Rate ◽  
Genetic Responses ◽  
In Cells

ABSTRACT In this paper we describe the growth, morphological, and genetic responses of Lactobacillus plantarum WCFS1 to bile. Growth experiments revealed that a stepwise increase in the porcine bile concentration led to a gradual decrease in the maximal growth rate. Moreover, the final density reached by an L. plantarum culture growing in MRS containing 0.1% bile was approximately threefold lower than that in MRS lacking bile. The morphology of the cells grown in MRS containing 0.1% bile was investigated by scanning electron microscopy, which revealed that cells clumped together and had rough surfaces and that some of the cells had a shrunken and empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of L. plantarum cells grown in MRS without bile. An alr complementation-based genome-wide promoter screening analysis was performed with L. plantarum, which led to identification of 31 genes whose expression was potentially induced by 0.1% porcine bile. Remarkably, 11 membrane- and cell wall-associated functions appeared to be induced by bile, as were five functions involved in redox reactions and five regulatory factors. Moreover, the lp_0237 and lp_0775 genes, identified here as genes that are inducible by bile in vitro, were previously identified in our laboratory as important for L. plantarum in vivo during passage in the mouse gastrointestinal tract (P. A. Bron, C. Grangette, A. Mercenier, W. M. de Vos, and M. Kleerebezem, J. Bacteriol. 186:5721-5729, 2004). A quantitative reverse transcription-PCR approach focusing on these two genes confirmed that the expression level of lp_0237 and lp_0775 was significantly higher in cells grown in the presence of bile and cells isolated from the mouse duodenum than in cells grown on laboratory medium without bile.

scholarly journals Modulation of RNA condensation by the DEAD-box protein eIF4A

2019 ◽  
Author(s):  
Devin Tauber ◽  
Gabriel Tauber ◽  
Anthony Khong ◽  
Briana Van Treeck ◽  
Jerry Pelletier ◽  
...  
Keyword(s):  
Stress Granules ◽  
Protein Aggregates ◽  
Stress Granule ◽  
List Type ◽  
Dependent Manner ◽  
Granule Formation ◽  
Dead Box ◽  
The Dead ◽  
In Cells

SUMMARYStress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is poorly understood. In vitro, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates. We demonstrate that the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a purpose for eIF4A to limit intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function. These results establish an important role for DEAD-box proteins as ATP-dependent RNA chaperones that can limit the intermolecular condensation and entanglement of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.eTOC BlurbStress granules are formed in part by the process of RNA condensation, which is mediated by and promotes trans RNA-RNA interactions. The essential DEAD-box protein and translation initiation factor eIF4A limits stress granule formation by reducing RNA condensation through its function as an ATP-dependent RNA binding protein, behaving analogously to how protein chaperones like HSP70 combat protein aggregates.HighlightsRNA condensates promote intermolecular RNA-RNA interactions at their surfaceseIF4A limits the recruitment of RNAs to stress granules in cellseIF4A reduces the nucleation of stress granules in cellsRecombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner

scholarly journals Poly(ADP-ribose) potentiates ZAP antiviral activity

2020 ◽  
Author(s):  
Guangai Xue ◽  
Klaudia Braczyk ◽  
Daniel Gonçalves-Carneiro ◽  
Daria M. Dawidziak ◽  
Katarzyna Zawada ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Stress Granules ◽  
Human Pathogens ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Dna And Rna ◽  
Cellular Compartments ◽  
Hiv 1

AbstractZinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of iso(ADP-ribose), a repeating structural unit of PAR. Mutation of the putative iso(ADP-ribose) binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.

DER EINFLUSS VON RESERPIN AUF DIE ANTITHYREOIDALE WIRKUNG VON KALIUMPERCHLORAT

1962 ◽  
Vol 39 (3) ◽  
pp. 423-430
Author(s):  
H. L. Krüskemper ◽  
F. J. Kessler ◽  
E. Steinkrüger
Keyword(s):  
Thyroid Gland ◽  
Male Rats ◽  
Normal Male ◽  
Tissue Respiration ◽  
List Type ◽  
Morphological Alterations ◽  
Hormone Synthesis ◽  
Stimulation Of

ABSTRACT 1. Reserpine does not inhibit the tissue respiration of liver in normal male rats (in vitro). 2. The decrease of tissue respiration of the liver with simultaneous morphological stimulation of the thyroid gland after long administration of reserpine is due to a minute inhibition of the hormone synthesis in the thyroid gland. 3. The morphological alterations of the thyroid in experimental hypothyroidism due to perchlorate can not be prevented with reserpine.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

ÉTUDE IN VITRO DU MÉTABOLISME DES HYDRATES DE CARBONE DANS LE LEUCOCYTE CIRCULANT DU COBAYE TRAITÉ À LA CORTISONE

1966 ◽  
Vol 51 (2) ◽  
pp. 193-202
Author(s):  
J. A. Antonioli ◽  
A. Vannotti
Keyword(s):  
Glucose Concentration ◽  
Intramuscular Injection ◽  
Acute Inflammation ◽  
Glycogen Content ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Glucose Consumption ◽  
List Type ◽  
Hydrates De Carbone

ABSTRACT 1. The metabolism of suspensions of circulating leucocytes has been studied after intramuscular injection of a dose of 50 mg/kg of a corticosteroid (cortisone acetate). The suspensions were incubated under aerobic conditions in the presence of a glucose concentration of 5.6 mm. Glucose consumption, lactate production, and variations in intracellular glycogen concentration were measured. After the administration of the corticosteroid, the anabolic processes of granulocyte metabolism were reversibly stimulated. Glucose consumption and lactate production increased 12 hours after the injection, but tended to normalize after 24 hours. The glycogen content of the granulocytes was enhanced, and glycogen synthesis during the course of the incubation was greatly stimulated. The action of the administered corticosteroid is more prolonged in females than in males. The injection of the corticosteroid caused metabolic modifications which resemble in their modulations and in their chronological development those found in circulating granulocytes of guinea-pigs suffering from sterile peritonitis. These results suggest, therefore, that, in the case of acute inflammation, the glucocorticosteroids may play an important role in the regulation of the metabolism of the blood leucocytes.

MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion

2020 ◽  
Vol 17 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Xuan Chen ◽  
Sumei Zhang ◽  
Peipei Shi ◽  
Yangli Su ◽  
Dong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Small Gtpase ◽  
Luciferase Reporter ◽  
Pathological Feature ◽  
In Cells

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

Biologic Aspects of Expression of Stably Integrated Transgenes in Cells of the Skin In Vitro and In Vivo

1999 ◽  
Vol 111 (3) ◽  
pp. 198-205 ◽  
Author(s):  
Gerald G. Krueger ◽  
Jeffery R. Morgan ◽  
Marta J. Petersen
Keyword(s):  
In Cells
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