scholarly journals Mapping biochemical states associated with HP1 target recognition at sites of heterochromatin formation in living cells

2021 ◽  
Author(s):  
Saikat Biswas ◽  
Joshua D. Karslake ◽  
Ziyuan Chen ◽  
Ali Farhat ◽  
Peter L. Freddolino ◽  
...  
Keyword(s):  
High Specificity ◽  
Living Cells ◽  
Biochemical Properties ◽  
Nucleic Acid Binding ◽  
Heterochromatin Formation ◽  
Individual Mobility ◽  
Biophysical Studies ◽  
Comprehensive Framework ◽  
Hp1 Proteins

ABSTRACTHP1 proteins bind with low affinity but high specificity to sites of histone H3 lysine 9 methylation (H3K9me) in the genome. HP1 binding to H3K9me compartmentalizes the genome into transcriptionally inactive heterochromatin and actively transcribed euchromatin. A characteristic feature of HP1 proteins is their dynamic and rapid turnover from sites of heterochromatin formation. How low-affinity H3K9me recognition enables HP1 proteins to rapidly and efficiently traverse a complex and crowded chromatin landscape on the millisecond timescale remains a paradox. Here, we visualize the real-time motions of an HP1 homolog, the fission yeast protein Swi6, in its native chromatin environment. By analyzing the motions of Swi6 with high spatial and temporal resolution, we map individual mobility states that are directly linked to discrete biochemical intermediates. We find that nucleic acid binding titrates Swi6 away from sites of heterochromatin formation, whereas increasing the valency of chromodomain-mediated H3K9me recognition promotes specific chromatin localization. We propose that Swi6 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for protein turnover. Our high-resolution biophysical studies provide a comprehensive framework for in vivo biochemistry and reveal how the competing biochemical properties of Swi6 affect H3K9me recognition in living cells.

scholarly journals HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization

2021 ◽  
Author(s):  
Saikat Biswas ◽  
Joshua Karslake ◽  
Ziyuan Chen ◽  
Ali Farhat ◽  
Peter Freddolino ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Biochemical Property ◽  
High Specificity ◽  
Nucleic Acid Binding ◽  
New Paradigm ◽  
Chromatin Interactions ◽  
Specific Localization ◽  
Order Complexes ◽  
Hp1 Proteins

Abstract HP1 proteins bind with low affinity but high specificity to histone H3 lysine 9 methylation (H3K9me), forming transcriptionally inactive genomic compartments referred to as heterochromatin. How HP1 proteins traverse a complex and crowded chromatin landscape on the millisecond timescale to bind H3K9me chromatin remains paradoxical. Here, we apply single-molecule imaging to visualize an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By analyzing Swi6 motions, we identify individual mobility states that map to discrete biochemical intermediates. Using mutants that perturb Swi6 H3K9me recognition, oligomerization, or nucleic acid binding, we mechanistically parse how each biochemical property affects protein dynamics. While nucleic acid binding titrates Swi6 away from heterochromatin, as few as four tandem chromodomains are sufficient to restore H3K9me-dependent localization. Our studies propose a new paradigm where HP1 oligomerization stabilizes higher-order complexes to outcompete inhibitory nucleic acid and non-specific chromatin interactions, enabling high specificity H3K9me recognition in cells.

scholarly journals High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme

2020 ◽  
Vol 48 (10) ◽  
pp. 5254-5267 ◽  
Author(s):  
Prince Kumar Lat ◽  
Kun Liu ◽  
Dev N Kumar ◽  
Kenneth K L Wong ◽  
Esther M Verheyen ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
High Specificity ◽  
Living Cells ◽  
Chain Reaction ◽  
Dna And Rna ◽  
Reaction Amplification ◽  
Polymerase Chain

Abstract Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ–heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn’t extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.

scholarly journals NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

2015 ◽  
Vol 81 (18) ◽  
pp. 6294-6301 ◽  
Author(s):  
Lingfeng Zhu ◽  
Xiaoling Xu ◽  
Limin Wang ◽  
Hui Dong ◽  
Bo Yu ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Catalytic Mechanism ◽  
High Specificity ◽  
Biochemical Properties ◽  
Enzymatic System ◽  
Content Type ◽  
Wide Range ◽  
Lactate Dehydrogenases

ABSTRACTHydroxy acid dehydrogenases, includingl- andd-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, ad-lactate dehydrogenase from aSporolactobacillus inulinusstrain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization andin vivoandin vitroenzymatic activity analyses. The residue Asn174was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

scholarly journals The molecular principles of Piwi-mediated co-transcriptional silencing through the dimeric SFiNX complex

2021 ◽  
Author(s):  
Jakob Schnabl ◽  
Juncheng Wang ◽  
Ulrich Hohmann ◽  
Maja Gehre ◽  
Julia Batki ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transcriptional Silencing ◽  
General Mechanism ◽  
Nucleic Acid Binding ◽  
Dynein Light Chain ◽  
Dimerization Domain ◽  
Heterochromatin Formation ◽  
Condensate Formation

Nuclear Argonaute proteins, guided to nascent target RNAs by their bound small RNAs, elicit co-transcriptional silencing through heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms involved in this process are incompletely understood. Here, we propose that the SFiNX complex, a silencing mediator downstream of nuclear Piwi-piRNA complexes in Drosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX dimerization, mediated by the dynein light chain protein, LC8/Cutup. LC8's function within SFiNX can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive feature of SFiNX. Mutations preventing LC8- mediated SFiNX dimerization result in loss of condensate formation in vitro and inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. Formation of molecular condensates might be a general mechanism that underlies effective heterochromatin establishment at small RNA target loci in a co-transcriptional manner.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 235-245
Author(s):  
Daimark Bennett ◽  
Balázs Szöőr ◽  
Sascha Gross ◽  
Natalia Vereshchagina ◽  
Luke Alphey
Keyword(s):  
Protein Phosphatase ◽  
Muscle Development ◽  
Ectopic Expression ◽  
High Specificity ◽  
Type I ◽  
Protein Phosphatase Type 1 ◽  
Mitotic Figures ◽  
Protein Phosphatase Type

Abstract We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFβ superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

A near-infrared fluorescence off–on probe for sensitive imaging of hydrogen polysulfides in living cells and mice in vivo

2017 ◽  
Vol 53 (62) ◽  
pp. 8759-8762 ◽  
Author(s):  
Yu Fang ◽  
Wei Chen ◽  
Wen Shi ◽  
Hongyu Li ◽  
Ming Xian ◽  
...  
Keyword(s):  
Near Infrared ◽  
Living Cells ◽  
Near Infrared Fluorescence

A new near-infrared fluorescence off–on probe with phenyl 2-(benzoylthio)benzoate as the recognition moiety is developed and applied in imaging H2Sn in living cells and mice in vivo.

scholarly journals Force generation by protein–DNA co-condensation

2021 ◽  
Author(s):  
Thomas Quail ◽  
Stefan Golfier ◽  
Maria Elsner ◽  
Keisuke Ishihara ◽  
Vasanthanarayan Murugesan ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Self Assembly ◽  
Spider Silk ◽  
Regulatory Elements ◽  
Heterochromatin Formation ◽  
First Order Phase Transition ◽  
Dna Strand ◽  
First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

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