scholarly journals AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

2006 ◽  
Vol 27 (1) ◽  
pp. 20-30 ◽  
Author(s):  
Mani Larijani ◽  
Alexander P. Petrov ◽  
Oxana Kolenchenko ◽  
Maribel Berru ◽  
Sergey N. Krylov ◽  
...  
Keyword(s):  
Half Life ◽  
Dissociation Constants ◽  
Hot Spot ◽  
Scaffolding Protein ◽  
Sequence Specificity ◽  
Independent Manner ◽  
Single Stranded Dna ◽  
Stem Loop ◽  
High Affinity

ABSTRACT Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements

1989 ◽  
Vol 9 (11) ◽  
pp. 5055-5061
Author(s):  
D J Haile ◽  
M W Hentze ◽  
T A Rouault ◽  
J B Harford ◽  
R D Klausner
Keyword(s):  
Binding Protein ◽  
Reduced Form ◽  
Scatchard Analysis ◽  
Stem Loop ◽  
Lower Affinity ◽  
Translational Repressor ◽  
High Affinity ◽  
Iron Responsive Element ◽  
Responsive Element

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

scholarly journals Single-Stranded DNA Structure and Positional Context of the Target Cytidine Determine the Enzymatic Efficiency of AID

2007 ◽  
Vol 27 (23) ◽  
pp. 8038-8048 ◽  
Author(s):  
Mani Larijani ◽  
Alberto Martin
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Single Stranded Dna ◽  
Stem Loop ◽  
Double Stranded Dna ◽  
Antibody Diversification ◽  
Multiple Structures ◽  
Optimal Target

ABSTRACT Activation-induced cytidine deaminase (AID) initiates antibody diversification processes by deaminating immunoglobulin sequences. Since transcription of target genes is required for deamination in vivo and AID exclusively mutates single-stranded DNA (ssDNA) in vitro, AID has been postulated to mutate transcription bubbles. However, since ssDNA generated by transcription can assume multiple structures, it is unknown which of these are targeted in vivo. Here we examine the enzymatic and binding properties of AID for different DNA structures. We report that AID has minimal activity on stem-loop structures and preferentially deaminates five-nucleotide bubbles. We compared AID activity on cytidines placed at various distances from the single-stranded/double-stranded DNA junction of bubble substrates and found that the optimal target consists of a single-stranded NWRCN motif. We also show that high-affinity binding is required for but does not necessarily lead to efficient deamination. Using nucleotide analogues, we show that AID's WRC preference (W = A or T; R = A or G) involves the recognition of a purine in the R position and that the carbonyl or amino side chains of guanosine negatively influence specificity at the W position. Our results indicate that AID is likely to target short-tract regions of ssDNA produced by transcription elongation and that it requires a fully single-stranded WRC motif.

scholarly journals Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

1989 ◽  
Vol 9 (11) ◽  
pp. 5055-5061 ◽  
Author(s):  
D J Haile ◽  
M W Hentze ◽  
T A Rouault ◽  
J B Harford ◽  
R D Klausner
Keyword(s):  
Binding Protein ◽  
Reduced Form ◽  
Scatchard Analysis ◽  
Stem Loop ◽  
Lower Affinity ◽  
Translational Repressor ◽  
High Affinity ◽  
Iron Responsive Element ◽  
Responsive Element

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

scholarly journals Identifying triplex binding rules in vitro leads to creation of a new synthetic regulatory tool in vivo

2019 ◽  
Author(s):  
Beate Kaufmann ◽  
Or Willinger ◽  
Noa Eden ◽  
Lisa Kermas ◽  
Leon Anavy ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Deep Sequencing ◽  
Mammalian Cells ◽  
Double Helix ◽  
Independent Manner ◽  
Sequencing Platform ◽  
High Affinity ◽  
Nucleic Acid Molecule

SummaryNature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes1. Recently, non-canonical structures gained renewed scientific and biotechnological interest2,3. One particularly intriguing form of such structures are triplexes4 in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix5. Despite extensive research in vitro6–9, the underlying rules for triplex formation remain debated and evidence for triplexes in vivo is circumstantial10–12. Here, we demonstrate the development of a deep-sequencing platform termed Triplex-Seq to systematically refine the DNA triplex code and identify high affinity triplex forming oligo (TFO) variants. We identified a preference for short G-rich motifs using an oligo-library with a mix of all four bases. These high-information content motifs formed specific high-affinity triplexes in a pH-independent manner and stability was increased with G-rich double-stranded molecules. We then conjugated one high-affinity and one low-affinity variant to a VP48 peptide and studied these synthetic biomolecules in mammalian cells. Using these peptide-oligo constructs (POCs), we demonstrated possible triplex-induced down-regulation activity in 544 differentially expressed genes. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules, which in turn has led to the development of a functional non-genetically encoded regulatory tool for in vivo applications.

scholarly journals Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

2021 ◽  
Vol 118 (38) ◽  
pp. e2108281118
Author(s):  
Aditya S. Vaidya ◽  
Francis C. Peterson ◽  
James Eckhardt ◽  
Zenan Xing ◽  
Sang-Youl Park ◽  
...  
Keyword(s):  
Seed Germination ◽  
Receptor Antagonist ◽  
Stress Responses ◽  
Dissociation Constants ◽  
Aba Signaling ◽  
Biotic And Abiotic Stress ◽  
High Affinity ◽  
Aba Receptor

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

scholarly journals Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

2020 ◽  
Author(s):  
Sandrine Coquille ◽  
Stéphane Thore
Keyword(s):  
Complex Formation ◽  
Rna Binding ◽  
Leigh Syndrome ◽  
Stable Complex ◽  
Specific Gene ◽  
Functional Domain ◽  
Independent Manner ◽  
Stem Loop

ABSTRACTMitochondria are essential organelles carrying their own genetic information which require specific gene expression processes. The leucine rich pentatricopeptide protein (LRPPRC) and its partner the SRA stem-loop interacting RNA binding protein (SLIRP) form a stable complex implicated in mRNA stability and polyadenylation. LRPPRC/SLIRP complex formation is still poorly characterized. We demonstrate that SLIRP interacts with the N-terminal region of LRPPRC in a RNA independent manner. We further show that the complex is stable in presence of high salt concentration. Point mutation and deletions found in the LRPPRC protein and responsible for the French-Canadian Leigh Syndrome (LSFC) are shown to affect complex formation in vitro. Our data are identifying the key region of LRPPRC involved in SLIRP association and showing the direct consequence of various LSFC mutations on the complex formation. Further experiments aiming at deciphering LRPPRC/SLIRP function(s) in vivo will benefit from our functional domain characterization.

scholarly journals Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two ConservedssoA Regions

1998 ◽  
Vol 180 (1) ◽  
pp. 83-89 ◽  
Author(s):  
M. Gabriela Kramer ◽  
Saleem A. Khan ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sequence Specificity ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Cell Extracts ◽  
Rolling Circle Mechanism ◽  
Derivative Plasmid ◽  
Lagging Strand

ABSTRACT The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RSB] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication inStreptococcus pneumoniae. Cells containing plasmids with mutations in the RSB accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RSB region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.

High Affinity Binding of FVIII to VWF Is Not Required for the Haemostatic Effect of FVIII In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1182-1182
Author(s):  
Heidi L Holmberg ◽  
Marianne Kjalke ◽  
Ditte Karpf ◽  
Ida Hilden ◽  
Hermann Pelzer ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Clot Formation ◽  
Pharmacokinetic Studies ◽  
Affinity Binding ◽  
Maximal Effect ◽  
High Affinity Binding ◽  
High Affinity

Abstract Abstract 1182 VWF protects FVIII from clearance in the circulation and is believed to ensure location of platelets to the site of injury. However, it is unknown if binding of FVIII to VWF has a role in localizing and thereby also facilitating the effect of FVIII in vivo. In the present study, a FVIII variant, FVIII-Y1680F, lacking the high affinity binding to VWF (Leyet et al. JCB 1991; 15; 740) was used to evaluate the binding of FVIII to VWF in clot formation hemophilia A mice in vivo. Binding of the FVIII variant to immobilized VWF was evaluated by surface plasmon resonance showing a 50–25 fold reduction in the Kd for FVIII-Y1680F compared to wt FVIII. Furthermore, pharmacokinetic studies in hemophilia A mice indicated that FVIII-Y1680F is basically devoid of VWF binding in vivo. The circulating half-life decreased from 7–8 hours for wt FVIII to 0.5 hours for FVIII-Y1680F (see figure) which is comparable to the half-life of wt FVIII in VWF knockout mice (0.5 hours). Using a chromogenic assay the specific activity of FVIII-Y1680F was 9200 IU/mg similar to that of wt FVIII confirming normal activity FVIII-Y1680F after cleavage with thrombin and removal of VWF. As the short half-life may influence the haemostatic effect of FVIII-Y1680F in vivo, a 40 kDa PEG moiety was attached to the O-glycan in the B-domain of the FVIII variant. This re-establishes the circulating half-life (6.1 hours) to that of wt FVIII without affecting the specific activity in vitro. The haemostatic effect of 40K-O-PEG FVIII-Y1680F was subsequently used to investigate if high affinity VWF binding of FVIII influences its haemostatic effect in vivo. The acute haemostatic effect of 40K-O-PEG-FVIII-Y1680F was compared to wt FVIII (Advate®) in the tail bleeding model in hemophilia A mice at doses equivalent to the 50% of the maximal effect and at maximal efficacy (20 and 280 IU/kg; Elm et al., Hemophilia 2011 epub). The blood loss was significantly reduced at both doses with comparable effect of 40K-O-PEG-FVIII-Y1680F and wt FVIII (see see figure, * indicates significant differences compared to vehicle treated hemophilia A mice). This indicates that the lack of VWF binding does not interfere with the haemostatic properties of FVIII in this particular model. To further support these data, the haemostatic effect of 40K-O-PEG-FVIII-Y1680F was tested in the FeCl3 injury model (2.5, 5 and 10 IU/kg) in hemophilia A mice. No clot formation was observed in vehicle mice and 40K-O-PEG-FVIII-Y1680F normalized dose dependently the clot formation time comparable to wt FVIII. In conclusion, the current data suggest that the haemostatic effect of FVIII in vivo is not dependent on high affinity binding of FVIII to VWF. Disclosures: Holmberg: Novo Nordisk A/S: Employment. Kjalke:Novo Nordisk A/S: Employment. Karpf:Novo NOrdisk A/S: Employment. Hilden:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Koefoed-Hansen:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Thim:Novo Nordisk A/S: Employment. Karlsson:Novo Nordisk A/S: Employment. Jespersgaard:Novo Nordisk A/S: Employment. Bolt:Novo Nordisk: Employment. Stennicke:Novo Nordisk A/S: Employment.

CHANGES IN BRAIN, PITUITARY AND UTERINE CYTOPLASMIC OESTROGEN RECEPTORS INDUCED BY OESTRADIOL-17β IN THE OVARIECTOMIZED RAT

1976 ◽  
Vol 71 (3) ◽  
pp. 343-349 ◽  
Author(s):  
I. D. MORRIS
Keyword(s):  
Dissociation Constants ◽  
Steroid Receptor ◽  
Posterior Hypothalamus ◽  
Ovariectomized Rat ◽  
Oestrogen Receptors ◽  
Differential Effects ◽  
High Affinity ◽  
Reproductive Processes

SUMMARY The oestrogen specific, high-affinity cytosol receptor (HAR) from amygdala, anterior, middle and posterior hypothalamus, pituitary and uterus was studied in the ovariectomized rat. A single in-vivo injection of oestradiol-17β produced significant changes in both the tissue HAR concentrations and the apparent dissociation constants (Kd) determined in vitro. Four hours after oestradiol-17β treatment (20 μg/kg), the HAR concentration was depleted in all tissues except the posterior hypothalamus. A lower dose of oestradiol-17β (4 μg/kg) produced similar changes in HAR concentration with the exception of those in the amygdala and posterior hypothalamus. Twenty-four hours after oestradiol-17β, HAR concentrations had returned to pre-injection levels in all tissues except the uterus. The uterine HAR concentrations were raised after both doses of oestradiol-17β. The apparent tissue cytosol Kd values were decreased by both doses of oestradiol-17β. The results suggest that brain, pituitary and uterine oestrogen cytosol HARs react to plasma oestrogen in a manner predictable by the steroid receptor hypothesis. The oestradiol-17β-induced differential effects upon the tissue cytosol concentration may contribute to the overall spectrum of action of oestrogen in the central and peripheral reproductive processes.

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