scholarly journals Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Yongming Dong ◽  
Yueyang Gou ◽  
Yi Li ◽  
Yan Liu ◽  
Jihong Bai
Keyword(s):  
Affinity Binding ◽  
Phosphatase Domain ◽  
High Affinity Binding ◽  
Bar Domain ◽  
High Affinity ◽  
Rich Domain ◽  
Phosphoinositide Phosphatase ◽  
Core Function ◽  
Membrane Bending

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin–Amphiphysin–Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.

RAT MYOMETRIAL ACTIVITY IN VIVO: EFFECTS OF OESTRADIOL-17β AND PROGESTERONE IN RELATION TO THE CONCENTRATIONS OF CYTOPLASMIC PROGESTERONE RECEPTORS

1978 ◽  
Vol 78 (1) ◽  
pp. 103-117 ◽  
Author(s):  
SANDRA J. DOWNING ◽  
S. J. LYE ◽  
JANE M. C. BRADSHAW ◽  
D. G. PORTER
Keyword(s):  
Binding Protein ◽  
Progesterone Receptors ◽  
Affinity Binding ◽  
Pregnant Rats ◽  
Pregnant Rat ◽  
High Affinity Binding ◽  
High Affinity ◽  
Repeated Doses ◽  
In Vivo Effects

The amplitude, frequency and rate of rise of intra-uterine pressure cycles in rats (postpartum, ovariectomized) were unaffected by treatment with progesterone. Amplitude was also unaffected by a combination of treatments with progesterone and oestradiol-17β, which was adequate to ensure the survival of 84% of foetuses in ovariectomized pregnant rats. The failure of progesterone to influence myometrial activity could not be attributed to a lack of 'true' progesterone receptors since these were present in the myometria of the test animals in concentrations exceeding those of oestrous animals. Evidence was obtained which suggested that a high-affinity binding protein, different from the 'true' receptor may predominate in the myometrium of the pregnant rat. Oestradiol-17β in single or repeated doses of from 0·25 to 5 μg, however, was found to reduce the frequency of pressure cycles but to increase significantly their rate of rise of pressure. There was a latency of 6–8 h in these effects of oestradiol. The possibility that inhibition of the myometrium by oestrogen may play a part in the preparation for parturition is discussed.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

2006 ◽  
Vol 188 (6) ◽  
pp. 2081-2095 ◽  
Author(s):  
Leah S. Feldman-Cohen ◽  
Yongping Shao ◽  
Derrick Meinhold ◽  
Charmi Miller ◽  
Wilfredo Colón ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Motif ◽  
Affinity Binding ◽  
Flanking Sequence ◽  
Binding Assays ◽  
High Affinity Binding ◽  
High Affinity

ABSTRACT Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd , ∼1 to 4 nM) and nonspecific (Kd , ∼4 μM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and λ attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the λ attR Fis site, and the role of R89 was dramatically altered by the λ attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

scholarly journals Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

2000 ◽  
Vol 182 (14) ◽  
pp. 4022-4027 ◽  
Author(s):  
Richard A. Fekete ◽  
Laura S. Frost
Keyword(s):  
Binding Sites ◽  
Integration Host Factor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Negative Dominance ◽  
Transfer Apparatus

ABSTRACT Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage atnic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage atnic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriTallowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

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.

scholarly journals Ligand size is a major determinant of high-affinity binding of fucose- and galactose-exposing (lipo)proteins by the hepatic fucose receptor

1994 ◽  
Vol 299 (1) ◽  
pp. 291-296 ◽  
Author(s):  
E A Biessen ◽  
H F Bakkeren ◽  
D M Beuting ◽  
J Kuiper ◽  
T J Van Berkel
Keyword(s):  
Kupffer Cell ◽  
Kupffer Cells ◽  
Asialoglycoprotein Receptor ◽  
Major Determinant ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Parenchymal Liver

Previous in vivo studies have demonstrated that small galactose-exposing particles are preferentially internalized by the asialoglycoprotein receptor on the parenchymal liver cell and large particles by the galactose-particle receptor on the Kupffer cell. In this study, we have investigated using in vitro binding studies whether the affinity for either receptor is affected by the ligand size. The asialoglycoprotein receptor appeared to bind and process lactosylated proteins irrespective of their size. In contrast, recognition of galactose-exposing proteins by the galactose-particle receptor on the Kupffer cell was strongly dependent on size. The affinity increased 3000-fold with protein sizes increasing from 5 to 15 nm, reaching its maximum at approx. 1 nM for ligands larger than 15 nm. Apparently, the preferential in vivo uptake of large galactose-exposing ligands by Kupffer cells does not result from an inability of the parenchymal liver cells to internalize these ligands, but from the high affinity of large ligands for the galactose-particle receptor and the strategic anatomical localization of the Kupffer cells in the liver. In the preceding paper [Kuiper, Bakkeren, Biessen and Van Berkel (1994) Biochem. J. 299, 285-290] the galactose-particle receptor on the Kupffer cell was suggested to be identical with the fucose receptor. 125I-Lac-LDL-binding studies clearly showed that the galactose-particle receptor exhibited high-affinity binding of fucose-exposing proteins also. The affinity of fucosylated proteins for the galactose-particle receptor was greatly affected by ligand size. The above data strongly support the hypothesis that the galactose-particle receptor is identical with the fucose receptor. The size of neoglycoproteins can be appreciated as a new major determinant of affinity for the fucose receptor.

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