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Nuclear magnetic resonance (NMR) spectroscopy shows interaction between twenty-amino acid cytoplasmic domain (TFCD) and Pin1 requires phosphorylation of Ser258 and trans-configuration of the pSer258-Pro259 peptide bond in the TFCD. (A) Superimposition of the assigned 1 H/ 15 N HSOC spectra of the Pin1 WW-domain with double phosphorylated TFCD (pSer253/pSer258) showing the chemical shift changes upon increasing amount of peptide to a 10x molar excess. A non-linear regression fit of the Ser18 (peak shift shown in black dotted box) was used to calculate the binding constant of the complex. (B) Bundle of 20 NMR conformers sampling into two major conformers. Sidechains of Pro259 (TFCD), Arg21 and Trp34 (Pin1 WW-domain) are indicated with circles. (C) Representative models of the two lowest-energy conformations from (B) are shown in green and magenta. The polypeptide backbones are shown as ribbons. Pin1 WW-domain residues are underlined. (D) Contact of the TFCD pSer258-Pro259 motif with the Pin1 WW-domain loop1 and comparison of NMR and X-ray structures.
Source publication
Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regula...
Contexts in source publication
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... our detailed structural analyses, we con- clude that the TFCD-Pin1 WW-domain complex shows a larger contact area than what was observed in previous WW-domain/phosphopeptide NMR structures, but simi- lar to the RNAP II-CTD:Pin1 crystal structure. 32 Residues in the anchoring zone of the Pin1 WW-domain-TFCD complex showed apparent chemical shift perturbation (e.g. Tyr23) ( Figure 3A), while these residues were stag- nant in previous Pin1 NMR titrations. 32 This discrepancy could potentially be the result of titration of the short pThr-Pro fragment of Cdc25 or tau in these experiments, rather than the full protein domain that was used here. Furthermore, the β1-β2 loop1 conformer shows structural similarity to the isolated ligand-free Pin1 WW-domain ...
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... next prepared 13 C/ 15 N-isotopically-labeled Pin1 WW-domain in complex with double phosphorylated pSer253/pSer258 TFCD peptide for sequential backbone assignment and NMR structure determination. Analysis of the Cα and Cβ values based upon the weighted chemical shift index highlighted the three β-strands, which are char- acteristic of WW-domains (Online Supplementary Figure S2A). A total of 203 unique intra-and intermolecular dis- tance constraints were derived from the analysis of the NOE spectroscopy (NOESY) experiments. We used these constraints together with the weighted chemical shift index (Online Supplementary Table S1 and Online Supplementary Figure S2A) to calculate the solution struc- ture of the Pin1 WW-domain and TFCD complex (see Methods for more details). The 20 lowest energy con- formers of the Pin1 WW-domain yielded a root-mean- square deviation (RMSD) of 1 Å (residues 6-39) ( Figure 3B and Online Supplementary Figure S2B). The Pin1 WW- domain is a canonical WW-domain 18 consisting of three twisted anti-parallel β-sheet strands with a conserved Trp- Trp motif located at the N-terminus of the first β-strand and C-terminus of the third β-strand, respective- ...
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... characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15 N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1 H/ 15 N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) ( Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) ( Figure 3A; residues in bold). During our NMR titra- tions, the 1 H/ 15 N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance ( Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale ( Figure 3B). A K d of 133 ± 13 µM was determined from the δg (ppm) for HN changes in Pin1 WW-domain residue Ser18 ( Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted K d value of ~137 mM (Online Supplementary Figure ...
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... characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15 N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1 H/ 15 N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) ( Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) ( Figure 3A; residues in bold). During our NMR titra- tions, the 1 H/ 15 N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance ( Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale ( Figure 3B). A K d of 133 ± 13 µM was determined from the δg (ppm) for HN changes in Pin1 WW-domain residue Ser18 ( Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted K d value of ~137 mM (Online Supplementary Figure ...
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... characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15 N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1 H/ 15 N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) ( Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) ( Figure 3A; residues in bold). During our NMR titra- tions, the 1 H/ 15 N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance ( Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale ( Figure 3B). A K d of 133 ± 13 µM was determined from the δg (ppm) for HN changes in Pin1 WW-domain residue Ser18 ( Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted K d value of ~137 mM (Online Supplementary Figure ...
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... characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15 N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1 H/ 15 N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) ( Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) ( Figure 3A; residues in bold). During our NMR titra- tions, the 1 H/ 15 N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance ( Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale ( Figure 3B). A K d of 133 ± 13 µM was determined from the δg (ppm) for HN changes in Pin1 WW-domain residue Ser18 ( Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted K d value of ~137 mM (Online Supplementary Figure ...
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... characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15 N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1 H/ 15 N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) ( Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) ( Figure 3A; residues in bold). During our NMR titra- tions, the 1 H/ 15 N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance ( Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale ( Figure 3B). A K d of 133 ± 13 µM was determined from the δg (ppm) for HN changes in Pin1 WW-domain residue Ser18 ( Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted K d value of ~137 mM (Online Supplementary Figure ...
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... | 2018; 103(6) ray structures, as well as to the Cdc25 pThr peptide com- plex NMR structure, suggesting that loop1 undergoes a thermodynamic switch between ligand-bound and ligand- free states ( Figure ...
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... of the Pin1 WW-domain-TFCD complex reveals that TFCD residues Asn257, pSer258 and Pro259 are involved in the binding interface of the Pin1 WW- domain, consistent with our pull-down assays. While the Pin1 WW-domain as a whole shows a well-defined, single conformation, the β1-β2 loop1 binding region of the WW- domain (residues 17 to 21) and the pSer258-Pro259 motif of TFCD show two distinct ensembles of conformers ( Figure 3B and C), consistent with the fact that less NOEs were detected for the β1-β2 loop1. The features that prin- cipally drive the complex formation are the charge-charge interaction and the hydrophobic interaction between Trp34 (the second invariant Trp of the WW motif) with invariant Pro259. The ionic interactions between the phosphate of pSer258 with the positively charged guani- dinium groups of Arg14, Arg17, and Arg21 also appeared to stabilize the complex. However, as mentioned earlier, the resonances for Arg17, located at position 1 of loop1, disappeared during the NMR titration due to intermediate exchange in the NMR timescale ( Figure 3A), suggesting high flexibility around loop1. Arg21 connects loop1 to the β2-strand into two conformations and involves multiple polar contacts with the double phosphorylated TFCD at pSer258 ( Figure 3B and C). Similarly, Trp34-driven hydrophobic packing of Pro259 induces two ensembles of Trp34 side chain rotamers and Pro259 configurations, and both ensemble states still adopt a trans-configuration of the pSer258-Pro259 peptide bond (Online Supplementary Table S1). For comparison, the previously determined interactions of Pin1 WW-domain with Cell division cycle 25 (Cdc25) and the C-terminal domain of the RNA poly- merase II largest subunit (RNAP II-CTD) are also shown ( Figure 3D). 29,30 In order to further confirm the reliability of our struc- tures, step-wise energy minimization was performed on all Pin1 WW-domain-TFCD complexes. The RMSD of heavy atoms/all atoms before and after energy minimiza- tion was less than 0.45 Å, while the RMSD of the back- bone was less than 0.3 Å (Online Supplementary Figure S2B). Therefore, the interactions between the Pin1 WW- domain and the TFCD, such as the electrostatic interac- tions with pSer258 and the hydrophobic interactions of Pro259 with the WW-domain are consistent with the NMR structures calculated. The trans conformation of the pSer258-Pro259 peptide bond in the TFCD was stable dur-ing 10,000 steps of energy minimization, further demon- strating that our structures satisfy energy-of-motion (EOM) criteria with reasonable convergence ...
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... of the Pin1 WW-domain-TFCD complex reveals that TFCD residues Asn257, pSer258 and Pro259 are involved in the binding interface of the Pin1 WW- domain, consistent with our pull-down assays. While the Pin1 WW-domain as a whole shows a well-defined, single conformation, the β1-β2 loop1 binding region of the WW- domain (residues 17 to 21) and the pSer258-Pro259 motif of TFCD show two distinct ensembles of conformers ( Figure 3B and C), consistent with the fact that less NOEs were detected for the β1-β2 loop1. The features that prin- cipally drive the complex formation are the charge-charge interaction and the hydrophobic interaction between Trp34 (the second invariant Trp of the WW motif) with invariant Pro259. The ionic interactions between the phosphate of pSer258 with the positively charged guani- dinium groups of Arg14, Arg17, and Arg21 also appeared to stabilize the complex. However, as mentioned earlier, the resonances for Arg17, located at position 1 of loop1, disappeared during the NMR titration due to intermediate exchange in the NMR timescale ( Figure 3A), suggesting high flexibility around loop1. Arg21 connects loop1 to the β2-strand into two conformations and involves multiple polar contacts with the double phosphorylated TFCD at pSer258 ( Figure 3B and C). Similarly, Trp34-driven hydrophobic packing of Pro259 induces two ensembles of Trp34 side chain rotamers and Pro259 configurations, and both ensemble states still adopt a trans-configuration of the pSer258-Pro259 peptide bond (Online Supplementary Table S1). For comparison, the previously determined interactions of Pin1 WW-domain with Cell division cycle 25 (Cdc25) and the C-terminal domain of the RNA poly- merase II largest subunit (RNAP II-CTD) are also shown ( Figure 3D). 29,30 In order to further confirm the reliability of our struc- tures, step-wise energy minimization was performed on all Pin1 WW-domain-TFCD complexes. The RMSD of heavy atoms/all atoms before and after energy minimiza- tion was less than 0.45 Å, while the RMSD of the back- bone was less than 0.3 Å (Online Supplementary Figure S2B). Therefore, the interactions between the Pin1 WW- domain and the TFCD, such as the electrostatic interac- tions with pSer258 and the hydrophobic interactions of Pro259 with the WW-domain are consistent with the NMR structures calculated. The trans conformation of the pSer258-Pro259 peptide bond in the TFCD was stable dur-ing 10,000 steps of energy minimization, further demon- strating that our structures satisfy energy-of-motion (EOM) criteria with reasonable convergence ...
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... of the Pin1 WW-domain-TFCD complex reveals that TFCD residues Asn257, pSer258 and Pro259 are involved in the binding interface of the Pin1 WW- domain, consistent with our pull-down assays. While the Pin1 WW-domain as a whole shows a well-defined, single conformation, the β1-β2 loop1 binding region of the WW- domain (residues 17 to 21) and the pSer258-Pro259 motif of TFCD show two distinct ensembles of conformers ( Figure 3B and C), consistent with the fact that less NOEs were detected for the β1-β2 loop1. The features that prin- cipally drive the complex formation are the charge-charge interaction and the hydrophobic interaction between Trp34 (the second invariant Trp of the WW motif) with invariant Pro259. The ionic interactions between the phosphate of pSer258 with the positively charged guani- dinium groups of Arg14, Arg17, and Arg21 also appeared to stabilize the complex. However, as mentioned earlier, the resonances for Arg17, located at position 1 of loop1, disappeared during the NMR titration due to intermediate exchange in the NMR timescale ( Figure 3A), suggesting high flexibility around loop1. Arg21 connects loop1 to the β2-strand into two conformations and involves multiple polar contacts with the double phosphorylated TFCD at pSer258 ( Figure 3B and C). Similarly, Trp34-driven hydrophobic packing of Pro259 induces two ensembles of Trp34 side chain rotamers and Pro259 configurations, and both ensemble states still adopt a trans-configuration of the pSer258-Pro259 peptide bond (Online Supplementary Table S1). For comparison, the previously determined interactions of Pin1 WW-domain with Cell division cycle 25 (Cdc25) and the C-terminal domain of the RNA poly- merase II largest subunit (RNAP II-CTD) are also shown ( Figure 3D). 29,30 In order to further confirm the reliability of our struc- tures, step-wise energy minimization was performed on all Pin1 WW-domain-TFCD complexes. The RMSD of heavy atoms/all atoms before and after energy minimiza- tion was less than 0.45 Å, while the RMSD of the back- bone was less than 0.3 Å (Online Supplementary Figure S2B). Therefore, the interactions between the Pin1 WW- domain and the TFCD, such as the electrostatic interac- tions with pSer258 and the hydrophobic interactions of Pro259 with the WW-domain are consistent with the NMR structures calculated. The trans conformation of the pSer258-Pro259 peptide bond in the TFCD was stable dur-ing 10,000 steps of energy minimization, further demon- strating that our structures satisfy energy-of-motion (EOM) criteria with reasonable convergence ...
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... of the Pin1 WW-domain-TFCD complex reveals that TFCD residues Asn257, pSer258 and Pro259 are involved in the binding interface of the Pin1 WW- domain, consistent with our pull-down assays. While the Pin1 WW-domain as a whole shows a well-defined, single conformation, the β1-β2 loop1 binding region of the WW- domain (residues 17 to 21) and the pSer258-Pro259 motif of TFCD show two distinct ensembles of conformers ( Figure 3B and C), consistent with the fact that less NOEs were detected for the β1-β2 loop1. The features that prin- cipally drive the complex formation are the charge-charge interaction and the hydrophobic interaction between Trp34 (the second invariant Trp of the WW motif) with invariant Pro259. The ionic interactions between the phosphate of pSer258 with the positively charged guani- dinium groups of Arg14, Arg17, and Arg21 also appeared to stabilize the complex. However, as mentioned earlier, the resonances for Arg17, located at position 1 of loop1, disappeared during the NMR titration due to intermediate exchange in the NMR timescale ( Figure 3A), suggesting high flexibility around loop1. Arg21 connects loop1 to the β2-strand into two conformations and involves multiple polar contacts with the double phosphorylated TFCD at pSer258 ( Figure 3B and C). Similarly, Trp34-driven hydrophobic packing of Pro259 induces two ensembles of Trp34 side chain rotamers and Pro259 configurations, and both ensemble states still adopt a trans-configuration of the pSer258-Pro259 peptide bond (Online Supplementary Table S1). For comparison, the previously determined interactions of Pin1 WW-domain with Cell division cycle 25 (Cdc25) and the C-terminal domain of the RNA poly- merase II largest subunit (RNAP II-CTD) are also shown ( Figure 3D). 29,30 In order to further confirm the reliability of our struc- tures, step-wise energy minimization was performed on all Pin1 WW-domain-TFCD complexes. The RMSD of heavy atoms/all atoms before and after energy minimiza- tion was less than 0.45 Å, while the RMSD of the back- bone was less than 0.3 Å (Online Supplementary Figure S2B). Therefore, the interactions between the Pin1 WW- domain and the TFCD, such as the electrostatic interac- tions with pSer258 and the hydrophobic interactions of Pro259 with the WW-domain are consistent with the NMR structures calculated. The trans conformation of the pSer258-Pro259 peptide bond in the TFCD was stable dur-ing 10,000 steps of energy minimization, further demon- strating that our structures satisfy energy-of-motion (EOM) criteria with reasonable convergence ...
Citations
... Pull-down assays using peptides corresponding to the cytoplasmic domain of TF in different phosphorylation states indicated that MAGI1 preferentially interacted with non-phosphorylated and Ser258phosphorylated TF, but the presence of the phosphate group associated with Ser253 hindered this interaction. In addition, lower ability of MAGI1 and MAGI3 to interact with Ser258-phosphorylated TF was detected which may arise from the interaction of the two ww domains within MAGI proteins with the phosphoserine-proline motif (termed an MPM-2 motif ) within the cytoplasmic domain of TF [25,40] and may be involved in the recycling of TF. We previously showed that the activation of PAR2 resulted in maximal phosphorylation of Ser253 at around 20 min in MDA-MB-231 cells [24,26]. ...
Background
Tissue factor (TF) activity is stringently regulated through processes termed encryption. Post-translational modification of TF and its interactions with various protein and lipid moieties allows for a multi-step de-encryption of TF and procoagulant activation. Membrane-associated guanylate kinase-with inverted configuration (MAGI) proteins are known to regulate the localisation and activity of a number of proteins including cell-surface receptors.
Methods
The interaction of TF with MAGI1 protein was examined as a means of regulating TF activity. MDA-MB-231 cell line was used which express TF and MAGI1, and respond well to protease activated receptor (PAR)2 activation. Proximity ligation assay (PLA), co-immunoprecipitation and pull-down experiments were used to examine the interaction of TF with MAGI1-3 proteins and to investigate the influence of PAR2 activation. Furthermore, by cloning and expressing the PDZ domains from MAGI1, the TF-binding domain was identified. The ability of the recombinant PDZ domains to act as competitors for MAGI1, allowing the induction of TF procoagulant and signalling activity was then examined.
Results
PLA and fluorescence microscopic analysis indicated that TF predominantly associates with MAGI1 and less with MAGI2 and MAGI3 proteins. The interaction of TF with MAGI1 was also demonstrated by both co-immunoprecipitation of TF with MAGI1, and co-immunoprecipitation of MAGI1 with TF. Moreover, activation of PAR2 resulted in reduction in the association of these two proteins. Pull-down assays using TF-cytoplasmic domain peptides indicated that the phosphorylation of Ser253 within TF prevents its association with MAGI1. Additionally, the five HA-tagged PDZ domains of MAGI1 were overexpressed separately, and the putative TF-binding domain was identified as PDZ1 domain. Expression of this PDZ domain in cells significantly augmented the TF activity measured both as thrombin-generation and also TF-mediated proliferative signalling.
Conclusions
Our data indicate a stabilising interaction between TF and the PDZ-1 domain of MAGI1 and demonstrate that the activation of PAR2 disrupts this interaction. The release of TF from MAGI1 appears to be an initial step in TF de-encryption, associated with increased TF-mediated procoagulant and signalling activities. This mechanism is also likely to lead to further interactions and modifications leading to further enhancement of procoagulant activity, or the release of TF.
... Peptidyl-prolyl isomerase (Pin1) Pin1 was found to increase TF-PCA in multiple cell types (Table 1) [74]. Although the mechanism is unclear, this effect is likely mediated by Pin1 binding to Ser 258 -Pro 259 in TF's short cytoplasmic domain (Fig. 2a, b) [75]. ...
... Although the mechanism is unclear, this effect is likely mediated by Pin1 binding to Ser 258 -Pro 259 in TF's short cytoplasmic domain (Fig. 2a, b) [75]. Pin1 also enhances TF stability by inhibiting its polyubiquitination and subsequent proteasomal degradation [74,75]. Pin1 also activates the F3 gene via the pro-inflammatory transcription factor complexes NFκB and AP-1 [74]. ...
... Pin1 also enhances TF stability by inhibiting its polyubiquitination and subsequent proteasomal degradation [74,75]. Pin1 also activates the F3 gene via the pro-inflammatory transcription factor complexes NFκB and AP-1 [74]. Overall, Pin1 likely regulates many TF functions to enhance PCA in normal cells, although whether these mechanisms are present across all cancers is unknown. ...
Venous and arterial thromboses, called as cancer-associated thromboembolism (CAT), are common complications in cancer patients that are associated with high mortality. The cell-surface glycoprotein tissue factor (TF) initiates the extrinsic blood coagulation cascade. TF is overexpressed in cancer cells and is a component of extracellular vesicles (EVs). Shedding of TF+EVs from cancer cells followed by association with coagulation factor VII (fVII) can trigger the blood coagulation cascade, followed by cancer-associated venous thromboembolism in some cancer types. Secretion of TF is controlled by multiple mechanisms of TF+EV biogenesis. The procoagulant function of TF is regulated via its conformational change. Thus, multiple steps participate in the elevation of plasma procoagulant activity. Whether cancer cell-derived TF is maximally active in the blood is unclear. Numerous mechanisms other than TF+EVs have been proposed as possible causes of CAT. In this review, we focused on a wide variety of regulatory and shedding mechanisms for TF, including the effect of SARS-CoV-2, to provide a broad overview for its role in CAT. Furthermore, we present the current technical issues in studying the relationship between CAT and TF.
... Pin1 belongs to the parvulin subfamily of peptidyl-prolyl cis-trans isomerase (PPIase) group of proteins. Pin1 is the only PPIase that specifically binds phosphorylated Ser/Thr-Pro protein motifs and catalyzes the cis/trans isomerization of the peptide bond [20][21][22][23]. Through protein-protein interactions and inducing conformational changes on the substrates, Pin1 regulates diverse cellular processes. ...
... Through protein-protein interactions and inducing conformational changes on the substrates, Pin1 regulates diverse cellular processes. Pin1 has been shown to modulate signal transduction by interacting with a diversity of transcription factors [20,[24][25][26][27]. Interestingly, Pin1 has been reported to interact with TGF-β/BMP-specific receptorregulated transcription factors Smad1, Smad2, and Smad3 but not with the common mediator Smad Smad4 [28]. ...
Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.
... Consequently, the procoagulant activity of TF is precisely regulated through various mechanisms that influence the TF protein [1][2][3]. Recently it was demonstrated that the activity and release of TF can be controlled through the action of peptidyl-prolyl trans/cis isomerase 1 (Pin1) [4,5]. Pin1 is a regulator of post-phosphorylation processes and binds to the phosphoserine-proline (termed an MPM-2) motif [6][7][8][9][10][11][12][13][14][15]. ...
... This extends the release of TF within microvesicles by preventing TF ubiquitination [4,20]. Pin1 has also been reported to prolong the activity of TF and also induce the de novo expression of TF mRNA [5]. In addition to the procoagulant activity, TF is known to promote signalling mechanisms that can give rise to cell proliferation [21][22][23] or alternatively cell apoptosis [24][25][26]. ...
... Therefore, while both compound 4b and 4d are capable of preventing the approach of the longer peptides, the presence of acidic tyrosine as the headgroup may also hinder the catalytic function of Pin1. Isomerisation of TF on the cell surface by Pin1 is assumed to prolong its presence on the cell surface [5] and allow its incorporation into microvesicles [4]. Therefore, the inhibition of Pin1 would be expected to accelerate the processing and endocytosis of TF into the cells [4,20,59]. ...
Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-D-alanine (4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound 4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.
... Consequently, the procoagulant activity of TF is precisely regulated through various mechanisms that influence the TF protein [1][2][3]. Recently it was demonstrated that the activity and release of TF can be controlled through the action of peptidyl-prolyl trans/cis isomerase 1 (Pin1) [4,5]. Pin1 is a regulator of post-phosphorylation processes and binds to the phosphoserine-proline (termed an MPM-2) motif [6][7][8][9][10][11][12][13][14][15]. ...
... This extends the release of TF within microvesicles by preventing TF ubiquitination [4,20]. Pin1 has also been reported to prolong the activity of TF and also induce the de novo expression of TF mRNA [5]. In addition to the procoagulant activity, TF is known to promote signalling mechanisms that can give rise to cell proliferation [21][22][23] or alternatively cell apoptosis [24][25][26]. ...
... Therefore, while both compound 4b and 4d are capable of preventing the approach of the longer peptides, the presence of acidic tyrosine as the headgroup may also hinder the catalytic function of Pin1. Isomerisation of TF on the cell surface by Pin1 is assumed to prolong its presence on the cell surface [5] and allow its incorporation into microvesicles [4]. Therefore, the inhibition of Pin1 would be expected to accelerate the processing and endocytosis of TF into the cells [4,20,59]. ...
Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-Dalanine
(4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound
4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase
activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.
... 36 In addition to NF-κB and AP-1, the TF promoter contains Sp1 binding sites 37 and is enhanced by Pin1. 38 Furthermore, in mouse macrophages loss of PARP14 was demonstrated to increase the levels of TF expression. 39 In addition, PARP14 is in part regulated by STAT6 as STAT6 activation induces a switch in PARP14 from a repressor to a promoter of STAT6 signaling via the ribosylation of histone deacetylases previously recruited to IL-4 response elements. ...
Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.
... flTF expression was slightly elevated in brain and lung of homozygous C213G/C213G TF mice compared to wt mice. In hearts of male C213G/C213G TF mice, flTF mRNA was significantly increased by 9.6 times compared to hearts of wt mice suggesting a compensatory transcriptional induction of a functionally insufficient protein that may involve recently demonstrated feedback loops of the TF cytoplasmic domain 34 . In C213G/C213G TF female hearts a similar induction was seen, however with a higher variability. ...
Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.
... Activation of endothelial PAR2 with an agonist peptide is a model system for characterizing TF + EV release. Phosphorylation of the TF cytoplasmic domain regulates incorporation into EVs [17,18] that involves interaction with peptidyl-proline isomerase 1 [19], which is a determinant for TF cellular fate also in smooth muscle cells [20]. Endothelial cell-derived EVs carry integrin b 1 , which supports extracellular matrix binding, thereby stimulating prothrombotic activity [21]. ...
Background
Cell injury signal‐induced activation and release of tissue factor (TF) on extracellular vesicles (EV) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EV occurs in concert with the release of cell adhesion receptors, including integrin β1 heterodimers which control trafficking of the TF‐FVIIa complex. Activation of the TF signaling partner, protease activated receptor (PAR) 2, also triggers TF release on integrin β1⁺ EV from endothelial cells, but the physiological signals for PAR2‐dependent EV generation at the vascular interface remain unknown.
Objective
The goal of this study was to define relevant protease ligands of TF contributing to PAR2‐dependent release on EV from endothelial cells.
Methods
In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EV by a combination of activity and antigen assays, immunocapture and confocal imaging.
Results and Conclusions
PAR2 stimulation generated time‐dependent release of distinct TF⁺ EV with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF‐FVIIa‐FXa complex triggered TF⁺ EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced complex formation of TF with integrin α5β1. Internalization of TF by FVIIa or anti‐TF and activating antibodies to β1 prevented PAR2 agonist‐induced release of TF on EV. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrin β1 regulates TF availability for release on procoagulant EV.
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... In contrast, the binary TF-FVIIa complex activates PAR2 and has been linked to a variety of pathological conditions. In many cases, pathological TF-FVIIa signaling involves the short cytoplasmic domain of TF that in human TF is phosphorylated at Ser 253 by protein kinase C (PKC) [51,52] and at Ser 258 by mitogen-activated protein (MAP) kinase p38 [53], resulting in conformational changes influencing ligand binding [54,55]. ...
... PAR2 cleavage and certain proximal signaling responses of TF-FVIIa do not require the TF cytoplasmic domain [56]. Instead, the TF cytoplasmic domain recruits adaptors for signaling complexes and protein trafficking (i.e. the regulatory subunit of phosphatidylinositol 3 kinase [PI3K]) [57], the actin binding protein filamin [13,[58][59][60] and the prolyl-isomerase Pin1 [55,61]. Interaction of the TF cytoplasmic domain with Pin1 not only influences TF protein half-life and incorporation into EV, but also Pin1-dependent transcriptional regulation of TF expression in human smooth muscle cells [55]. ...
... Instead, the TF cytoplasmic domain recruits adaptors for signaling complexes and protein trafficking (i.e. the regulatory subunit of phosphatidylinositol 3 kinase [PI3K]) [57], the actin binding protein filamin [13,[58][59][60] and the prolyl-isomerase Pin1 [55,61]. Interaction of the TF cytoplasmic domain with Pin1 not only influences TF protein half-life and incorporation into EV, but also Pin1-dependent transcriptional regulation of TF expression in human smooth muscle cells [55]. It is currently unclear whether Pin1 regulation involves PAR2, which is known to constitutively signal independent of proteolytic cleavage [62]. ...
The tissue factor (TF) pathway plays a central role in hemostasis and thrombo‐inflammatory diseases. Although structure‐function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities on cells continue to emerge. Cellular pathways that render TF non‐coagulant participate in signaling of distinct TF complexes with associated proteases through the protease‐activated receptor (PAR) family of G‐protein coupled receptors. Additional co‐receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol‐disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co‐receptors, and associated proteases are integrated in PAR‐dependent cell signaling pathways controlling innate immunity, cancer, and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.
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The unique prolyl isomerase Pin1 binds to and catalyzes cis–trans conformational changes of specific Ser/Thr-Pro motifs after phosphorylation, thereby playing a pivotal role in regulating the structure and function of its protein substrates. In particular, Pin1 activity regulates the affinity of a substrate for E3 ubiquitin ligases, thereby modulating the turnover of a subset of proteins and coordinating their activities after phosphorylation in both physiological and disease states. In this review, we highlight recent advancements in Pin1-regulated ubiquitination in the context of cancer and neurodegenerative disease. Specifically, Pin1 promotes cancer progression by increasing the stabilities of numerous oncoproteins and decreasing the stabilities of many tumor suppressors. Meanwhile, Pin1 plays a critical role in different neurodegenerative disorders via the regulation of protein turnover. Finally, we propose a novel therapeutic approach wherein the ubiquitin–proteasome system can be leveraged for therapy by targeting pathogenic intracellular targets for TRIM21-dependent degradation using stereospecific antibodies.
