Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-α activity

Phosphorylation of estrogen receptor-α (ERα) at specific residues in transcription activation function 1 (AF-1) can stimulate ERα activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERα activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERα activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERα activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERα activity. Collectively, these data indicate that the MAPK stimulation of ERα activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERα at these sites may contribute to resistance to tamoxifen in breast cancer

Reduced plasma magnesium levels in type-1 diabetes associate with prothrombotic changes in fibrin clotting and fibrinolysis

This research was funded by the British Heart Foundation, grant numbers PG/15/9/31270 and FS/15/42/3155. The study sponsor was not involved in the design of the study; the collection, analysis, and interpretation of data.Individuals with type-1 diabetes mellitus (T1DM) have a higher risk of thrombosis and low plasma magnesium concentrations. As magnesium is a known regulator of fibrin network formation, we investigated potential associations between fibrin clot properties and plasma magnesium concentrations in 45 individuals with T1DM and 47 age- and sex-matched controls without diabetes. Fibrin clot characteristics were assessed using a validated turbidimetric assay and associations with plasma magnesium concentration were examined. Plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1), and lipids were measured and fibrin fiber diameters assessed using scanning electron microscopy. Fibrin clot maximum absorbance was unchanged in subjects with T1DM compared with controls, while lysis time was prolonged (p = 0.0273). No differences in fibrin fiber diameters or in lipid profile were observed between T1DM and controls. PAI-1 concentration was lower in the T1DM group compared with the controls (p = 0.0232) and positively correlated with lysis time (p = 0.0023). Plasma magnesium concentration was lower in the T1DM group compared with controls (p < 0.0001). Magnesium concentration negatively correlated with clot maximum absorbance (p = 0.0215) and lysis time (p = 0.0464). A turbidimetric fibrin clot lysis assay performed in a purified system that included PAI-1 and 0 to 3.2 mM Mg2+ showed a shortening of lysis time with increasing Mg2+ concentrations (p = 0.0004). Our findings reveal that plasma magnesium concentration is associated with changes in fibrin clot and lysis parameters.Publisher PDFPublisher PDFPeer reviewe

Changes in fibrin clot properties in patients after Roux en-Y gastric bypass surgery

Background: Obesity is a complex condition associated with prothrombotic fibrin networks that are resistant to fibrinolysis. Altered fibrin clot properties enhance cardiovascular risk and associate with a poorer prognosis following acute ischemic events. Bariatric surgery is commonly employed to improve cardiometabolic outcomes in obese individuals. However, the effects of this surgical intervention on fibrin clot properties have not been comprehensively studied. Methods: The fibrin clot properties of 32 obese individuals before and 9-months after Roux en-Y gastric bypass (RYGB) surgery were determined using turbidimetric analysis. Correlation and regression analyses were used to identify relationships between clot properties and anthropomorphic and clinical measures. Results: RYGB surgery resulted in a significant reduction in adiposity-associated anthropometric measures as well as improvements in glycaemia and lipid profile. Clot maximum absorbance was reduced from 0.43 ± 0.11 at baseline to 0.29 ± 0.10 at 9 months post-surgery (p < 0.0001), while fibrin clot lysis time failed to show a difference. The change in maximum absorbance was not caused by alterations in fibrinogen levels, while PAI-1 concentration was significantly increased after surgery from 10,560 ± 6,681 pg/ml to 15,290 ± 6,559 pg/ml (p = 0.0093). Correlation and regression analysis indicated that maximum absorbance was influenced by markers of adiposity as well as HbA1c and hs-CRP concentrations. Conclusions: RYGB surgery led to a decrease in the maximum absorbance of the fibrin clot. Values of maximum absorbance were associated with measures of glycaemic control and inflammation. In contrast to previous reports, fibrin clot lysis time was not affected after surgery.Peer reviewe

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexe

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

BACKGROUND: Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. METHODOLOGY/PRINCIPAL FINDINGS: We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. CONCLUSIONS/SIGNIFICANCE: We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation

Changes in fibrin clot properties in patients after Roux en-Y gastric bypass surgery

Background: Obesity is a complex condition associated with prothrombotic fibrin networks that are resistant to fibrinolysis. Altered fibrin clot properties enhance cardiovascular risk and associate with a poorer prognosis following acute ischemic events. Bariatric surgery is commonly employed to improve cardiometabolic outcomes in obese individuals. However, the effects of this surgical intervention on fibrin clot properties have not been comprehensively studied.Methods: The fibrin clot properties of 32 obese individuals before and 9-months after Roux en-Y gastric bypass (RYGB) surgery were determined using turbidimetric analysis. Correlation and regression analyses were used to identify relationships between clot properties and anthropomorphic and clinical measures.Results: RYGB surgery resulted in a significant reduction in adiposity-associated anthropometric measures as well as improvements in glycaemia and lipid profile. Clot maximum absorbance was reduced from 0.43 ± 0.11 at baseline to 0.29 ± 0.10 at 9 months post-surgery (p &lt; 0.0001), while fibrin clot lysis time failed to show a difference. The change in maximum absorbance was not caused by alterations in fibrinogen levels, while PAI-1 concentration was significantly increased after surgery from 10,560 ± 6,681 pg/ml to 15,290 ± 6,559 pg/ml (p = 0.0093). Correlation and regression analysis indicated that maximum absorbance was influenced by markers of adiposity as well as HbA1c and hs-CRP concentrations.Conclusions: RYGB surgery led to a decrease in the maximum absorbance of the fibrin clot. Values of maximum absorbance were associated with measures of glycaemic control and inflammation. In contrast to previous reports, fibrin clot lysis time was not affected after surgery

The proposed role of MASP-1 in plasma clot formation.

<p>MASP-1 induces fibrin clot formation by converting prothrombin to thrombin which then cleaves fibrinogen. MASP-1 activates FXIII and TAFI independently albeit at a lower efficiency than thrombin. Changes in clot structure and TAFI activation by MASP-1 may be responsible for prolonged lysis time of clots made in the presence of MASP-1. The finer arrows for MASP-1-dependent reactions, as compared with thrombin-dependent reactions, represent the fact that in plasma MASP-1 is less efficient compared with thrombin. The mechanisms underlying the impact of MASP-1 on fibrin structure remain to be elucidated.</p