Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ.

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

The link between bone disease and cardiovascular complications in hemodialysis patients.

INTRODUCTION: The burden on the cardiovascular system is the main cause of mortality in chronic renal patients, and bone disease, which also may cause disability, is one of the most important complications in those patients. The aim of this study was to determine the link between cardiovascular and bone disease, which frequently occur together. METHODS: In this matched case-control study, 70 subjects were subjected for full laboratory assessment as well as estimation of parathyroid hormone (PTH) level, vitamin D level, complete echocardiography, and dual energy absorptiometry. Of the 70 patients, 50 were on regular hemodialysis, and there were 20 normal controls matched with the patients with respect to age and gender. RESULTS: There was a significant decrease in the mean value of serum vitamin D in the hemodialysis patients, i.e., their mean value was 20.47 ± 9.60 whereas the controls had a mean value of 37.15 ± 7.67. Thus, there was a highly-significant, negative correlation between vitamin D and left ventricular mass (LVM) in the patients. We found that there was a highly-significant increase in the mean PTH levels of the patients (820.22 ± 393.51), whereas it was 57.60 ± 13.72 for the controls. The statistical significance was less than 0.001, a highly-significant increase in the mean of the T score levels in the patients (-2.15 ± 2.56), whereas it was -0.47 ± 0.71 for the controls with a statistical significance of less than 0.001. There also was a highly-significant correlation between the T score and LVM. CONCLUSION: A significant correlation was found between bone disease and the occurrence of a left ventricular mass. We recommend early strict correction of the serum levels of vitamin D, PTH, calcium, and phosphorus.

Brain Natriuretic Peptide in Liver Cirrhosis and Fatty Liver: Correlation with Cardiac Performance.

OBJECTIVE: The aims of the present study were to assess the serum BNP level in patients with post hepatitis C liver cirrhosis and patients with fatty liver and to determine the correlation between BNP and the severity of liver disease and cardiac performance. METHODS: The study was conducted on 140 subjects subdivided into 3 groups: group 1 included 60 patients having post hepatitis C virus (HCV) liver cirrhosis; group 2 included 60 patients with nonalcoholic fatty liver disease (NAFLD); and group 3 included 20 healthy volunteers serving as a control group. All patients and volunteers were subjected to full physical examinations, laboratory evaluation of hemoglobin percent, liver and renal function tests, serum electrolytes, cholesterol, triglyceride, HBs antigen, HCV antibody and serum BNP levels, ECG, abdominal ultrasonography, and echocardiography. RESULTS: There was a significant increase in the BNP level in cirrhotic patients compared to the other two groups (p = 0.000), and it was correlated with the severity of liver disease assigned as Child's classification (p = 0.000). Also, there was a significant increase in the BNP level in cirrhotic patients with decompensation components compared to those without decompensation components (p = 0.000), history of hepatic encephalopathy (p = 0.000), history of variceal bleeding (p = 0.000), history of spontaneous bacterial peritonitis (p = 0.000), presence of ascites (p = 0.000) and portal vein diameter > 11 mm in abdominal ultrasound (p = 0.000), and prolonged QTc interval in ECG (p = 0.011). There was a significant increase in serum BNP in patients with cirrhosis with the following echocardiographic findings: IVST > 11 mm, PWT > 11 mm, LA diameter > 40 mm, EF% < 54%, and E/A ratio < 1 compared to those without these echocardiographic findings (p = 0.000). CONCLUSION: BNP level increases in post hepatitis C cirrhotic patients and tends to decrease in fatty liver disease patients, and it is correlated with both the severity of liver disease and the morpho-functional cardiac changes. Given the ever-increasing prevalence of liver cirrhosis and fatty liver disease worldwide, it is important to understand the benefits and limitations of BNP as a heart failure biomarker in hepatic patients, where the relationship between BNP level and myocardial function is complex and is altered by the liver disease.

Differential modes of peptide binding onto replicative sliding clamps from various bacterial origins.

Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

Phosphorylation of the retinoic acid receptor alpha induces a mechanical allosteric regulation and changes in internal dynamics.

Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9-10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.

Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation.

The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity.