Structural divergence is more extensive than sequence divergence for a family of intrinsically disordered proteins

The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain that undergoes coupled folding and binding when interacting with partner proteins like the E3 ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and dynamics of six closely related mammalian homologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Differences in both transient secondary structure and backbone dynamics were observed for the homologues. Many of these differences were localized to the binding sites for MDM2 and RPA70. The amount of transient helical secondary structure observed for the MDM2 binding site was lower for the dog and mouse homologues, compared with human, and the amount of transient helical secondary structure observed for the RPA70 binding site was higher for guinea pig and rabbit, compared with human. Differences in the amount of transient helical secondary structure observed for the MDM2 binding site were directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms during the p53TAD/MDM2 interaction. Differences in the amount of transient helical secondary structure were not as easily explained for the RPA70 binding site because of its extensive sequence divergence. Clustering analysis shows that the divergence in the transient secondary structure of the p53TAD homologues exceeds the amino acid sequence divergence. In contrast, strong correlations were observed between the backbone dynamics of the homologues and the sequence identity matrix, suggesting that the dynamic behavior of IDPs is a conserved evolutionary feature. Proteins 2013; 81:1686–1698. © 2013 Wiley Periodicals, Inc.     

A simplified method for the preparation of rat thyroxine-binding prealbumin. Factors influencing its circulating level

Rat thyroxine-binding prealbumin (TBPA) was isolated in three simple steps by means of a serum precipitation by a 5% phenol solution and two consecutive semi-preparative polyacrylamide gel electrophoreses. The overall yield was 15% and the TBPA preparation contained less than 1% impurities. In addition a monospecific antiserum was raised in the rabbit. In polyacrylamide gel, rat TBPA, as with its human counterpart, migrated anodally to albumin while in agarose gel, its electrophoretic mobility was similar to that of albumin. Serum TBPA measured in adult male Wistar rats did not exhibit a circadian rhythm. However, a significant 13% decrease was observed between 9 and 15 h, followed by the restoration of the initial value by 21 h. TBPA concentration was measured in 1-, 15- and 28-day-old male and female pups as well as in adult rats. The level of this protein increased from 1 to 28 days of age and did not display any sexual difference. Yet, while TBPA concentrations in adult males were similar to those recorded in the 28-day-old pups, for adult females, they returned to the levels measured in the 1-day-old pups.     

The membrane-associated lipoprotein-9 GmpC from Staphylococcus aureus binds the dipeptide GlyMet via side chain interactions

Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates. This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters. SBPs bind dipeptides with little regard for their amino acid content. Here, we report the 1.7 A resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine. Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein. This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter. Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues. Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions. Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP. Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not. Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine. For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein.     

Quantification of a cytochrome P450 3A4 substrate, buspirone, in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive HPLC-tandem mass spectrometry method was developed for determination of buspirone levels in human plasma. After solid phase extraction and reversed phase HPLC separation, detection of buspirone and the internal standard (prazosin) was performed using eletrospray ionization and selected reaction monitoring in the positive ion mode. Linear calibration curves were established over a concentration range of 0.025-2.5 ng/ml when 0.5 ml aliquots of plasma were used. Satisfactory results of within-day precision (RSD of 1.9-7.7%) and accuracy (% difference of 0.5-6.6%) and between-day precision (RSD of 3.7-11.1%) and accuracy (% difference of 2.2-6.8%) were obtained. The assay has been successfully applied to the analysis of buspirone levels in more than 500 human plasma samples collected from a drug interaction study.     

Human relaxin-2: historical perspectives and role in cancer biology

One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A-B heterodimer linked by three disulfide bonds. This mini-review is divided into three broad themes: an overview of all insulin superfamily members (including structural similarities); roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.     

Mi-2/NuRD complex function is required for normal S phase progression and assembly of pericentric heterochromatin

During chromosome duplication, it is essential to replicate not only the DNA sequence, but also the complex nucleoprotein structures of chromatin. Pericentric heterochromatin is critical for silencing repetitive elements and plays an essential structural role during mitosis. However, relatively little is understood about its assembly and maintenance during replication. The Mi2/NuRD chromatin remodeling complex tightly associates with actively replicating pericentric heterochromatin, suggesting a role in its assembly. Here we demonstrate that depletion of the catalytic ATPase subunit CHD4/Mi-2β in cells with a dampened DNA damage response results in a slow-growth phenotype characterized by delayed progression through S phase. Furthermore, we observe defects in pericentric heterochromatin maintenance and assembly. Our data suggest that chromatin assembly defects are sensed by an ATM-dependent intra-S phase chromatin quality checkpoint, resulting in a temporal block to the transition from early to late S phase. These findings implicate Mi-2β in the maintenance of chromatin structure and proper cell cycle progression.