Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

Crystallographic analysis of the pH-dependent binding of iminobiotin by streptavidin.

Streptavidin binds 2'-iminobiotin in a pH-dependent fashion--affinity decreases as the pH is lowered. This property makes the purification of compounds conjugated to streptavidin or immobiotin possible under mild conditions by affinity chromatography. In order to understand the molecular details of this pH-dependent binding, we analyzed the crystal structures of the complex of core streptavidin with 2'-iminobiotin at pH values 4.0 and 7.5. The two structures are very similar to each other even at their binding sites. Although the relative abundance of the protonated species of the ligand is increased more than 3,000-fold on going from pH 7.5 to pH 4.0, both structures contain only the nonprotonated from of the ligand. Streptavidin selects the nonprotonated form, which, at pH 4.0, is one part in 7.9 x 10(7).     

Phosphatidylinositol‐4 kinase III beta and oxysterol‐binding protein accumulate unesterified cholesterol on poliovirus‐induced membrane structure

Studies on anti‐picornavirus compounds have revealed an essential role of a novel cellular pathway via host phosphatidylinositol‐4 kinase III beta (PI4KB) and oxysterol‐binding protein (OSBP) family I in poliovirus (PV) replication. However, the molecular role for this pathway in PV replication has yet to be determined. Here, viral and host proteins modulating production of phosphatidylinositol 4‐phosphate (PI4P) and accumulation of unesterified cholesterol (UC) in cells were analyzed and the role of the PI4KB/OSBP pathway in PV replication characterized. Virus protein 2BC was identified as a novel interactant of PI4KB. PI4KB and VCP/p97 bind to a partially overlapped region of 2BC with different sensitivity to a 2C inhibitor. Production of PI4P and accumulation of UC were enhanced by virus protein 2BC, but suppressed by virus proteins 3A and 3AB. In PV‐infected cells, a PI4KB inhibitor suppressed production of PI4P, and both a PI4KB inhibitor and an OSBP ligand suppressed accumulation of UC on virus‐induced membrane structure. Inhibition of PI4KB activity caused dissociation of OSBP from virus‐induced membrane structure in PV‐infected cells. Synthesis of viral nascent RNA in PV‐infected cells was not affected in the presence of PI4KB inhibitor and OSBP ligand; however, transient pre‐treatment of PV‐infected cells with these inhibitors suppressed viral RNA synthesis. These results suggest that virus proteins modulate PI4KB activity and provide PI4P for recruitment of OSBP to accumulate UC on virus‐induced membrane structure for formation of a virus replication complex.     

Sequence and structure space of RNA-binding peptides

Studies of RNA-binding peptides, and recent combinatorial library experiments in particular, have demonstrated that diverse peptide sequences and structures can be used to recognize specific RNA sites. The identification of large numbers of sequences capable of binding to a particular site has provided extensive phylogenetic information used to deduce basic principles of recognition. The high frequency at which RNA-binding peptides are found in large sequence libraries suggests plausible routes to evolve sequence-specific binders, facilitating the design of new binding molecules and perhaps reflecting characteristics of natural evolution.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC₄) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-α-MSH and TAMRA-NDP-α-MSH, were used and compared in assays that monitored their binding to MC₄ receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC₄ receptors was saturable and with high affinity. All studied MC₄ receptor-specific nonlabeled ligands displaced fluoroligands’ binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

Synthesis, structural and electrochemical features of alicyclic and aromatic α,α′-N2- and-S2-dioximate macrobicyclic cobalt(II,III) and ruthenium(II) tris-complexes

The triribbed-functionalized cobalt(II,III) and ruthenium(II) clathrochelate derivatives of the vic-dioximes with two nitrogen or sulfur atoms in α-positions to π-conjugated diazomethine chelate fragments of a macrobicyclic framework were obtained in moderate yields under mild and high dilution conditions by nucleophilic substitution of six reactive chlorine atoms of the boron-capped macrobicyclic cobalt and ruthenium(II) precursors with N₂- and S₂-dinucleophiles (ethylenediamine and the corresponding α-dithiols in the presence of triethylamine, respectively). The complexes obtained were characterized using elemental analysis, MALDI-TOF mass spectrometry, IR, UV-Vis, ¹H and ¹³C{¹H} NMR and EPR spectroscopies, magnetochemistry and X-ray crystallography. The MN₆-coordination polyhedra of all the X-ray studied clathrochelates possess a slightly distorted trigonal prismatic geometry. The encapsulated cobalt(II) ions are shifted from the centers of the cavities formed by the macrobicyclic ligand due to the Jahn-Teller distortion, while the ruthenium and iron(II) ions in their clathrochelate analogs do occupy these centers. The main geometrical parameters of the macrobicyclic frameworks vary with Shannon radius of an encapsulated metal ion. In the case of the tris-ethylenediamine cobalt(III) clathrochelate, the field strength of the macrobicyclic amine ligand is essentially lower than those for their aromatic and aliphatic analogs because of the negative σ_para-effect of the ribbed alkylamine substituents. The magnetometry and EPR data confirmed the low-spin character of the cobalt(II) complexes synthesized. The electrochemically generated oxidized cobalt clathrochelates are stable in the CVA time scale, whereas their ruthenium- and iron-containing analogs as well as the reduced forms of all the cobalt, ruthenium and iron complexes obtained are unstable.