Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

Relationship Between Curved DNA Conformations and Slow Gel Migration

Abstract

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA₄T₄X)₁, and (V₂A₃X₂)₁ where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions a mong aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)_n · (A)_n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove, TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT₄A₄X)₁ might form a very tightly coiled super-helix whereas the sequences (VA₄T₄X)₁ form a broad super-helix of radius ~ 120 A for i = 25. The sequence (V₂A₃T₃X₂)₁ forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallesuiimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I₁ about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I₁)^1/5.     

Synthesis and Characterization of Binding of 5-[76Br] Bromo-3-[[2(S)-Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Interaction of non-intercalative drugs with DNA: Distamycin analogues

Distamycin and netropsin are two oligopeptides which bind to DNA in a nonintercalative manner. Analogues of distamycin have been synthesized and their binding with poly d(A-T) studied using ultraviolet absorption spectroscopy. Preliminary biological activity tests on a gram positive bacteria using these analogues have also been carried out Based on the lecture given by Dr. V. Sasisekharan at the Royal Society of Chemistry (Deccan Section) Bangalore, 26 June 1984.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila

Misexpression Suppressor of Ras 4 (MESR4), a plant homeodomain (PHD) finger protein with nine zinc‐finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. Here it is shown that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti‐MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E‐F region, which is known to contain the histone gene cluster. P‐element insertions in the MESR4 locus, which were homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation was identified. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene was greatly reduced, and the level of EGFR signal‐dependent double phosphorylated ERK (dp‐ERK) remained low. However, in the context of wing vein formation, genetic interaction experiments suggested that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggested that MESR4 is a novel chromatin‐binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development. genesis 53:701–708, 2015. © 2015 Wiley Periodicals, Inc.     

Electron Density Sharpening as a General Technique in Crystallographic Studies

Sharpening is a powerful method to restore the details from blurred electron density in crystals with high overall temperature factors (B-factors). This valuable technique is currently not optimally used because of the uncertainty in the scope of its application and ambiguities in practice. We performed an analysis of ~ 2000 crystal data sets deposited in the Protein Data Bank and show that sharpening improves the electron density map in many cases across all resolution ranges, often with dramatic enhancement for mid- and low-resolution structures. It is effective when used with either experimental or model phases without introducing additional bias. Our tests also provide a practical guide for optimal sharpening. We further show that anisotropic diffraction correction improves electron density in many cases but should be used with caution. Our study demonstrates that a routine practice of electron density sharpening may have a broad impact on the outcomes of structural biology studies.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.