Homologous and Heterologous Interactions between Hexokinase and Mitochondrial Porin: Evolutionary Implications

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase–mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

Glucansucrases: mechanism of action and structure-function relationships

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.     

Functional and genetic analysis of annexin VI

This study is concerned with the determination of the function of the 68kDa calcium-binding protein, annexin VI. Studies on the structure and regulation of the gene include a detailed analysis of annexin VI expressed heterologously in human A431 carcinoma cells. We have recently discovered that annexin VI is subject to a novel growth dependent post-translational modification. Interestingly, the protein exerts a negative effect on A431 cells. This effect was manifested as a partial reversal of the transformed phenotype. We are currently exploring the hypothesis that the post-translational modification of annexin VI is required for sub-cellular targeting, and that correct localisation within the cell is essential for function.     

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.     

基于蛋白质序列的泛素化位点预测研究进展

泛素化是目前广受关注的一种翻译后修饰过程,对蛋白质降解、DNA修复等多种细胞过程都具有重要的调控作用。本文根据国内外蛋白质泛素化位点预测的研究,分析了预测泛素化位点的特征属性,总结了对这些特征进行优化的特征选择方法,并对预测过程中所使用的各种机器学习分类器进行了概述。     

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.