Structure–function relationships in the neurotrophin family

The study of structure–function relationships in the neurotrophin family has in recent years increased our understanding of several important aspects of neurotrophin function. Site-directed mutagenesis studies have localized amino acid residues important for binding to the low-affinity (p75^LNGFR), as well as to the members of the Trk family of tyrosine kinase receptors. A cluster of positively charged residues has been shown to form a surface for binding to p75^LNGFR in all four neurotrophins. Differences in the spatial distribution of these charges among the different neurotrophins may explain some of their distinct binding properties. Elimination of these positive charges drastically reduces binding to P75^LNGFR but not to the Trk family members, and it does not impair the biological properties of the neurotrophins in vitro, arguing that binding to and activation of Trk receptors is sufficient to mediate the biological responses of neurotrophins. In contrast. the binding sites to Trk receptors appear to be formed by discontinuous stretches of amino acid residues distributed throughout the primary sequence of the molecule. These include the N-terminus, some of the variable loop regions and a β-strand. Despite their apparent distribution, when viewed in the three-dimensional structure of NGF, these residues appear grouped on one side of the neurotrophin dimer, delineating a continuous surface extending approximately parallel to the twofold symmetry axis of the molecule. Two symmetrical surfaces are formed along the axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. In the neurotrophin family, co-evolution of cognate ligands and Trk receptors has developed specific contacts through different residues in the same variable regions of the neurotrophins. Thus, binding specificity is determined by the cooperation of distinct active and inhibitory binding determinants that restrict ligand-receptors interactions. Binding determinants to the Trk receptors can be manipulated independently in a rational fashion to create neurotrophin analogues with novel ligand-binding properties. In this way, second-generation chimeric neurotrophins with multiple specificities (pan-neurotrophins) have been engineered which may have valuable applications in the treatment of neurodegeneration and nerve damage. 1994 John Wiley & Sons, Inc.     

Substrate tunnels in enzymes: Structure–function relationships and computational methodology

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. Proteins 2015; 83:599–611. © 2015 Wiley Periodicals, Inc.     

Structure‐function relationships of brazzein variants with altered interactions with the human sweet taste receptor

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non‐carbohydrate sweeteners. In an investigation of sequence‐dependent functional properties of the protein, we used NMR spectroscopy to determine the three‐dimensional structures and dynamic properties of two Brz variants: one with a single‐site substitution (D40K), which is three‐fold sweeter than wild‐type Brz, and one with a two‐residue insertion between residues 18 and 19 (ins₁₈RI₁₉), which is devoid of sweetness. Although the three‐dimensional folds of the two variants were very similar to wild‐type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter‐stand H‐bond between the side chains of residues Gln46 and Asp40 present in wild‐type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G‐protein coupled human sweet receptor. On the other hand, the Arg‐Ile insertion within Loop9–19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.     

Structure‐function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild‐type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high‐throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl‐terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.     

Structure–activity relationships of peptidomimetics that inhibit PPI of HER2‐HER3

Human epidermal growth factor receptor‐2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2‐mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure–activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N‐ and C‐terminal modification and cyclization as well as by substitution with d ‐amino acids at the N‐and C‐termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d ‐amino acid substitution and its N‐ and C‐termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2‐overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein–protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 693–702, 2014.     

Upregulation of telomerase function during tissue regeneration

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

Structure–stability relationships in networks combining mutualistic and antagonistic interactions

The relationship between the structure of ecological networks and community stability has been studied for decades. Recent developments highlighted that this relationship depended on whether interactions were antagonistic or mutualistic. Different structures promoting stability in different types of ecological networks, i.e. mutualistic or antagonistic, have been pointed out. However, these findings come from studies considering mutualistic and antagonistic interactions separately whereas we know that species are part of both types of networks simultaneously. Understanding the relationship between network structure and community stability, when mutualistic and antagonistic interactions are merged in a single network, thus appears as the next challenge to improve our understanding of the dynamics of natural communities. Using a theoretical approach, we test whether the structural characteristics known to promote stability in networks made of a single interaction type still hold for network merging mutualistic and antagonistic interactions. We show that the effects of diversity and connectance remain unchanged. But the effects of nestedness and modularity are strongly weakened in networks combining mutualistic and antagonistic interactions. By challenging the stabilizing mechanisms proposed for networks with a single interaction type, our study calls for new measures of structure for networks that integrate the diversity of interaction.     

Structure‐based barcoding of proteins

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha‐numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/ .     

Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.)

The Arabidopsis vacuolar H⁺‐pyrophosphatase (AVP1), when over‐expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over‐expressing AVP1 exhibited improved resistance to salinity than wild‐type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na⁺ uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na⁺, K⁺, Cl^‐ and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H⁺‐pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.     

Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development

Telomerase reintroduction in adult somatic tissues is envisioned as a way to extend their proliferative capacity. It is still a question, however, whether constitutive telomerase expression in adult tissues impacts the normal aging and spontaneous cancer incidence of an organism. Here, we studied the aging and spontaneous cancer incidence of mice with transgenic telomerase expression in a wide range of adult tissues, K5-Tert mice. For this, we maintained large colonies of K5-Tert mice for more than 2 years. K5-Tert mice showed a decreased life span compared to wild-type cohorts associated with a higher incidence of preneoplastic and neoplastic lesions in various tissue types. Neoplasias in K5-Tert mice were coincident with transgene expression in the affected tissues. These observations suggest that high telomerase activity may cooperate with genetic alterations that occur with age to promote tumorigenesis. Indeed, we demonstrate here that increased cancer incidence and the reduced viability of K5-Tert mice are aggravated in a p53(+/-) genetic background, indicating that telomerase cooperates with loss of p53 function in inducing tumorigenesis. Altogether, these results demonstrate that constitutive high levels of telomerase activity result in a decreased life span associated with an increased incidence of neoplasias as the organism ages.     

Inducible transgenic expression in the short‐lived fish Nothobranchius furzeri

This study demonstrates inducible transgenic expression in the exceptionally short‐lived turquoise killifish Nothobranchius furzeri, which is a useful vertebrate model for ageing research. Transgenic N. furzeri bearing a green fluorescent protein (Gfp) containing construct under the control of a heat shock protein 70 promoter were generated, heat shock‐induced and reversible Gfp expression was demonstrated and germline transmission of the transgene to the F1 and F2 generations was achieved. The availability of this inducible transgenic expression system will make the study of ageing‐related antagonistically pleiotropic genes possible using this unique vertebrate model organism.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Structure–function relationships in 3α-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3α-Hydroxysteroid dehydrogenases (3α-HSDs) inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone (5α-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3α-HSD isoforms exist and correspond to AKR1C1–AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3α-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3α-, 17β-, and 20α-hydroxysteroid oxidase activity. Their k_cat values are 50–100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3α-HSD, bile acid binding protein and peripheral 3α-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (β-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k_cat seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate “wobble” at the plastic active site.