On the Species Specificity of Acceptor RNA and Attachment Enzymes

One of the steps in protein biosynthesis appears to be the attachment of each amino acid to a specific acceptor (SRNA) molecule. According to the adaptor hypothesis, each SRNA molecule would then fit to a specific complementary base sequence on a linear RNA template, specifying the sequence of amino acids in the resultant protein [1,2]. An adaptor molecule thus could have two specificities: one recognizing the correct amino acid and activating enzyme; the other, the proper position on the template. The correctness of the amino-acid sequence therefore would depend upon the precision and constancy of the adaptors. However, the structures of the enzymes and adaptors are presumably under the genetic control of the organism and might be subject to heritable modifications. It is therefore conceivable that one or both ends of an adaptor might change sufficiently to cause occasional errors and, in the long run, an alteration of the genetic code might evolve. This notion, prompted by genetic observations [3] which suggested that mutation of a bacterium might modify its translation of genetic information, lead to the present comparison of the specificities of the acceptor RNA and activating enzymes of different organisms. Several differences in specificity have been reported previously. Berg et al. [4] demonstrated that SRNA from Escherichia coli contains two distinguishable acceptors for methionine. An enzyme prepared from yeast could attach methionine to one of these, while the enzyme from E. coli could attach to both. Webster found, in pig liver, a difference between the nuclear and cytoplasmic attachment enzymes for alanine. Rendi and Ochoa [6] noted that, for leucine, the enzymes in yeast and in E. coli could attach only to their homologous SRNA. Furthermore, in the case of leucine, rat liver enzyme and SRNA were interchangeable with those from E. coli. The observations presented below show that whether the enzymes and/or acceptors from two organisms are interchangeable depends upon not only the organisms in question but also the particular amino aci

Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit

The volatile compounds that constitute the fruit aroma of ripe tomato (Solanum lycopersicum) are often sequestered in glycosylated form. A homology-based screen was used to identify the gene SlUGT5, which is a member of UDP-glycosyltransferase 72 family and shows specificity towards a range of substrates, including flavonoid, flavanols, hydroquinone, xenobiotics and chlorinated pollutants. SlUGT5 was shown to be expressed primarily in ripening fruit and flowers, and mapped to chromosome I in a region containing a QTL that affected the content of guaiacol and eugenol in tomato crosses. Recombinant SlUGT5 protein demonstrated significant activity towards guaiacol and eugenol, as well as benzyl alcohol and methyl salicylate; however, the highest in vitro activity and affinity was shown for hydroquinone and salicyl alcohol. NMR analysis identified isosalicin as the only product of salicyl alcohol glycosylation. Protein modelling and substrate docking analysis were used to assess the basis for the substrate specificity of SlUGT5. The analysis correctly predicted the interactions with SlUGT5 substrates, and also indicated that increased hydrogen bonding, due to the presence of a second hydrophilic group in methyl salicylate, guaiacol and hydroquinone, appeared to more favourably anchor these acceptors within the glycosylation site, leading to increased stability, higher activities and higher substrate affinities

Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 β-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Å resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced α-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (β- and γ-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of α-CD. Glu258 is involved in catalysis in CGTases as well as α-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

Substrate specificity provides insights into the sugar donor recognition mechanism of O-GlcNAc transferase (OGT).

O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism

Heteroreceptor complexes and their allosteric receptor-receptor interactions in the central nervous system. Focus on examples from Dopamine D2 and Serotonin 5-HT1a receptors

GPCR interacting proteins (specially β- arrestin) and their receptor-protein interactions are also covered but their interactions with the allosteric receptor-receptor interactions in heteroreceptor complexes remain to be elucidated. The physiological and pathological relevance of the allosteric receptor-receptor interactions in heteroreceptor complexes is emphasized and novel strategies for treatment of mental and neurological disease are introduced based on this new biological principle of integration. This work gives further experimental evidences which strongly support the current view that allosteric receptor–receptor interactions in heteroreceptor complexes appear to represent a new principle in biology making possible integration of signals already at the level of the plasma membrane. These heteroreceptor complexes and their dynamics may be part of the molecular basis of learning and memory. The receptor protomers and their allosteric receptor-receptor interactions can be disturbed in neurological and mental disorders, and in diseases of peripheral tissues like the endocrine, cardiovascular and immune systems. The dopamine (DA) neuron system most relevant for schizophrenia and Parkinson s diseases is the meso-limbic-cortical DA system inter alia densely innervating subcortical limbic regions as well as the dorsal striatum. The field of dopamine D2Rs changed significantly with the discovery of many types of D2R heteroreceptor complexes in the ventral and dorsal striatum. The results indicate that the D2R is a hub receptor (www.gpcr-hetnet.com) which interacts not only with many other GPCRs including DA isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and DA transporters. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of schizophrenia and Parkinson s diseases through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the DA signal, especially to the ventral striato-pallidal GABA pathway. In schizophrenia, this will have consequences for the control of this pathway of the glutamate drive to the prefrontal cortex via the mediodorsal thalamic nucleus which can contribute to psychotic processes. Allosteric receptor-receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the GPCR protomers. In A2A-D2R heteroreceptor complexes allosteric A2A-D2R receptor-receptor interaction brings about a biased modulation of the D2R protomer signalling (Chapter 1). A conformational state of the D2R is induced which moves away from Gi/o signaling and instead favours b-arrestin2 mediated signalling which may be the main mechanism for its atypical antipsychotic properties especially linked to the limbic A2AR-D2R heteroreceptor complexes. Furthermore, D2R-NTS1R heterocomplexes also exist in the ventral and dorsal striatum (Chapter 2) and likely also in midbrain DA nerve cells as D2R-NTS1R autoreceptor complexes where neurotensin produces antipsychotic and propsychotic actions, respectively. D2R protomer appeared to bias the specificity of the NT orthosteric binding site towards neuromedin N vs neurotensin in the heteroreceptor complex. There is a new awareness that Receptor tyrosine kinases (RTK) and transmitter activated GPCR possess the capacity for transactivation not only via GPCR induced release of neurotrophic factors, but also during signal initiation and propagation, using shared signaling pathways or using themselves as signaling platforms via direct allosteric receptor–receptor interactions. RTK are a family of transmembrane- spanning receptors that mediate the signaling from ligands such as growth factors, like the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), the brain derived neurotrophic factor (BDNF), and the fibroblast growth factor (FGF). This hypothesis on direct GPCR-RTK receptor-receptor interactions in heteroreceptor complexes was introduced by Fuxe et al 1983. They also proposed the existence of 5- HT1A-FGFR1 heteroreceptor complexes having a role in depression. The hypothesis was introduced that the neurotrophic system FGF-2/FGFR1 may be a good candidate to mediate antidepressant induced improvement in 5-HT neuronal communication and neurotrophism with regeneration of connections lost during depression. RTK transactivation in response to antidepressant drug treatment was postulated to take place via a new allosteric receptor–receptor between distinct serotonin receptor subtypes and FGFR1 in heteroreceptor complexes. The discovery of brain FGFR1-5-HT1A heteroreceptor complexes and their enhancement of neuroplasticity offers an integration of the serotonin and the neurotrophic factor hypotheses of depression at the molecular level. These heteroreceptor complexes were found in the hippocampus and midbrain raphe 5-HT nerve cells, enriched in 5-HT1A autoreceptors. Based on the triplet puzzle theory several sets of triplet homologies were identified that may be part of the receptor interface. Combined FGF-2 and 5-HT1A agonist treatment increased the formation of these heterocomplexes and the facilitatory allosteric receptor-receptor interactions within them leading to an enhancement of FGFR1 signaling (Chapter 3). This integrative phenomenon is reciprocal and RTK signaling can be placed downstream of GPCRs. Formation of such heterocomplexes involving two major classes of membrane receptors can be involved in regulating all aspects of receptor protomer function including recognition, signaling, trafficking, desensitization, and downregulation (Chapter 3). These events were associated with development of rapid antidepressant effects. These heteroreceptor complexes are a novel target for antidepressant drugs. These examples, based on solid experimental evidences, serve to illustrate that allosteric receptor-receptor interactions in GPCR heteroreceptor complexes play a significant role in receptor diversity and bias of the participating GPCR protomers.G-protein coupled receptors (GPCR)-mediated signalling is a more complicated process than described previously since every GPCR and GPCR heteromer requires a set of G protein interacting proteins (GIP) which interacts with the receptor in an orchestrated spatio-temporal fashion. Therefore, there is a high interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time, and specially, their integration in GPCR heterocomplexes of the Central Nervous System (CNS). Also, pathological protein-protein interactions in homocomplexes and heterocomplexes of Aβ, Tau, and α-Syn are at the heart of the development of conformational protein disorders. Along this work, experimental evidences are given to illustrate that GPCR interactions have relevance for neurological and mental diseases and are targets for drug development. GPCR containing heteromers and higher order heteromers through allosteric receptor- receptor interactions have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. They have become exciting new targets for neurotherapeutics in e.g. Parkinson’s disease, schizophrenia, drug addiction, anxiety and depression opening up a new field in neuropsychopharmacology. Along this work, the allosteric receptor-receptor interactions over the interfaces in A2AR-D2R, D2R-NTS1R, D2R-Sigma1R and 5-HT1A-FGFR1 heteroreceptor complexes will be explored and their biochemical, pharmacological and functional integrative implications in the CNS described. Methodologies for studies on receptor- receptor interactions are discussed including the use of FRET and BRET-based techniques in the analysis of G protein coupled receptor (GPCR) dimerization in living cells. In situ proximity ligation assay is performed to establish the existence of native heteroreceptor complexes in the CNS

αCP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome.

Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5' to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5' to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome

Sterol 3β-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone

The main objectives of this work were to characterise a range of purified recombinant sterol 3β-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3β-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3β-glucosyltransferase was used to mine putative sterol 3β-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3β-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17β-hydroxysteroid. This study therefore reports for the first time sterol 3β-glucosyltransferases with selectivity for both 3β- and 17β-hydroxysteroids and is also the first report of recombinant 3β-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest