Veronica Gonzalez

Identification and Characterization of Nucleolin as a c-myc G-quadruplex-binding Protein

myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85–90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.

The c-MYC NHE III1: Function and Regulation

c-MYC is an important regulator of a wide array of cellular processes necessary for normal cell growth and differentiation, and its dysregulation is one of the hallmarks of many cancers. Consequently, understanding c-MYC transcriptional activation is critical for understanding developmental and cancer biology, as well as for the development of new anticancer drugs. The nuclease hypersensitive element (NHE) III(1) region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. This review focuses on what is known about the formation of a G-quadruplex in the NHE III(1) region of the c-MYC promoter, as well as on those factors that are known to modulate its formation. Last, we discuss the development of small molecules that stabilize or induce the formation of G-quadruplex structures and could potentially be used as anticancer agents.

The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity.

Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85-90% of c-MYCs transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroism spectropolarimetry, thermal stability, and in vitro transcription. Here we report that nucleolins RNA binding domains 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription.

Probing eudesmane cation-π interactions in catalysis by aristolochene synthase with non-canonical amino acids.

Stabilization of the reaction intermediate eudesmane cation (3) through interaction with Trp 334 during catalysis by aristolochene synthase from Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical aromatic amino acids. The amount of germacrene A (2) generated by the mutant enzymes served as a measure of the stabilization of 3. 2 is a neutral intermediate, from which 3 is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp 334 with para-substituted phenylalanines of increasing electron-withdrawing properties led to a progressive accumulation of 2 that showed a good correlation with the interaction energies of simple cations such as Na(+) with substituted benzenes. These results provide compelling evidence for the stabilizing role played by Trp 334 in aristolochene synthase catalysis for the energetically demanding transformation of 2 to 3.

A 1,6-Ring Closure Mechanism for (+)-δ-Cadinene Synthase?

Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1-(2)H(1)](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways.

Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti

The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13-(2)H(6)]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H(2)O and D(2)O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of with PR-AS in D(2)O led to the production of (6R)-[6-(2)H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.

Temperature-Switched Binding of a RuII(dppz)/DNA Light-Switch Complex†

MLCT-based emission of this chiral complex is only “switched on” when it intercalates into the duplex. Numerous studies designed to identify the exact nature of the binding site of the complex, to outline the binding differences between its L and D enantiomers, and to delineate the photophysical details of its binding-induced optical response have been carried out. In order to facilitate the construction of mixed-motif and oligonuclear DNA binding substrates based on the Ru(dppz) moiety, we investigated the properties of achiral [Ru(tpy)(L)(dppz)] complexes (tpy = tris(pyrazolyl)methane, L = monodentate N-donor ligand). Recently, we demonstrated that the DNA binding properties of such systems are highly dependent on small changes in the structure of L; for example, while complex 1 is a typical light-switch DNA intercalator with high binding affinities for duplexes (Kb> 10 m ), complex 2 shows reduced interaction with DNA. Through experimental and computational studies, we ascribed this effect to unfavorable steric interactions involving the amino group of the pyridyl-based pyNH2 ancillary ligand preventing intercalation of the dppz moiety. To explore this unusual behavior more fully, the thermodynamic interaction of 2 with calf thymus (CT)-DNA at 25 8C as well as 10 8C and 35 8C were initially investigated through isothermal calorimetry (ITC). To aid comparisons, the interaction of 1 with CT-DNA under the same conditions was also studied. Although we have previously reported an ITC study on the interaction of 1 with synthetic homopolymers, this is the first study on genomic B-DNA. At all three temperatures, complex 1 binds to CT-DNA through an endothermic, entropically driven interaction (see Table 1 and the Supporting Information). This kind of thermodynamic signature was seen for several other Ru-

Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers

Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar ‘Sweet Laura’ is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. ‘Sweet Laura’ with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. ‘Sweet Laura’ and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. ‘Sweet Laura’ placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R28(R)X8W and D321DXXD are the putative Mg2+-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. ‘Sweet Laura’ flowers.

Chemoenzymatic preparation of germacrene analogues

A small library of novel germacrenes was generated using a combination of two plant enzymes, germacrene A synthase, and D synthase and modified farnesyl diphosphate (FDP) analogues. This chemoenzymatic approach allows the preparation of potentially valuable volatiles for biological studies.

Evolutionary and Mechanistic Insights from the Reconstruction of α‑Humulene Synthases from a Modern (+)-Germacrene A Synthase

Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.