Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1 or TGF-1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1 induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-1 stimulation. Inhibition of IL-1-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed upregulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic doublestranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation.The linear non-sulfated glycosaminoglycan hyaluronan (HA) 5 is a ubiquitous component of the vertebrate extracellular matrix with a multiplicity of cellular functions in both physiological and pathophysiological contexts (1-8). HA is synthesized at the cell membrane by the HA synthase (HAS) enzymes, encoded in humans by the corresponding multigene family HAS1-3 (9 -11).Frequently associated with the fibrotic response in major organs, HA is a highly effective biomarker for liver fibrosis (12,13). Similarly, in lung fibrosis, accumulation of HA has been observed (14, 15), and in some cases, up-regulated HAS2 transcription has also been reported (14). For all progressive renal diseases, the rate of progression correlates with the degree of corticointerstitial fibrosis. Increased HA synthesis and extracellular matrix expansion in the renal corticointerstitium are common features of kidney fibrosis (16), and our recent studies on renal biopsy samples from diabetic nephropathy patients demonstrate that HA is a correlate of interstitial fibrosis in vivo (17). A number of our in vitro studies have suggested that HA may contribute to renal fibrosis via regulation of the renal proximal tubular epithelia...
The coordination chemistry of a Rh(III) porphyrin building block was investigated with a view to the construction of heterometallic arrays of porphyrins. The Rh(III) porphyrin was found to coordinate methanol in the solid state and weakly in CDCl(3) solution. Crystallization afforded five coordinate pi stacked Rh(III) porphyrins. The distribution of products from reaction of Rh(III) porphyrin with DABCO, 4,4'-bipyridine, and 4,4'-bipyrimidine could be displaced toward dimeric species by silica gel column chromatography or recrystallization which served to remove excess ligand. Weak coordination to nitriles was observed, although it was sufficiently strong to organize a dimeric complex of 5,5'-dicyano-2,2'-bipyridine in the solid state. Complexes with 4,4'-bipyrimidine and 5,5'-dicyano-2,2'-bipyridine possess uncoordinated chelating nitrogen atoms. Larger heterometallic porphyrin arrays were assembled using a combination of Sn(IV) and Rh(III) porphyrin coordination chemistry. A Sn(IV) porphyrin acted as a core around which were coordinated two isonicotinate groups, carboxylic acid functionalized porphyrins, or porphyrin trimer dendrons. Rh(III) porphyrins were coordinated to pyridyl groups at the periphery of these entities. In this way an eleven porphyrin array, with four different porphyrin metalation states, was assembled. The diamagnetic nature of both the Rh(III) and Sn(IV) porphyrins, the slow ligand exchange kinetics on the NMR time scale, and tight ligand binding permitted the porphyrin arrays to be analyzed by two-dimensional (1)H NMR techniques.
Cyclic peptides have come under scrutiny as potential antimicrobial therapeutic agents. Combinatorial split-and-pool synthesis of cyclic peptides can afford single compound per well libraries for antimicrobial screening, new lead identification, and construction of quantitative structure-activity relationships (QSAR). Here, we report a new sequencing protocol for rapid identification of the members of a cyclic peptide library based on automated computer analysis of mass spectra, obviating the need for library encoding/decoding strategies. Furthermore, the software readily integrates with common spreadsheet and database packages to facilitate data visualization and archiving. The utility of the new MS-sequencing approach is demonstrated using sonic spray ionization ion trap MS and MS/MS spectrometry on a single compound per bead cyclic peptide library and validated with individually synthesized pure cyclic D,L-alpha-peptides.
The synthesis and characterization of a butadiyne linked Rh(III) porphyrin dimer containing a 4,4‘-bipyridine ligand are presented. The crystal structure is compared to the crystal structures of complexes of a monomeric Rh porphyrin with 4,4‘-bipyridine and pyridine, and with the results of molecular modeling.
We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface plasmon resonance measurements revealed that all compounds bound to the human telomeric quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were used to model the effects of substituent orientation and peptide sequence. Modelling and experiment were in agreement that placement of the peptide over the face of the acridine is detrimental to binding affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that bound best displayed quadruplex affinities in the 1-5 nM range and at least 10-fold discrimination between the quadruplexes studied.
We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5′-(GGTTAG) 5 -3′, and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.The Cys 2 -His 2 zinc finger proteins represent one of the largest classes of proteins encoded in the genomes of all eukaryotes that have been sequenced to date. These proteins typically have multidomain architectures in which individual zinc binding domains are connected by relatively short linker sequences to form arrays of two or more. The biological roles of specific zinc finger proteins have been extensively investigated, and in most cases these proteins function by binding to double-stranded nucleic acids in a sequence specific manner (1-3). The crystal structure of a complex of the murine transcription factor Zif268 with its double-stranded DNA 1 target sequence (4) has provided a structural framework for understanding a wide set of zinc finger protein-DNA interactions. This three zinc finger protein was shown to specifically recognize nine consecutive base pairs, with each zinc finger domain interacting with a triplet of base pairs. † This work was supported by grants from the BBSRC and Cancer Research U.K.
Nine crystal structures of free-base 5,15-diarylporphyrin derivatives are reported, and systematic analysis is made of the molecular conformations and intermolecular interactions in these and other comparable structures. In all cases, the porphyrins show minimal out-of-plane distortion in the solid state, but significant inplane distortion, consistent with previous observations. The simplest 5,15-diarylporphyrin (with no further substitution on the macrocycle periphery) crystallizes as a solvate with features comparable to the structure of porphine itself, and also in an unsolvated form in which edge-to-face interactions between the phenyl substituent and the porphyrin faces give rise to one-dimensional porphyrin chains. The majority of the structures reported here are derived from a balance between these latter interactions and the common offset face-to-face π-stacking interaction. Introduction of substituents such as hydroxyl and methoxy groups on the phenyl rings does not disrupt the edgeto-face interactions. Introduction of bulky groups on both sides of the phenyl substituent can disrupt chain formation, and the structures in these cases are dominated by offset π-stacking. In structures where the porphyrins bear solubilizing n-alkyl groups on the macrocycle periphery, the porphyrin chains generally may be considered to form layers that are stacked with n-alkyl groups filling space between them.
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