Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous sequences, such as the human telomeric repeat, and RPA’s ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ forming DNA sequences (PQS) to their stability against RPA mediated unfolding using single molecule FRET and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two located in the promoter region of tyrosine hydroxylase gene are highly heterogeneous sequences, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. Our measurements showed that the most thermally stable structure (Tm= 86 °C) was also the most stable against RPA mediated unfolding, although the least thermally stable structure (Tm= 69 °C) had at least an order of magnitude higher stability against RPA mediated unfolding compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these, we conclude that thermal stability is not necessarily an adequate criterion for predicting physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single molecule stopped-flow type method. All three GQ structures were unfolded within Δt≈0.30±0.10 sec, a surprising result as the unfolding time does not correlate with thermal stability or stability against RPA mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.

The insulin-linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5'-ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b-n). Here, we have investigated the structural polymorphism of G-quadruplexes formed from the most common repeat sequences (a-c) and their effect on insulin protein binding. We first established that the ILPR repeats "b" and "c" can form quadruplex structures. Insulin has previously been shown to bind a G-quadruplex formed by a dimer of the repeat "a". Our findings show that insulin binds preferentially to the repeat "a" G-quadruplex (K(d) = 0.17 + or - 0.03 microM) over G-quadruplexes formed from other ILPR repeats that were tested (K(d)s from 0.71 + or - 0.15 to 1.07 + or - 0.09 microM). Additionally, the Watson-Crick complementary relationship between the loop regions of repeat "a" (ACA and TGT) seemingly play an important role in favoring a specific G-quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat "a" G-quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G-quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G-quadruplexes and begin to provide molecular details on such interactions.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.