Antisense Peptides: Tools for Receptor Isolation? Lack of Antisense Msh and Acth to Interact with their Sense Peptides and to Induce Receptor-Specific Antibodies

Abstract

The use of antisense peptides for receptor isolation as proposed by Blalock and his colleages (e.g. TIBTECH 8, 140–144, 1990) was tested for human ACTH as well as α- and β-MSH. We synthesized the corresponding antisense peptides HTCA_h, HSM-α and HSM-β and analyzed them for specific interaction with the sense peptides using several types of binding assay and bioassay. Similarly HTCA_h antibodies were tested for binding to ACTH receptors and ACTH antibodies. All these experiments were negative, i.e. there was no specific interaction between sense and antisense peptides nor between the corresponding antibodies. Receptor binding of the sense peptides was not affected by the antisense peptides or HTCA_h antibodies. Unexpectedly, HTCA_h but not HSM-α or HSM-β was a weak MSH agonist acting through a site independent of the MSH receptor. A detailed analysis of the concept of antisense peptides revealed that the theoretical background of the hypothesis of the ‘molecular recognition theory’ is rather weak, explaining the failure of various attempts to obtain specific receptor antibodies.     

Mismatch Recognition Protein MutS�� Does Not Hijack (CAG)n Hairpin Repair in Vitro

CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)_n expansion is caused by abnormal binding of the MutSβ mismatch recognition protein to (CAG)_n hairpins, leading to hijacking mismatch repair function during (CAG)_n hairpin repair. We demonstrate here that MutSβ displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)_n hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSβ does not inhibit (CAG)_n hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSβ is involved in CAG repeat instability in humans.Expansion of trinucleotide repeats (TNRs)³ causes hereditary neurological disorders such as Huntington disease and myotonic dystrophy, whose clinical symptoms are directly linked to expansion of CAG and CTG repeats, respectively (1–3). The precise mechanisms by which TNR expansion occurs and the factors that promote it are not fully understood. It has been proposed that CAG and CTG repeats form thermostable hairpins that include A-A and T-T mispairs in the hairpin stem (4, 5). Therefore, cellular mechanisms that process DNA hairpin/loop structures and/or A-A or T-T mispairs may influence TNR stability.Recent studies have identified and characterized a DNA hairpin repair (HPR) system in human cells that promotes CAG/CTG repeat stability (6, 7). The mechanism of human HPR involves incision and removal of CAG/CTG repeat hairpins in a nick-directed and proliferating cell nuclear antigen-dependent manner, followed by DNA resynthesis using the continuous strand as a template (6). In addition to human HPR, the human mismatch repair (MMR) system is well known for its role in stabilizing simple repetitive sequences called microsatellites, which are prone to forming small loops or insertion/deletion (ID) mispairs. In human cells, MutSα (MSH2–MSH6) and MutSβ (MSH2–MSH3) both bind to 1–2-nt ID mispairs, but MutSβ has higher affinity for these small loops (8). Defects in MMR genes cause microsatellite instability and predisposition to cancer (9), demonstrating that MMR is essential for genetic stability in human cells. Surprisingly, genetic studies in mice suggest that MutSβ promotes (CAG)_n expansion and TNR instability. These studies show that expansion of a heterologous (CAG)_n tract occurs in wild type and MSH6^−/− mice but that expansion of the (CAG)_n tract is suppressed in MSH2^−/− and MSH3^−/− mice (10, 11). Recently, Owens et al. (11) reported that binding to a (CAG)_n hairpin influences the protein conformation, nucleotide binding, and hydrolysis activities of MutSβ so that they are different from what has been reported for MutSα during mismatch recognition. It is therefore hypothesized that (CAG)_n hairpins, through their ability to alter the biochemical properties of MutSβ, hijack the MMR process, leading to CAG repeat expansion instead of CAG hairpin removal (11). However, it is not clear why MMR, a major genome maintenance system, would promote TNR instability instead of TNR stability. We, therefore, have developed a novel functional assay and examined the validity of this hypothesis. Our results reveal that MutSβ displays normal biochemical activities when binding to CAG hairpins and does not inhibit (CAG)_n hairpin repair. The observations presented here provide novel thoughts on whether or not or how MutSβ is involved in CAG repeat instability in human cells.     

The Comparative Genomics of Polyglutamine Repeats: Extreme Difference in the Codon Organization of Repeat-Encoding Regions Between Mammals and Drosophila

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes. Received: 17 August 2000 / Accepted: 20 November 2000