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Altered biochemical specificity of G-quadruplexes with mutated tetrads.

ABSTRACT: A fundamental motif in canonical nucleic acid structure is the base pair. Mutations that disrupt base pairs are typically destabilizing, but stability can often be restored by a second mutation that replaces the original base pair with an isosteric variant. Such concerted changes are a way to identify helical regions in secondary structures and to identify new functional motifs in sequenced genomes. In principle, such analysis can be extended to non-canonical nucleic acid structures, but this approach has not been utilized because the sequence requirements of such structures are not well understood. Here we investigate the sequence requirements of a G-quadruplex that can both bind GTP and promote peroxidase reactions. Characterization of all 256 variants of the central tetrad in this structure indicates that certain mutations can compensate for canonical G-G-G-G tetrads in the context of both GTP-binding and peroxidase activity. Furthermore, the sequence requirements of these two motifs are significantly different, indicating that tetrad sequence plays a role in determining the biochemical specificity of G-quadruplex activity. Our results provide insight into the sequence requirements of G-quadruplexes, and should facilitate the analysis of such motifs in sequenced genomes.

SUBMITTER: Svehlova K

PROVIDER: S-EPMC5159562 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Altered biochemical specificity of G-quadruplexes with mutated tetrads.

Švehlová Kateřina K Lawrence Michael S MS Bednárová Lucie L Curtis Edward A EA

Nucleic acids research 20161026 22

A fundamental motif in canonical nucleic acid structure is the base pair. Mutations that disrupt base pairs are typically destabilizing, but stability can often be restored by a second mutation that replaces the original base pair with an isosteric variant. Such concerted changes are a way to identify helical regions in secondary structures and to identify new functional motifs in sequenced genomes. In principle, such analysis can be extended to non-canonical nucleic acid structures, but this ap ...[more]

PMID: 27789695

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Project description:BackgroundCaveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage. So far, more than 40 dominant pathogenic mutations have been described leading to several phenotypes many of which are associated with a mis-localization of the mutant protein to the Golgi. Golgi retention and endoplasmic reticulum (ER) stress has been demonstrated for the CAV3 p.P104L mutation, but further downstream pathophysiological consequences remained elusive so far.MethodsWe utilized a transgenic (p.P104L mutant) mouse model and performed proteomic profiling along with immunoprecipitation, immunofluorescence and immunoblot examinations (including examination of α-dystroglycan glycosylation), and morphological studies (electron and coherent anti-Stokes Raman scattering (CARS) microscopy) in a systematic investigation of molecular and subcellular events in p.P104L caveolinopathy.ResultsOur electron and CARS microscopic as well as immunological studies revealed Golgi and ER proliferations along with a build-up of protein aggregates further characterized by immunoprecipitation and subsequent mass spectrometry. Molecular characterization these aggregates showed affection of mitochondrial and cytoskeletal proteins which accords with our ultra-structural findings. Additional global proteomic profiling revealed vulnerability of 120 proteins in diseased quadriceps muscle supporting our previous findings and providing more general insights into the underlying pathophysiology. Moreover, our data suggested that further DGC components are altered by the perturbed protein processing machinery but are not prone to form aggregates whereas other sarcolemmal proteins are ubiquitinated or bind to p62. Although the architecture of the ER and Golgi as organelles of protein glycosylation are altered, the glycosylation of α-dystroglycan presented unchanged.ConclusionsOur combined data classify the p.P104 caveolinopathy as an ER-Golgi disorder impairing proper protein processing and leading to aggregate formation pertaining proteins important for mitochondrial function, cytoskeleton, ECM remodeling and sarcolemmal integrity. Glycosylation of sarcolemmal proteins seems to be normal. The new pathophysiological insights might be of relevance for the development of therapeutic strategies for caveolinopathy patients targeting improved protein folding capacity.

Dataset Information

Altered biochemical specificity of G-quadruplexes with mutated tetrads.

Publications

Altered biochemical specificity of G-quadruplexes with mutated tetrads.

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