Structural Perturbations of Exon-Skipping Edits within the Dystrophin D20:24 Region.

Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.

Empirical and Computational Comparison of Alternative Therapeutic Exon Skip Repairs for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common and devastating genetic disease primarily caused by exon deletions that create a genetic frameshift in dystrophin. Exon skipping therapy seeks to correct this by masking an exon during the mRNA maturation process, restoring dystrophin expression, but creating an edited protein missing both the original defect and the therapeutically skipped region. Crucially, it is possible to correct many defects in alternative ways, by skipping an exon either before or after the patient's defect. This results in alternatively edited, hybrid proteins that might have different properties and therapeutic consequences. We examined three such dystrophin exon-skipped edits, Δe45-53, Δe46-54, and Δe47-55, comprising two pairs of alternative repairs of Δe46-53 and Δe47-54 DMD defects. We found that in both cases, Δe46-54 was the more stable repair as determined by a variety of thermodynamic and biochemical measurements. We also examined the origin of these differences with molecular dynamics simulations, which showed that these stability differences were the result of different types of structural perturbations. For example, in one edit there was partial unfolding at the edit site that caused domain-localized perturbations while in another there was unfolding at the protein domain junctions distal to the edit site that increased molecular flexibility. These results demonstrate that alternative exon skip repairs of the same underlying defect can have very different consequences at the level of protein structure and stability and furthermore that these can arise by different mechanisms, either locally or by more subtle long-range perturbations.

Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.

Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main ß-sheet and its specific C-terminal ß-finger.

Exon edited dystrophin rods in the hinge 3 region.

We have studied the properties of a panel of proteins engineered to be end-products of envisioned exon skipping therapy by antisense oligonucleotides, AONs, directed at exon 51 applied to relevant dystrophin defects causing Duchenne muscular dystrophy, DMD. Exon skipping therapy is a leading therapeutic strategy being investigated for the treatment of this devastating genetic disease. AONs targeting exon 51 have progressed furthest in human clinical trials. Exon 51 skipping is applicable to a variety of dystrophin defects found in different patients. Due to the differences in original defect, the end result of the therapy will be different in each case. An open question is whether these differences will produce significant differences in the dystrophin protein so edited. In this study we have identified differences in the stability, structure and lipid binding properties of these end-product proteins produced by exon 51 skipping repair.

Analysis of gene translation using a communications theory approach.

Rapid advances in both genomic data acquisition and computational technology have encouraged the development and use of advanced engineering methods in the field of bioinformatics and computational genomics. Processes in molecular biology can be modeled through the use of these methods. Such processes include identification and annotation of all the functional elements in the genome, including genes and regulatory sequences, which are a fundamental challenge in genomics and computational biology. Since regulatory elements are often short and variable, their identification and discovery using computational algorithms is difficult. However, significant advances have been made in the computational methods for modeling and detection of DNA regulatory elements. This paper proposes a novel use of techniques and principles from communications engineering, coding, and information theory for modeling, identification, and analysis of genomic regulatory elements and biological sequences. The methods proposed are not only able to identify regulatory elements (REs) at their exact locations, but can also "interestingly" distinguish coding from non-coding regions. Therefore, the proposed methods can be utilized to identify genes in the mRNA sequence.

A biophysical map of the dystrophin rod.

We have conducted a biophysical scan of the rod region of dystrophin, targeting all 24 single spectrin type repeat, STR, motifs and 23 2-STR tandem motifs. Of these 47 targets, we were able to express and purify 39 and have characterized them with regard to various stability metrics: thermodynamic stability as assessed by thermal and solvent denaturation, as well as resistance to proteolysis. We find that while all measured parameters varied greatly throughout the rod, there was no general stabilization of the 2-STR motifs over single STR motifs. However, stabilization by thermodynamic interaction was seen in six regions: strongly in D16:17 and D21:22 and to a lesser extent in D2:3, D4:5, D6:7 and D20:21. This indicates that these STRs interact structurally. In the rest of the rod, no cooperativity was seen and STRs appear to be thermodynamically independent. Stability also varied widely along the rod, with some motifs that are barely stable, beginning to unfold at physiological temperatures; these are largely found in the central rod region from D7 to D15. Regions of high stability were found in the interacting motifs, as well as a general trend toward increasing stability at the C-terminus of the rod. Interestingly, the rod region nNOS binding site occurs at such an interacting, very stable site, D16:17. Overall this describes a highly heterogeneous rod region.

Effect of mutations on the detection of translational signals based on a communications theory approach.

Gene and regulatory sequence identification is the first step in the functional annotation of any genome. Identification and annotation of such elements in the genome is a fundamental challenge in genomics and computational biology. Since regulatory elements are often short and variable, their identification and discovery using computational algorithms is difficult. However, significant advances have been made in the computational methods for modeling and detection of DNA regulatory elements. This paper proposes a novel use of techniques and principles from communications engineering and coding theory for modeling, identification and analysis of genomic regulatory elements and biological sequences. The last 13 bases sequence in the 16S rRNA molecule was used as a test sequence and was detected using the proposed models. Results show that the proposed models are not only able to identify this regulatory element (RE) in the mRNA sequence, but also can help identify coding from noncoding regions. The models described in this work where used to study the effect of mutations in the last 13 bases sequence of the 16S rRNA molecule. The obtained results showed total agreement with published investigations on mutations which further certify the biological relevance of the proposed models.

Differential stabilities of alternative exon-skipped rod motifs of dystrophin.

Exon skipping repair is a strategy being investigated in early stage clinical trials to treat Duchenne muscular dystrophy. This is most applicable to the majority of cases which arise when genetic defects cause frame shift mutations, and induced exon skipping of out-of-phase exons restores the reading frame. However, the consequences to the edited protein so produced have not been considered. In many cases alternative routes to restoring the reading frame are possible, and we show in a test case involving exon 44 that the resulting differently edited proteins greatly vary in stability, with one of them very similar to normal unskipped dystrophin, and the other much less stable as assessed by the thermodynamics of folding as well as resistance to proteolysis. This has implications for the design of optimal therapeutic exon skipping strategies, which presumably wish to result repairs with as much fidelity to normal dystrophin as possible.

Stability of dystrophin STR fragments in relation to junction helicity.

Dystrophin is a rod shaped protein consisting of amino- and carboxy-terminal binding domains linked by a large central rod composed of 24 homologous copies of the STR motif and 4 non-homologous regions termed hinges. These hinges are proposed to confer local flexibility; conversely, the tacit implication is that the STR regions away from the hinges are comparatively rigid. This, and the repeating nature of this rod, has contributed to the view that the STR region of the rod is uniform and monolithic. However, we have produced various 2 STR fragments, chosen to have high and low alpha-helix content at their junctions with each other, and show that they exhibit markedly different stabilities. In contrast to a related protein, spectrin, these differences are not correlated with the calculated helicity, but appear to be an intrinsic property of the motifs themselves. A full understanding of how these properties vary along the length of the rod has implications for the engineering of these rods regions in exon skipping and minidystrophin therapies.

Hybrid spectrin type repeats produced by exon-skipping in dystrophin.

Dystrophin is the protein whose defect underlies Duchenne Muscular Dystrophy, DMD, a common (1:3500 male births) and fatal condition in which muscle tissue deteriorates leading to death in the second or third decade of life. Dystrophin is coded for by the largest human gene, and one of the most complex. It is translated from at least 7 distinct promoters, with the largest transcripts (which are the ones involved in DMD) containing 79 exons over >2.5 Mbp [K.F. O'Brien, L.M. Kunkel, Dystrophin and muscular dystrophy: past, present, and future, Mol. Genet. Metab. 74 (2001) 75-88, H.M. Sadoulet-Puccio, L.M. Kunkel, Dystrophin and its isoforms, Brain Pathol. 6 (1996) 25-35]. Exacerbating this complexity, it has recently been shown that dystrophin is subject to extensive alternative RNA processing, potentially producing a wide variety dystrophin variants [M. Sironi, R. Cagliani, U. Pozzoli, A. Bardoni, G.P. Comi, R. Giorda, N. Bresolin, The dystrophin gene is alternatively spliced throughout its coding sequence FEBS Lett 517 (2002) 163-166]. The structure of the dystrophin protein is highly modular, with the most common module being a motif termed the spectrin type repeat, or STR, of which there are 24. Each STR is roughly coded for by two exons, and the most common type of multiple exon-skipping events start and end at introns in the middle of STRs [R.G. Roberts, A.J. Coffey, M. Bobrow, D.R. Bentley, Exon structure of the human dystrophin gene Genomics 16 (1993) 536-538, M. Koenig, L.M. Kunkel, Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility, J. Biol. Chem. 265 (1990) 4560-4566]. This would produce fractional STR modules, however, the concept of STRs as proteins domains makes the viability of such fractional motifs questionable. However, certain of these events produce pairs of potentially complementary fractional domain that might reassemble into a hybrid STR motif. We have constructed model fragment corresponding to one such exon-skipping event, and show that the hybrid STR so produced is viable, and furthermore that some of the properties of the protein containing it differ substantially of the native, un-skipped parent.

Structural cooperativity in spectrin type repeats motifs of dystrophin.

Dystrophin is a member of the spectrin family of proteins, which are characterized as being predominantly composed the spectrin-type-repeat, a triple alpha-helical bundle motif present in multiple tandem copies, producing a rod-like shape. Whether or not this motif, which is determined by sequence homology, is correlated with biophysical domains in the intact protein is uncertain. The nature of the domain structure impacts the flexibility and shape of the rod region of this protein, which is a target for modification in several therapeutic approaches aimed at Duchenne Muscular Dystrophy, a common and fatal genetic disease caused by defective dystrophin. We examined three such motifs in dystrophin, expressing them recombinantly both singly and in tandem, and studying their thermodynamic properties by solvent and thermal denaturation. We have found that the degree to which they are independently stable and expressible varies considerably. The fourth motif appears to be largely stable and independent, whereas the third and second motifs interact strongly.

Identification of TrkA on living PC12 cells by atomic force microscopy.

In neural cells, nerve growth factor (NGF) initiates its survival signal through the binding to its cell surface receptor tyrosine kinase A (TrkA). Understanding the pattern of TrkA distribution and association in living cells can provide a fingerprint for the diagnostic comparison with alterations underlying ligand-receptor dysfunction seen in various neurological diseases. In this study, we use the NGF-TrkA-specific interaction as a probe to identify TrkA on living PC12 cell by atomic force microscopy (AFM). An NGF-modified AFM tip was used to perform force volume (FV) imaging, generating a 2D force map to illustrate the distribution and association of TrkA on PC12 cell membrane. It is found that TrkA is highly aggregated at local regions of the cell. This unique protein association may be required to promote its function as a receptor of NGF. The methodology that we developed in this study can be adapted by other systems, thus providing a general tool for investigating protein association in its natural environment.

Liquid-chromatography-coupled SAXS for accurate sizing of aggregating proteins.

Small-angle X-ray scattering and size-exclusion chromatography have been combined within a unified experimental set-up to obtain molecular size information. Besides providing simultaneous corroborative data bearing on the same question from two distinct experimental techniques, passing the samples over a gel filtration column immediately prior to illumination by X-rays provides both a more homogeneous sample and a continuous set of data as the concentration is extrapolated to zero. This greatly facilitates analysis of data from oligomerizing or aggregating proteins and increases the reliability of the results.