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Disease-Proportional Proteasomal Degradation of Missense Dystrophins

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Disease-Proportional Proteasomal Degradation of Missense Dystrophins Disease-proportional proteasomal degradation of missense dystrophins Dana M. Talsness, Joseph J. Belanto, and James M. Ervasti1 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota–Twin Cities, Minneapolis, MN 55455 Edited by Louis M. Kunkel, Children’s Hospital Boston, Harvard Medical School, Boston, MA, and approved September 1, 2015 (received for review May 5, 2015) The 427-kDa protein dystrophin is expressed in striated muscle insertions or deletions (indels) represent ∼7% of the total DMD/ where it physically links the interior of muscle fibers to the BMD population (13). When indel mutations cause a frameshift, they extracellular matrix. A range of mutations in the DMD gene encod- can specifically be targeted by current exon-skipping strategies (15). ing dystrophin lead to a severe muscular dystrophy known as Du- Patients with missense mutations account for only a small percentage chenne (DMD) or a typically milder form known as Becker (BMD). of dystrophinopathies (<1%) (13), yet they represent an orphaned Patients with nonsense mutations in dystrophin are specifically tar- subpopulation with an undetermined pathomechanism and no cur- geted by stop codon read-through drugs, whereas out-of-frame de- rent personalized therapies. letions and insertions are targeted by exon-skipping therapies. Both The first missense mutation reported to cause DMD was L54R treatment strategies are currently in clinical trials. Dystrophin mis- in ABD1 of an 8-y-old patient (16). Another group later reported sense mutations, however, cause a wide range of phenotypic se- L172H, a missense mutation in a structurally analogous location of verity in patients. The molecular and cellular consequences of such ABD1 (17), yet this patient presented with mild symptoms at 42 mutations are not well understood, and there are no therapies spe- years of age. We first hypothesized that differential effects on cifically targeting this genotype. Here, we have modeled two rep- actin-binding affinity could account for differences in disease se- resentative missense mutations, L54R and L172H, causing DMD and verity between L54R and L172H. However, biochemical assays BMD, respectively, in full-length dystrophin. In vitro, the mutation reported only nominal effects of six different missense mutations, associated with the mild phenotype (L172H) caused a minor de- including L54R and L172H, on actin-binding affinity (18). In crease in tertiary stability, whereas the L54R mutation associated vitro biophysical analyses of the full-length proteins instead with a severe phenotype had a more dramatic effect. When stably revealed that each of the mutations rendered dystrophin more expressed in mammalian muscle cells, the mutations caused steady- insoluble and less stable during thermal denaturation. Missense state decreases in dystrophin protein levels inversely proportional mutations analyzed in purified ABD1 fragments also exhibited to the tertiary stability and directly caused by proteasomal degra- significant aggregation as assessed by thioflavin T fluorescence, dation. Both proteasome inhibitors and heat shock activators were Congo red staining, and electron microscopy (19). able to increase mutant dystrophin to WT levels, establishing the Although previous research indicates that missense mutations new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. in dystrophin ABD1 cause protein instability and aggregation, no in vitro measurements were able to distinguish between missense Duchenne muscular dystrophy | missense mutation | protein folding | mutations that cause severe DMD versus those that cause mild proteasome inhibitor | heat shock activator BMD symptoms. There is also currently no understanding of the in vivo cellular consequences of dystrophin instability. If such protein instability does indeed cause protein aggregation in the n striated muscle cells, the contraction of sarcomeres is coupled cell, therapeutic methods may aim at removal or resolubilization Ito the sarcolemma through the costamere protein network (1). One substructure of the costamere, known as the dystrophin– glycoprotein complex (DGC), physically links the cytoskeleton to Significance the extracellular matrix, contributing to myofiber integrity during contraction and relaxation (2). One of the key components of the Duchenne and Becker muscular dystrophies occur at a frequency DGC, dystrophin, is a 427-kDa structural protein composed of an of 1:4,000 live male births. Patients experience progressive muscle N-terminal actin binding domain (ABD1), a central rod region weakness and often succumb to cardiac or respiratory failure. made of 24 spectrin-like repeats that contain binding domains for Many laboratories are investigating potential treatments, in- several proteins (3–6), a cysteine-rich (CR) globular domain, and cluding several therapies that target subsets of patients with an intrinsically disordered C-terminal tail (CT) (7). Dystrophin is certain types of mutations. Yet there are currently no treatments encoded by the DMD gene, which is located on the X chromo- specific to patients with missense mutations in the affected pro- some and spans over 2.4 Mb of DNA, making it the largest in the tein, dystrophin. Here, we have shown missense mutant dystro- human genome (8). phin is degraded by the cell, which can be prevented by several Males who harbor a disease-causing mutation in their single copy small molecules targeting the cellular degradation machinery. In of the DMD gene develop a muscular dystrophy either with severe addition to determining disease mechanism, this work has gen- erated new cell-based models that may aid in the discovery of symptoms known as Duchenne (DMD) or typically milder symp- personalized medicines for dystrophinopathy patients afflicted toms referred to as Becker (BMD) (9), occurring with an incidence with missense mutations. of ∼1 in 4,000 live male births (10). Muscle degeneration is pro- gressive and boys lose ambulation in their teenage years. Death Author contributions: J.M.E. designed research; D.M.T. performed research; D.M.T. and occurs in the second or third decade of life, commonly due to J.J.B. contributed new reagents/analytic tools; D.M.T. analyzed data; and D.M.T. and J.M.E. cardiac or respiratory failure (11). The types of mutations causing wrote the paper. DMD and BMD include insertions, deletions, point mutations, and The authors declare no conflict of interest. splice site mutations (12). A recent collaborative data bank has This article is a PNAS Direct Submission. calculated that patients with nonsense mutations account for ∼10% 1To whom correspondence should be addressed. Email: [email protected]. of dystrophinopathies (13), all of whom are the target population This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for stop codon read-through therapies (14). Patients with small 1073/pnas.1508755112/-/DCSupplemental. 12414–12419 | PNAS | October 6, 2015 | vol. 112 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1508755112 Downloaded by guest on September 25, 2021 of aggregates akin to treatments for polyglutamine diseases or protein submissions to the SNP database (rs145668843). To determine the aggregate myopathies (20, 21). If, however, the unstable dystrophin stability of the tertiary protein structures, each purified protein was protein is degraded, then more effective disease treatments may in- measured by DSF over a temperature gradient from 20 to 90 °C (Fig. stead focus on stabilizing the mutant protein. Here, we use differ- 1B). WT dystrophin showed a cooperative unfolding transition (Tm = ential scanning fluorimetry (DSF) to assess the in vitro tertiary 43.36 °C) and the SNP I232M gave a similar melt curve. Mutant structure of L54R and L172H mutant dystrophins, and found that the dystrophin L172H was slightly left shifted (Tm = 42.08 °C), indicating more severe mutation (L54R) caused greater tertiary instability. asmalldecreaseinproteinstability. L54R was more left-shifted Additionally, we generated stable myoblast lines expressing L54R and (Tm = 40.03 °C), suggesting either greater thermal instability or the L172H dystrophin, which revealed that the mutant proteins are de- presence of a partially unfolded state of the protein at initiation of the graded by the proteasome to a degree that is inversely proportional to experiment. These DSF results indicate that L54R and L172H cause their in vitro stabilities. Flow cytometry used to detect dystrophin improper folding of dystrophin, with the mutation leading to severe showed several small molecules capable of increasing the mutant disease symptoms (L54R) having a greater deleterious effect. protein levels, supporting the cell lines’ potential for future high- As a sensitive measure of protein aggregation, the hydrodynamic throughput screens for dystrophin stabilizers. radius of full-length protein was measured by dynamic light scat- tering (DLS) (Fig. 1 C and D). DLS analysis of dystrophin isoforms Results Dp71 and Dp260, and full-length dystrophin yielded z-average sizes L54R and L172H Cause Different Degrees of Protein Instability in that correlated positively with increasing molecular weight as Vitro. We expressed and purified full-length WT, L54R, and expected (Fig. S1). Boiling full-length WT dystrophin protein in- L172H dystrophins (Fig. 1A) using the previously established bacu- creased its z-average size by approximately fourfold (Fig. 1C), lovirus
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