Modular dispensability of dysferlin C2 domains reveals rational design for mini-dysferlin molecules.

Dysferlin is a large transmembrane protein composed of a C-terminal transmembrane domain, two DysF domains, and seven C2 domains that mediate lipid- and protein-binding interactions. Recessive loss-of-function mutations in dysferlin lead to muscular dystrophies, for which no treatment is currently available. The large size of dysferlin precludes its encapsulation into an adeno-associated virus (AAV), the vector of choice for gene delivery to muscle. To design mini-dysferlin molecules suitable for AAV-mediated gene transfer, we tested internally truncated dysferlin constructs, each lacking one of the seven C2 domains, for their ability to localize to the plasma membrane and to repair laser-induced plasmalemmal wounds in dysferlin-deficient human myoblasts. We demonstrate that the dysferlin C2B, C2C, C2D, and C2E domains are dispensable for correct plasmalemmal localization. Furthermore, we show that the C2B, C2C, and C2E domains and, to a lesser extent, the C2D domain are dispensable for dysferlin membrane repair function. On the basis of these results, we designed small dysferlin molecules that can localize to the plasma membrane and reseal laser-induced plasmalemmal injuries and that are small enough to be incorporated into AAV. These results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models.

Factors influencing the inhibition of protein kinases.

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

mTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4.

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.