Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E 90 K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E 90 K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.protein trafficking | protein misrouting | intracellular trafficking

The SMYD (SET and MYND domain) family of lysine methyltransferases harbor a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein-protein interaction MYND domain. SMYD proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Smyd1 is critical for development of the right ventricle in mice. In zebrafish, Smyd1 is necessary for sarcomerogenesis in fast-twitch muscles. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. To determine the role of Smyd1 in mammalian myogenesis, we conditionally eliminated Smyd1 from the skeletal muscle lineage at the myoblast stage using Myf5(cre). Deletion of Smyd1 impaired myoblast differentiation, resulted in fewer myofibers and decreased expression of muscle-specific genes. Muscular defects were temporally restricted to the second wave of myogenesis. Thus, in addition to the previously described functions for Smyd1 in heart development and skeletal muscle sarcomerogenesis, these results point to a novel role for Smyd1 in myoblast differentiation.

The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity.

GnRH, produced in the hypothalamus, acts on pituitary gonadotropes to stimulate release of the gonadotropins LH and FSH. Reduced responsiveness of gonadotropes to GnRH is a primary cause of hypogonadotropic hypogonadism (HH), a disease characterized by gonadal dysfunction and low blood levels of gonadotropins. Loss-of-function mutations in the gene encoding the receptor for GnRH (GNRHR) are a common cause of HH. Sequencing of the GNRHR gene in patients with HH revealed mainly point mutations producing single amino acid substitutions that cause misfolding and misrouting of this G protein-coupled receptor. To generate a mouse model that mimics the human disease, we introduced a single amino acid substitution (E90K) into the mouse Gnrhr gene, which is identical to a known human recessive mutation. In humans, E90K causes severe HH by preventing formation of the E90-K121 salt bridge, which is essential for correct folding. In cell cultures, E90K causes misfolding that leads to almost complete retention by the protein quality control system and subsequent degradation. Here we report that the primary phenotype of mice homozygous for E90K is female infertility due to ovulation failure. Mutant males are fertile despite reduced gonadotropin levels and smaller testes. These results suggest decreased GnRH receptor signaling in the mutant animal, compared with wild type. Our findings suggest that a threshold level of GnRH receptor activity is required for ovulation.

A new and efficient strategy for labeling of RNA sequences prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the 3'-phosphate of cleaved RNA fragments with a fluorescent molecule bearing aromatic bromomethyl function.