Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Analysis of gene networks in white adipose tissue development reveals a role for ETS2 in adipogenesis.

Obesity is characterized by an expansion of white adipose tissue mass that results from an increase in the size and the number of adipocytes. However, the mechanisms responsible for the formation of adipocytes during development and the molecular mechanisms regulating their increase and maintenance in adulthood are poorly understood. Here, we report the use of leptin-luciferase BAC transgenic mice to track white adipose tissue (WAT) development and guide the isolation and molecular characterization of adipocytes during development using DNA microarrays. These data reveal distinct transcriptional programs that are regulated during murine WAT development in vivo. By using a de novo cis-regulatory motif discovery tool (FIRE), we identify two early gene clusters whose promoters show significant enrichment for NRF2/ETS transcription factor binding sites. We further demonstrate that Ets transcription factors, but not Nrf2, are regulated during early adipogenesis and that Ets2 is essential for the normal progression of the adipocyte differentiation program in vitro. These data identify ETS2 as a functionally important transcription factor in adipogenesis and its possible role in regulating adipose tissue mass in adults can now be tested. Our approach also provides the basis for elucidating the function of other gene networks during WAT development in vivo. Finally these data confirm that although gene expression during adipogenesis in vitro recapitulates many of the patterns of gene expression in vivo, there are additional developmental transitions in pre and post-natal adipose tissue that are not evident in cell culture systems.

A fission yeast-based platform for phosphodiesterase inhibitor HTSs and analyses of phosphodiesterase activity.

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors that possess "drug-like" characteristics, displaying activity in a growth stimulation assay over a 48-h period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme's potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify whether different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.

Identification of biologically active PDE11-selective inhibitors using a yeast-based high-throughput screen.

The biological roles of cyclic nucleotide phosphodiesterase 11 (PDE11) enzymes are poorly understood, in part due to the lack of selective inhibitors. To address the need for such compounds, we completed an ~200,000 compound high-throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay, and identified 4 potent and selective PDE11 inhibitors. One compound, along with two structural analogs, elevates cAMP and cortisol levels in human adrenocortical cells, consistent with gene association studies that link PDE11 activity to adrenal function. As such, these compounds can immediately serve as chemical tools to study PDE11 function in cell culture, and as leads to develop therapeutics for the treatment of adrenal insufficiencies. Our results further validate this yeast-based HTS platform for the discovery of potent, selective, and biologically active PDE inhibitors.

Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases.

The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1-ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5' AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1-ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2-GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1-ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC(50) of 232nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.

New classes of PDE7 inhibitors identified by a fission yeast-based HTS.

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor alpha from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.