A novel mutation in STXBP1 causing epileptic encephalopathy (late onset infantile spasms) with partial respiratory chain complex IV deficiency.

STXBP1 (MUNC18.1), encoding syntaxin binding protein 1, has been reported in Ohtahara syndrome, a rare epileptic encephalopathy with suppression burst pattern on EEG, in patients with infantile spasms and in a few patients with nonsyndromic mental retardation without epilepsy. We report a patient who presented late onset infantile spasms. Epilepsy was controlled but the patient developed severe mental delay. A first diagnosis of mitochondrial disease was based on clinical presentation and on a partial deficit of respiratory chain complex IV, but molecular screening for mitochondrial genes was negative. The sequencing of STXBP1 gene found a de novo nonsense mutation (c.585C>G/p.Tyr195X). This observation widens the clinical spectrum linked to STXBP1 mutations with the description of a patient with late onset infantile spasms. It raises the question of the value of epilepsy genes screening in patients with uncertain, partial or unconfirmed mitochondrial dysfunction.

Looking for an insulin pill? Use the BRET methodology!

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.

Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer.

We have developed a procedure based on bioluminescence resonance energy transfer (BRET) to monitor the activation state of the insulin receptor in vitro. Human insulin receptor cDNA was fused to either Renilla luciferase (Rluc) or enhanced yellow fluorescent protein (EYFP) coding sequences. Fusion insulin receptors were partially purified by wheat-germ lectin chromatography from human embryonic kidney 293 cells cotransfected with these constructs. The conformational change induced by insulin on its receptor could be detected as an energy transfer (BRET signal) between Rluc and EYFP. BRET signal parallels insulin-induced autophosphorylation of the fusion receptor. Dose-dependent effects of insulin, insulin-like growth factor 1, and epidermal growth factor on BRET signal are in agreement with known pharmacological properties of these ligands. Moreover, antibodies that activate or inhibit the autophosphorylation of the receptor have similar effects on BRET signal. This method allows for rapid analysis of the effects of agonists on insulin receptor activity and could therefore be used in a high-throughput screening test for discovery of molecules with insulin-like properties.