measured in a multilabel counter with a TR FRET option The count

measured in a multilabel counter with a TR FRET option. The counter setting was 340 nm excitation, 100 us delay, and dual emission collection for 200 us at 495 and 520 nm. The energy transfer signal data were used to calcu late the percentage inhibition and IC50 values. To moni tor the assay system and to compare the hit compounds, Bayer compound was used as a positive control. Lafora disease is an selleck compound autosomal recessive, neurode generative disorder resulting in myoclonus, epilepsy, de mentia, and death. Affected individuals experience an initial seizure during adolescence, followed by severe neuro logical decline until the patients death approximately ten years after the first seizure. Characteristic of the dis ease is the cytoplasmic accumulation of hyperphosphory lated glycogen like particles called Lafora bodies in various tissues including brain, muscle and liver.

Approximately 50% of Lafora disease cases are caused by mutations in the EPM2A gene that encodes the protein laforin. EPM2A is conserved in all vertebrate ge nomes, but Inhibitors,Modulators,Libraries it is absent from the genome of most non vertebrate organisms including standard model organ isms such as Saccharomyces Inhibitors,Modulators,Libraries cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. An exception to this rule is a small subgroup of protists that synthesize floridean starch, an insoluble carbohydrate similar to LBs. Five protozoan laforin orthologs have been identified, however, sequence identity between these proteins and human laforin Inhibitors,Modulators,Libraries is 37% and the genes have major inser tions and Inhibitors,Modulators,Libraries deletions. Thus, these proteins are not opti mal orthologs to utilize for modeling human laforin.

Laforin is a bimodular protein with a carbohydrate binding module at its amino terminus and a dual specificity phosphatase domain at its carboxy terminus. CBMs are most commonly found in glyco syl hydrolases and glucosyl transferases from bacteria, fungi or plants, and there are over 39 families of CBMs that bind a variety of carbohydrate GSK-3 substrates. Laforin belongs to the CBM20 family according to the Carbohydrate Active En zymes database. CBM20s are closely related to CBM48s, and both are classified as starch binding domains with similar folds and binding sites. Typical of DSPs, laforin is capable of hydrolyzing phosphotyrosine and phosphoserine phos phothreonine substrates, however, laforin is unique among phosphatases in that it is the only phosphatase in humans containing a CBM, which targets laforin to glycogen.

Laforin has been shown to bind and de phosphorylate glycogen and other glucans in vitro and in vivo. Glycogen is an energy storage molecule namely synthesized by bacterial, fungal and animal species consisting of 1,4 and 1,6 linked residues of glucose, with 12 14 residues per branch. Glycogen has been shown to contain small amounts of phosphate, but the regulation and ef fects of this phosphorylation event are currently under debate. While the source of phosphorylation is disputable, data from multiple labs has clearly estab lished tha

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