{"id":294,"date":"2015-01-15T23:01:37","date_gmt":"2015-01-15T23:01:37","guid":{"rendered":"http:\/\/alectos.com\/content\/?p=294"},"modified":"2015-01-15T23:01:37","modified_gmt":"2015-01-15T23:01:37","slug":"substrates-imaging-glucocerebrosidase","status":"publish","type":"post","link":"https:\/\/alectos.com\/alectos-content\/index.php\/2015\/01\/15\/substrates-imaging-glucocerebrosidase\/","title":{"rendered":"Fluorescence-quenched substrates for live cell imaging of human glucocerebrosidase activity"},"content":{"rendered":"<p>Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher&#8217;s disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson&#8217;s disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of <span class=\"highlight\">live<\/span> cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within <span class=\"highlight\">live<\/span> mammalian cells. Here we describe the successful modular design and synthesis of <span class=\"highlight\">fluorescence-quenched<\/span> <span class=\"highlight\">substrates<\/span> for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of <span class=\"highlight\">substrates<\/span> that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (\u223c99.9%) fluorescent <span class=\"highlight\">substrates<\/span> also permit assessment of GCase inhibition in <span class=\"highlight\">live<\/span> cells by either confocal microscopy or high content <span class=\"highlight\">imaging<\/span>. Such <span class=\"highlight\">substrates<\/span> should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate <span class=\"highlight\">fluorescence-quenched<\/span> <span class=\"highlight\">substrates<\/span> for other mammalian glycoside hydrolases for use in <span class=\"highlight\">live<\/span> <span class=\"highlight\">cell<\/span> <span class=\"highlight\">imaging<\/span>.<\/p>\n<p>Source: <a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/25562638\" target=\"_blank\" rel=\"noopener\">Yadav, A.K. et al. <em>J Am Chem Soc<\/em> <strong>137,<\/strong> 1181-1189 (2015).<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to  [&#8230;]<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[19],"tags":[48,45,20],"class_list":["post-294","post","type-post","status-publish","format-standard","hentry","category-publications","tag-gcase","tag-parkinsons","tag-sfu"],"_links":{"self":[{"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/posts\/294","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/comments?post=294"}],"version-history":[{"count":0,"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/posts\/294\/revisions"}],"wp:attachment":[{"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/media?parent=294"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/categories?post=294"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/alectos.com\/alectos-content\/index.php\/wp-json\/wp\/v2\/tags?post=294"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}