{"57713":{"#nid":"57713","#data":{"type":"news","title":"New Technique Detects Enzyme Implicated in Cancer, Atherosclerosis","body":[{"value":"\u003Cp\u003EAn enzyme implicated in osteoporosis, arthritis, atherosclerosis and cancer metastasis -- cathepsin K -- eluded reliable detection in laboratory experiments in the past. Now, a research team at the Georgia Institute of Technology has developed an assay that reliably detects and quantifies mature cathepsin K using a technique called gelatin zymography. \u003C\/p\u003E\u003Cp\u003E\u0022This assay is important because researchers and pharmaceutical companies need a dependable method for sensitively detecting a small amount of cathepsin K and quantifying its activity to develop inhibitors to the enzyme that can fight the diseases while minimizing side effects,\u0022 said Manu Platt, an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. \u003C\/p\u003E\u003Cp\u003ECathepsin K is required to maintain adequate calcium levels in the body, but it can be highly destructive because it has the ability to break down bone by degrading collagen and elastin. \u003C\/p\u003E\u003Cp\u003EPlatt described the cathepsin K detection protocol in the June issue of the journal \u003Cem\u003EAnalytical Biochemistry\u003C\/em\u003E. This research was funded by new faculty support from Georgia Tech, and the Facilitating Academic Careers in Engineering and Science Scholars (FACES) and Summer Undergraduate Research in Engineering (SURE) programs at Georgia Tech. \u003C\/p\u003E\u003Cp\u003EThe benefits of this assay over existing techniques are numerous, according to Platt. The major advantage of this protocol, he said, is the definitive knowledge that mature cathepsin K is being detected in cells and tissues -- and not its immature form or one of the other 10 cathepsin varieties: B, H, L, S, C, O, F, V, X or W. \u003C\/p\u003E\u003Cp\u003EAnother advantage of this technique is that it is more sensitive and less expensive than current, less reliable techniques. The new assay allows cathepsin K to be detected in quantities as small as a few femtomoles and does not require antibodies, which can be expensive and cannot be used across different species. \u003C\/p\u003E\u003Cp\u003E\u0022In our experiments we were able to detect mature cathepsin K activity in quantities as small as 3.45 femtomoles with zymography, which was 10 to 50 times more sensitive at detecting the enzyme than conventional Western blotting,\u0022 noted Platt, who is also a Georgia Cancer Coalition Distinguished Cancer Scholar. \u003C\/p\u003E\u003Cp\u003EIn addition, zymography allowed the researchers to measure the activity of the enzyme, whereas Western blotting just measured its presence. \u003C\/p\u003E\u003Cp\u003ETo detect mature cathepsin K with gelatin zymography, Platt and Georgia Tech undergraduate student Weiwei Li first separated the enzymes present in cells by their molecular weights. This allowed them to distinguish the mature form of cathepsin K from the immature form and other cathepsin varieties. \u003C\/p\u003E\u003Cp\u003EThen, to verify the identity and presence of mature cathepsin K, the team activated the enzymes in the gel. They created the perfect acidic environment for cathepsin K to thrive and added inhibitors to block the activity of all enzymes except mature cathepsin K. \u003C\/p\u003E\u003Cp\u003ETo validate the cathepsin K activity detected in the laboratory experiments, Platt and Georgia Tech undergraduate student Zachary Barry developed a computational kinetic model of the enzyme\u0027s activity. By solving a system of differential equations, they were able to calculate the concentrations of immature, mature and inactive cathepsin K present at all times during the experimental procedure. \u003C\/p\u003E\u003Cp\u003E\u0022It is more challenging to work with enzymes than proteins because enzymes have to be functional, which means they have to be folded correctly to be active,\u0022 explained Platt. \u0022The model suggested that even after the slight denaturation and refolding required by our assay, the cathepsin K activity determined by zymography reflected what happens in nature and was not an artifact of the experimental procedure.\u0022 \u003C\/p\u003E\u003Cp\u003EThe model also predicted something unexpected -- the inactive form of cathepsin K commonly purchased from supply houses contained 20 percent mature enzyme. \u003C\/p\u003E\u003Cp\u003E\u0022Cathepsins are implicated in many different diseases and the value of this assay is that it enables the measurement of previously undeterminable cathepsin activity in normal and diseased cells and tissues,\u0022 noted Platt. \u003C\/p\u003E\u003Cp\u003EWith this assay, Platt\u2019s team is currently investigating whether cathepsin K activity is different in the cells of individuals with metastatic and non-metastatic breast and prostate cancers, and the role of cathepsin K in cardiovascular diseases, such as stroke, in children with sickle cell anemia. They are also examining whether cathepsin K plays a role in the inflammation associated with HIV. \u003C\/p\u003E\u003Cp\u003E\u0022This research should provide new information on a number of existing pathophysiological conditions where cathepsin K activity had been previously undetectable,\u0022 added Platt. \u003C\/p\u003E\u003Cp\u003EAdditional contributors to this work included Georgia Tech research technologists Catera Wilder and Philip Keegan; former graduate student Rebecca Deeds; and Joshua Cohen, a summer researcher at Georgia Tech and currently an undergraduate at the Massachusetts Institute of Technology. \u003C\/p\u003E\u003Cp\u003E\u003Cstrong\u003EResearch News \u0026amp; Publications Office\u003Cbr \/\u003EGeorgia Institute of Technology\u003Cbr \/\u003E75 Fifth Street, N.W., Suite 314\u003Cbr \/\u003EAtlanta, Georgia 30308 USA\u003C\/strong\u003E \u003C\/p\u003E\u003Cp\u003E\u003Cstrong\u003EMedia Relations Contacts\u003C\/strong\u003E: Abby Vogel (\u003Ca href=\u0022mailto:avogel@gatech.edu\u0022\u003Eavogel@gatech.edu\u003C\/a\u003E)(404-385-3364) or John Toon (\u003Ca href=\u0022mailto:jtoon@gatech.edu\u0022\u003Ejtoon@gatech.edu\u003C\/a\u003E)(404-894-6986) \u003C\/p\u003E\u003Cp\u003E\u003Cstrong\u003EWriter\u003C\/strong\u003E: Abby Vogel \u003C\/p\u003E","summary":null,"format":"limited_html"}],"field_subtitle":"","field_summary":[{"value":"\u003Cp\u003EA Georgia Tech research team has developed a new technique that reliably detects and quantifies an enzyme implicated in osteoporosis, arthritis, atherosclerosis, cancer metastasis and other disease processes.\u003C\/p\u003E","format":"limited_html"}],"field_summary_sentence":[{"value":"An enzyme important to many disease processes can now be detected"}],"uid":"27303","created_gmt":"2010-06-01 00:00:00","changed_gmt":"2016-10-08 03:06:39","author":"John Toon","boilerplate_text":"","field_publication":"","field_article_url":"","dateline":{"date":"2010-06-01T00:00:00-04:00","iso_date":"2010-06-01T00:00:00-04:00","tz":"America\/New_York"},"extras":[],"hg_media":{"57714":{"id":"57714","type":"image","title":"Manu Platt detecting key enzyme","body":null,"created":"1449176051","gmt_created":"2015-12-03 20:54:11","changed":"1475894506","gmt_changed":"2016-10-08 02:41:46","alt":"Manu Platt detecting key enzyme","file":{"fid":"190638","name":"ths97847.jpg","image_path":"\/sites\/default\/files\/images\/ths97847_0.jpg","image_full_path":"http:\/\/tlwarc.hg.gatech.edu\/\/sites\/default\/files\/images\/ths97847_0.jpg","mime":"image\/jpeg","size":1624014,"path_740":"http:\/\/tlwarc.hg.gatech.edu\/sites\/default\/files\/styles\/740xx_scale\/public\/images\/ths97847_0.jpg?itok=YlTZ3YIC"}},"57715":{"id":"57715","type":"image","title":"Manu Platt detecting key enzyme","body":null,"created":"1449176051","gmt_created":"2015-12-03 20:54:11","changed":"1475894506","gmt_changed":"2016-10-08 02:41:46","alt":"Manu Platt detecting key enzyme","file":{"fid":"190639","name":"trg97847.jpg","image_path":"\/sites\/default\/files\/images\/trg97847_0.jpg","image_full_path":"http:\/\/tlwarc.hg.gatech.edu\/\/sites\/default\/files\/images\/trg97847_0.jpg","mime":"image\/jpeg","size":1277779,"path_740":"http:\/\/tlwarc.hg.gatech.edu\/sites\/default\/files\/styles\/740xx_scale\/public\/images\/trg97847_0.jpg?itok=1s1L0xrr"}}},"media_ids":["57714","57715"],"related_links":[{"url":"http:\/\/www.bme.gatech.edu\/","title":"Wallace H. Coulter Department of Biomedical Engineering"},{"url":"http:\/\/www.bme.gatech.edu\/facultystaff\/faculty_record.php?id=111","title":"Manu Platt"}],"groups":[{"id":"1188","name":"Research Horizons"}],"categories":[{"id":"140","name":"Cancer Research"},{"id":"146","name":"Life Sciences and Biology"},{"id":"135","name":"Research"}],"keywords":[{"id":"9686","name":"assay"},{"id":"7270","name":"atherosclerosis"},{"id":"385","name":"cancer"},{"id":"1108","name":"detection"},{"id":"7735","name":"enzyme"}],"core_research_areas":[],"news_room_topics":[],"event_categories":[],"invited_audience":[],"affiliations":[],"classification":[],"areas_of_expertise":[],"news_and_recent_appearances":[],"phone":[],"contact":[{"value":"\u003Cp\u003E\u003Cstrong\u003EJohn Toon\u003C\/strong\u003E\u003Cbr \/\u003EResearch News \u0026amp; Publications Office\u003Cbr \/\u003E\u003Ca href=\u0022http:\/\/www.gatech.edu\/contact\/index.html?id=jt7\u0022\u003EContact John Toon\u003C\/a\u003E\u003Cbr \/\u003E\u003Cstrong\u003E404-894-6986\u003C\/strong\u003E\u003C\/p\u003E","format":"limited_html"}],"email":["jtoon@gatech.edu"],"slides":[],"orientation":[],"userdata":""}}}