J K Chen1, W S Lane, S L Schreiber. 1. Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Abstract
BACKGROUND: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography. RESULTS: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis. CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.
BACKGROUND:Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography. RESULTS:Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murineLCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis. CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.
Authors: Volker Brinkmann; Andreas Billich; Thomas Baumruker; Peter Heining; Robert Schmouder; Gordon Francis; Shreeram Aradhye; Pascale Burtin Journal: Nat Rev Drug Discov Date: 2010-10-29 Impact factor: 84.694
Authors: Stuart L Schreiber; Joanne D Kotz; Min Li; Jeffrey Aubé; Christopher P Austin; John C Reed; Hugh Rosen; E Lucile White; Larry A Sklar; Craig W Lindsley; Benjamin R Alexander; Joshua A Bittker; Paul A Clemons; Andrea de Souza; Michael A Foley; Michelle Palmer; Alykhan F Shamji; Mathias J Wawer; Owen McManus; Meng Wu; Beiyan Zou; Haibo Yu; Jennifer E Golden; Frank J Schoenen; Anton Simeonov; Ajit Jadhav; Michael R Jackson; Anthony B Pinkerton; Thomas D Y Chung; Patrick R Griffin; Benjamin F Cravatt; Peter S Hodder; William R Roush; Edward Roberts; Dong-Hoon Chung; Colleen B Jonsson; James W Noah; William E Severson; Subramaniam Ananthan; Bruce Edwards; Tudor I Oprea; P Jeffrey Conn; Corey R Hopkins; Michael R Wood; Shaun R Stauffer; Kyle A Emmitte Journal: Cell Date: 2015-06-04 Impact factor: 41.582
Authors: Vishal C Mehra; Elias Jackson; Xian M Zhang; Xian-Cheng Jiang; Lawrence W Dobrucki; Jun Yu; Pascal Bernatchez; Albert J Sinusas; Gerald I Shulman; William C Sessa; Timur O Yarovinsky; Jeffrey R Bender Journal: Am J Pathol Date: 2014-03-05 Impact factor: 4.307
Authors: Jihui Ren; Justin Snider; Michael V Airola; Aaron Zhong; Nadia A Rana; Lina M Obeid; Yusuf A Hannun Journal: J Lipid Res Date: 2017-11-01 Impact factor: 5.922
Authors: Marine C C Raman; Kenneth A Johnson; Beverley A Yard; Jonathan Lowther; Lester G Carter; James H Naismith; Dominic J Campopiano Journal: J Biol Chem Date: 2009-04-17 Impact factor: 5.157