Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy.
Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC₅₀ values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulin-degradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells.
Center for Integrated Protein Science Munich CIPSM, Department of Chemistry, Institute of Advanced Studies IAS, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
Jürgen
J
Eirich
Jens L
JL
Burkhart
Angelika
A
Ullrich
Georg C
GC
Rudolf
Angelika
A
Vollmar
Stefan
S
Zahler
Uli
U
Kazmaier
Stephan A
SA
Sieber
D002460:Cell Line; D019295:Computational Biology; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000378:metabolism*; D015335:Molecular Probes / Q000378:metabolism*; D015394:Molecular Structure; D009842:Oligopeptides / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D015335:Molecular Probes; D009842:Oligopeptides; D014404:Tubulin; C575012:pretubulysin