Photocrosslinkers illuminate interactions in living cells

Tanaka, Yoshihito; Bond, Michelle; Kohler, Jennifer J.

doi:10.1039/b803218a

Cited by 163 publications

(165 citation statements)

References 65 publications

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“…Second, both cell surface glycan-mediated interactions (35) and FG-repeat nucleoporin-nuclear transport factor interactions are typically multivalent (34,36), which may enhance interaction specificity. Thus, the use of photocrosslinking groups offers an important strategy to covalently trap these transient and multivalent interactions (37).…”

Section: Discussionmentioning

confidence: 99%

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Boyce

Wands

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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141

269

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O-linked β- N -acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

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Section: Discussionmentioning

confidence: 99%

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Boyce

Wands

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

141

269

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“…Therefore, it is often used in vitro but rarely found in the literature for in cellulo or in vivo experiments except for a few examples using formaldehyde, a non-selective and also toxic reagent, resulting in extensive and complex cross-linking [2]. To overcome these issues, photoinduced cross-linking has been developed in chemical biology by using photo-reactive groups, which can now be routinely incorporated into the proteins either chemically or metabolically [3,4] and then used in the native environment [5]. Scheme A1 (Electronic Supplemental Material) shows three commonly used photoprobes.…”

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confidence: 99%

Photocross-Linked Peptide–Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes

Clavier

Bolbach

Sachon

2015

J. Am. Soc. Mass Spectrom.

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Abstract. Protein-protein interactions are among the keys to organizing cellular processes in space and time. One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique. The feasibility of in cellulo photocross-linking reactions has been shown and mass spectrometry is a tool of choice to analyze photocross-linked proteins. However, the interpretation of the MS and MS/MS spectra of photocross-linked peptides remains one of the most important bottlenecks of the method and still limits its potential for large-scale applications (interactomics). Fundamental studies are still necessary to understand and characterize the fragmentation behavior of photocross-linked peptides. Here, we report the successful identification of the interaction sites in a well-characterized model of in vitro interaction between a protein and a peptide. We describe in detail the fragmentation pattern of these photocrosslinked species in order to identify trends that could be generalized. In particular, we compare CID and ETD fragmentation modes (and HCD in a lesser extent), demonstrating the complementarity of both methods and the advantage of ETD for the analysis of photocross-linked species. The information should help further development of dedicated software to properly score MS/MS spectra of photocross-linked species.

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“…2). 29,30 Therefore, a careful chemical design is required in order to incorporate the photolinker in a way that it is at least partially embedded into the compound scaffold. As pretubulysin 2 exhibits a C-terminal tubuphenylalanine (Tup) moiety which can be modified with only little loss of activity according to SAR 14-17 studies we decided to expand its aromatic system by carbonylative cross coupling to a photoactive benzophenone moiety.…”

Section: Resultsmentioning

confidence: 99%

Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

et al. 2012

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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 EC 50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation 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.

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Photocrosslinkers illuminate interactions in living cells

Cited by 163 publications

References 65 publications

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Photocross-Linked Peptide–Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes

Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

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