Photoactivable peptides for identifying enzyme ...

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Photoactivable peptides for identifying enzyme–substrate and protein–protein interactionsw Dante Rotili,ab Mikael Altun,c Refaat B. Hamed,zad Christoph Loenarz,za Armin Thalhammer,za Richard J. Hopkinson,a Ya-Min Tian,c Peter J. Ratcliffe,c Antonello Mai,b Benedikt M. Kessler*c and Christopher J. Schofield*a

Downloaded by University of Oxford on 15 March 2011 Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0CC04457A

Received 16th October 2010, Accepted 19th November 2010 DOI: 10.1039/c0cc04457a Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme–substrate and protein–protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system. A major challenge in cellular research is to define the interactions occurring between bio-molecules. Significant advances in protein–protein interactions have been enabled by linking affinity purification techniques to mass spectrometric analyses.1 However, it is difficult to maintain transient interactions during purification, particularly when working at, often low, endogenous levels. One solution is to irreversibly cross-link molecules, e.g. by a photoactivated process.2 Elegant genetic methodologies for the introduction of unnatural residues into proteins have been developed, but these are presently limited in their applications.3 Photocross-linking reactions coupled to affinity purification have been used for profiling small molecule interactions.2,4 One approach has also employed a peptide-based probe for capturing Netrimethylated lysine binding proteins.5 Here, we report a relatively simple strategy that involves photoactivated cross-linking of ‘‘bait’’ peptide fragments of proteins to other proteins in crude cell lysates (Fig. S1, ESIw). We tested this method by studies on the role of posttranslational hydroxylation in the human hypoxic response system. In animals, the hypoxic response is mediated by the hypoxia inducible transcription factor (HIF) which coordinates expression of many genes.6 Human HIF levels are regulated by the hydroxylation of two prolyl-residues in the HIF-1a/2a domains, reactions that are catalyzed by the prolylhydroxylase domain (PHD) isoforms of which PHD2 is the most important.7 Prolyl-hydroxylation at either the N- or

C-terminal oxidation dependent degradation domain (NODD or CODD) increases binding of HIF-a to the von Hippel Lindau elongin C/B (VCB) complex which signals for HIF-a degradation via the proteasome.7 HIF hydroxylases require Fe(II) as a cofactor and 2-oxoglutarate (2-OG) and oxygen as cosubstrates.6 We are interested in defining protein–protein interactions in the HIF system and substrates for other 2-OG oxygenases catalysing post-translational modifications. To test whether a peptide photo cross-linking approach is useful for identifying protein–protein, including enzyme– substrate, interactions we prepared two peptide probes, one containing an unhydroxylated CODD fragment of HIF-1a (CODD-based Probe, CP) and one containing its Pro-564 hydroxylated analogue (HO–CODD-based Probe, HCP). Their common scaffold contains a phenyl azide as a photoactivable cross-linking group and a biotin group to enable purification and is based on previously reported photo cross-linking work with small molecules (Fig. 1A).4 The phenyl azide group was linked to the peptide by a Michael reaction between a cysteine residue and a substituted maleimide (Fig. 1B, Schemes S1 and S2, ESIw). We used this method, rather than the incorporation of p-azidophenylalanine, because it is efficient and the azido

a

University of Oxford, Department of Chemistry and the Oxford Centre for Integrative Systems Biology, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, United Kingdom. E-mail: Christopher.schofi[email protected] b Pasteur Institute-Cenci Bolognetti Foundation, Department of Chemistry and Technologies of Drugs, University of Rome ‘‘La Sapienza’’, P.le A. Moro 5, 00185 Rome, Italy c Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom. E-mail: [email protected] d Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, 71256, Egypt w Electronic supplementary information (ESI) available: Overview of the photo cross-linking strategy; Details for: Synthesis of CP and HCP; Production of PHD2 and VCB; Cell cultures and preparation of lysates; Photo cross-linking and affinity enrichment experiments; MS, western blot analysis of photo-affinity tagging; Protein digestion; Nano LC-MS/MS; Table of identified proteins. See DOI: 10.1039/ c0cc04457a z These authors contributed equally to the present work.

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Fig. 1 (A) The photoactivable peptide probes CP and HCP. (B) Michael reaction for the introduction of the photoactivable cross-linker.

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Fig. 2 Photoactivated cross-linking of CP to PHD2181–426. (A) MALDI-TOF MS analysis (for entire spectrum Fig. S3A, ESIw), (B) anti-PHD2 and (C) anti-biotin blots for the cross-linking of PHD2181–426 by CP in the presence of MnCl2 and NOG. (D) Anti-PHD2, (E) anti-biotin and (F) silver stained SDS-PAGE gel (for entire gel Fig. S6A, ESIw) for the same experiment as in (A) plus HEK293T cell lysate proteins as background.

group is sensitive towards the piperidine used in Fmoc-mediated solid phase peptide synthesis. The position of the cysteine at the N-terminus of the peptide was based on structures for the PHD2–HIF-1a and HIF-1a–pVHL complexes,6,7 and was intended to maintain binding whilst allowing cross-linking. The N-terminal aspartyl- of the HIF-1a peptides was substituted with an alaninyl-residue to minimize reaction of the carboxy group with the photo-activated group (see ESIw for synthetic details). Mass spectrometric (MS) analyses revealed that CP undergoes a 16 Da mass shift (from 2896 Da to 2912 Da) upon incubation with the catalytic domain of PHD2 under appropriate conditions, demonstrating that the modifications to CODD do not ablate PHD2 binding. HCP was shown to bind to VCB, similarly to Pro-564 hydroxylated HIF-1a, by using a fluorescence based assay (Fig. S2, ESIw). We then tested the utility of CP to cross-link to PHD2181–426 (B28 kDa) using Mn(II)8 and N-oxalylglycine9 as substitutes for Fe(II) and 2-OG, respectively. After irradiation, a peak with a mass shift of B2900 Da relative to PHD2 was observed, consistent with cross-linking (m/z 2868 for [CP–N2]) (Fig. 2A). Following purification using avidin beads, there was enrichment in the CP–PHD2 complex (Fig. 2A) (the unmodified PHD2 present after purification is likely due to non-specific capture), as shown by Western blot analyses (Fig. 2B and C). To test whether the cross-linking works within a complex environment we performed experiments with human (HEK293T) cell lysates (Fig. 2D–F, Fig. S3, ESIw) supplemented with recombinant PHD2181–426. As before, clear evidence for cross-linking was observed. Evidence for selectivity comes from controls, in which the cross-linking was reduced by competition with unmodified CODD (Fig. 2B–F), and not observed with HCP (the prolyl-hydroxylated product of PHD2 catalysis, HO–CODD, binds less strongly than the CODD substrate, Fig. 2B and C).6 Cross-linking was substantially reduced in the absence of irradiation (Fig. 2B–F). This journal is

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A low level of unmodified (i.e. unbiotinylated) PHD2 was observed after purification without irradiation (Fig. 2B and D); this is likely due to non-specific binding to the avidin beads (Fig. 2C and E). SDS-PAGE followed by MS/MS analysis of the B28–31 kDa band (Fig. 2F), supported the formation of a CP–PHD2 complex in cell lysates (Table S1, ESIw). We then investigated cross-linking of HCP to VCB in HEK293T cell lysates. The results clearly demonstrated that selective covalent cross-linking can be achieved (Fig. 3, Fig. S3, ESIw); elongins B/C were co-purified with the cross-linked HCP–pVHL, likely due to non-covalent interaction with pVHL (Fig. 3A and F). MS/MS analysis of tryptic fragments of the protein bands at B18–21 kDa (two bands), 13–16 kDa (one broad band) and 11–12 kDa (one broad band) (Fig. 3F) confirmed them as cross-linked pVHL, elongins B and C, respectively (Table S1, ESIw). The absence of cross-linking between HCP and elongins B and C was evident in the antibiotin blots (Fig. 3C and E) where only one band corresponding to HCP–pVHL was observed. Western blots using anti-pVHL antibody revealed the affinity purification of apparently unmodified pVHL (Fig. 3B and D); this may reflect instability of the HCP–pVHL complex during release of proteins from the beads or higher order pVHL-elongin B/C complexes [MS-analysis revealed the B13–16 kDa band (Fig. 3F) to consist not only of elongin B but also of pVHL (Table S1, ESIw)]. To investigate the use of the probes in more biologically relevant contexts, we then carried out analyses with human cell extracts without addition of recombinant proteins. For CP, we used lysates deriving from HEK293T cells (Fig. 4A); for HCP, we used lysates from the same cells but expressing an Nterminally ‘‘Flag’’-tagged version10 of pVHL (Fig. 4B) (ESIw). The anti-PHD2 blot (Fig. 4A, left panel) showed CP-mediated capture of full length endogenous PHD2 which was reduced by CODD competition and absent without Chem. Commun., 2011, 47, 1488–1490

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Fig. 3 Photoactivated cross-linking of HCP to pVHL. (A) MALDI-TOF MS analysis (for magnification Fig. S4A, ESIw), (B) anti-VHL and (C) anti-biotin blots for the cross-linking of pVHL by HCP. (D) Anti-VHL, (E) anti-biotin and (F) silver stained SDS-PAGE gel (for entire gel Fig. S6B, ESIw) for cross-linking of pVHL by HCP in the presence of HEK293T lysates.

and protein–protein interactions that are dependent on posttranslational modifications. They support previous proposals regarding the role of HIF-a prolyl-hydroxylation, i.e. increasing binding to pVHL and decreasing PHD binding.7,8 We are currently applying analogous approaches to search for other HIF-a modifying proteins and unidentified substrates for the PHDs and other 2-OG oxygenases. The approach demonstrated here is simple from an experimental perspective and readily adaptable to other systems. C.J.S. was supported by European Union, Wellcome Trust, and Biotechnology and Biological Research Council, A.T. by CRUK, B.M.K. by Biomedical Research Centre (NIHR), A.M. by Fondazione Roma, and M.A. by Swedish Research Council, Loo and Hans Ostermans Foundation and Foundation for Geriatric Diseases.

Notes and references

Fig. 4 (A) Capture of endogenous PHD2 by CP in HEK293T cell lysates in the presence of MnCl2 and NOG. (B) Capture of Flag-tagged pVHL in HEK293T cell lysates by HCP.

irradiation. The anti-Flag tag blot (Fig. 4B, left panel) confirmed that HCP captures pVHL, with a reduction in the presence of HO–CODD as competitor, and near complete absence of labelling without irradiation. As before, we observed the presence of some unmodified pVHL. The antibiotin blots (Fig. 4A and B, right panels) revealed few bands in the ‘‘capture’’ experiment, with some being substantially reduced in the competition controls so indicating selectivity in the enrichment of the target proteins (PHD2 and pVHL). Overall, our results reveal the potential of a photoactivated peptide cross-linking approach for ‘‘capturing’’ enzyme–substrate 1490

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