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In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification.

ABSTRACT: Small ubiquitin-like modifier (SUMO) is conjugated to its substrates via an enzymatic cascade consisting of three enzymes, E1, E2, and E3. The active site of the E2 enzyme, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide PsiKXE. However, recent proteomics studies suggested that a considerable part of sumoylation occurs on non-consensus sites. Current unbiased sumoylation site identification techniques typically require high stoichiometry in vitro sumoylation, mass spectrometry, and complex data analysis. To facilitate in vivo analysis, we have designed a mass spectrometric method based on an engineered human SUMO-1 construct that creates a signature tag on SUMO substrates. This construct enables affinity purification by covalent binding to cysteine residues in LysC/trypsin-cleaved peptides and site identification by diglycyl lysine tagging of sumoylation sites. As a proof of concept, site-specific and substrate-unbiased in vivo sumoylation analysis of HeLa cells was performed. We identified 14 sumoylation sites, including well known sites, such as Lys(524) of RanGAP1, and novel non-consensus sites. Only 3 of the 14 sites matched consensus sites, supporting the emerging view that non-consensus sumoylation is a common event in live cells. Six of the non-consensus sites had a nearby SUMO interaction motif (SIM), which emphasizes the role of SIM in non-consensus sumoylation. Nevertheless, the lack of nearby SIM residues among the remaining non-consensus sites indicates that there are also other specificity determinants of non-consensus sumoylation. The method we have developed proved to be a useful tool for sumoylation studies and will facilitate identification of novel SUMO substrates containing both consensus and non-consensus sites.

SUBMITTER: Blomster HA

PROVIDER: S-EPMC2885211 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification.

Blomster Henri A HA Imanishi Susumu Y SY Siimes Jenny J Kastu Juha J Morrice Nick A NA Eriksson John E JE Sistonen Lea L

The Journal of biological chemistry 20100413 25

Small ubiquitin-like modifier (SUMO) is conjugated to its substrates via an enzymatic cascade consisting of three enzymes, E1, E2, and E3. The active site of the E2 enzyme, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide PsiKXE. However, recent proteomics studies suggested that a considerable part of sumoylation occurs on non-consensus sites. Current unbiased sumoylation site identification techniques typically require high stoichiometry in vitro sumoylation, mass spec ...[more]

PMID: 20388717

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Project description:Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications.

Project description:Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A, a small, bispecific molecule with affinity for both HSA and the novel target was identified. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination.

Dataset Information

In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification.

Publications

In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification.

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