Identification of protein ubiquitination sites Yiquan Liu

Advisor: Dr. Tadhg P. Begley

Background

Posttranslational modification is a chemical modification of a protein after its

translation1. Such modification includes acylation, phosphorylation, glycosylation and so

on. Ubiquitination is one of the posttranslational modifications.

Ubiquitin is a small protein containing 76 amino acid residues, which has a

characteristic glycine-glycine C-terminus.

During ubiquitination, the C-terminal

glycine residue of ubiquitin can form an

isopeptide bond with lysine residue in the

substrate protein. And the ubiquitinated

protein will then be directed into different

cell processes, such as degradation, DNA

repair and endocytosis2-3. Figure 1 The ubiquitination process of a substrate protein

E1, E2 and E3 are the three enzymes participate in the ubiquitination process. Shown

in Figure 1, the three steps involved are activation, conjugation and ligation4-6. After that,

the substrate protein will be ubiquitinated and such process can be repeated so that the

substrate protein can be modified with plenty of ubiquitin molecules. There are various

ubiquitination types according to the different ubiquitination sites7-8.

Qualitative identification through mutation method

Since ubiquitination is carried out on the lysine residue in substrate proteins, if the

ubiquitinated lysine residue is mutated, the ubiquitination will stop, which can be

detected through immunoplotting assay.

One example is applying this method to identify the ubiquitination site in Rpn49, a

transcription activator in Saccharomyces cerevisiae. The ten lysine residues near N-

terminus might play an important role in ubiquitination, and this was confirmed by

comparing the degradation rate of the wild type protein and the one with ten N-terminal

lysine residues mutated.

The ten lysines were then divided into two groups: the four residues nearer the N-

terminus and the six residues nearer the C-terminus. And it was found that when the six

residues nearer C-terminus were mutated, the degradation rate decreased, indicating that

the preferential ubiquitination site lies among these six lysine residues. Hence, each of

these lysine residues was mutated back, and only the one with K187 exhibited a

degradation rate as the wild type protein.

Shown in Figure 2, solid square, hollow

square, solid diamond and hollow diamond

represent wild type Rpn4, Rpn4 with 10

lysines mutated, Rpn4 with K187 mutated

back and Rpn4 with K132 mutated back,

respectively. In this way, K187 was found to

be the most essential ubiquitination site. Figure 2 Degradation rate of different Rpn4 species

Qualitative identification through affinity chromatography – shotgun sequencing

Shotgun sequencing is a way for automated identification and cataloguing of proteins

directly from complex mixtures10. In this method, protein mixture can first be separated

through gel electrophoresis, following by in-gel trypsin digest and LC-MS/MS analysis

(Figure 3). An alternative way is to digest

protein mixture first, then pass the peptide

mixture through strong cation exchange

chromatography (SCX). The fractions are

collected and sent for LC-MS/MS test. In

both experiments, the results are searched

through database and the protein

sequences can be obtained. Figure 3 Workflow of shotgun sequencing

For ubiquitinated proteins, it can generate a signature peptide after trypsin digestion. It

has a glycine-glycine modification on lysine residue and has a mass shift of 114.1 Da. In

this way, the ubiquitination site can be determined10-11.

To enrich ubiquitinated proteins before identification, affinity chromatography is

usually used. Peng and coworkers applied this method by using His-tagged ubiquitin and

enriched the ubiquitinated proteins with Ni-NTA column12. They were able to identify 72

proteins with 110 ubiquitination sites. Such identification was confirmed by

immunoplotting assay. The polyubiquitination sites were also determined by signature

peptides. And it’s found that most polyubiquitin chains were assembled through K48.

Ubiquitin-associated domain could also be used for enrichment. Mayor and

coworkers designed two-step purification13. They used ubiquitin-associated domain for

the first step enrichment and Ni-NTA column for the second step. In this way, they were

able to identify 176 polyubiquitinated proteins. And such result was also confirmed by

testing whether these proteins were the substrate of Rpn10, a polyubiquitin receptor.

Quantitative analysis through stable isotope labeling

In quantitative analysis, stable isotope labeling is the most commonly used way14-15.

When peptide mixture is obtained before LC-MS/MS experiment, internal standard

peptides with isotope incorporated are added with known concentrations. In this way, the

concentration of proteins can be calculated through the intensity ratio of their

representative peptides and the standard peptides.

Kirkpatrick and coworkers applied this method to investigate whether different E2

enzymes (Ubc4 and UbcH10) could cause different assembly patterns of polyubiquitin

chains on cyclin B116.

Figure 4 Different assembly of cyclin B1 polyubiquitin chains in reactions catalyzed by Ubc4 and UbcH10

They designed ten internal standard peptides according to the different signature

peptides generated because of different linkage sites in polyubiquitination. Then for the

reactions catalyzed by Ubc4 and UbcH10, different gel regions were cut and sent for

quantitative analysis. The two E2 enzymes were found to perform differently in

polyubiquitination. Comparing with UbcH10, Ubc4 had a preferential site with lysine 48,

while UbcH10 achieved polyubiquitination more through lysine 11 (Figure 4).

Consequently, the topology of the chains varied depending on different E2 enzymes.

Protein ubiquitination is an important process in cell, which participate in protein

degradation, DNA repair, endocytosis and so on. To qualitatively identify the

ubiquitination sites, lysine to arginine mutation and affinity chromatography combined

with shotgun sequencing are both widely used. The former is specific and reliable for

single protein analysis, whereas the latter is powerful in identifying complex mixtures.

Stable isotope labeling is widely used for quantitative analysis, and this helps the

quantitation and comparison of different ubiquitin linkage patterns.

 Reference 1. Walsh, C.T. et al. Angew. Chem. Int. Ed. 2005, 44, 7342

2. Pickart, C.M.; Fushman, D. Curr. Opin. Chem. Biol. 2004, 8, 610

3. Kaiser, P.; Huang, L. Genome Biol. 2005, 6, 233

4. Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998. 67, 425

5. Pickart, C.M. Annu. Rev. Biochem. 2001. 70, 503

6. Komander, D. Biochem. Soc. Trans. 2009, 37, 937

7. Miranda, M.; Sorkin, A. Mol. Interv. 2007, 7, 157

8. Dikic, I.; Wakatsuki, S.; Walters, K.J. Nature Rev. Mol. Cell Biol. 2009, 10, 659

9. Ju, D.H.; Xie, Y. J. Biol. Chem. 2006, 281, 10657

10. Zhu, H.; Bilgin, M.; Snyder, M. Annu. Rev. Biochem. 2003. 72, 783

11. Kirkpatrick, D.S. et al. Nature Cell Biol. 2005, 7, 750

12. Peng, J. et al. Nature Biotech. 2003, 21, 921

13. Mayor, T. et al. Mol. Cell Proteomics 2005, 4, 741

14. Gygi, S.P. et al. Nature Biotech. 1999, 17, 994

15. Xu, P. et al. Cell 2009, 137, 133

16. Kirkpatrick, D. et al. Nature Cell Biol. 2006, 8, 700