Identification of protein ubiquitination...
Transcript of Identification of protein ubiquitination...
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.
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