In this work, we have employed an integrated proteomic approach for the study of Nedd8 associated and conjugated proteins. The combined use of efficient single-step affinity purification, 1-D gel electrophoresis, and two different tandem mass spectrometers, that is, LTQ and QSTAR XL MS, has provided the identification of a broad spectrum of proteins captured during the preparation. The high sensitivity of LTQ MS enables the identification of low-abundance proteins, and the usage of two different mass spectrometers greatly increases the detection confidence, especially for those identified by only one or two peptides. Furthermore, the information generated by both the LTQ and QSTAR XL MS is complimentary, providing the identification of an increased number of proteins. Separation of samples by 1-D SDS-PAGE prior to enzymatic digestion improves the dynamic range because several abundant components in the mixture are separated from the low-abundance protein bands prior to their mass spectrometric analyses. In addition, 1-D gel profiling provides a visual display of copurified proteins and direct observation of the same proteins migrating at different molecular weights due to post-translational modifications and/or processing. Purification of the GST-Gus protein effectively provides the elimination of nonspecific background. Therefore, the methods described here would be a valuable strategy for investigating protein interactions in general, especially for other ubiquitin-like proteins and their modified and associated proteins.
In total, 496 putative Nedd8 modified and associated proteins were identified. Among them are neddylation and deneddylation enzymes, proteasome components, polyubiquitin, transcriptional factors, DNA repair complexes, DNA replication complexes, cell cycle regulatory complexes, and histone complexes. The identification of such a diverse array of proteins is significant since the majority of them have not been reported from previous proteomic analyses of the Nedd8 pathway.20,21
For the first time, all cullin proteins were identified in a single analysis. On the basis of the relative abundance analysis using NSAF, it appears that Cul-2 and Cul-4B are at similar levels, but they are more abundant than Cul-1, Cul-3, and Cul-4A. Cul-5, Parc, and Cul-7 are much less abundant, among which Cul-7 is the least abundant. In addition to cullin proteins, various components of cullin containing E3 ubiquitin ligases were copurified and identified including F-box proteins, BTB family proteins, and WD40 repeat proteins. It is worth noting that several recently assigned and noncullin Nedd8 substrates, that is, pVHL, mdm2, p53, EGFR receptor, and BCA3,15–18
were not identified. Coincidently, they have not been identified by MS in any previous reports.20,21
This may be because (1) the newly assigned substrates have extremely low abundance in their neddylated forms and (2) the highly dynamic nature of neddylation/deneddylation yields very low level of neddylated species at steady state. In this regard, purification of Nedd8 modified proteins under fully denaturing conditions may prove to be beneficial.
The neddylation sites of all of the cullins (i.e., Cul-1, -2, -3, -4A/B, and -5), with the exception of Parc and Cul-7, were determined by MS/MS analyses. The lack of identification of neddylation sites of Parc and Cul-7 is due to their low abundance in the sample. So far, the neddylation sites of cullins have been only determined by mutagenesis.10
Our mass spectrometric data have provided the first evidence to support the mutagenesis results. In addition to cullin neddylation, one of the most exciting findings in this work is the observation of chain assembly on Nedd8 in vivo
and in vitro
. Four of the nine lysine residues of Nedd8, that is, K11, K22, K48, and K60, have been determined to be modified by GG after the trypsin digestion in GST-Nedd8 sample, suggesting the covalent attachment of another Nedd8 on these lysines for chain assembly in vivo
. This view has been further supported by MS analysis of in vitro
neddylation of Cul-1(324–776), revealing that both K48 and K22 of Nedd8 are able to covalently link to another Nedd8 and form a chain in vitro
. It appears that K48 of Nedd8 is the dominant site for chain formation both in vivo
and in vitro
due to its occurrence during MS analysis. Previous studies have shown that multiple Nedd8-containing bands were observed when affinity purification of EGFR, p53, or BCA3 was carried out from cells expressing tagged Nedd8.16–18
Although these authors suggested that these bands may be due to multiple mononeddylation events, we speculate polyneddylation is most likely the cause for these observations.
Ubiquitin chain formation is best known for its role in targeting conjugated proteins to the proteasome for degradation, a process that involves chain formation via K48 or K29.1,2
For yeast ubiquitin, it has been reported that chain formation can occur via all seven internal lysines.35
In contrast to the extensive information on ubiquitin chain formation,35–37
very little is known about polymerization of ubiquitin-like proteins. Recent studies have demonstrated that SUMO can form polysumoylation in vitro38,39
as well as in vivo
With our results, we speculate that chain formation of ubiquitin-like proteins may potentially be a general phenomenon in cell biology. The biological significance of ubiquitin-like protein chain formation such as SUMO and Nedd8, however, needs to be elucidated.
It is noteworthy that both neddylation and ubiquitination lead to the remnant of GG from either Nedd8 or ubiquitin on the modified internal lysine of their substrates after trypsin digestion. This is due to the high sequence similarity between ubiquitin and Nedd8. Since ubiquitin is present in the GST-Nedd8 sample, we cannot rule out the possibility that Nedd8 could be ubiquitinated, though the low abundance of ubiquitin relative to Nedd8 makes this scenario unlikely. Because of the overexpression of GST-Nedd8 in the 293 stable cell lines, it is possible that Nedd8 may replace ubiquitin to form mixed chains. In addition, it is possible that the Nedd8 chain assembly may be attributed to its overexpression in cells.
In this work, an extensive array of proteins in transcription, replication, DNA repair, and chromatin organization and remodeling have been identified as components that may potentially participate in Nedd8-related regulatory pathways. This is not surprising given the accumulation of evidence that cullin-based E3s are involved in regulating DNA replication and genome stability. However, it should be cautioned that these components were identified in cells that overexpress Nedd8. Further studies to confirm and extend these findings are necessary.
In summary, our proteomic analysis of the Nedd8 pathway has revealed a plethora of proteins involved in this pathway and provided the first hand evidence that Nedd8 can form chains both in vivo
and in vitro
. The important findings in Nedd8 chain assembly and its interaction network provide a strong basis for future studies to be steered toward understanding the extent of cellular processes influenced by neddylation events and how these events are regulated. Quantitative mass spectrometry such as SILAC41
combined with double affinity purification under fully denaturing conditions36
may be useful for future experiments to examine changes among Nedd8 substrates under different cellular conditions and genetic backgrounds.