Quantitative Proteomics Reveals Ubiquitin Linkages And Site-specific Modification On Substrate Proteins

Ubiquitin-proteasome system(UPS),as the main pathway mediated protein degradation,plays pivotal regulatory roles in a broad spectrum of cellular processes such as cell cycle,apoptosis,signal transduction and immune response.Ubiquitination is catalyzed by a cascade of enzymatic reactions through Ub activating enzyme(E1),Ub conjugating enzyme(E2),and Ub ligase(E3).The modification is reversely trimmed through deubiquitinating enzymes(DUB).The precise regulatory mechanism of UPS enzymes and substrates constitutes the complex but steady ubiquitin signaling network.The deregulation of UPS network leads to human pathogenesis,including the development of tumors and neurodegenerative diseases.Therefore,systematic study on the specificity of ubiquitination helps us get into profound understanding of molecular mechanism of pathogenicity and develop novel strategies to combat diseases.All seven lysine residues and N terminus of Ub can form polymeric Ub chains with distinct topologies to implement distinct physiological functions in cells.The most well studied K48-linked chains are the primary signals for proteasome degradation;K11-linked chains play important roles in the ERAD pathway and APC/C substrate degradation in cells mitotic exit;K63-linked chains mostly act as non-degradation signals but participate in endosomal trafficking,intracellular signaling and DNA repair.This linkage functional diversity may be originated from the different topological structures to transit distinct signals,which helps us understand the―ubiquitin language‖.Accumulating evidences have demonstrated that DUBs are responsible to distinguish diverse ubiquitin linkages and regulate a specific set of targets to execute specific functions.For example,yeast Ubp2 prefers K63 over K48 chains and antagonizes E3 Rsp5-mediated ubiquitination of K63chains.Conversely,the proteasome-associated USP14 shows preferences for K48 to K63 chains.While systematic assessment of specificity between DUBs and linkages in vivo are still less studied.Ubiquitination macroheterogeneity results that multiple sites on substrate protein are modified with different linkages,which may need precise regulation to execute their proper functions.However technically it is remaining challenge to directly display the modification sites on substrates and their corresponding ubiquitin linkages.In this study,we employed quantitative proteomics approaches to systematically evaluate the specificity of DUBs on all seven types of ubiquitin linkages.Based on the linkage specificity of DUBs,we developed a Ubiquitin linkage differential display strategy for ubiquitinated site on protein substrate with certain ubiquitin linkage(DDUbSL).We aimed to construct―Ub linkages-substrate-DUB-function‖signal transition to understand the precise regulation of ubiquitin networks.To systematically evaluate the specificity of DUB and linkages in vivo,we assumed that all existing DUBs maintained the ubiquitin chains homeostasis,while deletion one certain DUB will result in the accumulation of its preferred chains.First we constructed a series of 19 single DUB deletion strains.In summary,diverse DUB deletion strains differed from each other in terms of growth abnormalities and ubiquitination.We applied swapped SILAC strategy to quantify seven ubiquitin chains in each DUB deletion strain with WT and proved that the accumulation of Ub chains in each DUB deletion strain distinguished obviously with each other.For example,Ubp2showed a strong preference for K63 chains and may modulate a significant fraction of K63 chain modified substrates in budding yeast;Ubp14 deletion resulted in the huge accumulation of unanchored K29 and K48 chains,reaching up to 30 folder change,while for K63 linkage with no significant change;Otu1 showed highly linkage bias trend with that of Out2,but Otu1 prefered for K11 linkage obviously.Through hierarchical clustering of linkages quantification of USP DUBs deletion mutants,some paralog DUBs such as Ubp4-Ubp5,Ubp9-Ubp13 were clustered together.The seven linkages quantification between these paralog DUBs appeared highly co-related.However,the co-relationship of paralog Ubp7 and Ubp11 appeared not so good and R~2 was only 0.44.The domain structure analysis showed that Ubp7 has RHOD(Rhodanese homology domain)domain which Ubp4 and Ubp5 both shared,while Ubp11 does not have this domain.We compared quantification of Ubp4 and Ubp5 respectively with Ubp7,and the R~2 was 0.78 and 0.70,which was higher than that of Ubp7 and Ubp11.This indicated that the intermolecular regulatory domains besides catalytic domain of DUB contributes to the specificity of DUBs for linkages.In addition,the topologies of linkages to some extent affecting the specificity of DUBs.The cluster analysis showed that K11 and K48 were clustered together,of which shared compact conformations,while K63 chains shared open conformation and obviously outliered with other linkages.This may indicate that K48 and K11 differs remarkably from K63 chain in their three-dimensional structure.Interestingly,K29 and K33 were clustered together and recent research had proved that K29 and K33 chains adopt open conformations in solution.We concluded that the linkage topology contributes to the specificity of DUBs to some extent.In this study,we developed a strategy to combine DUB specificity for linkages and ubiquitination heterogeneity to identify the site-specific modification of certain linkage on substrate.This is the first time to combine protein and modified peptides quantification to verify substrates.To prove this strategy viable and powerful,we based on the specificity of Ubp2 and K63 chain and identified more than 100 potential K63 linkage modified substrates regulated through Ubp2.We successfully identified 11 site-specific modified peptides which could be candidates regulated through Ubp2.This proved that Ubp2 specifically regulated certain but not all modified sites on the substrates.We demonstrated that there existed K63 chains on K151 site and K48 chains mainly on non-K151 sites of the cyclophilin A(Cpr1).To profile the interactome of Cpr1,we employed SILAC-tandem affinity purification(TAP)strategy to effectively enrich the positive interacted proteins and reduce the background contaminants.Ubp2,Ubp3 and Ubp7 were also enriched in the interacted list.We had proved K63chains on K151 site of Cpr1 was regulated through Ubp2.Through SILAC quantification,we proved that Ubp3 participated in regulation of K48 chains on non-K151 sites and K63 chain on K151 site,while Ubp7 regulated mostly the K48 chains on non-K151 sites.Finally we proved which DUBs specifically regulated the linkages and revealed divergent functions of linkages on Cpr1.The K48 linkages on non-K151 sites of Cpr1,regulated through Ubp3 and Ubp7,were proved to be mediated with 26S proteasome degradation.In addition,Ubp2working cooperatively with Ubp3 participated in the regulation of K63 linkages on the K151 site of Cpr1.We demonstrated that the absence of K63 linkages on K151 site on Cpr1 will significantly decrease the interaction with zinc finger protein(Zpr1),which affecting the translocation of the Zpr1 from cytoplasm to the nucleus after treatment of the starved cells with glucose.In conclusion,we present here a novel screening of DUB specificity with ubiquitin linkages in vivo,which proved the domain of DUB and ubiquitin linkage topologies contributed to the specificity.Using DUB specificity,we developed a novel strategy to determine site-specific modification on substrates of DUBs,which may be widely used in the substrates identification through quantitative proteomics.We proved the precise editing and regulation of ubiquitin modification on substrates through DUBs.The signal"Ub chains-substrate-DUBs-function"become the basis of precise regulation mechanism of ubiquitin networks.