Dataset Information

Ubiquitinome UPS - Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila

ABSTRACT: In eukaryotic cells, short-lived, regulatory and misfolded or denatured proteins are degraded by the ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins for proteasomal degradation by ubiquitin. The UPS is critical in regulating proteostasis and dysfunctioning of this system has been implicated in diseases such as cancer and neurodegenerative disorders. We have investigated the effects of proteasome malfunctioning in Drosophila by monitoring the global proteome and ubiquitinome using SILAC proteomics. The proteasome was inactivated by either chemical inhibitors or by dsRNA mediated knockdown of specific proteasome subunit constituents. Upon proteasome perturbation both the global proteome and the ubiquitinome were remodeled to a great extent, albeit that the overall impact on the ubiquitinome was much more dramatic. The abundances of ~10% of all observable proteins were increased as a result of both de novo synthesis and of accumulation of ubiquitinated proteins. In the ubiquitinome analysis ~14,000 diGly peptides were identified and most of these could also be quantified. The far majority of diGly peptides showed increased levels, indicating that the pool of ubiquitinated proteins is highly dynamic. Interestingly, because of the high level of detail the ubiquitination dynamics of individual proteins could be explored. Several examples (e.g., Rps3 and H2B) will be discussed, in which different target lysine residues show either increased or decreased ubiquitination on the same protein, suggesting the occurrence of simultaneous and possibly functionally different ubiquitination events. In conclusion, the combination of quantitative proteomic methods with dsRNA mediated knockdown of individual subunits of the 26S proteasome offers a powerful tool to study the dynamics of the (modified) proteome upon perturbation of the UPS. This strategy opens up new avenues for the dissection of the mode of action of this cellular machinery, both under proteostasis and proteotoxic conditions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Jeroen Demmers

LAB HEAD: Jeroen Demmers

PROVIDER: PXD002552 | Pride | 2017-07-03

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	1081_KarenSap_hilic_8dec11_03.raw	Raw
	1081_KarenSap_hilic_8dec11_03_111227145857.raw	Raw
	1081_KarenSap_hilic_8dec11_03_111228161512.raw	Raw
	1081_KarenSap_hilic_8dec11_09.raw	Raw
	1081_KarenSap_hilic_8dec11_09_111227170954.raw	Raw

Items per page:

1 - 5 of 292

Publications

Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in Drosophila.

Sap Karen A KA Bezstarosti Karel K Dekkers Dick H W DHW Voets Olaf O Demmers Jeroen A A JAA

Journal of proteome research 20170711 8

The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits ...[more]

PMID: 28665616

Similar Datasets

Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

Project description:Ubiquitination is a post-translational modification that has been shown to have a range of effects, such as regulating protein function, protein:protein interactions, protein localization, and protein abundance by targeting proteins for degradation by the 26S proteasome. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2, is down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effect of increased ASB2 expression on skeletal muscle mass and function, we used 2-dimensional nano-ultra-high-performance mass spectrometry to investigate ASB2-induced changes to the mouse skeletal muscle proteome, and whether these were associated with changes in protein ubiquitination. The results show that in vivo recombinant adeno-associated viral vector-mediated ASB2β overexpression induces impaired intrinsic force production and progressive muscle atrophy over 12 weeks, while ASB2 knockdown induces mild muscle hypertrophy. ASB2-induced muscle atrophy and dysfunction were associated with the early down regulation of mitochondrial and contractile proteins, and the upregulation of proteins involved in proteasome-mediated protein degradation, protein synthesis and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination, however, there was no simple relationship between changes in ubiquitination status and protein abundance. To gain insights into proteins that interact with ASB2, and potential ASB2 targets, in muscle cells, flag-tagged wild type ASB2, and a mutant ASB2 with a deleted C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, we identified several novel potential ASB2β target substrates that await further investigation. Overall, this study reveals for the first time the complexity of changes to the proteome and ubiquitinome during E3 ligase-mediated skeletal muscle atrophy and dysfunction.

Project description:Ubiquitination has crucial roles in many cellular processes and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of E2 ubiquitin-conjugating enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, a very promiscuous E2 enzyme that affects a broad range of processes. Here, we set out to identify in vivo targets of UBE2D3 by using SILAC-based quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent and, interestingly, identified pathways related to mRNA translation as top pathways being affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control (RQC), is dependent on UBE2D3. Moreover, we find that UBE2D3 limits monoubiquitination of histone H2B at lysine 120, mediated by the E3 ligase RNF20. This identifies UBE2D3 as a potential regulator of the diverse and critical cellular processes that are controlled by H2B monoubiquitination. Collectively, our findings show that depletion of an E2 ubiquitin-conjugating enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Together with our identification of RQC factors and H2B as novel in vivo targets of UBE2D3, our work provides an important resource for further studies on the in vivo functions of UBE2D3.