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Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

ABSTRACT: Ageing is driven by a loss of cellular integrity¹. Given the major role of ubiquitin modifications in cell function², here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome³, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament⁴ and the EPS-8 modulator of RAC signalling⁵. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

SUBMITTER: Koyuncu S

PROVIDER: S-EPMC8357631 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Koyuncu Seda S Loureiro Rute R Lee Hyun Ju HJ Wagle Prerana P Krueger Marcus M Vilchez David D

Nature 20210728 7871

Ageing is driven by a loss of cellular integrity<sup>1</sup>. Given the major role of ubiquitin modifications in cell function<sup>2</sup>, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitinatio ...[more]

PMID: 34321666

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Project description:Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only approximately 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of approximately 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), approximately 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.

Dataset Information

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Publications

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

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