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The length of a ubiquitin chain is a general determinant for selective recognition by ubiquitin-binding proteins.

ABSTRACT: The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. The fate of a modified protein is determined by Ub-binding proteins (UBPs) that interact with Ub chains in a linkage-selective manner. Therefore, proteome-wide interaction studies using differently linked Ub chains have become a focus of research activities. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive, due to a lack of available tools and sufficient amounts of pure, length-defined Ub chains. Here we generated linkage- and length-defined Ub chains using click-chemistry and gel-free fractionation and employed such defined polymers in affinity-based enrichment assays to identify length- and linkage-selective interactors on a proteome-wide scale. For the first time, this revealed that the length of a Ub chain has generally a major impact on its ability to be selectively recognized by UBPs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Florian Stengel

LAB HEAD: Florian Stengel

PROVIDER: PXD015877 | Pride | 2020-04-20

REPOSITORIES: Pride

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Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.

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The length of a ubiquitin chain is a general determinant for selective recognition by ubiquitin-binding proteins.

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