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K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

ABSTRACT: Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly-ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell-based ubiquitin replacement strategy to enable selective and conditional abrogation of K27-linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27-linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97-proteasome pathway model substrate (Ub(G76V)-GFP) is directly modified by K27-linked ubiquitylation, and that disabling the formation of K27-linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage-specific binder UCHL3 impedes Ub(G76V)-GFP turnover at the level of p97 function. Our findings suggest a critical role of K27-linked ubiquitylation in supporting cell fitness by facilitating p97-dependent processing of ubiquitylated nuclear proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Fabian Coscia

LAB HEAD: Niels Mailand

PROVIDER: PXD023579 | Pride | 2022-04-14

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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K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

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