Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

Heuvel, Jasmin van den; Ashiono, Caroline; Gillet, Ludovic; Dörner, Kerstin; Wyler, Emanuel; Zemp, Ivo; Kutay, Ulrike

doi:10.7554/elife.70560

Cited by 19 publications

(27 citation statements)

References 104 publications

(150 reference statements)

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“…Next, we sought to extend the available substrate toolbox to investigate catalytic activity toward Ubls for which isopeptide-linked substrates had previously not been synthesized. We therefore turned to the Ubl Fubi which like ubiquitin is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30) and cleaved by the nucleolar DUB USP36 to release free Fubi and S30 . In addition to USP36, the mainly cytosolic DUB USP16 has Fubi protease activity, which may give rise to a two-tier system of Fubi-S30 maturation .…”

Section: Resultsmentioning

confidence: 99%

“…There are approximately 100 DUBs known in humans which can be grouped into 7 different classes of which the ubiquitin-specific proteases (USPs) are the largest and most heterogeneous family (Figure C) . While the bulk of USP DUBs are considered to be indeed ubiquitin-specific yet rather promiscuous with regards to the ubiquitinated substrates, some members notably feature preferences for distinct ubiquitin chains (CYLD and USP30), display Ub/Ubl cross-reactivity (USP16 and USP36 for Ub and the Ubl Fubi; , USP2, USP5, USP14 and USP21 for Ubiquitin and ISG15 − ), or are specific for a Ubl without ubiquitin activity (USP18 for ISG15, USPL1 for SUMO, Figure D). Ub/Ubl cross-reactivity has also been observed in other DUB families (e.g., UCHL3 with activity for Ub and NEDD8) and is particularly prevalent in viral and bacterial effector DUBs. − Moreover, additional examples exist where a member of a particular Ubl protease fold has evolutionarily been co-opted to provide cleavage activity for a distinct Ubl.…”

Section: Introductionmentioning

confidence: 99%

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Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Zhao,

O’Dea,

Wendrich

et al. 2023

J. Am. Chem. Soc.

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Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Zhao,

O’Dea,

Wendrich

et al. 2023

J. Am. Chem. Soc.

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“…Interestingly, in humans and other holozoan organisms, a second RP of the small subunit, RPS30/eS30, is synthesized as a fusion with a ubiquitin‐like protein called FUBI. Release of FUBI from the FUBI‐eS30 fusion protein is required for 40S subunit maturation, likely linked to its nuclear incorporation into pre‐40S subunits, and promoted by the deubiquitinase USP36 (van den Heuvel et al , 2021 ).…”

Section: Chaperones Of Ribosomal Proteinsmentioning

confidence: 99%

Ribosome biogenesis factors—from names to functions

et al. 2023

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The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre‐ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.

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“…Early studies have implicated UCHL3 and UCHL1, two members of ubiquitin C-terminal hydrolase family of DUBs, in the processing of ubiquitin-ribosomal protein fusions UBA52 and UBA80 [22]. More recently, studies with human cells have proved that the processing of the hybrid protein formed between eS40 and FUBI (a ubiquitin-like protein) is carried out by USP36 protease which is necessary for the maturation of the 40S ribosomal subunit [24]. In fact, the transient association and posterior cleavage of ubiquitin and ribosomal proteins is necessary for efficient ribosome biogenesis [25][26][27].…”

Section: Ubiquitination and Ribosome Biogenesismentioning

confidence: 99%