Biology subjects

Scheffner, M.

Publications and source records attributed to Scheffner, M..

4 recordsLinked to original sources

bioRxiv2025

Non-hydrolyzable acetyllysine analogs to study protein acetylation in vitro and in cells

Lysine acetylation plays a prominent regulatory role in eukaryotic cells. Yet, determining the functional consequences of acetylation for a given protein represents a considerable challenge. For instance, lysine residues are subject to various posttranslational modifications, rendering interpretation of mutational studies difficult. The genetic code expansion technology enables site-specific incorporation of acetyllysine (AcK) into proteins, but the applicability of AcK is limited, as within cells, the acetyl group is removed by deacetylases. Here, we show that site-specific incorporation of the non-hydrolyzable AcK analog ketolysine (KeK) into ubiquitin closely resembles the structural and functional effects of AcK incorporation. Furthermore, AcK and KeK can be efficiently incorporated into the tumor suppressor p53 in cells. However, whereas AcK becomes deacetylated, KeK remains stable. Accordingly, incorporation of KeK, but not AcK, affects p53-mediated transcription. Thus, we propose that KeK is the AcK surrogate of choice for studying acetylation of a given protein in cells.

biochemistry↗

bioRxiv2022

A human RNA ligase that operates via auto- and RNA-AMPylation

Different forms of life are known to express RNA ligases that catalyse the condensation of a 3-hydroxy group and a 5-terminal phosphate of RNA. No such RNA ligases have yet been identified in vertebrates. Here, we report that the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29), which we identified by a chemical proteomics approach, is a 5-3 RNA ligase. C12orf29 catalyses RNA ligation via auto-AMPylation of a critical lysine residue by using ATP as a cosubstrate and subsequent AMP transfer to the 5-phosphate of an RNA substrate followed by phosphodiester bond formation. Studies at the cellular level reveal the involvement of C12orf29 in maintaining RNA integrity upon cellular stress induced by reactive oxygen species. These findings highlight the importance of RNA ligation for cellular fitness.

biochemistry↗

bioRxiv2021

The interactome of site-specifically acetylated linker histone H1

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by posttranslational modifications (PTMs) is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification-mass spectrometry (AP-MS) and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together our data suggests that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

biochemistry↗

bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗