Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1 . Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.

The fate of poly-ubiquitylated proteins is determined by the linkage type of the ubiquitin chains attached. In their Communication on page 12925 ff., T. U. Mayer, M. Scheffner, A. Marx et al. report a straightforward method that relies on codon expansion and bioorthogonal polymerization to generate linkage-defined and protease-resistant ubiquitin chains. These chains can be used to analyze ubiquitin signaling in cell extracts, as demonstrated by a study of linkage-dependent effects on cell-cycle progression. The fate of poly-ubiquitylated proteins is determined by the linkage type of the ubiquitin chains attached. In their Communication on page 12925 ff., T. U. Mayer, M. Scheffner, A. Marx et al. report a straightforward method that relies on codon expansion and bioorthogonal polymerization to generate linkage-defined and protease-resistant ubiquitin chains. These chains can be used to analyze ubiquitin signaling in cell extracts, as demonstrated by a study of linkage-dependent effects on cell-cycle progression. Catalyst Stability In their Communication on page 12718 ff., J. A. Dumesic and co-workers show that microenvironments formed around the catalytic sites of supported metal hydrogenation catalysts can mitigate deactivation caused by biogenic impurities.1 “Cluster Linker” Approach The luminescence properties of clusters can be introduced into a bifunctional porous metal–organic framework as described by Q.-M. Wang et al. in their Communication on page 12771 ff.1 Biosensors S. Huan, Y. Li et al. describe in their Communication on page 12799 ff. how a DNAzyme, urease, and magnetic beads can be used to detect bacteria. The resulting increase in the pH value enables colorimetric detection using the litmus test.1

AbstractUbiquitylierung ist eine komplexe posttranslationale Modifikation, und die Deregulierung dieses Signalweges ist mit verschiedenen menschlichen Erkrankungen assoziiert. Ubiquitylierung kommt in unterschiedlichen Varianten vor: Neben Monoubiquitylierung werden auch Ubiquitinketten mit verschiedenartiger Verknüpfungen an Substratproteinen gebildet. Das Schicksal der ubiquitylierten Proteine wird dabei durch den Verknüpfungstyp der konjugierten Ubiquitinkette bestimmt. Der zugrundeliegende Mechanismus ist bisher nur dürftig charakterisiert. Wir zeigen hier ein neues Konzept, basierend auf Kodonerweiterung und Klick‐Chemie‐vermittelter Polymerisation, um Ubiquitinketten mit definierten Verknüpfungen herzustellen, die widerstandsfähig gegenüber ubiquitinspezifischen Proteasen sind und natürliche Funktionen haben. Das Potenzial dieser künstlichen Ketten für die Analyse des Ubiquitincodes wird durch verknüpfungsspezifische Auswirkungen auf den Verlauf des Zellzyklus gezeigt.

ADP-ribosyltransferases (ARTs) use NAD(+) as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP-ribose units onto target proteins to form poly(ADP-ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP-ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD(+) analogues that are efficiently processed by wild-type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD(+) analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono-ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage-type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click-chemistry-based polymerization to generate linkage-defined ubiquitin chains that are resistant to ubiquitin-specific proteases and adopt native-like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage-specific effects on cell-cycle progression.

AbstractADP‐Ribosyltransferasen (ARTs) nutzen NAD+ als Substrat und spielen aufgrund der Übertragung von mehreren ADP‐Ribose‐Einheiten sowie der Bildung von Poly(ADP‐Ribose)(PAR)‐Ketten unterschiedlicher Länge an Zielproteinen eine wichtige Rolle in vielen biologischen Prozessen wie der DNA‐Schadensantwort und der Zellzyklusregulierung. Bestrebungen, die Zielproteine der PARylierung sowie deren spezifischen ADP‐Ribose‐Akzeptorstellen zu identifizieren, müssen die Komplexität der PAR überwinden. Wir berichten hier von neuen NAD+‐Analoga, die effizient durch Wildtyp‐ARTs verarbeitet werden und infolge der Abwesenheit benötigter Hydroxygruppen zum Kettenabbruch führen, was zu einer Verringerung der Komplexität dieser Proteinmodifikation führt. Die Anwesenheit einer Alkingruppe in den hier vorgestellten NAD+‐Analoga ermöglicht die anschließende Markierung mit Farbstoffen oder Affinitätsmarkern mittels Click‐Chemie. Diese Studie bietet Einsicht in das ART‐Substratspektrum und könnte den Weg für weitere Entwicklungen chemischer Werkzeuge zur Erforschung des PAR‐Metabolismus bereiten.

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.