Light-activatable TET-dioxygenases reveal dynamics of 5-Methylcytosine oxidation and transcriptome reorganization

Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dy- namically reshapes epigenome and transcriptome by deposit- ing oxidized 5mC derivatives, and initiating active DNA de- methylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with tem- poral control in cells. We report activation of TETs in mam- malian cells by incorporation of genetically encoded 4,5- dimethoxy-2-nitrobenzyl-L-serine as transient active site block, and its subsequent deprotection with light. Our ap- proach enables precise insights into the impact of cancer- associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA de- methylation to chromatin and transcription regulation

Light-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization

Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes

Deciphering context-dependent amber suppression efficiency in mammalian cells with an expanded genetic code

The genetic code of organisms can be expanded by introducing orthogonal translation systems (OTSs). One of the most commonly applied OTSs in mammalian cells is the archaeal pyrrolysyl-tRNA synthetase/tRNA_Pyl_CUA (PylRS/PylT) pair from Methanosarcina species. Thereby, usually in-frame amber stop codons (UAG) are suppressed to site-specifically incorporate non-canonical amino acids (ncAAs) into target proteins. These ncAAs can harbor unique chemical moieties, allowing to probe or engineer protein structure and function with high precision. To date, applicability of an expanded genetic code has been particularly advanced in bacteria by optimizing OTS components, modifying host translation, and developing mutually orthogonal translation systems. In mammalian cells, development of genetic code expansion tools has been largely focused on intrinsic properties of the OTS itself, for instance by engineering OTS components or tuning their expression levels. However, several-fold differences in ncAA incorporation efficiency are frequently observed between different amber stop codon positions within a target protein. These unpredictable variations in incorporation efficiencies substantially hamper the theoretical advantage of ncAAs to modify any user-defined site within a target protein. Here, applying a proteomics-based approach and fluorescent reporter system, we compute and validate a linear regression model that predicts ncAA incorporation efficiency in mammalian cells based on the nucleotide context. Thereby, we demonstrate that the immediate context directly modulates the competition between ncAA incorporation and termination at UAG. Moreover, our data support a molecular model in which the identity of up- and downstream nucleotides influences translational efficiency independent of amino acid and tRNA identity. Instead, base stacking of neighboring nucleotides might uniquely affect codon-anticodon base pairing during decoding of UAG. Additionally, context-specific ribosomal pausing and speed could contribute to varying ncAA incorporation efficiency. Furthermore, treatment with aminoglycosides and inhibition of nonsense mediated decay are proposed to improve yields of ncAA-modified proteins in mammalian cells. Taken together, our strategy not only facilitates the applicability of an expanded genetic code in mammalian cells, but should also prove useful in further deciphering the molecular mechanisms that govern context effects in translational efficiency. A better general understanding of context effects in translation would in turn benefit synthetic expansion of the genetic code.Der genetische Code von Organismen kann durch die Einbringung orthogonaler Translationssysteme (OTSe) erweitert werden. Das Pyrrolysyl-tRNA Synthetase/tRNA_Pyl_CUA (PylRS/PylT) Paar der Spezies Methanosarcina ist eines der am häufigsten angewendeten OTSe in Säugerzellen. Üblicherweise wird damit das amber Stoppcodon (UAG) innerhalb eines Leserasters supprimiert, um an spezifischen Stellen eines Zielproteins nicht-kanonische Aminosäuren (nkASn) einzubauen. Diese nkASn können einzigartige chemische Motive enthalten, die es ermöglichen die Struktur und Funktion von Proteinen mit hoher Präzision zu untersuchen und zu manipulieren. Bisher wurde insbesondere in Bakterien die Anwendbarkeit eines erweiterten genetischen Codes verbessert, indem OTS Komponenten optimiert, die Translation in Wirtsorganismen modifiziert und wechselseitig orthogonale Translationssysteme entwickelt wurden. Die Weiterentwicklung von Methoden, um den genetischen Code in Säugerzellen zu erweitern, fokussierte sich überwiegend auf intrinsische Eigenschaften der OTSe selbst, zum Beispiel der Modifizierung von OTS Komponenten oder der Anpassung ihrer Expressionslevel. Häufig unterscheiden sich jedoch verschiedene UAG Positionen in ihrer Effizienz eine nkAS einzubauen in mehrfacher Höhe. Diese unvorhersehbaren Schwankungen in der Einbaueffizienz schränken den Vorteil von nkASn erheblich ein, theoretisch jede benutzerdefinierte Position innerhalb eines Zielproteins modifizieren zu können. In dieser Publikation berechnen und validieren wir mit Hilfe einer proteomischen Methode und eines fluoreszierenden Reportersystems ein lineares Regressionsmodell, das anhand des Nukleotidkontextes die Effizienz des nkAS Einbaus in Säugerzellen vorhersagt. Wir zeigen dadurch, dass der unmittelbare Kontext direkt das Verhältnis zwischen nkAS Einbau und Termination an UAG moduliert. Unsere Daten unterstützen zudem ein molekulares Modell, in dem die Identität der vorherigen und nachfolgenden Nukleotide die Effizienz der Translation unabhängig von der Identität der Aminosäure und tRNA beeinflusst. Hingegen könnte sich ein Basen-Stacking über benachbarte Nukleotide in einzigartiger Weise auf die Codon-Anticodon Basenpaarung während der Dekodierung von UAG auswirken. Zusätzlich könnten ein Pausieren sowie die Geschwindigkeit des Ribosoms in Abhängigkeit vom Kontext zu der uneinheitlichen Effizienz des nkAS Einbaus beitragen. Des Weiteren werden ein Behandlungsverfahren mit Aminoglycosiden und eine Inhibierung des Nonsense-mediated Decay vorgeschlagen, um die Ausbeute an nkAS-modifizierten Proteinen zu verbessern. Zusammenfassend vereinfacht unsere Strategie nicht nur die Anwendbarkeit eines erweiterten genetischen Codes in Säugerzellen, sondern sollte sich auch als nützlich erweisen, um die molekularen Mechanismen, über die der Kontext die Translationseffizienz beeinflusst, weiter zu entschlüsseln. Ein besseres allgemeines Verständnis der Kontexteffekte bei der Translation würde wiederum die synthetische Erweiterung des genetischen Codes fördern

Light-activateable apoptosis via genetic code expansion as an in-vivo single-cell ablation tool in Caenorhabditis elegans

Natural proteins are biopolymers built from a limited variety of canonical amino acids that are encoded by corresponding triplet codons. Genetic code expansion via amber suppression enables me to install (incorporate) in a target protein a “designer” amino acid beyond canonical amino acids, at the site of a pre-introduced amber stop codon. This is through expression in the host cell of an orthogonal pair, consisting of an aminoacyl-tRNA synthetase and an amber-suppressing tRNA (tRNACUA) evolved for the non-canonical amino acids (NCAA). Site-specific incorporation of NCAA endows target proteins with new properties, enabling protein measurement and/or manipulation in ways that are otherwise impossible. Photo-caged cysteine (PCCys) as a useful NCAA has not been used in any animal before. By using a PCCRS/tRNAPylCUA pair evolved from a PylRS/tRNAPylCUA pair, I introduced (PCCys) into protein synthesis of multicellular model species Caenorhabditis elegans (C. elegans). I demonstrated this incorporation of PCCys by expressing a fluorescent reporter either throughout the nematode or in two different neuronal classes. I used site-specific PCCys incorporation to develop a light-activatable caspase for precisely ablating cells (especially neurons) in living worms. Cell ablation has been widely adopted in studies on C. elegans cell lineage and cell functions. Common ablation methods include high-powered laser ablation, genetic ablation and optogenetic ablation. However, they are unable to ablate single cells in fully developed worms. Caspase is a core executor of apoptosis of both C. elegans and human cells. I designed and engineered a photo-caged caspase from human Caspase-3 by replacing its catalytic cysteine with PCCys. 365-nm UVA illumination removes the caging group of PCCys in the caged caspase, thereby activating the caspase to induce apoptosis of the cell(s) targeted. I succeeded in using global UV illumination to activate respective apoptosis events of oxygen-sensing neurons, touch receptor neurons and muscular cells in adult worms. Also, I demonstrate that individual adult neurons can be selectively targeted and efficiently killed with the use of a microscope-mounted 365-nm laser. With this better spatiotemporal control than other ablation methods, our approach is likely to facilitate future C. elegans studies with unprecedented specificity and precision

Towards more rational approaches of membrane protein stabilisation and novel structures of membrane-bound pyrophosphatase

Membrane proteins have a range of crucial biological functions and are targeted by most prescribed drugs despite lagging behind soluble proteins when it comes to their biochemical and biophysical characterisation. A major bottleneck in membrane protein research is protein instability upon extraction. Protein stabilisation strategies are typically expensive and labour-intensive. Therefore, I contributed to the development and evaluation of two new general-purpose tools, both designed for the streamlined and rational stabilisation of membrane proteins. The first tool, the integral membrane protein stability selector (IMPROvER), predicts stabilising point-mutations in membrane proteins using three individual approaches with additive prediction power. The second tool, a novel pre-prepared and easy-to-use screen for the high-throughput identification of stabilising lipids, facilitates the structural and functional analysis of stable and physiologically relevant protein sample. Both tools were successfully employed to stabilise a range of membrane proteins with different folds, topologies and modes of action at significantly reduced cost and work effort. Moreover, engineered or natively thermostable membrane-bound pyrophosphatases (M-PPase), were studied in more detail using conventional and time-resolved X-ray crystallography. Based on structural data obtained on a pyrophosphate-energised K+-independent H+-pump, I derived an updated model of ion-selectivity that is centred on a glutamate-serine interplay at the ion-gate. This is the first model that explains ion selectivity in all M PPase subclasses when considering functional asymmetry. Indeed, complementary time-resolved structural studies of a K+-dependent Na+-pump revealed asymmetric substrate binding to M-PPase active sites. These findings give valuable mechanistic insights into key processes of M-PPase biochemistry, which are of upmost importance for structure-guided drug discovery. Ultimately, tweaking M-PPase function has the potential to address existing and emerging challenges to human health and global food security as M-PPases play a vital role in the stress resistance of pathogens or salt and drought resistance in plants