Conditional Cell Penetration of Masked CPPs by an ADEPT-like Approach.

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro. Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.

A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo.

Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.

Genetic Code Expansion Tools to Study Lysine Acylation.

Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function, such as the activity, localization, and stability of enzymes. Mass spectrometric identification of lysine acylations has witnessed tremendous improvements in sensitivity over the last decade, facilitating the discovery of thousands of lysine acylation sites in proteins involved in all essential cellular functions across organisms of all domains of life. However, the vast majority of currently known acylation sites are of unknown function. Semi-synthetic methods for installing lysine derivatives are ideally suited for in vitro experiments, while genetic code expansion (GCE) allows the installation and study of such lysine modifications, especially their dynamic properties, in vivo. An overview of the current state of the art is provided, and its potential is illustrated with case studies from recent literature. These include the application of engineered enzymes and GCE to install lysine modifications or photoactivatable crosslinker amino acids. Their use in the context of central metabolism, bacterial and viral pathogenicity, the cytoskeleton and chromatin dynamics, is investigated.

Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution.

In addition to acetylation, histones are modified by a series of competing longer-chain acylations. Most of these acylation marks are enriched and co-exist with acetylation on active gene regulatory elements. Their seemingly redundant functions hinder our understanding of histone acylations' specific roles. Here, by using an acute lymphoblastic leukemia (ALL) cell model and blasts from individuals with B-precusor ALL (B-ALL), we demonstrate a role of mitochondrial activity in controlling the histone acylation/acetylation ratio, especially at histone H4 lysine 5 (H4K5). An increase in the ratio of non-acetyl acylations (crotonylation or butyrylation) over acetylation on H4K5 weakens bromodomain containing protein 4 (BRD4) bromodomain-dependent chromatin interaction and enhances BRD4 nuclear mobility and availability for binding transcription start site regions of active genes. Our data suggest that the metabolism-driven control of the histone acetylation/longer-chain acylation(s) ratio could be a common mechanism regulating the bromodomain factors' functional genomic distribution.

Discovery of Dihydro-1,4-Benzoxazine Carboxamides as Potent and Highly Selective Inhibitors of Sirtuin-1.

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.

Spanning the gap: unraveling RSC dynamics in vivo.

Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

14-3-3 Protein Bmh1 triggers short-range compaction of mitotic chromosomes by recruiting sirtuin deacetylase Hst2.

During mitosis, chromosomes are compacted in length by more than 100-fold into rod-shaped forms. In yeast, this process depends on the presence of a centromere, which promotes condensation in cis by recruiting mitotic kinases such as Aurora B kinase. This licensing mechanism enables the cell to discriminate chromosomal from noncentromeric DNA and to prohibit the propagation of the latter. Aurora B kinase elicits a cascade of events starting with phosphorylation of histone H3 serine 10 (H3S10ph), which signals the recruitment of lysine deacetylase Hst2 and the removal of lysine 16 acetylation in histone 4. The unmasked histone 4 tails interact with the acidic patch of neighboring nucleosomes to drive short-range compaction of chromatin, but the mechanistic details surrounding the Hst2 activity remain unclear. Using in vitro and in vivo assays, we demonstrate that the interaction of Hst2 with H3S10ph is mediated by the yeast 14-3-3 protein Bmh1. As a homodimer, Bmh1 binds simultaneously to H3S10ph and the phosphorylated C-terminus of Hst2. Our pull-down experiments with extracts of synchronized cells show that the Hst2-Bmh1 interaction is cell cycle dependent, peaking in the M phase. Furthermore, we show that phosphorylation of C-terminal residues of Hst2, introduced by genetic code expansion, stimulates its deacetylase activity. Hence, the data presented here identify Bmh1 as a key player in the mechanism of licensing of chromosome compaction in mitosis.

A Directed Evolution System for Lysine Deacetylases.

Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different enzymatic mechanisms, which differ mainly by the need for NAD+ as stoichiometric co-substrate. KDACs are often found as catalytic subunit in protein complexes involved in cell cycle regulation, chromatin organization and transcription. Their promiscuity with respect to sequence context and type of lysine acylation convolutes the network of functional and physical connections.Here we present an efficient selection method for KDACs in E. coli, which allows for the creation of acyl-type specific KDAC variants, which greatly facilitate the investigation of their physiological function . The selection system builds on the incorporation of acylated lysines by genetic code expansion in reporter enzymes with essential lysine residues. We describe the creation of KDAC mutant libraries by saturation mutagenesis of active site residues, the isolation of individual mutants from this library using the selection system, and their biochemical characterization with acylated firefly luciferase.

Interaction of RSC Chromatin Remodeling Complex with Nucleosomes Is Modulated by H3 K14 Acetylation and H2B SUMOylation In Vivo.

Chromatin remodeling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair, and transcription. To understand how these complexes find their target sites on chromatin, we use genetically encoded photo-cross-linker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC complex, on nucleosomes in living yeast. We find that H3 K14 acetylation induces the interaction of the Sth1 bromodomain with the H3 tail and mediates the interaction of RSC with neighboring nucleosomes rather than recruiting it to chromatin. RSC preferentially resides on H2B SUMOylated nucleosomes in vivo and shows a moderately enhanced affinity due to this modification in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but changes its mode of nucleosome binding. Our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications likely in combination with histone variants and transcription factors.

Evolved, Selective Erasers of Distinct Lysine Acylations.

Lysine acylations, a family of diverse protein modifications varying in acyl-group length, charge, and saturation, are linked to many important physiological processes. Only a small set of substrate-promiscuous lysine acetyltransferases and deacetylases (KDACs) install and remove this vast variety of modifications. Engineered KDACs that remove only one type of acylation would help to dissect the different contributions of distinct acylations. We developed a bacterial selection system for the directed evolution of KDACs and identified variants up to 400 times more selective for butyryl-lysine compared to crotonyl-lysine. Structural analyses revealed that the enzyme adopts different conformational states depending on the type of acylation of the bound peptide. We used the butyryl-selective KDAC variant to shift the cellular acylation spectrum towards increased lysine crotonylation. These new enzymes will help in dissecting the roles of different lysine acylations in cell physiology.

Reliability and Correlation Between Indoor Allergen Concentrations from Vacuumed Surface Samples and Electrostatic Dust Collectors.

OBJECTIVES: Most studies on indoor allergen exposure used vacuumed surface samples for quantification. One alternative is electrostatic dust collectors (EDCs), which sample previously airborne settled dust. The aim of this study was to compare allergen quantification using two different sampling methods, with respect to repeatability, and to determine how well the results agree with one another. METHODS: Four times a year, measurements were made from samples that were either collected from the vacuuming of surfaces, or from EDCs, from 20 German day-care centers totaling 167 rooms. Overall, 504 vacuumed samples collected from smooth floors, 435 samples from carpets, 291 samples from upholstered furniture and beds, and 605 EDC samples were analyzed using six fluorescence enzyme immunoassays recognizing Fel d 1, Can f 1, Mus m 1, domestic mite (DM), Dermatophagoides pteronyssinus (Dp), and Tyrophagus putrescentiae (Tp) antigens. Variances and correlations among the repeat measurements over the course of the year within each sample type, and the correlations between surface samples and the corresponding EDC samples were calculated. RESULTS: Repeat measurements over the year correlated significantly with one another. However, only Fel d 1, Can f 1, and DM in the EDC samples; DM, Dp, Tp, and Fel d 1 in the upholstered furniture samples; and DM in the carpet samples show representative results of single measurements according to their variance ratios (within-room/between-room variance <1). The highest correlation between surface and EDC samples was found for Fel d 1 on the upholstered furniture (r 0.52), followed by Can f 1 on the upholstered furniture and Can f 1 on carpets (r 0.47 and 0.45, respectively). The maximum correlation for mite antigens was between carpet samples and EDC (DM r 0.27, Dp r 0.33). Mus m 1 and Tp antigens for the most part did not correlate to the EDC results. CONCLUSIONS: Both vacuumed dust from upholstered furniture and EDC samples were suitable for repeatable quantification of several allergens in day-care centers within a year. However, there was little agreement among the different collection methods, especially for Mus m 1 and certain mite antigens. Therefore, the method and location used for collection may greatly influence allergen exposure assessment and study results.

Bypassing Kinase Signaling in Mammalian Cells.

Serine phosphorylation is frequently used to control the activity of proteins. Eukaryotic cells employ cascades of these phosphorylation events to encode and distribute information. In this issue of Cell Chemical Biology, Beránek et al. (2018) report the creation of a system to genetically incorporate phosphoserine in mammalian cells, thereby circumventing upstream kinase signaling.

Highly Sensitive Lysine Deacetylase Assay Based on Acetylated Firefly Luciferase.

Lysine deacetylases (KDACs) play important roles in many physiological processes and are implicated in many human diseases. Hence, the search for modulators of KDACs is very active, and reliable assays for monitoring their activity are key to success. Here, we describe a new KDAC assay based on Firefly luciferase harboring an acetylation on an essential active site lysine. We show that several KDACs can reverse this modification and hence activate luciferase. This new assay is extremely sensitive, reliable, and fast and can be performed in a continuous format. We used this assay to screen a small library of compounds and identified several novel effectors of SirT2 with low micromolar activity.

Cellular substrate limitations of lysine acetylation turnover by sirtuins investigated with engineered futile cycle enzymes.

Metabolic activity and epigenetic regulation of gene expression are intimately coupled. The mechanisms linking the two are incompletely understood. Sirtuins catalyse the removal of acetyl groups from lysine side chains of proteins using NAD+ as a stoichiometric cofactor, thereby connecting the acetylation state of histones to energy supply of the cell. Here, we investigate the impact of lysine acetylation turnover by sirtuins on cell physiology by engineering Sirtase, an enzyme that self-acetylates and deacetylates in futile cycles. Expression of Sirtase in E. coli leads to the consumption of the majority of the cellular NAD+ supply, indicating that there is little negative feedback from reaction products, O-acetyl-ADP-ribose and nicotinamde, on sirtuin activity. Targeting Sirtase to a partially defective E silencer of the budding yeast mating type locus restores silencing, indicating that lysine acetylation turnover stabilizes heterochromatin in yeast. We speculate that this could be the consequence of local acetyl-CoA depletion because the effect is equally pronounced if the sirtuin moiety of Sirtase is exchanged with Hos3, a NAD+-independent deacetylase. Our findings support the concept that metabolism and epigenetic regulation are linked via modulation of heterochromatin stability by lysine acetylation turnover.

Epigenetic chromatin modification by amber suppression technology.

The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technology provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.

Trapping Chromatin Interacting Proteins with Genetically Encoded, UV-Activatable Crosslinkers In Vivo.

The installation of unnatural amino acids into proteins of living cells is an enabling technology that facilitates an enormous number of applications. UV-activatable crosslinker amino acids allow the formation of a covalent bond between interaction partners in living cells with nearly perfect spatial and temporal control. Here, we describe how this method can be employed to map chromatin interactions and to follow these interactions across the cell cycle in synchronized yeast populations. This method thereby provides unprecedented insights into the molecular events controlling chromatin reorganization in mitosis. As similar tools are available for other organisms, it should be possible to derive similar strategies for these and for other synchronizable processes.

Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome.

Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions.

Allergen quantification in surface dust samples from German day care centers.

Indoor allergens are among the main causes of allergic rhinitis and asthma. Allergen exposure is not limited to private homes. Mite, cat, and dog allergens were measured in day care centers to determine whether these concentrations detected might exert significant influence on human health. In 20 day care centers across North Rhine-Westphalia, Germany, the surfaces of 171 rooms were vacuumed 4 times a year to collect dust (1340 samples in total). In all samples, domestic mite antigens (DM) and the main allergens of cats (Fel d 1) and dogs (Can f 1) were quantified using enzyme immunoassays. Provisional threshold limits (PTL) for increased risks of sensitization and allergic symptoms were estimated according to published values and conversion factors. The influence of room characteristics on allergen concentrations was analyzed in mixed linear models, also considering values below the limit of detection (LOD). Nearly all samples contained allergens (99% DM, 96% Fel d 1, and 96% Can f 1). The concentrations rarely exceeded levels that were previously found to induce symptoms in home environments, but were frequently higher than estimates for enhanced sensitization risk (13% DM, 43% Fel d 1, and 27% Can f 1). Upholstered furnishings had the highest dust and allergen loads, followed by carpets and smooth floors. Allergen concentrations on different surface types that were sampled in the same room at the same time were significantly correlated and analyzed in separate models. The highest DM concentrations were present in bedrooms and in autumn. Further, DM loads on floors decreased significantly in rooms that were renovated within the last 5 years. If there were no records that furnishings were vacuumed, there were then significantly higher Can f 1 loads. Sweeping floors elevated DM and cat allergen concentrations. In addition to mite allergens, cat and dog allergens were detected in nearly all samples from day care centers. Overall, the present results indicate that allergen concentrations may be reduced by renovation and appropriate cleaning procedures.

The use of unnatural amino acids to study and engineer protein function.

The expansion of the genetic code for the incorporation of unnatural amino acids (UAAs) in proteins of bacteria, yeasts, mammalian cells or whole animals provides molecular and structural biologists with an amazing kit of novel tools. UAAs can be used to investigate the structure and dynamics of proteins, to study their interactions or to control their activity in living cells. Incorporation of UAAs with bioorthogonal reactivity facilitates the site-specific installation of labels for spectroscopy and microscopy. Light-activatable crosslinker UAAs can be used to trap interacting molecules in living cells with a precision almost at the structural level. Post-translational modifications such as lysine acetylation and serine phosphorylation can be directly encoded to analyse their impact on protein function, and caging groups can be installed on critical residues to create light-activatable proteins. In this review we highlight recent applications of this technology to investigate protein function.

Organelle size control - increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion.

Cells control the size of their compartments relative to cell volume, but there is also size control within each organelle. Yeast vacuoles neither burst nor do they collapse into a ruffled morphology, indicating that the volume of the organellar envelope is adjusted to the amount of content. It is poorly understood how this adjustment is achieved. We show that the accumulating content of yeast vacuoles activates fusion of other vacuoles, thus increasing the volume-to-surface ratio. Synthesis of the dominant compound stored inside vacuoles, polyphosphate, stimulates binding of the chaperone Sec18/NSF to vacuolar SNAREs, which activates them and triggers fusion. SNAREs can only be activated by lumenal, not cytosolic, polyphosphate (polyP). Control of lumenal polyP over SNARE activation in the cytosol requires the cytosolic cyclin-dependent kinase Pho80-Pho85 and the R-SNARE Nyv1. These results suggest that cells can adapt the volume of vacuoles to their content through feedback from the vacuole lumen to the SNAREs on the cytosolic surface of the organelle.