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.

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.

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.

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.

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.

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.

A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation.

Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

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.

MD simulations and FRET reveal an environment-sensitive conformational plasticity of importin-ß.

The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-ß in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-ß. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes.

Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.

Concentration of bioaerosols in composting plants using different quantification methods.

BACKGROUND: Bioaerosols (organic dusts) containing viable and non-viable microorganisms and their metabolic products can lead to adverse health effects in exposed workers. Standard quantification methods of airborne microorganisms are mainly based on cultivation, which often underestimates the microbial burden. The aim of the study was to determine the microbial load in German composting plants with different, mainly cultivation-independent, methods. Second purpose was to evaluate which working areas are associated with higher or lower bioaerosol concentrations. METHODS: A total of 124 inhalable dust samples were collected at different workplaces in 31 composting plants. Besides the determination of inhalable dust, particles, and total cell numbers, antigen quantification for moulds (Aspergillus fumigatus, Aspergillus versicolor, Penicillium chrysogenum, and Cladosporium spp.) and mites was performed. Concentrations of ß-glucans as well as endotoxin and pyrogenic activities were also measured. The number of colony forming units (cfu) was determined by cultivation of moulds and actinomycetes in 36 additional dust samples. RESULTS: With the exception of particle numbers, concentrations of all determined parameters showed significant correlations (P < 0.0001; r Spearman: 0.40-0.80), indicating a close association between these exposure markers. Colony numbers of mesophilic moulds and actinomycetes correlated also significantly with data of cultivation-independent methods. Exposure levels showed generally large variations. However, all parameters were measured highest in dusty working areas like next to the shredder and during processing with the exception of Cladosporium antigens that were found in the highest concentrations in the delivery area. The lowest concentrations of dust, particles, antigens, and pyrogenic activity were determined in wheel loader cabins (WLCs), which were equipped with an air filtration system. CONCLUSION: It was possible to assess the microbial load of air in composting plants with different quantification methods. Since allergic and toxic reactions may be also caused by nonliving microorganisms, cultivation-independent methods may provide additional information about bioaerosol composition. In general, air filtration reduced the bioaerosol exposure shown in WLCs. Due to the fact that the mechanical processing of compost material, e.g. by shredding or sieving is associated with the generation of high bioaerosol concentrations, there is still a need of improved risk assessment and state-of-the-art protective measures in composting plants.

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.

Acetylation regulates cyclophilin A catalysis, immunosuppression and HIV isomerization.

Cyclophilin A (CypA) is a ubiquitous cis-trans prolyl isomerase with key roles in immunity and viral infection. CypA suppresses T-cell activation through cyclosporine complexation and is required for effective HIV-1 replication in host cells. We show that CypA is acetylated in diverse human cell lines and use a synthetically evolved acetyllysyl-tRNA synthetase/tRNA(CUA) pair to produce recombinant acetylated CypA in Escherichia coli. We determined atomic-resolution structures of acetylated CypA and its complexes with cyclosporine and HIV-1 capsid. Acetylation markedly inhibited CypA catalysis of cis to trans isomerization and stabilized cis rather than trans forms of the HIV-1 capsid. Furthermore, CypA acetylation antagonized the immunosuppressive effects of cyclosporine by inhibiting the sequential steps of cyclosporine binding and calcineurin inhibition. Our results reveal that acetylation regulates key functions of CypA in immunity and viral infection and provide a general set of mechanisms by which acetylation modulates interactions to regulate cell function.

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.

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.

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.

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.

De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs.

The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by aminoacyl-tRNA synthetase/tRNA pairs, which direct the unique coupling of specific amino acids with specific anticodons. The evolutionary record suggests that a primitive genetic code expanded into the current genetic code, over billions of years, through duplication and specialization (neofunctionalization) of aminoacyl-tRNA synthetases and tRNAs from common ancestral synthetase/tRNA pairs. This process produced the current set of mutually orthogonal aminoacyl-tRNA synthetases and tRNAs that direct natural protein synthesis. Here we demonstrate the creation of new orthogonal pairs, which are mutually orthogonal with existing orthogonal pairs, de novo, by a logical series of steps implemented in the laboratory, via the de novo generation of orthogonality in RNA-RNA interactions, protein-RNA interactions, and small molecule substrate selection by protein catalysts. Our laboratory evolution experiments provide experimental evidence for duplication and specialization as a plausible route to the current set of synthetases and tRNAs via natural evolution. Moreover our experiments extend billions of years of natural evolution and demonstrate that the small number of naturally occurring orthogonal aminoacyl-tRNA synthetase/tRNA pairs do not place an intrinsic limitation on the scope of synthetic genetic code expansion for the incorporation of multiple distinct unnatural amino acids into proteins or the synthesis and evolution of unnatural polymers in cells.