SuFEx Chemistry Enables Covalent Assembly of a 280-kDa 18-Subunit Pore-Forming Complex.

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein-protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.

Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response.

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1-controlled polyamine export.

Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species.

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC-MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast.

Global analysis of cytosine and adenine DNA modifications across the tree of life.

Interpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species, Raphanus sativus. In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa, Helianthus annuus, or Andropogon gerardi, more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications.

LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.

Machine Learning Predicts the Yeast Metabolome from the Quantitative Proteome of Kinase Knockouts.

A challenge in solving the genotype-to-phenotype relationship is to predict a cell's metabolome, believed to correlate poorly with gene expression. Using comparative quantitative proteomics, we found that differential protein expression in 97 Saccharomyces cerevisiae kinase deletion strains is non-redundant and dominated by abundance changes in metabolic enzymes. Associating differential enzyme expression landscapes to corresponding metabolomes using network models provided reasoning for poor proteome-metabolome correlations; differential protein expression redistributes flux control between many enzymes acting in concert, a mechanism not captured by one-to-one correlation statistics. Mapping these regulatory patterns using machine learning enabled the prediction of metabolite concentrations, as well as identification of candidate genes important for the regulation of metabolism. Overall, our study reveals that a large part of metabolism regulation is explained through coordinated enzyme expression changes. Our quantitative data indicate that this mechanism explains more than half of metabolism regulation and underlies the interdependency between enzyme levels and metabolism, which renders the metabolome a predictable phenotype.

Properties and exploitation of oleosins.

Oleosins stabilize oil bodies in seeds and other tissues and contain a unique hydrophobic domain which appears to be inserted into the oil matrix as an alpha-helical hairpin. The oleosin proteins may be exploited to stabilize emulsions while the ease of oil body preparation has led to the expression of bioactive proteins as oleosin fusions in molecular farming.

The metabolic background is a global player in Saccharomyces gene expression epistasis.

The regulation of gene expression in response to nutrient availability is fundamental to the genotype-phenotype relationship. The metabolic-genetic make-up of the cell, as reflected in auxotrophy, is hence likely to be a determinant of gene expression. Here, we address the importance of the metabolic-genetic background by monitoring transcriptome, proteome and metabolome in a repertoire of 16 Saccharomyces cerevisiae laboratory backgrounds, combinatorially perturbed in histidine, leucine, methionine and uracil biosynthesis. The metabolic background affected up to 85% of the coding genome. Suggesting widespread confounding, these transcriptional changes show, on average, 83% overlap between unrelated auxotrophs and 35% with previously published transcriptomes generated for non-metabolic gene knockouts. Background-dependent gene expression correlated with metabolic flux and acted, predominantly through masking or suppression, on 88% of transcriptional interactions epistatically. As a consequence, the deletion of the same metabolic gene in a different background could provoke an entirely different transcriptional response. Propagating to the proteome and scaling up at the metabolome, metabolic background dependencies reveal the prevalence of metabolism-dependent epistasis at all regulatory levels. Urging a fundamental change of the prevailing laboratory practice of using auxotrophs and nutrient supplemented media, these results reveal epistatic intertwining of metabolism with gene expression on the genomic scale.

Using the canary genome to decipher the evolution of hormone-sensitive gene regulation in seasonal singing birds.

BACKGROUND: While the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity. RESULTS: We present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch. CONCLUSIONS: The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics.

The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides.

Peptide display on Potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles.

The potexvirus Potato virus X (PVX) can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused to coat protein (CP) subunits. A spontaneous PVX mutant expressing a truncated, but functional, form of the CP has been isolated. With the aim of exploiting this virus to display peptides useful for vaccine formulations, two novel viral expression vectors based on pPVX201 (bearing the wild-type PVX genome) were constructed encoding the truncated CP. Both vectors were able to produce infectious virus particles in planta and were used to insert a panel of sequences encoding peptides of biopharmaceutical interest as N-terminal fusions to the truncated cp gene. The analysis of infection progression induced by the different constructs enabled identification of two important structural features of the fused peptide, namely tryptophan content and isoelectric point, critically affecting the formation of PVX CVPs and virus movement through the plant. These results are discussed in view of the rising interest in engineered plant viruses for development of peptide-based epitope vaccines.