A 'writer-reader-eraser' post-translational modification regulatory system consisting of a large number of methyltransferases 8,9 , methyl-recognition domain-containing proteins 10 and putative demethylases 11 are expressed in different subcellular locations in humans, an indication that protein methylation is involved in processes other than epigenetic regulation.We prepared 82 Y2H bait strains spanning human R-methyltransferases (PRMT1-PRMT8) 8 , 16 SET domaincontaining K-methyltransferases (PKMTs) 9 , 9 members of the JMJD domain-containing protein family of protein demethylases and AOF2 (LSD1) 11 (Supplementary Table 1). In our current matrix screening protocol 4,12 , we perform four replicates, testing every set of baits individually against each of the ~13,000 prey contained in the matrix. Interacting prey are identified by their position in the matrix. To increase the sensitivity of the approach while also reducing the workload, we used a pooled strategy to test each protein pair substantially more than four times. Baits were pooled with all prey strains and then assayed for interaction in more than 100,000 separate spots ( Fig. 1a and Supplementary Fig. 1). Using Y2H-seq, we obtained 4-10 times the number of positive colonies obtained with the matrix approach. To reveal the prey identities, we collected all colonies and performed a 36-base parallel sequencing run. More than 20 million reads mapped perfectly to human RefSeq coding sequences (open reading frames, ORFs), corresponding to more than 500,000 unique 36-base reads (Supplementary Table 2). To rank the potentially interacting proteins for subsequent interaction retesting, we calculated a 'SeqScore' that incorporates the number of total mappings and the number of unique reads matching the ORF ( Supplementary Fig. 2). Notably, >99.7% of the RefSeq mappings matched to the 400 top-ranked genes, thus allowing the identification of potentially interacting ORFs with an extremely high signal-tobackground ratio (Supplementary Table 2).We performed four biological replicates and demonstrated in statistical pairwise comparisons that they result in very similar ranked prey orders (Supplementary Fig. 3). Top-ranked prey in at least two replicate screens were retested against all baits in a pairwise manner (Supplementary Fig. 4) and yielded 463 protein interactions (Supplementary Table 3). The success rate of the retest-that is, the probability that the prey is interactingdecreased with decreasing SeqScore (Fig. 1b).We also performed a matrix screen in quadruplicate with a subset of the protein methyltransferase (PMT) and protein demethylase (PDeM) baits for direct method comparison. With the matrix approach, we found 151 interactions (Supplementary to accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2h-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2h-seq to investigate enzymes in...
Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.
The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulation in all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.Keywords: oligonucleotide-peptide conjugates; cellular uptake; cell-penetrating peptides.The effectiveness of antisense oligonucleotides and peptide nucleic acids in modifying mammalian cell function can be improved substantially by covalent attachment or complexing with natural cell-penetrating peptide sequences [1][2][3][4]. This increase in biological activity has been commonly attributed to an enhanced cellular uptake of the conjugates [5][6][7]. The peptide components used to date have been protein-derived sequences that exhibit very different structural properties, ranging from lipophilic to unstructured and highly positively charged sequences [5,[7][8][9][10][11] as well as to strongly structured amphipathic ones [12][13][14][15]. The structural requirements for the peptide moiety and the necessity for covalent attachment remain controversial.In the present study we investigated the influence of the complexing or covalent tagging of phosphorothioate oligonucleotides with cationic model peptides of different structure forming properties (Table 1, Fig. 1) upon the cellular uptake. The a-helical amphipathic 18-mer model peptide used here (I) and its derivatives (exhibiting reduced helicity or amphipathicity) were previously shown to possess analogous cell penetrating properties to the above mentioned natural sequences [16][17][18]. We observed extensive cellular uptake of naked oligonucleotides as well as of their peptide derivatives. The uptake rates were all within an order of magnitude for a given cell type and oligonucleotide length irrespective of the mode of peptide binding or peptide structural properties. Conjugation or complexing of the oligonucleotides with the most widely used natural vector peptide, derived from the homeodomain of Antennapedia [19], led to comparable results. Our results therefore imply other aspects than an improved translocation across mammalian plasma membranes such as increased affinity to target structures or interactions with oligonucleotide bind...
The ability of the glucocorticoid receptor (GR) to regulate the transcriptional output of genes relies on its interactions with transcriptional coregulators. However, which coregulators are required for GR-dependent activation is context-dependent and can be influenced by the sequence of the DNA bound by GR and by the nature of the GR isoform responsible for the regulation of a gene. Here, we screened for GR-interacting proteins for which the interaction signal differed between two GR isoforms GRα and GRγ. These isoforms diverge by a single amino acid insertion in a domain, the lever arm, which adopts DNA sequence-specific conformations. We identify Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), an AP-1 family transcription factor, as a GR coregulator whose interaction with GR is modulated by the lever arm. Further, a combination of experiments uncovered that BATF3 acts as a gene-specific coactivator of GR whose coactivator potency is influenced by the sequence of the GR binding site. Together, our findings suggest that GR isoform and the sequence of GR binding site influence the interaction of GR with BATF3, which might direct the assembly of gene-specific regulatory complexes to fine-tune the expression of individual GR target genes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.