Protein–protein interactions (PPIs) govern diverse cellular processes, which raise demands for better methods to investigate them. We have herein developed a method for high-resolution mapping of endogenous PPIs – Enzyme-Activated Proximity of Oligonucleotides Sensing (EPOS) – an antibody-based proximity assay that integrates enzymatic activation with the hybridization chain reaction (HCR). EPOS utilizes deoxyuridine-containing DNA hairpins conjugated to antibodies; following target binding, uracil-DNA glycosylase and endonuclease IV degrade blocking regions, thereby unmasking initiator sequences that drive HCR amplification. This design generates compact, fluorophore-dense products, by that improving subcellular localization compared with rolling circle amplification-based in situ proximity ligation assays (in situ PLA). Benchmarking against in situ PLA across multiple cellular models demonstrated EPOS’s superior spatial resolution and broader dynamic range. EPOS offers a robust and versatile platform for quantitative, high-resolution visualization of protein complexes in situ.

The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 × 10-6 with an enrichment in active promoter regions.

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.

IntroductionDevelopment of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.Areas coveredOver the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.Expert opinionUnderstanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.

The ability to analyze whether DNA includes lesions is important in identifying mitogenic substances. Until now, the detection of single-stranded DNA breaks (SSBs) has lacked precise methods. To overcome this limitation, we have engineered a chimeric DNA polymerase, Sloppymerase, that is able to replicate DNA in the absence of one nucleotide. In addition to polymerase activity, Sloppymerase demonstrates 5´-3´exonuclease activity. We characterized the activity of Sloppymerase and utilized the enzyme to develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq) that is relevant to the mapping of SSBs. Following the omission of a specific nucleotide, e.g., dATP, from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions that should contain deoxyadenosine. The ability to retain sequence information after end-labelling ensures that hits are bona fide SSBs. STEEL-seq works with a variety of sequencing technologies, shown by our successful experiments using Sanger, Illumina, PacBio and Nanopore systems.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.

Platelet-derived growth factor (PDGF)-induced signalling via PDGF receptor β (PDGFRβ) leads to activation of downstream signalling pathways which regulate multiple cellular responses. It is unclear how PDGFRβ is degraded; both lysosomal and proteasomal degradation have been suggested. In this study, we have characterised the proteolytic cleavage of ligand-activated PDGFRβ, which results in two fragments: a larger fragment containing the extracellular domain, the transmembrane segment, and a part of the intracellular juxtamembrane region with a molecular mass of ∼130 kDa, and an intracellular ∼70 kDa fragment released into the cytoplasm. The proteolytic processing did not take place without internalisation of PDGFRβ. In addition, chelation of intracellular Ca2+ inhibited proteolytic processing. Inhibition of the proteasome affected signal transduction by increasing the phosphorylation of PDGFRβ, PLCγ, and STAT3 while reducing it on Erk1/2 and not affecting Akt. The proteolytic cleavage was observed in fibroblasts or cells that had undergone epithelial-mesenchymal transition.

This study explored the signaling interplay between the vitamin D receptor (VDR) and receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB promotes cell proliferation in normal and cancer cells. At the same time, the active form of vitamin D (1,25(OH)2-vitamin D3) inhibits proliferation in some cells. Although EGF receptors (EGFR) and PDGF receptors (PDGFR) activate similar downstream pathways, we found that they interact with VDR signaling in distinct ways. We confirmed that 1,25(OH)2-vitamin D3 induces CYP24A1 gene expression in U2OS, T98G, and U251 cells. We found this to be potentiated when combined with EGF. In contrast, PDGF-BB did not impact 1,25(OH)2-vitamin D3-induced CYP24A1 expression in U2OS cells. The increase in CYP24A1 expression due to the combined action of EGF and 1,25(OH)2-vitamin D3 was dependent on AKT and ERK1/2 activation. Another VDR-responsive gene, CYP27B1, was unaffected by the addition of EGF, suggesting that EGF may have gene-specific effects on VDR signaling. While PDGF-BB did not influence CYP24A1 expression, 1,25(OH)2-vitamin D3 significantly influenced PDGF-BB-induced receptor phosphorylation and cell proliferation. In summary, we found that EGF, but not PDGF-BB, influenced the expression of the VDR-dependent gene CYP24A1, while 1,25(OH)2-vitamin D3 had an inhibitory effect on PDGFR signaling and proliferation. These findings highlight unique crosstalk between 1,25(OH)2-vitamin D3 signaling and EGF or PDGF-BB.

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

Supplementary Fig. S1: Tissue microarray VN:IGFBP-3 PLA immunoreactivity and patient information; Supplementary Fig. S2: VN peptide array amino acid sequence information; Supplementary Fig. S3: IGFBP-3 peptide array amino acid sequence information; Supplementary Fig. S4: VN and IGFBP-3 immunoreactivity of HMLER-FOXC2 primary tumour xenografts; Supplementary Fig. S5: Single channel images of in situ PLA; Supplementary Fig. S6: IGF-II-induced cell signalling in MCF-7 breast cancer cells; Supplementary Fig. S7: Breast cell migration measured using the xCELLigence assay; Supplementary Fig. S8: Optimisation of ligand binding to VN and its subsequent immunodetection; Supplementary Fig. S9: Identification of ligand binding sites on VN using information from VN peptide arrays; Supplementary Fig. S10: Binding sites on IGFBP-3 for VN using the IGFBP-3 peptide arrays

Supplementary Table 1 from Novel and Highly Recurrent Chromosomal Alterations in Sézary Syndrome

Supplementary Table 3 from Novel and Highly Recurrent Chromosomal Alterations in Sézary Syndrome

Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on every hairpin being metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which puts a strong demand on oligonucleotide quality. We show how further purification can greatly increase polymerization potential. It was found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved poly-merization, yielding in situ immunoHCR stains at least 3.4-times stronger than a non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.

Supplementary Figure 1 from Novel and Highly Recurrent Chromosomal Alterations in Sézary Syndrome

Supplementary Table 2 from Novel and Highly Recurrent Chromosomal Alterations in Sézary Syndrome

Supplementary materials and methods

Supplementary Table 4 from Novel and Highly Recurrent Chromosomal Alterations in Sézary Syndrome