Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions

Abstract: Intrinsically Disordered Regions (IDRs) are enriched in disease-linked proteins known to have multiple post-translational modifications, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than ordered protein regions, if they retain their flexibility in human cells. Indeed, we observed increased biotinylation density in predicted IDRs in several cellular compartments >20,000 biotin sites from four proximity proteomics studies. We show that in a biotin ‘painting’ time course experiment, biotinylation events in Escherichia coli ribosomes progress from unfolded and exposed regions at 10 s, to structured and less accessible regions after five minutes. We conclude that biotin proximity tagging favours sites of local disorder in proteins and suggest the possibility of using biotin painting as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins

Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions

Abstract: Intrinsically Disordered Regions (IDRs) are enriched in disease-linked proteins known to have multiple post-translational modifications, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than ordered protein regions, if they retain their flexibility in human cells. Indeed, we observed increased biotinylation density in predicted IDRs in several cellular compartments >20,000 biotin sites from four proximity proteomics studies. We show that in a biotin ‘painting’ time course experiment, biotinylation events in Escherichia coli ribosomes progress from unfolded and exposed regions at 10 s, to structured and less accessible regions after five minutes. We conclude that biotin proximity tagging favours sites of local disorder in proteins and suggest the possibility of using biotin painting as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins

PLK1 inhibition dampens NLRP3 inflammasome–elicited response in inflammatory disease models

Unabated activation of the NLR family pyrin domain–containing 3 (NLRP3) inflammasome is linked with the pathogenesis of various inflammatory disorders. Polo-like kinase 1 (PLK1) has been widely studied for its role in mitosis. Here, using both pharmacological and genetic approaches, we demonstrate that PLK1 promoted NLRP3 inflammasome activation at cell interphase. Using an unbiased proximity-dependent biotin identification (Bio-ID) screen for the PLK1 interactome in macrophages, we show an enhanced proximal association of NLRP3 with PLK1 upon NLRP3 inflammasome activation. We further confirmed the interaction between PLK1 and NLRP3 and identified the interacting domains. Mechanistically, we show that PLK1 orchestrated the microtubule-organizing center (MTOC) structure and NLRP3 subcellular positioning upon inflammasome activation. Treatment with a selective PLK1 kinase inhibitor suppressed IL-1β production in in vivo inflammatory models, including LPS-induced endotoxemia and monosodium urate–induced peritonitis in mice. Our results uncover a role of PLK1 in regulating NLRP3 inflammasome activation during interphase and identify pharmacological inhibition of PLK1 as a potential therapeutic strategy for inflammatory diseases with excessive NLRP3 inflammasome activation.This work was supported by British Heart Foundation (BHF) fellowship grants (FS/14/28/30713 and FS/SBSRF/22/31036, to XL); a BHF project grant (PG/17/69/33229, to XL); a BHF PhD studentship (FS/17/5/32531, to XL); and a Cambridge BHF Centre of Research Excellence grant (RE/18/1/34212). MB was supported by a BHF PhD studentship (BHF FS/17/5/32531). CD was supported by a BHF 4-year PhD student programme (FS/16/53/32729). EW was supported by a BHF project grant (PG/17/69/33229). ZM is supported by a BHF chair grant (CH/10/001/27642). The BR team was supported by the Region Centre Val de Loire (2003-00085470) and the Conseil Général du Loiret and the European Regional Development Fund (FEDER no. 2016-00110366 and EX005756). CB was supported by a Wellcome Trust Investigator Award (108045/Z/15/Z). This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. We thank Mike Deery, Renata Feret, and Konstantin Barylyuk at the Cambridge Centre for Proteomics.Peer reviewe

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Biotin proximity tagging favours unfolded proteins

Intrinsically Disordered Regions (IDRs) are enriched in disease-linked proteins known to have multiple post-translational modifications, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than ordered protein regions, if they retain their flexibility in vivo. Indeed, we observed increased biotinylation density in predicted IDRs in the disorder-enriched compartments of the nucleus and cytoplasm among >20 000 biotin sites from four proximity proteomics studies. We conclude that biotin proximity tagging favours sites of local disorder in proteins and suggest the possibility of using biotin ‘painting’ as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins.Thermo Fisher gold level TMT awar

Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions

Abstract: Intrinsically Disordered Regions (IDRs) are enriched in disease-linked proteins known to have multiple post-translational modifications, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than ordered protein regions, if they retain their flexibility in human cells. Indeed, we observed increased biotinylation density in predicted IDRs in several cellular compartments >20,000 biotin sites from four proximity proteomics studies. We show that in a biotin ‘painting’ time course experiment, biotinylation events in Escherichia coli ribosomes progress from unfolded and exposed regions at 10 s, to structured and less accessible regions after five minutes. We conclude that biotin proximity tagging favours sites of local disorder in proteins and suggest the possibility of using biotin painting as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.UKRI-BBSRC (BB/P021107/1

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical science. © The Author(s) 2019. Published by Oxford University Press