PhosIDP: a web tool to visualize the location of phosphorylation sites in disordered regions

Nicolaou, Sonia T.; Hebditch, Max; Jonathan, Owen J.; Verma, Chandra; Warwicker, Jim

doi:10.1038/s41598-021-88992-0

Cited by 8 publications

(7 citation statements)

References 43 publications

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“…Previous charge distribution analysis showed that IDRs of proteins involved in LLPS are biased towards neutral net charge [17]. Human proteins in our dataset were divided into scaffold, client, and regulator proteins, or unannotated (null), based on DrLLPS, an online database that lists proteins involved in LLPS [46].…”

Section: Resultsmentioning

confidence: 99%

“…The charge-hydrophobicity relationship of IDPs is clearly demonstrated in the charge-hydropathy plot, also known as the Uversky plot [5, 14]. Several disorder prediction servers utilise this principle to describe IDPs [15–17]. A diagram-of-states to classify the predicted conformational properties of IDPs based on their charge-hydropathy relationship was initially created by Mao and co-workers and was later improved by Das and Pappu [18, 19].…”

Section: Introductionmentioning

confidence: 99%

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Enrichment of charge-absent regions in phase separated proteins

Nicolaou

Verma

Warwicker

2022

Preprint

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Many studies focus on the relationship between protein charge and liquid-liquid phase separation (LLPS), generally finding that a large degree of charge neutralisation is involved for condensate formation. Here, sequences within human proteins that lack the charge-bearing residues Asp, Glu, Lys, and Arg (termed charge-absent) are analysed alongside annotation for involvement in LLPS. Scaffold proteins, central to condensate formation, on average possess longer charge-absent regions than those not key for LLPS. Charge-absent regions tend to have relatively high hydropathy scores. Overall, they are enriched in Ala, Gly, Pro, and Ser with more specific groupings evident when the subset is clustered by amino acid composition. For several proteins, segments with charge-absent regions have been identified as modulators of LLPS. It is hypothesised that for at least some of the charge-absent regions, a lack of charged group desolvation energy, together with a relatively hydrophobic sequence composition, may facilitate condensation through homomeric interactions. If this is the case, it should be relatively easy to modulate through incorporation of charge through engineering, potentially including pH-sensing.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enrichment of charge-absent regions in phase separated proteins

Nicolaou

Verma

Warwicker

2022

Preprint

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“…Although we have demonstrated this capability using a single phosphorylation site in tau, we anticipate this method will be widely applicable to phospho-specific antibody characterization and screening. Due to the fact that protein phosphorylation sites tend to be located in disordered regions (Iakoucheva et al, 2004;Nicolaou et al, 2021), synthetic peptides containing phosphorylated residues have been extremely effective antigens for generating phospho-site specific antibodies suitable for a wide range of applications, including various cell and tissue labeling, immunoblotting, and immunoassays.…”

Section: Discussionmentioning

confidence: 99%

Yeast biopanning for detecting antibody binding to site-specific phosphorylations in tau

Arbaciauskaite¹,

Pirhanov²,

Liu³

et al. 2022

Preprint

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The detection of phosphorylated tau (p-tau) levels in clinical samples is of extreme importance for the detection of Alzheimer′s Disease (AD) as well as other neurodegenerative diseases. Recent reports show that detecting low levels of p-tau in plasma can be used as a reliable biomarker for detecting AD prior to the onset of memory loss. The ability to detect such low levels of p-tau is dependent on antibodies specific to the post translationally modified protein. However, the need for reliable phospho-site specific antibodies persists due to a lack of approaches for identifying monoclonal antibodies and characterizing non-specific binding. Here, we report a novel approach using the principles of yeast biopanning to create a robust platform that uses synthetic peptides as target antigens. Using peptides as antigens enables screening antibodies against defined post-translational modification sites, particularly for targeting intrinsically disordered proteins such as the human tau protein. To readily assess yeast binding and distinguish non-specific binding, we developed bi-directional expression vectors that allow antibody fragment surface display and intracellular fluorescent protein expression. We show that our platform can specifically and robustly detect a specific site within the p-tau target peptide when compared against non-phosphorylated controls. By improving biopanning parameters, we enabled phospho-specific capture of yeast cells displaying single-chain variable region fragments (scFvs) against p-tau with a wide range of affinities (KD = 0.2 to 60 nM). These results demonstrate that yeast biopanning can robustly capture yeast cells based on phospho-site specific antibody binding, opening doors for facile identification of high-quality monoclonal antibodies.

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“…This IDR of Src, which shares low sequence identity with other SFKs, interacts weakly with its targets through its limited and transient structural characteristics. It is thought that post-translational modifications additionally affect these interactions to confer the role of a protein regulation hub on IDR [41,83,84]. The FIMC composed of the SH4/UD scaffold with a structured SH3 in Src is conserved among SFKs.…”

Section: Phosphorylation Of Usrc In Srcmentioning

confidence: 99%

Regulatory Roles of the N-Terminal Intrinsically Disordered Region of Modular Src

Kato

2022

IJMS

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Src, the prototype of Src family kinases (SFKs), is a modular protein consisting of SH4 (SH4) and unique (UD) domains in an N-terminal intrinsically disordered region (IDR), and SH3, SH2, and kinase (KD) folded domains conserved among SFKs. Src functions as a pleiotropic signaling hub in proliferating and post-mitotic cells, and it is related to cancer and neurological diseases. However, its regulatory mechanism is unclear because the existing canonical model is derived from crystallographic analyses of folded constructs lacking the IDR. This work reviews nuclear magnetic resonance analyses of partially structured lipid-binding segments in the flexible UD and the fuzzy intramolecular complex (FIMC) comprising IDR and SH3 domains, which interacts with lipid membranes and proteins. Furthermore, recently determined IDR-related Src characteristics are discussed, including dimerization, SH4/KD intramolecular fastener bundling of folded domains, and the sorting of adhesive structures. Finally, the modulatory roles of IDR phosphorylation in Src activities involving the FIMC are explored. The new regulatory roles of IDRs are integrated with the canonical model to elucidate the functions of full-length Src. This review presents new aspects of Src regulation, and provides a future direction for studies on the structure and function of Src, and their implications for pathological processes.

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PhosIDP: a web tool to visualize the location of phosphorylation sites in disordered regions

Cited by 8 publications

References 43 publications

Enrichment of charge-absent regions in phase separated proteins

Enrichment of charge-absent regions in phase separated proteins

Yeast biopanning for detecting antibody binding to site-specific phosphorylations in tau

Regulatory Roles of the N-Terminal Intrinsically Disordered Region of Modular Src

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