Temporal validation of a risk prediction model for breast cancer-related lymphoedema in European population: A retrospective study.

AIMS: To perform temporal validation of a risk prediction model for breast cancer-related lymphoedema in the European population. DESIGN: Temporal validation of a previously developed prediction model using a new retrospective cohort of women who had undergone axillary lymph node dissection between June 2018 and June 2020. METHODS: We reviewed clinical records to identify women who did and did not develop lymphoedema within 2 years of surgery and to gather data regarding the variables included in the prediction model. The model was calibrated by calculating Spearman's correlation between observed and expected cases. Its accuracy in discriminating between patients who did versus did not develop lymphoedema was assessed by calculating the area under the receiver operating characteristic curve (AUC). RESULTS: The validation cohort comprised 154 women, 41 of whom developed lymphoedema within 2 years of surgery. The value of Spearman's coefficient indicated a strong correlation between observed and expected cases. Sensitivity of the model was higher than in the derivation cohort, as was the value of the AUC. CONCLUSION: The model shows a good capacity to discriminate women at risk of lymphoedema and may therefore help in developing improved care pathways for individual patients. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Identifying risk factors for lymphoedema secondary to breast cancer treatment is vital given its impact on women's physical and emotional well-being. IMPACT: What problem did the study address? Risk of BCRL. What were the main findings? The prediction model has a good capacity to discriminate women at risk of lymphoedema. Where and on whom will the research have an impact? In clinical practice with women at risk of BCRL. REPORTING METHOD: STROBE checklist. WHAT DOES THIS PAPER CONTRIBUTE TO THE WIDER GLOBAL CLINICAL COMMUNITY?: It presents a validated risk prediction model for BCRL. NO PATIENT OR PUBLIC CONTRIBUTION: There was no patient or public contribution in the conduct of this study.

Quantitative fragmentomics allow affinity mapping of interactomes.

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained by High-Throughput Holdup Assay.

The holdup assay is an automated high-throughput comparative chromatographic retention approach that allows to measure quantitative binding intensities (BI) for a large number of domain-motif pairs and deduce equilibrium binding affinity constants. We routinely apply this approach to obtain quantitative binding specificity profiles of particular PDZ-binding motifs (PBMs) toward the full library of known human PDZ domains (the PDZome). The quality of the electropherograms extracted from the capillary electrophoresis instrument at the final step of the holdup assay may vary, influencing the accuracy and reproducibility of the measurement. By using bioinformatic tools, we can solve these issues to extract more reliable BIs by means of a better superimposition of the electropherograms. The protocol presented in this chapter describes the main principles and strategies of our curated method to process holdup data and new ways to plot and compare the BIs for the PBM-PDZ interactions. For this particular protocol, all the necessary computing commands are freely available in open Python packages.

Interactomic affinity profiling by holdup assay: Acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase.

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study.

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches.

Rewiring of RSK-PDZ Interactome by Linear Motif Phosphorylation.

Phosphorylation of short linear peptide motifs is a widespread process for the dynamic regulation of protein-protein interactions. However, the global impact of phosphorylation events on the protein-protein interactome is rarely addressed. The disordered C-terminal tail of ribosomal S6 kinase 1 (RSK1) binds to PDZ domain-containing scaffold proteins, and it harbors a phosphorylatable PDZ-binding motif (PBM) responsive to epidermal growth factor stimulation. Here, we examined binding of two versions of the RSK1 PBM, either phosphorylated or unphosphorylated at position -3, to almost all (95%) of the 266 PDZ domains of the human proteome. PBM phosphorylation dramatically altered the PDZ domain-binding landscape of RSK1, by strengthening or weakening numerous interactions to various degrees. The RSK-PDZome interactome analyzed in this study reveals how linear motif-based phospho-switches convey stimulus-dependent changes in the context of related network components.