Structural vulnerability in EPCR suggests functional modulation.

The endothelial protein C receptor (EPCR) is a fundamental component of the vascular system in mammals due to its contribution in maintaining blood in a non-prothrombotic state, which is crucial for overall life development. It accomplishes this by enhancing the conversion of protein C (PC) into the anticoagulant activated protein C (APC), with this property being dependent on a known EPCR conformation that enables direct interaction with PC/APC. In this study, we report a previously unidentified conformation of EPCR whereby Tyr154, critical for PC/APC binding, shows a striking non-canonical configuration. This unconventional form is incompatible with PC/APC binding, and reveals, for the first time, a region of structural vulnerability and potential modulation in EPCR. The identification of this malleability enhances our understanding of this receptor, prompting inquiries into the interplay between its plasticity and function, as well as its significance within the broader framework of EPCR's biology, which extends to immune conditions.

Impact of AlphaFold on structure prediction of protein complexes: The CASP15-CAPRI experiment.

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem.

Discriminating physiological from non-physiological interfaces in structures of protein complexes: A community-wide study.

Reliably scoring and ranking candidate models of protein complexes and assigning their oligomeric state from the structure of the crystal lattice represent outstanding challenges. A community-wide effort was launched to tackle these challenges. The latest resources on protein complexes and interfaces were exploited to derive a benchmark dataset consisting of 1677 homodimer protein crystal structures, including a balanced mix of physiological and non-physiological complexes. The non-physiological complexes in the benchmark were selected to bury a similar or larger interface area than their physiological counterparts, making it more difficult for scoring functions to differentiate between them. Next, 252 functions for scoring protein-protein interfaces previously developed by 13 groups were collected and evaluated for their ability to discriminate between physiological and non-physiological complexes. A simple consensus score generated using the best performing score of each of the 13 groups, and a cross-validated Random Forest (RF) classifier were created. Both approaches showed excellent performance, with an area under the Receiver Operating Characteristic (ROC) curve of 0.93 and 0.94, respectively, outperforming individual scores developed by different groups. Additionally, AlphaFold2 engines recalled the physiological dimers with significantly higher accuracy than the non-physiological set, lending support to the reliability of our benchmark dataset annotations. Optimizing the combined power of interface scoring functions and evaluating it on challenging benchmark datasets appears to be a promising strategy.

pyDockDNA: A new web server for energy-based protein-DNA docking and scoring.

Proteins and nucleic acids are essential biological macromolecules for cell life. Indeed, interactions between proteins and DNA regulate many biological processes such as protein synthesis, signal transduction, DNA storage, or DNA replication and repair. Despite their importance, less than 4% of total structures deposited in the Protein Data Bank (PDB) correspond to protein-DNA complexes, and very few computational methods are available to model their structure. We present here the pyDockDNA web server, which can successfully model a protein-DNA complex with a reasonable predictive success rate (as benchmarked on a standard dataset of protein-DNA complex structures, where DNA is in B-DNA conformation). The server implements the pyDockDNA program, as a module of pyDock suite, thus including third-party programs, modules, and previously developed tools, as well as new modules and parameters to handle the DNA properly. The user is asked to enter Protein Data Bank files for protein and DNA input structures (or suitable models) and select the chains to be docked. The server calculations are mainly divided into three steps: sampling by FTDOCK, scoring with new energy-based parameters and the possibility of applying external restraints. The user can select different options for these steps. The final output screen shows a 3D representation of the top 10 models and a table sorting the model according to the scoring function selected previously. All these output files can be downloaded, including the top 100 models predicted by pyDockDNA. The server can be freely accessed for academic use (https://model3dbio.csic.es/pydockdna).

New Insights into the Evolution of the Electron Transfer from Cytochrome f to Photosystem I in the Green and Red Branches of Photosynthetic Eukaryotes.

In cyanobacteria and most green algae of the eukaryotic green lineage, the copper-protein plastocyanin (Pc) alternatively replaces the heme-protein cytochrome c6 (Cc6) as the soluble electron carrier from cytochrome f (Cf) to photosystem I (PSI). The functional and structural equivalence of 'green' Pc and Cc6 has been well established, representing an example of convergent evolution of two unrelated proteins. However, plants only produce Pc, despite having evolved from green algae. On the other hand, Cc6 is the only soluble donor available in most species of the red lineage of photosynthetic organisms, which includes, among others, red algae and diatoms. Interestingly, Pc genes have been identified in oceanic diatoms, probably acquired by horizontal gene transfer from green algae. However, the mechanisms that regulate the expression of a functional Pc in diatoms are still unclear. In the green eukaryotic lineage, the transfer of electrons from Cf to PSI has been characterized in depth. The conclusion is that in the green lineage, this process involves strong electrostatic interactions between partners, which ensure a high affinity and an efficient electron transfer (ET) at the cost of limiting the turnover of the process. In the red lineage, recent kinetic and structural modeling data suggest a different strategy, based on weaker electrostatic interactions between partners, with lower affinity and less efficient ET, but favoring instead the protein exchange and the turnover of the process. Finally, in diatoms the interaction of the acquired green-type Pc with both Cf and PSI may not yet be optimized.

Sentinel Lymph Node Biopsy in Breast Cancer with 99mTc-Tilmanocept: A Multicenter Study on Real-Life Use of a Novel Tracer.

99mTc-tilmanocept is a novel radiopharmaceutical for sentinel lymph node (SLN) biopsy in breast cancer. Our aim was to describe results with 99mTc-tilmanocept in a heterogeneous group of breast cancer patients scheduled for SLN biopsy. Methods: Radiotracer preparation followed the manufacturer's indications. Local protocols for SLN detection within 9 participant centers were not changed for the entire duration of the study. In total, 344 patients with T1-T4, N0-N2 breast cancer (352 lesions) were included. Superficial (intradermal or periareolar) or deep (peritumoral or intratumoral) injections were performed. The doses were adjusted depending on the scheduled time for surgery. Results: Lymphoscintigraphy was able to depict at least 1 SLN in 339 of 352 breast lesions (96.3%), and the intraoperative SLN detection rate reached 97.2%. On univariable analysis, SLN detection rates did not differ by age, clinical T or N stage, tumor location, histologic subtype, or prior neoadjuvant therapy. Lymphoscintigraphy showed higher SLN detection in patients with a normal weight (body mass index < 25) than in those who were overweight or obese (body mass index ≥ 25), at 99.2% versus 94.6%, respectively (P = 0.031). The proportion of patients with preoperative lymphoscintigraphic detection or excised SLNs was higher with superficial than deep injections. Reinjected cases were significantly lower when superficial injection was chosen first (P < 0.001). Injection site and the tumor markers human epidermal growth factor receptor 2 and estrogen receptor had an impact on preoperative SLN visualization and intraoperative localization. In 80 cases, SLN biopsy resulted in a positive lymph node. During a mean follow-up of 19 mo, no axillary recurrences were observed. Conclusion: Whatever the protocol, 99mTc-tilmanocept showed good results in a heterogeneous breast cancer population, although the best results were achieved when a superficial injection was chosen.

Modeling of Protein Complexes and Molecular Assemblies with pyDock.

The study of the 3D structural details of protein interactions is essential to understand biomolecular functions at the molecular level. In this context, the limited availability of experimental structures of protein-protein complexes at atomic resolution is propelling the development of computational docking methods that aim to complement the current structural coverage of protein interactions. One of these docking approaches is pyDock, which uses van der Waals, electrostatics, and desolvation energy to score docking poses generated by a variety of sampling methods, typically FTDock or ZDOCK. The method has shown a consistently good prediction performance in community-wide assessment experiments like CAPRI or CASP, and has provided biological insights and insightful interpretation of experiments by modeling many biomolecular interactions of biomedical and biotechnological interest. Here, we describe in detail how to perform structural modeling of protein assemblies with pyDock, and the application of its modules to different biomolecular recognition phenomena, such as modeling of binding mode, interface, and hot-spot prediction, use of restraints based on experimental data, inclusion of low-resolution structural data, binding affinity estimation, or modeling of homo- and hetero-oligomeric assemblies.

Integrative modeling of protein-protein interactions with pyDock for the new docking challenges.

The seventh CAPRI edition imposed new challenges to the modeling of protein-protein complexes, such as multimeric oligomerization, protein-peptide, and protein-oligosaccharide interactions. Many of the proposed targets needed the efficient integration of rigid-body docking, template-based modeling, flexible optimization, multiparametric scoring, and experimental restraints. This was especially relevant for the multimolecular assemblies proposed in the CASP12-CAPRI37 and CASP13-CAPRI46 joint rounds, which were described and evaluated elsewhere. Focusing on the purely CAPRI targets of this edition (rounds 38-45), we have participated in all 17 assessed targets (considering heteromeric and homomeric interfaces in T125 as two separate targets) both as predictors and as scorers, by using integrative modeling based on our docking and scoring approaches: pyDock, IRaPPA, and LightDock. In the protein-protein and protein-peptide targets, we have also participated with our webserver (pyDockWeb). On these 17 CAPRI targets, we submitted acceptable models (or better) within our top 10 models for 10 targets as predictors, 13 targets as scorers, and 4 targets as servers. In summary, our participation in this CAPRI edition confirmed the capabilities of pyDock for the scoring of docking models, increasingly used within the context of integrative modeling of protein interactions and multimeric assemblies.

Blind prediction of homo- and hetero-protein complexes: The CASP13-CAPRI experiment.

We present the results for CAPRI Round 46, the third joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of 20 targets including 14 homo-oligomers and 6 heterocomplexes. Eight of the homo-oligomer targets and one heterodimer comprised proteins that could be readily modeled using templates from the Protein Data Bank, often available for the full assembly. The remaining 11 targets comprised 5 homodimers, 3 heterodimers, and two higher-order assemblies. These were more difficult to model, as their prediction mainly involved "ab-initio" docking of subunit models derived from distantly related templates. A total of ~30 CAPRI groups, including 9 automatic servers, submitted on average ~2000 models per target. About 17 groups participated in the CAPRI scoring rounds, offered for most targets, submitting ~170 models per target. The prediction performance, measured by the fraction of models of acceptable quality or higher submitted across all predictors groups, was very good to excellent for the nine easy targets. Poorer performance was achieved by predictors for the 11 difficult targets, with medium and high quality models submitted for only 3 of these targets. A similar performance "gap" was displayed by scorer groups, highlighting yet again the unmet challenge of modeling the conformational changes of the protein components that occur upon binding or that must be accounted for in template-based modeling. Our analysis also indicates that residues in binding interfaces were less well predicted in this set of targets than in previous Rounds, providing useful insights for directions of future improvements.

Exactitud diagnóstica de la gammagrafía de perfusión miocárdica en una población femenina con sospecha de cardiopatía isquémica / Diagnostic accuracy of myocardial perfusion gammagraphy in a female cohort with suspected ischemic heart disease

Introducción y objetivos: determinar los valores diagnósticos del estudio de perfusión miocárdica con 99mTc-tetrofosmina en protocolo de un día tras estímulo farmacológico con dipiridamol en una población de mujeres. Métodos: se estudiaron, con carácter retrospectivo, 202 historias clínicas de mujeres con sospecha de cardiopatía isquémica que fueron sometidas a una prueba de perfusión miocárdica. Se consideraron las variables: tratamiento farmacológico, factores de riesgo cardiovascular, parámetros bioquímicos, electrocardiograma basal, diagnóstico definitivo y parámetros de la gammagrafía de perfusión miocárdica. Resultados: los valores diagnósticos en general para la población objeto de estudio fueron de una sensibilidad de 87,16% y una especificidad de 81,48%. Conclusiones: la prueba de perfusión miocárdica estimulación/reposo con 99mTc-tetrofosmina y dipiridamol como vasodilatador, utilizando el protocolo de un día, presenta una elevada validez diagnóstica en mujeres con sospecha de cardiopatía isquémica.

Introduction and Objectives: to determine the diagnostic values of myocardial perfusion with 99mTc-tetrophosmine in a one-day protocol after pharmacological stimulation with dipyridamole in a women population. Methods: 202 medical records of women with suspected ischemic cardiopathy who underwent a myocardial perfusion test were studied. The following variables were considered: drug treatment, cardiovascular risk factors, biochemical parameters, baseline electrocardiogram, definitive diagnosis and myocardial perfusion scan parameters. Results: the diagnostic values for the studied population had in general a sensitivity of 87,16% and a specificity of 81,48%. Conclusions: The myocardial stimulation/rest perfusion test with 99mTc-tetrophosmine and dipyridamole as vasodilator using the one-day protocol, has a high diagnostic accuracy in women with suspected ischemic heart disease.

Valor diagnóstico de la perfusión miocárdica SPECT con dipiridamol en una población femenina / Diagnostic Value of Myocardial Perfusion SPECT with Dipyridamole in a Female Population

Introducción La gammagrafía de esfuerzo es un método ampliamente utilizado para el diagnóstico de la cardiopatía isquémica, que ofrece además una gran seguridad. Los estudios con estrés farmacológico son una alternativa importante para evocar una situación de estrés. La presentación más tardía de cardiopatía isquémica en mujeres que en hombres y las limitaciones relacionadas con el sexo para la realización de la ergometría han generado interés en el beneficio potencial añadido de la imagen de perfusión de esfuerzo en mujeres. Objetivo Determinar los valores diagnósticos del estudio de perfusión miocárdica con 99mTc-tetrofosmina en protocolo de un día tras estímulo farmacológico con dipiridamol en una población de mujeres, así como la relación con los territorios vasculares coronarios tomando como referencia la coronariografía. Material y métodos Se estudiaron con carácter retrospectivo 149 historias clínicas de mujeres con sospecha de cardiopatía isquémica sometidas a una prueba de perfusión miocárdica y posteriormente a una coronariografía. Resultados Se obtuvieron valores de sensibilidad y especificidad del 94% (93,47%-94,53%) y el 82% (80,94%-83,06%), respectivamente. Por territorio arterial, se obtuvieron valores de sensibilidad y especificidad del 71,62% (70,88%-72,36%) y el 76% (75,27%-76,73%) para la descendente anterior (DA), del 69,09% (68,11%-70,07%) y el 76,84% (76,26%-77,42%) para la circunfleja (Cx) y del 87,23% (86,11%-88,36%) y el 74,51% (73,97%-75,05%) para la coronaria derecha (CD), respectivamente. Conclusiones La gammagrafía de perfusión miocárdica estimulación/reposo con 99mTc-tetrofosmina y dipiridamol, utilizando el protocolo de un día, tiene alta sensibilidad y especificidad para el diagnóstico en mujeres con sospecha de cardiopatía isquémica.

Background Exercise stress scintigraphy is a safe procedure widely used for the diagnosis of ischemic heart disease. Pharmacologic stress testing is an important alternative. The delayed presentation of ischemic heart disease in women, together with a lower diagnostic accuracy of exercise stress testing in this population, has generated interest in the potential benefits provided by myocardial perfusion imaging tests. Objective To determine the diagnostic value of myocardial perfusion images with 99mTc-tetrofosmin in a one day protocol after a pharmacologic stress with dipyridamole in a female population, and the relation with the coronary territories using coronary angiography as a reference technique. Material and Methods In total, 149 clinical charts of women with suspected ischemic heart disease undergoing myocardial perfusion imaging tests and coronary angiography were retrospectively analyzed. Results Sensitivity and specificity were 94% (93.47%-94.53%) and 82% (80.94%-83.06%), respectively. Values of sensitivity and specificity according to coronary territories were 71.62% (70.88%-72.36%) and 76% (75.27%-76.73%) for the left anterior descending (LAD) artery, 69.09% (68.11%-70.07%) and 76.84% (76.26%-77.42%) for the left circumflex (LCx) coronary artery, and 87.23% (86.11%-88.36%) and 74.51% (73.97%-75.05%) for the right coronary artery (RCA), respectively. Conclusion Myocardial perfusion scintigraphy with 99mTc-tetrofosmin and dipyridamole using a one day stress-rest protocol has high sensitivity and specificity for the diagnosis of ischemic heart disease in women.