Determining folding and binding properties of the C-terminal SH2 domain of SHP2

SH2 domains are a class of protein–protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras-ERK1/2 signaling pathway that possess two SH2 domains, namely, N-SH2 and C-SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the SH2 domains of SHP2 is Gab2, a scaffolding protein with critical role in determining cell differentiation. Despite their key biological role and the importance of a correct native fold to ensure it, the mechanism of binding of SH2 domains with their ligands and the determinants of their stability have been poorly characterized. In this article, we present a comprehensive kinetic study of the folding of the C-SH2 domain and the binding mechanism with a peptide mimicking a region of Gab2. Our data, obtained at different pH and ionic strength conditions and supported by site-directed mutagenesis, highlight the role of electrostatic interactions in the early events of recognition. Interestingly, our results suggest a key role of a highly conserved histidine residue among SH2 family in the interaction with negative charges carried by the phosphotyrosine of Gab2. Moreover, the analysis of the equilibrium and kinetic folding data of C-SH2 describes a complex mechanism implying a change in rate-limiting step at high denaturant concentrations. Our data are discussed under the light of previous works on N-SH2 domain of SHP2 and other SH2 domains

Experimental characterization of the interaction between the n-terminal sh3 domain of crkl and c3g

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains

Unveiling induced folding of intrinsically disordered proteins – Protein engineering, frustration and emerging themes

Intrinsically disordered proteins (IDPs) can be generally described as a class of proteins that lack a well-defined ordered structure in isolation at physiological conditions. Upon binding to their physiological ligands, IDPs typically undergo a disorder-to-order transition, which may or may not lead to the complete folding of the IDP. In this short review, we focus on some of the key findings pertaining to the mechanisms of such induced folding. In particular, first we describe the general features of the reaction; then, we discuss some of the most remarkable findings obtained from applying protein engineering in synergy with kinetic studies to induced folding; and finally, we offer a critical view on some of the emerging themes when considering the structural heterogeneity of IDPs vis-à-vis to their inherent frustration

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure–function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, in-fluencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts

Mini-extracorporeal circulation minimizes coagulation abnormalities and ameliorates pulmonary outcome in coronary artery bypass grafting surgery

Hemostasis is impaired during CABG and coagulation abnormalities often result in clinically relevant organ dysfunctions, eventually increasing morbidity and mortality rates. Fifteen consecutive patients with coronary artery disease submitted to conventional extracorporeal circulation (cECC) have been compared with 15 matched patients, using mini-ECC (MECC). Postoperative lung function was evaluated according to gas exchange, intubation time and lung injury score. In the MECC group, thrombin-antithrombin complex levels (TaTc), prothrombin fragments (PF1+2) formation and thromboelastography (TEG) clotting times were lower compared to the cECC group (p=0.002 and p<0.001, respectively) whereas postoperative blood loss was higher in the cECC group (p=0.030) and more patients required blood transfusion (p=0.020). In the MECC group, postoperative gas exchange values were better, intubation time shorter and lung injury score lower (p<0.001 for all comparisons). Our study suggests that MECC induces less coagulation disorders, leading to lower postoperative blood loss and better postoperative lung function. This approach may be advantageous in high-risk patients. © The Author(s) 2013

The effect of bilateral internal thoracic artery harvesting on superficial and deep sternal infection: The role of skeletonization

Objective: To determine the relative risk of sternal dehiscence in patients undergoing bilateral internal thoracic artery harvesting and to assess whether and to what extent the technique of artery skeletonization might reduce this risk. Methods: Prospectively collected data on patients undergoing coronary artery bypass operations with at least a single internal thoracic artery were reviewed. The last 450 patients receiving bilateral internal thoracic artery grafts were compared with 450 patients who received a single internal thoracic artery during the same period. The left internal thoracic artery was always harvested in a pedicled fashion. Among patients receiving a bilateral internal thoracic artery, both arteries were harvested in a pedicled fashion in 300 cases, whereas both internal thoracic arteries were skeletonized in the remaining 150 cases. Results: Compared with a single internal thoracic artery, harvesting both internal thoracic arteries either in a skeletonized or in a pedicled fashion increased the chance of deep (1.1% vs 3.3% vs 4.7%; P =. 01) or superficial (4.8% vs 7.8% vs 12%; P =. 002) sternal infection. However, the technique of artery harvesting (odds ratio, 4.1; 95% confidence interval, 1.4-12.1); the presence of peripheral arteriopathy (odds ratio, 3.1; 95% confidence interval, 1.2-8.5), and resternotomy for bleeding (odds ratio, 8.2; 95% confidence interval, 2.0-33.6) were the only independent predictors for deep sternal infection, whereas the technique of artery harvesting (odds ratio, 3.0; 95% confidence interval, 1.6-5.4), female sex (odds ratio, 2.2; 95% confidence interval, 1.2-4.2), and diabetes (odds ratio, 1.7; 95% confidence interval, 1.0-2.9) were the only independent predictors of superficial sternal infection. In diabetic patients, there was no difference in the incidence of deep sternal infection among patients receiving a single internal thoracic artery or double skeletonized internal thoracic arteries (P =. 4). Conclusions: Bilateral internal thoracic artery harvesting carries a higher risk of sternal infection than harvesting a single internal thoracic artery. Skeletonization of both internal thoracic arteries significantly decreases this risk. A strategy of bilateral thoracic artery grafting can also be offered to patients at high risk for wound infection. Copyright © 2005 by The American Association for Thoracic Surgery

Study of the susceptibility to benznidazole in a Trypanosoma cruzi isolated from a patient of Mendoza

La enfermedad de Chagas es una patología endémica en 21 países de América, transportada a través de migraciones a países no endémicos como Estados Uni-dos y algunos países de Europa, Oceanía y Asia. En Argen-tina, la enfermedad de Chagas afecta de 6 a 8 millones de personas y provoca, en promedio, 12.000 muertes al año. Mendoza es una de las 6 provincias que aún figura con alto riesgo de trasmisión vectorial. Hasta el momento, Benznida-zol (BNZ) y Nifurtimox son las únicas drogas aprobadas para el tratamiento del Chagas. Si bien son muy eficaces para el tratamiento de la infección aguda, existen pocos anteceden-tes del efecto de las mismas en la etapa crónica. Objetivos: estudiar la susceptibilidad de un aislado de T. cruzi de Mendoza (TcM) frente a la droga BNZ comparando con la susceptibilidad de la cepa TcY mantenida en labora-torio, la cual es considerada me-dianamente resistente a la droga BNZ. Nos propusimos estudiar nuevas alternativas para mejorar el trata-miento de la enfermedad mediante el uso combinado de BNZ y otra droga utilizada para el trata-miento del parásito T. brucei, agente etiológico de la tripano-somiasis africana conocida como Eflornitina (DFMO)

Targeting PDZ domains as potential treatment for viral infections, neurodegeneration and cancer

The interaction between proteins is a fundamental event for cellular life that is generally mediated by specialized protein domains or modules. PDZ domains are the largest class of protein–protein interaction modules, involved in several cellular pathways such as signal transduction, cell–cell junctions, cell polarity and adhesion, and protein trafficking. Because of that, dysregulation of PDZ domain function often causes the onset of pathologies, thus making this family of domains an interesting pharmaceutical target. In this review article we provide an overview of the structural and functional features of PDZ domains and their involvement in the cellular and molecular pathways at the basis of different human pathologies. We also discuss some of the strategies that have been developed with the final goal to hijack or inhibit the interaction of PDZ domains with their ligands. Because of the generally low binding selectivity of PDZ domain and the scarce efficiency of small molecules in inhibiting PDZ binding, this task resulted particularly difficult to pursue and still demands increasing experimental efforts in order to become completely feasible and successful in vivo

A Bio-Conjugated Fullerene as a Subcellular-Targeted and Multifaceted Phototheranostic Agent

Fullerenes are candidates for theranostic applications because of their high photodynamic activity and intrinsic multimodal imaging contrast. However, fullerenes suffer from low solubility in aqueous media, poor biocompatibility, cell toxicity, and a tendency to aggregate. C70@lysozyme is introduced herein as a novel bioconjugate that is harmless to a cellular environment, yet is also photoactive and has excellent optical and optoacoustic contrast for tracking cellular uptake and intracellular localization. The formation, water-solubility, photoactivity, and unperturbed structure of C70@lysozyme are confirmed using UV-visible and 2D 1H, 15N NMR spectroscopy. The excellent imaging contrast of C70@lysozyme in optoacoustic and third harmonic generation microscopy is exploited to monitor its uptake in HeLa cells and lysosomal trafficking. Last, the photoactivity of C70@lysozyme and its ability to initiate cell death by means of singlet oxygen (1O2) production upon exposure to low levels of white light irradiation is demonstrated. This study introduces C70@lysozyme and other fullerene-protein conjugates as potential candidates for theranostic applications