RESUMEN
Protein (un)folding rates depend on the free-energy barrier separating the native and unfolded states and a prefactor term, which sets the timescale for crossing such barrier or folding speed limit. Because extricating these two factors is usually unfeasible, it has been common to assume a constant prefactor and assign all rate variability to the barrier. However, theory and simulations postulate a protein-specific prefactor that contains key mechanistic information. Here, we exploit the special properties of fast-folding proteins to experimentally resolve the folding rate prefactor and investigate how much it varies among structural homologs. We measure the ultrafast (un)folding kinetics of five natural WW domains using nanosecond laser-induced temperature jumps. All five WW domains fold in microseconds, but with a 10-fold difference between fastest and slowest. Interestingly, they all produce biphasic kinetics in which the slower phase corresponds to reequilibration over the small barrier (<3 RT) and the faster phase to the downhill relaxation of the minor population residing at the barrier top [transition state ensemble (TSE)]. The fast rate recapitulates the 10-fold range, demonstrating that the folding speed limit of even the simplest all-ß fold strongly depends on the amino acid sequence. Given this fold's simplicity, the most plausible source for such prefactor differences is the presence of nonnative interactions that stabilize the TSE but need to break up before folding resumes. Our results confirm long-standing theoretical predictions and bring into focus the rate prefactor as an essential element for understanding the mechanisms of folding.
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Proteínas/química , Secuencia de Aminoácidos , Cinética , Pliegue de Proteína , Proteínas/genética , Proteínas/metabolismo , Termodinámica , Dominios WWRESUMEN
OBJECTIVE: The Expert Consensus Guidelines initiative on MIDH for LDLT was organized with the goal of safe implementation and development of these complex techniques with donor safety as the main priority. BACKGROUND: Following the development of minimally invasive liver surgery, techniques of MIDH were developed with the aim of reducing the short- and long-term consequences of the procedure on liver donors. These techniques, although increasingly performed, lack clinical guidelines. METHODS: A group of 12 international MIDH experts, 1 research coordinator, and 8 junior faculty was assembled. Comprehensive literature search was made and studies classified using the SIGN method. Based on literature review and experts opinions, tentative recommendations were made by experts subgroups and submitted to the whole experts group using on-line Delphi Rounds with the goal of obtaining >90% Consensus. Pre-conference meeting formulated final recommendations that were presented during the plenary conference held in Seoul on September 7, 2019 in front of a Validation Committee composed of LDLT experts not practicing MIDH and an international audience. RESULTS: Eighteen Clinical Questions were addressed resulting in 44 recommendations. All recommendations reached at least a 90% consensus among experts and were afterward endorsed by the validation committee. CONCLUSIONS: The Expert Consensus on MIDH has produced a set of clinical guidelines based on available evidence and clinical expertise. These guidelines are presented for a safe implementation and development of MIDH in LDLT Centers with the goal of optimizing donor safety, donor care, and recipient outcomes.
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Hepatectomía/métodos , Hepatectomía/normas , Trasplante de Hígado , Recolección de Tejidos y Órganos/normas , Humanos , Donadores Vivos , Procedimientos Quirúrgicos Mínimamente InvasivosRESUMEN
Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR-, isothermal calorimetry-, and X-ray crystallography-based analyses of the p12-PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12 N terminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively; and is stabilized by an aspartate at +6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested.
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ADN Polimerasa III/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Secuencias de Aminoácidos , Calorimetría , Dominio Catalítico , ADN Polimerasa III/química , ADN Polimerasa III/aislamiento & purificación , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificaciónRESUMEN
BACKGROUND: Acute kidney injury (AKI) after liver transplantation (LT) is a common problem with complex management. The aims were to analyze the profile of AKI-RIFLE categories in the post-transplant setting of a wide multicentre cohort of patients in the MELD era and to specifically determine the effect of tacrolimus-based (TACRO) immunosuppressive regimes on the development of AKI. METHODS: A retrospective analysis of 550 (2007-2012) consecutive patients transplanted at Reina Sofia, Cordoba, and King's College Hospital, London, was performed. Inclusion criterion was to have CNI as part of initial immunosuppression immediately after LT. RESULTS: After exclusion criteria, a total of 477 patients were analyzed. Incidence of AKI within the first 2 weeks after LT was 65.8% (AKI-Risk), 41.3% (AKI-Injury), and 12.3% (AKI-Failure). The development of any type of AKI had no impact on short- and/or long-term survival up to 3 years after the transplant. Moreover, AKI was almost universal in the early post-transplant period and TACRO trough concentrations during the first 2 weeks after the transplant were not predictors of AKI in none of its categories in the multivariate analyses. CONCLUSIONS: Low-TACRO-based regimes were not as useful as expected in the prevention of AKI when analyzed in the context of a large contemporary LT series.
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Lesión Renal Aguda , Trasplante de Hígado , Lesión Renal Aguda/epidemiología , Lesión Renal Aguda/etiología , Humanos , Inmunosupresores/efectos adversos , Trasplante de Hígado/efectos adversos , Estudios Retrospectivos , Factores de Riesgo , Tacrolimus/efectos adversosRESUMEN
BACKGROUND: The laparoscopic approach to liver resection has experienced exponential growth in recent years. However, evidence-based guidelines are needed for its safe future progression. The main aim of our study was to perform a systematic review and meta-analysis comparing the short- and long-term outcomes of laparoscopic and open liver resections for colorectal liver metastases (CRLM). METHODS: To identify all the comparative manuscripts between laparoscopic and open liver resections for CRLM, all published English language studies with more than ten cases were screened. In addition to the primary meta-analysis, 3 specific subgroup analyses were performed on patients undergoing minor-only, major-only and synchronous resections. The quality of the studies was assessed using the Scottish Intercollegiate Guidelines Network (SIGN) methodology and Newcastle-Ottawa Score. RESULTS: From the initial 194 manuscripts identified, 21 were meta-analysed, including results from the first randomized trial comparing open and laparoscopic resections of CRLM. Five of these were specific to patients undergoing a synchronous resection (399 cases), while six focused on minor (3 series including 226 cases) and major (3 series including 135 cases) resections, respectively. Thirteen manuscripts compared 2543 cases but could not be assigned to any of the above sub-analyses, so were analysed independently. The majority of short-term outcomes were favourable to the laparoscopic approach with equivalent rates of negative resection margins. No differences were observed between the approaches in overall or disease-free survival at 1, 3 or 5 years. CONCLUSION: Laparoscopic liver resection for CRLM offers improved short-term outcomes with comparable long-term outcomes when compared to open approach.
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Neoplasias Colorrectales/patología , Hepatectomía/métodos , Laparoscopía , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Humanos , Resultado del TratamientoRESUMEN
BACKGROUND: The laparoscopic approach to liver resection has experienced exponential growth in recent years; however, its application is still under debate and objective, evidence-based guidelines for its safe future progression are needed. OBJECTIVE: The aim of this study was to perform a systematic review and meta-analysis comparing the short- and long-term outcomes of laparoscopic and open liver resections for hepatocellular carcinoma (HCC). METHODS: To identify all the comparative manuscripts reporting on laparoscopic and open liver resection for HCC, all published English-language studies with more than 10 cases were screened. In addition to the primary meta-analysis, four specific subgroup analyses were performed on patients with Child-Pugh A cirrhosis, resections for solitary tumors, and those undergoing minor and major resections. The quality of the studies was assessed using the Scottish Intercollegiate Guidelines Network (SIGN) methodology and the Newcastle-Ottawa Scale. RESULTS: From the initial 361 manuscripts, 28 were included in the meta-analysis. Five of these 28 manuscripts were specific to patients with Child-Pugh A cirrhosis (321 cases), 11 focused on solitary tumors (1003 cases), 16 focused on minor resections (1286 cases), and 3 focused on major resections (164 cases). Three manuscripts compared 1079 cases but could not be assigned to any of the above subanalyses. In general terms, short-term outcomes were favorable when using a laparoscopic approach, especially in minor resections. The only advantage seen with an open approach was reduced operative time during major liver resections. No differences in long-term outcomes were observed between the approaches. CONCLUSIONS: Laparoscopic liver resection for HCC is feasible and offers improved short-term outcomes, with comparable long-term outcomes as the open approach.
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Carcinoma Hepatocelular/cirugía , Hepatectomía/mortalidad , Laparoscopía/mortalidad , Tiempo de Internación/estadística & datos numéricos , Neoplasias Hepáticas/cirugía , Complicaciones Posoperatorias , Guías de Práctica Clínica como Asunto/normas , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
BACKGROUND AND AIMS: Biliary anastomosis is a frequent area of complications after liver transplantation (LT) and a potential area of "microangiopathy". The concept of a "marginal bile duct" is unexplored. The main aim was to make a preliminary evaluation of the utility of an innovative real-time oxygen microtension (pO2mt) testing device for the assessment of bile duct viability during LT and to correlate these pO2mt values with microvascular tissue quality by histopathology and outcomes. PATIENTS AND METHODS: Observational prospective cohort study with 23 patients. Oxygen microtension measurements were made placing a micropO2 probe in different areas of recipient and donor's bile duct intraoperative. RESULTS: Mean pO2mt in the graft bile duct at the level of the anastomosis 103.82 (31-157) mm Hg, being 121.52 (55-174) mm Hg 1.5 cm proximal to the hilar plate (P < 0.001). Mean pO2mt in the recipient's bile duct was 117.87 (62-185) mm Hg, while a value of 137.30 (81-198) mm Hg was observed 1.5 cm distal to the anastomosis (P < 0.001). Cystic duct resection (12 cases) was also related with higher pO2mt values at anastomosis [117.8 (93-157) vs 88.54 (31-124) mm Hg] and distal to anastomosis [135.6 (111-174) vs 106.2 (55-133) mm Hg; P < 0.001]. Patients with 1-, 3-, and 12-month biliary complications had significantly lower pO2mt in the intraoperative measurements. CONCLUSION: Our preliminary results show that distal borders of donor and recipient bile ducts may be low-vascularized areas. Tissue pO2mt is significantly higher in areas close to the hilar plate and to the duodenum in donor and recipient's sides, respectively. Bile duct injury and biliary complications are associated with worse tissue pO2mt.
Asunto(s)
Anastomosis Quirúrgica/efectos adversos , Enfermedades de las Vías Biliares/diagnóstico , Sistema Biliar/metabolismo , Complicaciones Intraoperatorias/diagnóstico , Trasplante de Hígado/efectos adversos , Donadores Vivos/provisión & distribución , Oxígeno/metabolismo , Sistema Biliar/patología , Enfermedades de las Vías Biliares/etiología , Enfermedades de las Vías Biliares/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Complicaciones Intraoperatorias/etiología , Complicaciones Intraoperatorias/metabolismo , Masculino , Persona de Mediana Edad , Oxígeno/efectos adversos , Oxígeno/análisis , Pronóstico , Estudios Prospectivos , Factores de RiesgoRESUMEN
Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis.
Asunto(s)
Dominios Homologos src , Secuencia de Aminoácidos , Animales , Biología Computacional , Simulación por Computador , Evolución Molecular , Humanos , Ligandos , Modelos Moleculares , Mutación , Unión Proteica , Alineación de Secuencia , Termodinámica , Dominios Homologos src/genéticaRESUMEN
The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.
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Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/química , Dominios Homologos src , Algoritmos , Regulación Alostérica , Rastreo Diferencial de Calorimetría , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , TermodinámicaRESUMEN
The WW domains are the smallest modular domains known. The study of the structural basis of their stability is important to understand their physiological role. These domains are intrinsically flexible, which makes them difficult to crystallize. The first WW domain of the human Yes tyrosine kinase Associated Protein (YAP) has been crystallized and its structure has been solved by X-ray diffraction at 1.6 Å resolution. Crystals belong to the orthorhombic space group P21212 with unit cell parameters a=42.67, b=43.10 and c=21.30. The addition of proline and other small-molecule additives improves drastically the quality of the crystals. The interactions that stabilize this minimal modular domain have been analysed. This crystal structure reveals that, besides the stabilization of the hydrophobic core of the protein by the aromatic cluster formed by Trp177-Phe189-Pro202, some salt-bridges interactions might affect the stability of the domain.
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Proteínas Adaptadoras Transductoras de Señales/química , Fosfoproteínas/química , Isoformas de Proteínas/química , Secuencia de Aminoácidos , Cristalización/métodos , Cristalografía por Rayos X/métodos , Humanos , Datos de Secuencia Molecular , Prolina/química , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción , Difracción de Rayos X/métodos , Proteínas Señalizadoras YAPRESUMEN
Colonic diverticular disease is a chronic disorder presenting with a variety of abdominal symptoms and recurrent episodes of acute diverticulitis. It is close linked to age so its prevalence has risen notably during the last decades in western countries, increasing costs related to medical attention. Recently, several works have provided evidence to a series of measures that could improve the outcomes as well as reduce expenses associated to this process.The aim of the present review is to expose a view of the new trends in the management of diverticulitis and colonic diverticular disease, based on the highest clinical evidence available.
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Enfermedades del Colon/terapia , Manejo de la Enfermedad , Diverticulitis del Colon/cirugía , Diverticulitis del Colon/terapia , Diverticulitis/terapia , Diverticulosis del Colon/cirugía , Diverticulosis del Colon/terapia , Anastomosis Quirúrgica , Enfermedades del Colon/cirugía , Diverticulitis/cirugía , Humanos , Laparoscopía , Lavado PeritonealRESUMEN
Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low µm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.
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Flavonoides/biosíntesis , Fragaria/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catequina/metabolismo , Cristalografía por Rayos X , Fragaria/genética , Ligandos , Redes y Vías Metabólicas , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Molecular Dynamics (MD) simulations are essential in analyzing the physical movement of molecules, with GROMACS being a widely recognized open-source package for this purpose. However, conducting analyses individually in GROMACS can take excessive time and effort. Addressing this challenge, we introduce ASGARD, an innovative workflow designed to streamline and automate the analysis of MD simulation of protein or protein-ligand complex. Unlike the traditional, manual approach, ASGARD enables researchers to generate comprehensive analyses with a single command line, significantly accelerating the research process and avoiding the laborious task of manual report generation. This tool automatically performs a range of analyses post-simulation, including system stability and flexibility assessments through RMSD Fluctuation and Distribution calculations. It further provides dynamic analysis using SASA, DSSP method graphs, and various interaction analyses. A key feature of ASGARD is its user-friendly design; it requires no additional installations or dependencies, making it highly accessible for researchers. In conclusion, ASGARD simplifies the MD simulation analysis process and substantially enhances efficiency and productivity in molecular research by providing an integrated, one-command analysis solution.Communicated by Ramaswamy H. Sarma.
RESUMEN
The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disrupting TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of novel host-oriented antivirals with a broad spectrum of action. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. Here we present a detailed thermodynamic, structural, and dynamic characterization viral PTAP Late domain recognition by TSG101-UEV, combining isothermal titration calorimetry, X-ray diffraction structural studies, molecular dynamics simulations, and computational analysis of intramolecular communication pathways. Our analysis highlights key contributions from conserved hydrophobic contacts and water-mediated hydrogen bonds at the PTAP binding interface. We have identified additional electrostatic hotspots adjacent to the core motif that modulate affinity. Using competitive phage display screening we have improved affinity by 1-2 orders of magnitude, producing novel peptides with low micromolar affinities that combine critical elements found in the best natural binders. Molecular dynamics simulations revealed that optimized peptides engage new pockets on the UEV domain surface. This study provides a comprehensive view of the molecular forces directing TSG101-UEV recognition of PTAP motifs, revealing that binding is governed by conserved structural elements yet tuneable through targeted optimization. These insights open new venues to design inhibitors targeting TSG101-dependent pathways with potential application as novel broad-spectrum antivirals.
Asunto(s)
Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Simulación de Dinámica Molecular , Unión Proteica , Termodinámica , Factores de Transcripción , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Humanos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Ligandos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Péptidos/química , Péptidos/metabolismo , Sitios de Unión , Dominios Proteicos , Técnicas de Visualización de Superficie Celular/métodosRESUMEN
Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.
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Proteínas Portadoras , Neoplasias Colorrectales , Cinesinas , Proteínas de Microfilamentos , Invasividad Neoplásica , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Portadoras/metabolismo , Cinesinas/metabolismo , Cinesinas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Metástasis de la Neoplasia/prevención & control , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tionas/farmacología , Antineoplásicos/farmacologíaRESUMEN
In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 (Src homology 3) complexes and their thermodynamic behaviour calls for a revision of the SH3-binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database in the present study reveals that this dual binding mode is universal to SH3 domains. Tightly bound buried-interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high percentage of the free SH3 domains. A detailed analysis of the pattern of water-mediated interactions enabled the identification of conserved hydration sites in the polyproline-recognition region and the establishment of relationships between hydration profiles and the sequence of both ligands and SH3 domains. Water-mediated interactions were also systematically observed in WW (protein-protein interaction domain containing two conserved tryptophan residues), UEV (ubiquitin-conjugating enzyme E2 variant) and EVH-1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] structures. The results of the present study clearly indicate that the current description of proline-rich sequence recognition by protein-protein interaction modules is incomplete and insufficient for a correct understanding of these systems. A new binding paradigm is required that includes interfacial water molecules as relevant elements in polyproline recognition.
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Péptidos/química , Dominios Homologos src , Bases de Datos de Proteínas , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Agua/químicaRESUMEN
Syntenin-1 is a multidomain protein containing a central tandem of two PDZ domains flanked by two unnamed domains. Previous structural and biophysical studies show that the two PDZ domains are functional both isolated and in tandem, occurring a gain in their respective binding affinities when joined through its natural short linker. To get insight into the molecular and energetic reasons of such a gain, here, the first thermodynamic characterization of the conformational equilibrium of Syntenin-1 is presented, with special focus on its PDZ domains. These studies include the thermal unfolding of the whole protein, the PDZ-tandem construct and the two isolated PDZ domains using circular dichroism, differential scanning fluorimetry and differential scanning calorimetry. The isolated PDZ domains show low stability (ΔG < 10 kJ·mol-1) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20-30 kJ·mol-1) and a fully cooperative behaviour, with energetics similar to that previously described for archetypical PDZ domains. The high-resolution structures suggest that this remarkable increase in cooperativity is associated to strong, water-mediated, interactions at the interface between the PDZ domains, associated to nine conserved hydration regions. The low Tm value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol-1), and native heat capacity values (above 40 kJ·K-1·mol-1), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics.
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Pliegue de Proteína , Sinteninas , Humanos , Calorimetría , Rastreo Diferencial de Calorimetría , Termodinámica , Dicroismo Circular , Desnaturalización ProteicaRESUMEN
The hepatitis C virus nonstructural 5A (NS5A) protein is a large zinc-binding phosphoprotein that plays an important role in viral RNA replication and is involved in altering signal transduction pathways in the host cell. This protein interacts with Fyn tyrosine kinase in vivo and regulates its kinase activity. The 1.5 Å resolution crystal structure of a complex between the SH3 domain of the Fyn tyrosine kinase and the C-terminal proline-rich motif of the NS5A-derived peptide APPIPPPRRKR has been solved. Crystals were obtained in the presence of ZnCl(2) and belonged to the tetragonal space group P4(1)2(1)2. The asymmetric unit is composed of four SH3 domains and two NS5A peptide molecules; only three of the domain molecules contain a bound peptide, while the fourth molecule seems to correspond to a free form of the domain. Additionally, two of the SH3 domains are bound to the same peptide chain and form a ternary complex. The proline-rich motif present in the NS5A protein seems to be important for RNA replication and virus assembly, and the promiscuous interaction of the Fyn SH3 domain with the NS5A C-terminal proline-rich peptide found in this crystallographic structure may be important in the virus infection cycle.
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Proteínas Proto-Oncogénicas c-fyn/química , Proteínas no Estructurales Virales/química , Secuencias de Aminoácidos , Calorimetría/métodos , Clonación Molecular , Cristalografía por Rayos X/métodos , Hepacivirus/metabolismo , Humanos , Ligandos , Luz , Péptidos/química , Prolina/química , Unión Proteica , Dispersión de Radiación , Transducción de Señal , Zinc/química , Dominios Homologos src/genéticaRESUMEN
Within the modular protein domains there are five families that recognize proline-rich sequences: SH3, WW, EVH1, GYF and UEV domains. This chapter reviews the main strategies developed for the design of ligands for these families, including peptides, peptidomimetics and drugs. We also describe some studies aimed to understand the molecular reasons responsible for the intrinsic affinity and specificity of these domains.
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Péptidos , Prolina , Sitios de Unión , Humanos , Ligandos , Péptidos/química , Prolina/química , Prolina/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions.