Unequivocal single-molecule force spectroscopy of proteins by AFM using pFS vectors

Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages, applications, and potential of this ready-to-go system are illustrated through the analysis of representative proteins. © 2012 Biophysical Society.Peer Reviewe

Structural Evidence of Amyloid Fibril Formation in the Putative Aggregation Domain of TDP-43

TDP-43 can form pathological proteinaceous aggregates linked to ALS and FTLD. Within the putative aggregation domain, engineered repeats of residues 341-366 can recruit endogenous TDP-43 into aggregates inside cells; however, the nature of these aggregates is a debatable issue. Recently, we showed that a coil to β-hairpin transition in a short peptide corresponding to TDP-43 residues 341-357 enables oligomerization. Here we provide definitive structural evidence for amyloid formation upon extensive characterization of TDP-43(341-357) via chromophore and antibody binding, electron microscopy (EM), solid-state NMR, and X-ray diffraction. On the basis of these findings, structural models for TDP-43(341-357) oligomers were constructed, refined, verified, and analyzed using docking, molecular dynamics, and semiempirical quantum mechanics methods. Interestingly, TDP-43(341-357) β-hairpins assemble into a novel parallel β-turn configuration showing cross-β spine, cooperative H-bonding, and tight side-chain packing. These results expand the amyloid foldome and could guide the development of future therapeutics to prevent this structural conversion.Peer Reviewe

The nanomechanics of neurotoxina proteins reveals common features at the start of the neurodegeneration cascade.

1 pags. -- 56th Annual Meeting of the Biophysical-Society, FEB 25-29, 2012, San Diego, CAAmyloidogenic neurodegenerative diseases are incurable conditions caused by specific largely disordered proteins. However, the underlying molecular mechanism remains elusive. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these ‘‘neurotoxic proteins’’ triggers the pathogenic cascade. Using force spectroscopy with unequivocal singlemolecule identification we demonstrate a rich conformational polymorphism at their monomer level. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the monomeric b-conformational change and neurodegeneration. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt or reverse multiple neurodegenerative disease

Aging and Brain Deterioration

Carlos Dotti and Vicente Rodríguez (coordinators).Advanced age significantly increases the risk of developing chronic diseases such as cancer, diabetes, cardiovascular, immune and mental disease. Regarding the latter, advanced age is a necessary factor for the development of non-hereditary forms of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Despite years of intense research, we still do not know how these diseases occur, this being one of the main reasons for the lack of adequate interventions to prevent or cure these pathologies. To overcome the current limitations in the field, we plan to: 1) generate basic knowledge on the mechanisms responsible for cognitive, behavioral, motor, metabolic and sociability disorders that occur with age, 2) define the mechanisms that determine individual susceptibility to neurodegeneration, 3) design and develop strategies to improve brain aging, and 4) explore social and environmental conditions of the older population to know their influence in brain degeneration. Individual, social and policy interventions must be considered for future research.Peer reviewe

Caracterización, purificación y clonaje de la PSD-95, una proteína específica de las densidades postsinápticas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Sección de Biología. Fecha de lectura: 7-11-199

Use of QBP1peptide for the inhibition of memory consolidation

The present invention relates to a composition that comprises the peptide QBP1 for use in the inhibition of memory consolidation, after a trauma, more preferably in Post-Traumatic Stress Disorder or related diseases. [EN]Uso del péptido QBP1 para la prevención o el tratamiento del trastorno del estrés postraumático, el síndrome de estrés agudo y/o el síndrome de adaptación general. La invención se refiere a una composición que comprende el péptido QBP1 para su uso en prevenir el Trastorno de Estrés Postraumático y trastornos relacionados (como el trastorno de estrés agudo y el trastorno general de adaptación). La invención puede ser circunscrita al campo de la Medicina. [ES]Peer reviewedConsejo Superior de Investigaciones Científicas (España)T3 Traducción de patente europe

Uso del péptido QBP1 para la inhibición de la consolidación de la memoria

[EN] The present invention relates to a composition that comprises the peptide QBP for use in the inhibition of memory consolidation, after a trauma, more preferably in Post-Traumatic Stress Disorder or related diseases[ES] La presente invención se refiere a una composición que comprende el péptido QBP para su uso en la inhibición de la consolidación de la memoria, después de un trauma, más preferiblemente en el trastorno por estrés postraumático o enfermedades relacionadas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Uso del péptido QBP1 para la inhibición de la consolidación de la memoria

[EN] The present invention relates to a composition that comprises the peptide QBP for use in the inhibition of memory consolidation, after a trauma, more preferably in Post-Traumatic Stress Disorder or related diseases[ES] La presente invención se refiere a una composición que comprende el péptido QBP para su uso en la inhibición de la consolidación de la memoria, después de un trauma, más preferiblemente en el trastorno por estrés postraumático o enfermedades relacionadas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Uso del péptido QBP1 para la inhibición de la consolidación de la memoria

[EN] The present invention relates to a composition that comprises the peptide QBP for use in the inhibition of memory consolidation, after a trauma, more preferably in Post-Traumatic Stress Disorder or related diseases[ES] La presente invención se refiere a una composición que comprende el péptido QBP para su uso en la inhibición de la consolidación de la memoria, después de un trauma, más preferiblemente en el trastorno por estrés postraumático o enfermedades relacionadas.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Dispositivo interfase digital para la integración del modo de imagen en espectroscopio de fuerza monomolecular

Dispositivo interfase digital para la integración del modo de imagen en espectroscopio de fuerza monomolecular. Cuenta con un cabezal de detección de AFM (4) conectado a un controlador de espectroscopia (9) que se une a una tarjeta de obtención de datos (10) instalada en un ordenador (11) unido a un bloque procesador DSP (12) que conecta con un bloque de conversión DAC-ADC (13). La muestra a analizar (1) se monta en un elemento oscilante (2) colocado sobre un posicionador 3D (3) empujado por un motor y unido a un sensor de posición (8) con sensores en tres direcciones. Un controlador de imagen (7) conecta con la tarjeta (10), el bloque de conversión (13), el sensor de posición (8), el posicionador (3), el elemento oscilante (2), el cabezal de detección (4) y el controlador de espectroscopia (9), facilitando el conexionado una caja integradora. El cabezal de detección (4) se ubica en la parte superior de una mesa antivibraciones.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridB1 Patente con informe sobre el estado de la ténic