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ABSTRACT: β-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of β-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the β-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous α-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.
Biophysical Journal 10/2012; 103(8):1744-52. · 3.65 Impact Factor
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Rubén Hervás,
Javier Oroz,
Albert Galera-Prat,
Oscar Goñi, Alejandro Valbuena,
Andrés M Vera,
Angel Gómez-Sicilia,
Fernando Losada-Urzáiz,
Vladimir N Uversky,
Margarita Menéndez,
Douglas V Laurents,
Marta Bruix,
Mariano Carrión-Vázquez
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ABSTRACT: Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these "neurotoxic proteins" triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. 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 critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. 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 diseases.
PLoS Biology 05/2012; 10(5):e1001335. · 11.45 Impact Factor
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Rubén Hervás,
Javier Oroz,
Albert Galera-Prat,
Oscar Goñi, Alejandro Valbuena,
Andrés Manuel Vera,
Ángel Gómez-Sicilia,
Douglas V. Laurents,
Margarita Menéndez,
Marta Bruix,
Mariano Carrión-Vázquez
[show abstract]
[hide abstract]
ABSTRACT: Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these "neurotoxic proteins" triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. 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 critical monomeric beta-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. 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 diseases.
PLoS Biology 01/2012; 10(5):e1001335. · 11.45 Impact Factor
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ABSTRACT: Intrinsically disordered proteins (IDPs) are predicted to represent about one third of the eukaryotic proteome. The dynamic ensemble of conformations of this steadily growing class of proteins has remained hardly accessible for bulk biophysical techniques. However, single-molecule techniques provide a useful means of studying these proteins. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is one of such techniques, which has certain peculiarities that make it an important methodology to analyze the biophysical properties of IDPs. However, several drawbacks inherent to this technique can complicate such analysis. We have developed a protein engineering strategy to overcome these drawbacks such that an unambiguous mechanical analysis of proteins, including IDPs, can be readily performed. Using this approach, we have recently characterized the rich conformational polymorphism of several IDPs. Here, we describe a simple protocol to perform the nanomechanical analysis of IDPs using this new strategy, a procedure that in principle can also be followed for the nanomechanical analysis of any protein.
Methods in molecular biology (Clifton, N.J.) 01/2012; 896:71-87.
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Rubén Hervás,
Javier Oroz,
Albert Galera-Prat,
Oscar Goñi, Alejandro Valbuena,
Andrés Manuel Vera,
Ángel Gómez-Sicilia,
Douglas V. Laurents,
Margarita Menéndez,
Marta Bruix,
Mariano Carrión-Vázquez
[show abstract]
[hide abstract]
ABSTRACT: Amyloidogenic 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 single-molecule 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 β-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 diseases.
Biophysical Journal 01/2012; 102(3):633a. · 3.65 Impact Factor
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ABSTRACT: Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis,
differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the
cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although
their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy,
molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin
ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain
extends through a defined (“canalized”) pathway that involves two mechanical resistance elements: a mechanical clamp from
the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions (“calcium rivet”) that
is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence
of calcium, the mechanical response of the ectodomain becomes largely “decanalized” and destabilized. The cadherin ectodomain
may therefore behave as a calcium-switched “mechanical antenna” with very different mechanical responses depending on calcium
concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design
of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that,
we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity,
and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the
door to understanding the mechanical bases of its mechanotransduction.
Journal of Biological Chemistry 03/2011; 286(11):9405-9418. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.
Journal of Biological Chemistry 12/2010; 286(11):9405-18. · 4.77 Impact Factor
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ABSTRACT: Protein mechanostability is a fundamental biological property that can only be measured by single-molecule manipulation techniques. Such studies have unveiled a variety of highly mechanostable modules (mainly of the Ig-like, beta-sandwich type) in modular proteins subjected to mechanical stress from the cytoskeleton and the metazoan cell-cell interface. Their mechanostability is often attributed to a "mechanical clamp" of secondary structure (a patch of backbone hydrogen bonds) fastening their ends. Here we investigate the nanomechanics of scaffoldins, an important family of scaffolding proteins that assembles a variety of cellulases into the so-called cellulosome, a microbial extracellular nanomachine for cellulose adhesion and degradation. These proteins anchor the microbial cell to cellulose substrates, which makes their connecting region likely to be subjected to mechanical stress. By using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and computer simulations, here we show that the cohesin I modules from the connecting region of cellulosome scaffoldins are the most robust mechanical proteins studied experimentally or predicted from the entire Protein Data Bank. The mechanostability of the cohesin modules studied correlates well with their mechanical kinetic stability but not with their thermal stability, and it is well predicted by computer simulations, even coarse-grained. This extraordinary mechanical stability is attributed to 2 mechanical clamps in tandem. Our findings provide the current upper limit of protein mechanostability and establish shear mechanical clamps as a general structural/functional motif widespread in proteins putatively subjected to mechanical stress. These data have important implications for the scaffoldin physiology and for protein design in biotechnology and nanotechnology.
Proceedings of the National Academy of Sciences 08/2009; 106(33):13791-6. · 9.68 Impact Factor
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ABSTRACT: Most of studies in protein nanomechanics have used the atomic force microscope (AFM) in its force-measuring mode on immobilized protein repeats (polyproteins) as single-molecule markers. Here, we add imaging capabilities to a standard, state-of-the-art AFM "puller" and integrate the most powerful programs of analysis available for both AFM modes. This unique instrument allows high-resolution, quasi-simultaneous imaging/force spectroscopy in aqueous solution. We demonstrate its capabilities using polyproteins of a model system (titin I27 domain). This tool should greatly facilitate the development of a much needed universal functionalization system for AFM, one that should allow better sample control and an improved efficiency of protein immobilization.
Review of Scientific Instruments 12/2007; 78(11):113707. · 1.37 Impact Factor
