Nanomecánica del celulosoma. Implicaciones para la actividad del sistema

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 05-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 05-02-2017Cellulose is the major biopolymer and carbon source on the biosphere. It is composed of glucose, a sugar that can be processed by several microorganisms to produce added-value chemicals such as biofuels. The major bottleneck towards its utilization in industry is the saccharification step, i. e. the breakdown of the polymer to release the constituent soluble sugars. In nature, this process is carried out by several microorganisms with specialized enzymatic machinery. In particular, some anaerobic bacteria have developed a high molecular weight complex known as the cellulosome. This comprises a large usually non-catalytic protein called scaffoldin, capable of docking several enzymes and targeting them to the cellulosic substrate while binding it to the cell surface. As an adhesion system, we expect that the cellulosome is subjected to mechanical stress since the relative movement of the cell and its substrate would stretch the connecting region: the portion of the scaffoldin located between the two anchoring points. We hypothesized that the mechanical stability, i. e. the resistance to forced unfolding, of this region might be important to understand cellulosome activity since the unfolding of cohesins, which are the scaffoldin modules capable of binding enzymes, might result in enzyme release and activity loss. We have used atomic force microscopy-based single molecule force spectroscopy in combination with molecular dynamics simulations to study the mechanical properties of several cohesins from different cellulosomes. We found that those cohesins located in the connecting region of scaffoldins that bind enzymes are more mechanically stable than those located outside the two anchoring points, in the external region. This principle applies for cohesins of different cellulosomes despite the sequence difference of these modules. We also study the effects of both the linker sequences located between cohesins and enzyme binding on the mechanical stability of cohesins which are elements that are found in natural cellulosomes. We did not observe any effect on cohesin mechanical stability nor on their mechanical clamp positionLa celulosa es el biopolímero y la fuente de carbono más abundante de la biosfera. Está compuesta por glucosa, un azúcar que puede ser procesado por microorganismos para producir productos químicos con valor añadido como los biocombustibles. El mayor cuello de botella para su utilización industrial se encuentra en el paso de la sacarificación, es decir, la degradación de este polímero para liberar los azúcares solubles constituyentes. En la naturaleza este proceso lo llevan a cabo algunos microorganismos que presentan una maquinaria enzimática especializada. En particular, algunas bacterias anaerobias han desarrollado un complejo conocido como celulosoma que comprende una proteína de elevado peso molecular y generalmente no catalítica llamada escafoldina, capaz de unir varias enzimas y dirigirlas al sustrato a la vez que las ancla a la superficie celular. Por el hecho de tratarse de un sistema de adhesión, es de esperar que el celulosoma esté sometido a estrés mecánico dado que el movimiento relativo de la bacteria respecto al sustrato podría estirar la región conectora: la porción de la escafoldina que se encuentra entre los dos puntos de anclaje. Propusimos la hipótesis de que la estabilidad mecánica, es decir, la resistencia al desplegamiento por fuerza, de esta región puede ser importante para entender la actividad del celulosoma. Esto es debido a que el desplegamiento de las cohesinas, los módulos de la escafoldina capaces de unir enzimas, podría conducir a una pérdida de actividad. Hemos empleado espectroscopia de fuerza de moléculas individuales mediante microscopía de fuerza atómica en combinación con simulaciones de dinámica molecular para estudiar las propiedades mecánicas de varios módulos cohesina de distintos celulosomas. Hemos observado que las cohesinas situadas en la región conectora presentan una mayor estabilidad mecánica comparada con la de aquellas que no se espera que estén sometidas a estrés mecánico. Este principio es aplicable a las cohesinas de distintos celulosomas a pesar de las diferencias de secuencia entre ellas. También hemos estudiado el efecto de las secuencias intermodulares y de la unión a enzimas sobre la estabilidad mecánica de las cohesinas, que son elementos que se encuentran en los celulosomas naturales. Hemos visto que ni la estabilidad mecánica de las cohesinas ni la posición de su parche mecánico se ven afectados por estos factore

Nanomechanics of tip-link cadherins

Hearing and balance rely on the transduction of mechanical stimuli arising from sound waves or head movements into electrochemical signals. This archetypal mechanoelectrical transduction process occurs in the hair-cell stereocilia of the inner ear, which experience continuous oscillations driven by undulations in the endolymph in which they are immersed. The filamentous structures called tip links, formed by an intertwined thread composed of an heterotypic complex of cadherin 23 and protocadherin 15 ectodomain dimers, connect each stereocilium to the tip of the lower sterocilium, and must maintain their integrity against continuous stimulatory deflections. By using single molecule force spectroscopy, here we demonstrate that in contrast to the case of classical cadherins, tip-link cadherins are mechanoresilient structures even at the exceptionally low Ca2+ concentration of the endolymph. We also show that the D101G deafness point mutation in cadherin 23, which affects a Ca2+ coordination site, exhibits an altered mechanical phenotype at the physiological Ca2+ concentration. Our results show a remarkable case of functional adaptation of a protein’s nanomechanics to extremely low Ca2+ concentrations and pave the way to a full understanding of the mechanotransduction mechanism mediated by auditory cadherinsThis work was supported by the BIO2010-22275 grant from the Spanish Ministry of Science and Innovation (MICINN) to M.C.-V

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

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair

Abstract ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents

BMC Infect Dis

BackgroundFrom June to November 2005, 18 cases of community-acquired Legionnaires' disease (LD) were reported in Rapid City South Dakota. We conducted epidemiologic and environmental investigations to identify the source of the outbreak.MethodsWe conducted a case-control study that included the first 13 cases and 52 controls randomly selected from emergency department records and matched on underlying illness. We collected information about activities of case-patients and controls during the 14 days before symptom onset. Environmental samples (n = 291) were cultured for Legionella. Clinical and environmental isolates were compared using monoclonal antibody subtyping and sequence based typing (SBT).ResultsCase-patients were significantly more likely than controls to have passed through several city areas that contained or were adjacent to areas with cooling towers positive for Legionella. Six of 11 case-patients (matched odds ratio (mOR) 32.7, 95% CI 4.7- 1e) reported eating in Restaurant A versus 0 controls. Legionella pneumophila serogroup 1 was isolated from four clinical specimens: 3 were Benidorm type strains and 1 was a Denver type strain. Legionella were identified from several environmental sites including 24 (56%) of 43 cooling towers tested, but only one site, a small decorative fountain in Restaurant A, contained Benidorm, the outbreak strain. Clinical and environmental Benidorm isolates had identical SBT patterns.ConclusionThis is the first time that small fountain without obvious aerosol-generating capability has been implicated as the source of a LD outbreak. Removal of the fountain halted transmission.2007663

An Evolutionary Perspective on the Origin, Conservation and Binding Partner Acquisition of Tankyrases

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species may exist. In this work, we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identify the single-celled choanoflagellates as earliest origin of tankyrases. We further show that the sequences and structural aspects of TNKSs are well-conserved even between distantly related species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners varies greatly between species, indicating that tankyrases may have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, and that their regulatory functions in cells have likely changed considerably throughout evolution

The cohesin module is a major determinant of cellulosome mechanical stability

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.This work was supported by a Seventh Framework Programme in Nanosciences, Nanotechnologies, Materials, and New Production Technologies (7PM-NMP 2013–17 and 604530-2) and ERA-IB-ERANET-2013–16 (EIB.12.022) through the Spanish MINECO (PCIN-2013-011-C02-01). The authors declare that they have no conflicts of interest with the contents of this article

Efficient and simplified nanomechanical analysis of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) lack a tertiary structure. Amyloidogenic IDPs (aIDPs) in particular have attracted great interest due to their implication in several devastating diseases as well as in critical biological functions. However, the conformational changes that trigger amyloid formation in aIDPs are largely unknown. aIDPs' conformational polymorphism at the monomer level encumbers their study using bulk techniques. Single-molecule techniques like atomic force microscopy-based single-molecule force spectroscopy represent a promising approach and a >carrier-guest> strategy, in which the protein of interest is mechanically protected, was developed to overcome the spurious signals from the noisy proximal region. However, since the carrier and single-molecule markers have similar mechanostabilities, their signals can intermingle in the force-extension recordings, making peak selection and analysis very laborious, cumbersome and prone to error for the non-expert. Here we have developed a new carrier, the c8C module from the CipC scaffoldin, with a higher mechanostability so that the signals from the protected protein will appear at the end of the recordings. This assures an accurate, more efficient and expert-independent analysis, simplifying both the selection and analysis of the single-molecule data. Furthermore, this modular design can be integrated into any SMFS polyprotein-based vector, thus constituting a useful utensil in the growing toolbox of protein nanomechanics.Peer Reviewe

Understanding biology by stretching proteins: recent progress

Single molecule manipulation techniques combined with molecular dynamics simulations and protein engineering have enabled, during the last decade, the mechanical properties of proteins to be studied directly, thereby giving birth to the field of protein nanomechanics. Recent data obtained from such techniques have helped gain insight into the structural bases of protein resistance against forced unfolding, as well as revealing structural motifs involved in mechanical stability. Also, important technical developments have provided new perspectives into protein folding. Eventually, new and exciting data have shown that mechanical properties are key factors in cell signaling and pathologies, and have been used to rationally tune these properties in a variety of proteins. Crown Copyright © 2009.Peer Reviewe