Solution Structure of Human p8 MTCP1 , a Cysteine-rich Protein Encoded by the MTCP1 Oncogene, Reveals a New a a a-Helical Assembly Motif

International audienceMature-T-Cell Proliferation) is the ®rst gene unequivocally identi®ed in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8 MTCP1 protein encoded by the MTCP1 oncogene was determined by homonuc-lear proton two-dimensional NMR methods at 600 MHz. After sequence speci®c assignments, a total of 931 distance restraints and 57 dihedral restraints were collected. The location of the three previously unassigned disul®de bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of p8 MTCP1 is presented as a set of 30 DIANA structures, further re®ned by restrained molecular dynamics using a simulated annealing protocol with the AMBER force ®eld. The r.m.s.d. values with respect to the mean structure for the backbone and all heavy atoms for a family of 30 structures are 0.73(AE0.28) and 1.17(AE0.23) A Ê , when the structured core of the protein (residues 5 to 63) is considered. The solution structure of p8 MTCP1 reveals an original scaffold consisting of three a helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disul®de bridges, forming an a-hairpin which resembles an antiparallel coiled-coil. The third helix is oriented roughly parallel to the plane de®ned by the a-antiparallel motif and its axis forms an angle of %60 with respect to the main axis of this motif

Probing the relationship between Gram-negative and Gram-positive S1 proteins by sequence analysis

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine–Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed ‘S1 proteins’. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their β-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4–5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway

NMR-spektroskopia monidomeenisten proteiinien tutkimuksessa : ihmisen filamiini A -proteiinin immunoglobuliinimaiset domeenit

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.Proteiinit ovat suuria, rakenteeltaan monimutkaisia biomolekyylejä, jotka vastaavat fysiologisista prosesseista kaikissa elävissä organismeissa. Proteiinit toimivat soluissa rakenteellisina yksiköinä, entsyymeinä, kuljettimina, prosessien säätelijöinä sekä signaalinvälittäjinä. Välttämättömien proteiinien toiminnan häiriintyminen ilmenee sairauksina. Usein toimintahäiriöiden taustalla on geneettinen mutaatio, joka muuttaa proteiinin rakennetta. Proteiinit toimivat vuorovaikuttamalla muiden molekyylien kanssa ja kaikki proteiinien toiminnot ovat riippuvaisia niiden rakenteesta. Jotta ymmärtäisimme fysiologisia ja patologisia mekanismeja molekyylitasolla, on meidän tunnettava niihin osallistuvien proteiinien rakenne ja vuorovaikutusmekanismit. Tässä väitöskirjassa on tutkittu ihmisen filamiinien rakennetta ja toimintaa. Filamiinit ovat solun tukirangan aktiinisäikeitä sitovia proteiineja, jotka koostuvat N-terminaalisen osan aktiinia sitovasta domeenista sekä 24 peräkkäisestä immunoglobuliinimaisesta (IgFLN) domeenista. Filamiinit vuorovaikuttavat aktiinin lisäksi lukuisten muiden proteiinien kanssa ja useimmat näistä vuorovaikutuksista on paikannettu C-terminaalisiin IgFLN domeeneihin. Filamiinit osallistuvat solun kiinnittymiseen ja liikkumiseen sekä solukalvon reseptorimolekyylien ja solun sisäisten viestintäreittien koordinointiin. Filamiinimutaatiot aiheuttavat ihmisissä vakavia kehityshäiriöitä ja sairauksia, joiden oireet ilmenevät aivoissa, luustossa, raajoissa sekä sydän- ja verenkiertoelimistössä. Ydinmagneettinen resonanssispekrtroskopia eli NMR-spektroskopia on monipuolinen menetelmä proteiinien rakenteen, vuorovaikutuksen ja dynamiikan tutkimuksessa. Tässä väitöskirjassa NMR-spektroskopiaa hyödynnettiin filamiinin C-terminaalisten IgFLN domeenien rakenteen ja vuorovaikutusmekanismien tutkimuksessa. Työssä ratkaistiin IgFLN domeenien 17 ja 23 sekä IgFLN domeeniparien 16 17 ja 18 19 rakenteet NMR-spektroskopiaa käyttäen. NMR-mittausten avulla selvitettiin myös filamiinin vuorovaikutusta useiden solujen toiminnan kannalta tärkeiden proteiinien kanssa. Väitöskirjassa tutkittiin myös IgFLN domeenisekstetti 16 21:n rakennetta jäännösdipolikytkentöjen ja NMR-relaksaatioanalyysin avulla. Tämä väitöskirja havainnollistaa NMR-spekrtroskopian monipuolisuutta ja mahdollisuuksia monidomeenisten proteiinien rakenteen ja toiminnan tutkimuksessa

Structure and function of the PABC domain

The poly (A)-binding protein (PABP) is an essential protein found in all eukaryotes and functions in mRNA metabolic and translational processes. Structurally, PABP consists of two distinct regions. The N-terminal half contains four RNA recognition motifs that bind to the poly (A)-tail of mRNA, while the C-terminal segment contains a unique peptide binding module referred to as the PABC domain. The function of this domain in PABP is to recruit proteins containing a very specific 'PAM-2' motif to the mRNP complex. Unique to metazoans, a PABC domain is also found in the hyperplastic discs tumor suppressor (HYD), which is an E3 ubiquitin ligase.This thesis completes a structural investigation of PABC domains from various species by nuclear magnetic resonance spectroscopy. In particular, we report the solution structure of PABC from the parasite Trypanosoma cruzi and plant Triticum aevestium PABP. Both domains consist of five alpha-helices which fold into a structure highly comparable to the human PABC domain from PABP and HYD. All four PABCs interact with a similar PAM-2 sequence and show comparable peptide binding surfaces. The human PABC-PAM-2 complex structure displays a PAM-2 peptide interacting with specific residues within the domain. Sequence analyses and peptide surface mapping studies show that these residues are highly conserved, which indicates an analogous mechanism of peptide recognition throughout animal, parasite, and plant species. An exception to these observations was found in the PABC domain from Saccharomyces cerevisiae PABP. Yeast PABC recognizes a variation of the typical PAM-2 motif but mediates its interaction through a similar mechanism as human PABC.The PAM-2 motif encloses a signature sequence which was used to successfully identify new interacting partners for the PABC domain via a bioinformatics screen. In mammalian systems, the identified proteins are implicated in either RNA metabolic, translational, or ubiquitin associated functions. This thesis concludes with a model illustrating a unique cross-talk between major gene expression pathways mediated by the PABC domain and its binding partners

Le COP9 signalosome : activité et régulation

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) is an eight-subunit-containing multiprotein complex (320 kDa) implicated in diverse cellular processes including cell cycle progression, gene expression and DNA repair via its function in the ubiquitin-proteasome pathway. It is a highly evolutionary conserved protein complex in higher eukaryotes for which its activity is essential. Over years of biochemical and biological studies to elucidate the role of the CSN, its most studied and best understood function is linked to the control of protein ubiquitylation (post-translational modification corresponding to the covalent conjugation of an ubiquitin molecule) by a class of E3 ubiquitin ligases. The CSN exhibits catalytic activity to regulate E3-cullin RING ubiquitin ligases (CRLs) by the removal of an ubiquitin-like protein, Nedd8 (cullin-neural precursor cell expressed developmentally downregulated gene 8), from CRLs. Cycles of neddylation/deneddylation are essential for CRL function and the CSN is central in this process through its activity as a CRL deneddylase.Structural and functional similarities link the CSN, the 19S lid of the 26S proteasome and the eukaryotic initiation factor-3 (elF3). These multi-subunit assemblies comprise six PCI (proteasome COP9 eIF3) domain subunits and two MPN (Mpr1–Pad1–N-terminal) domain-containing subunits. The catalytic activity of the CSN is centred on its subunit 5 (CSN5/Jab1), which hydrolyses the Nedd8-CRL isopeptide bond. CSN5 contains a zinc-dependent isopeptidase catalytic centre constituted of a JAMM (Jab1/MPN/Mov34) motif. CSN5 incorporation within the CSN complex unleashes its isopeptidase activity, whereas it remains inactive in isolation. The work presented in this manuscript led to five main findings. (i) Having elucidated the crystal structure of CSN5 catalytic domain, biochemical and in silico investigations that furthered the understanding of CSN5 molecular regulation, led to the identification of a potential molecular trigger enabling CSN5 to be active and the design of a constitutively active CSN5 variant form. (ii) The ability of CSN5 to homodimerise was investigated in solution, in silico and in cellular extracts and brought information that could be important for its function. (iii) Further to that work, to address CSN5 activity within the CSN, the contribution of another CSN subunit, mainly CSN6, shown to interact directly with CSN5 was evaluated and this led to the identification of CSN6 as the CSN5 activating subunit. (iv) The biochemical and biophysical characterisation of the CSN5-CSN6 complex was exploited to explore at the molecular level this complex in the context of its binding to Nedd8 and of its integration within the holo-CSN assembly through an integrated approach that includes biochemical, structural, biophysical and computational techniques. (v) Finally the CSN5-CSN6 complex was shown to be able in vitro to pursue peptidase activity on the Nedd8 precursor protein, pro-Nedd8 by cleaving its C-terminal extension (-G75G76GGLRQ) and preliminary results relating to the exploration of this new activity are presented.Overall this work allowed to gain an in-depth understanding of the activity determinants and of the regulatory mechanisms that the CSN catalytic subunit CSN5 is subjected to.Le COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) est un complexe multiprotéique contenant huit sous-unités (320 kDa), impliqué dans des processus cellulaires divers allant de la progression du cycle cellulaire, à l'expression des gènes et la réparation de l'ADN, à travers sa fonction au sein du système ubiquitine-protéasome. Il s'agit d'un complexe fortement conservé au cours de l'évolution chez les eucaryotes supérieurs chez qui son activité catalytique est essentielle. Au cours des années d'études biologiques et biochimiques qui ont permis d'élucider le rôle du CSN, sa fonction la mieux étudiée et la mieux comprise est celle liée au contrôle de l'ubiquitylation des protéines (une modification post-traductionnelle qui implique la liaison covalente d'une protéine cible par une molécule d'ubiquitine) par une classe d'E3 ubiquitine ligases. L'activité catalytique du CSN régule spécifiquement les E3 cullin RING ubiquitin ligases (CRLs) via la suppression d'une molécule ressemblant à l'ubiquitine, Nedd8 (cullin-neural precursor cell expressed developmentally downregulated gene 8) des CRLs au cours d'une réaction de déneddylation. Les cycles de neddylation/déneddylation sont essentiels au fonctionnement correct des CRLs et le CSN joue un rôle central dans ce processus à travers sa fonction de déneddylase. Une similarité globale lie le CSN, le chapeau du protéasome (19S) et le complexe elF3 (eukaryotic initiation factor-3). Ces assemblages multi-protéiques comprennent tous six sous-unités contenant un domaine PCI (proteasome COP9 eIF3) et deux sous-unités contenant un domaine MPN (Mpr1–Pad1–N-terminal). L'activité catalytique du CSN est portée par la sous-unité 5, CSN5 qui hydrolyse la liaison isopeptidique entre Nedd8 et la CRL. CSN5 contient un cœur catalytique dépendent d'un zinc et comprenant un motif JAMM (Jab1/MPN/Mov34).L'incorporation de CSN5 dans le CSN révèle son activité isopeptidasique, alors qu'à l'état isolé, CSN5 n'est pas actif. Le travail réalisé au cours de ces trois ans a abouti à cinq aspects principaux qui ont contribué à une meilleure compréhension globale du système CSN. (i) S'appuyant sur la structure du domaine catalytique de CSN5, des études in vitro et in silico ont abouti à l'identification d'un élément moléculaire permettant à CSN5 de passer de la forme inactive à la forme active. Ceci a débouché sur la conception et validation d'un variant constitutivement actif de CSN5. (ii) La capacité de CSN5 à homodimériser a été étudié en solution, in silico et dans des extraits cellulaires et a apporté des perspectives potentiellement intéressantes concernant la fonction de CSN5. (iii) Au-delà de ce travail et pour aborder la question de la régulation de l'activité de CSN5 dans le CSN, la contribution de la sous-unité 6, CSN6 qui interagit directement avec CSN5 a été évaluée et ceci a abouti à l'identification de CSN6 comme sous-unité activatrice de CSN5. (iv) La caractérisation biochimique et biophysique du complexe CSN5-CSN6 a été utilisée pour explorer les bases moléculaires de cette association, non seulement, dans le contexte de son interaction avec Nedd8, mais aussi, de son intégration au sein du CSN, à travers une approche intégrée alliant des techniques biochimiques, structurales, biophysiques et computationnelles. (v) La dernière partie de ce travail est focalisée sur l'activité de maturation du précurseur de Nedd8 par le complexe CSN5-CSN6 mise en évidence in vitro et une exploration préliminaire de cette activité décrite pour la première fois est présentée. En résumé, ce travail a permis d'améliorer la compréhension des déterminants de l'activité et des mécanismes de régulation auxquels la sous-unité catalytique du CSN, CSN5 est soumise

Estrategias para el análisis de datos metabolómicos dirigidos al diagnóstico clínico

La metabolómica es un área de investigación emergente y puede ser considerada, a nivel bioquímico, como el final de la cascada “ómica” (genómica → transcriptómica →proteómica → metabolómica) ya que los cambios en el metaboloma constituyen la última respuesta del organismo a alteraciones genéticas, químicas, ambientales, etc. En ese sentido, el metaboloma está muy ligado al fenotipo y puede constituir una herramienta sumamente útil para diagnosticar enfermedades y evaluar el efecto de los tratamientos. Los metabolitos son los productos finales de todos los procesos que se producen en las células, y las concentraciones de metabolitos en los procesos patológicos reflejan la adaptación de los sistemas biológicos a las alteraciones bioquímicas características de cada enfermedad. La firma metabólica de un paciente, generalmente obtenida de forma no-invasiva a partir del análisis de biofluidos, contiene información relacionada con el genotipo, pero también con otros factores como la progresión de la enfermedad o la respuesta a los tratamientos. Esto explica por qué la metabolómica está atrayendo tanto interés para la identificación de biomarcadores de valor diagnóstico y para el seguimiento de pacientes de distintas patologías. En particular, los procesos oncológicos, que implican la desregulación de múltiples vías bioquímicas, son excelentes candidatos para la realización de estudios metabolómicos. La variabilidad en la fisiopatología de la enfermedad, junto con las diferencias individuales en la respuesta a los tratamientos, constituye la base de los esfuerzos por personalizar este tipo de terapias. Para la obtención de información biológica relevante y llevar a cabo con éxito un estudio metabolómico es necesario establecer una metodología experimental adecuada que debe incluir un buen diseño experimental, una adecuada selección y almacenamiento de las muestras, así como una adecuada técnica analítica y un correcto tratamiento e interpretación de los datos. Este estudio se divide en tres partes que intentan abordar los elementos adecuados y necesarios para una correcta aproximación experimental en un estudio de metabolómica enfocado a la búsqueda de nuevos biomarcadores de utilidad clínica. En la primera parte se evalúa la estabilidad de las muestras durante los procesos de la fase preanalítica para así poder identificar posibles biomarcadores de calidad de las muestras. La heterogeneidad de los procedimientos de muestreo y almacenamiento puede introducir una variabilidad significativa en la composición molecular de las muestras biológicas y, en consecuencia, interferir en el resultado experimental o afectar a su reproducibilidad. Tanto el suero como el plasma son biofluidos usados ampliamente como matrices biológicas en la investigación biomédica para identificar biomarcadores clínicamente relevantes. En este contexto es importante conocer la viabilidad de las muestras y las condiciones específicas que son necesarias para el uso de esta tecnología tanto para la investigación como para la práctica clínica diaria. La Resoncia Magnética Nuclear (RMN) de protón (1H-RMN) es una técnica no-invasiva que tiene multitud de aplicaciones en el análisis de sistemas biológicos y puede resultar extremadamente útil. Una de las principales limitaciones en el análisis metabolómico es la ausencia de metodologías estandarizadas que permitan desarrollar estudios en profundidad y fácilmente reproducibles en otros laboratorios. La segunda parte del proyecto analiza las diferentes estrategias y herramientas que se emplean en el análisis de los perfiles metabolómicos. Cómo desde un biofluido, usando como plataforma la 1H-RMN, podemos obtener información global de las señales correspondientes a los perfiles metabolómicos presentes en la muestra (metabolomic fingerprinting), pudiéndose identificar diferencias y/o similitudes entre los individuos a través de un análisis conjunto y multivariante de estas señales. En este contexto, se plantean dos aplicaciones prácticas dirigidas a la búsqueda de nuevos biomarcadores. Por un lado, ser realizó un estudio para evaluar la variabilidad biología y la búsqueda de biomarcadores en muestras de orina de pacientes con Cáncer de Próstata frente a individuos con Hiperplasia Benigna de Próstata. Por otro lado, se realizó un estudio de validación de biomarcadores de suero en pacientes Cáncer de Pulmón No Microcítico (CPNM). En este contexto, tras la realización de un estudio previo en nuestro laboratorio de un conjunto de metabolitos útiles en el diagnóstico precoz de CPNM, se realizó un estudio de validación de dichos metabolitos con un conjunto de muestras independiente

Advances in Ginsenosides

This book collected recent innovative research and review articles on analytical techniques, production protocols, biotechnological tools, and new insights into bioactivities of ginsenosides including the effects on epithelial-mesenchymal transition, hippocampal neurogenesis and inflammation as well as on diseases such as ischemic stroke, autoimmune diseases, and allergic disorders. Additionally, the analysis through molecular docking and an overview of the Panax ginseng pharmacopuncture were also presented