The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

International audienceDNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated

BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-nucleotide oligomer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.</p

Analyse structurale de complexes protéiques impliqués dans des pathologies génétiques

La lamina nucléaire qui tapisse la membrane interne du noyau contient des protéines très diverses, soit ancrées à la membrane nucléaire, soit filamentaires. Il a été montré depuis une dizaine d années que des mutations dans des gènes codant pour ces protéines causent un grand nombre de pathologies humaines touchant des tissus très différents. Récemment, il a été observé que certaines de ces protéines sont importantes pour conférer aux cellules leur capacité à proliférer ou à se différencier. En particulier, les lamines et MAN1 interagissent avec des régulateurs de la transcription appartenant à des voies de signalisation impliquées dans la prolifération et la différenciation cellulaire et influencent ainsi ces voies. Cependant, aucune donnée structurale n est aujourd hui disponible qui permettrait de connaître les détails moléculaires des interactions entre protéines de l enveloppe nucléaire et facteurs de transcription. En effet, la nature même des protéines de l enveloppe nucléaire (membranaires ou filamentaires) en fait des molécules difficiles à manipuler biochimiquement. De plus, les interactions entre ces protéines et les régulateurs de la transcription sont nécessairement transitoires, ce qui rend les complexes d intérêt difficiles à purifier. Le laboratoire de Biologie Structurale et Radiobiologie a résolu la structure 3D de nombreux domaines de protéines de l enveloppe nucléaire. Sur cette base, j ai débuté l analyse structurale des interactions entre ces domaines et des régulateurs de la transcription. J ai travaillé d une part sur le complexe MAN1 / Smad2 et, d autre part, sur le complexe lamines de type A / SREBP1. Dans les deux cas, j ai utilisé diverses techniques de biochimie et de biophysique pour caractériser les résidus impliqués dans la formation du complexe et à partir de là proposer un mécanisme pour la régulation transcriptionnelle par la protéine de l enveloppe nucléaire. En particulier, mes résultats suggèrent que la région C-terminale de MAN1 a une structure 3D proche de celle trouvée chez plusieurs facteurs de transcription, et reconnait Smad2 par un motif en partie retrouvé chez ces facteurs de transcription. L interaction MAN1 / Smad2 entrerait ainsi en compétition avec les interactions facteurs de transcription / Smad2, ce qui est cohérent avec l observation que la surexpression de MAN1 inhibe la voie de signalisation TGFb.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Chemical engineering of a three-fingered toxin with anti-α7 neuronal acetylcholine receptor activity

International audienc

Protein Structural and Mechanistic Basis of Progeroid Laminopathies

Progeroid laminopathies are characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. They are caused by mutations in genes coding for A-type lamins or lamin binding proteins. Here, we review how different mutations causing progeroid laminopathies alter protein structure or protein-protein interactions and how these impact on mechanisms that protect cell viability and function. One group of progeroid laminopathies, which includes Hutchinson-Gilford progeria syndrome, is characterized by accumulation of unprocessed prelamin A or variants. These are caused by mutations in the A-type lamin gene (LMNA), altering prelamin A itself, or in ZMPSTE24, encoding an endoprotease involved in its processing. The abnormally-expressed farnesylated proteins impact on various cellular processes that may contribute to progeroid phenotypes. Other LMNA mutations lead to the production of non-farnesylated A-type lamin variants with amino acid substitutions in solvent-exposed hot spots located mainly in coil 1B and the immunoglobulin fold domain. Dominant missense mutations might reinforce interactions between lamin domains, thus giving rise to excessively stabilized filament networks. Recessive missense mutations in A-type lamins and barrier-to-autointegration factor (BAF) causing progeroid disorders are found at the interface between these interacting proteins. The amino acid changes decrease the binding affinity of A-type lamins for BAF, which may contribute to lamina disorganization, as well as defective repair of mechanically-induced nuclear envelope rupture. Targeting these molecular alterations in A-type lamins and associated proteins identified through structural biology studies could facilitate the design of therapeutic strategies to treat patients with rare but severe progeroid laminopathies

Internal motion time scales of a small, highly stable and disulfide rich protein. An 15N, 13C NMR and molecular dynamics study.

International audienceMotions of the backbone C alpha H alpha and threonine C beta H beta bonds of toxin alpha were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H-->13C NOE were obtained, as well as the variations of R1 rho (90 degrees) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several C alpha H alpha and threonine C beta H beta experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin alpha, a highly stable protein (Tm = 75 degrees C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 microseconds to 10 ms