57 research outputs found
Intermédiaires réactionnels de la fructose-1,6-biphosphate aldolase de Leishmania mexicana : comparaison avec l'enzyme de mammifÚre et recrutement de la région C-terminale
Mémoire numérisé par la Direction des bibliothÚques de l'Université de Montréal
CaractĂ©risation structurale et fonctionnelle des interactions impliquant TFIIH et la machinerie de rĂ©paration de lâADN
La rĂ©paration de lâADN par excision des nuclĂ©otides (NER) est un mĂ©canisme capable de retirer une large variĂ©tĂ© de lĂ©sions causant une distorsion de la double hĂ©lice, comme celles causĂ©es par les rayons ultraviolets (UV). Comme toutes les voies de rĂ©paration de lâADN, la NER contribue Ă la prĂ©vention de la carcinogĂ©nĂšse en prĂ©venant la mutation de lâADN. Lors de ce processus, il y a dâabord reconnaissance de la lĂ©sion par la protĂ©ine XPC/Rad4 (humain/levure) qui recrute ensuite TFIIH. Ce complexe dĂ©roule lâADN par son activitĂ© hĂ©licase et recrute lâendonuclĂ©ase XPG/Rad2 ainsi que dâautres protĂ©ines nĂ©cessaires Ă lâexcision de lâADN. Lors de son arrivĂ©e au site de lĂ©sion, XPG/Rad2 dĂ©place XPC/Rad4.
TFIIH agit Ă©galement lors de la transcription de lâADN, entre autres par son activitĂ© hĂ©licase. Outre cette similaritĂ© de la prĂ©sence de TFIIH lors de la transcription et la rĂ©paration, il est possible de se demander en quoi les deux voies sont similaires. Nous nous sommes donc intĂ©ressĂ©s aux interactions impliquant TFIIH et la machinerie de rĂ©paration de lâADN.
Nous avons donc entrepris une caractĂ©risation structurale et fonctionnelle de ces interactions. Nous avons dĂ©couvert que Rad2 et Rad4 possĂšdent un motif dâinteraction en nous basant sur dâautres interactions de la sous-unitĂ© Tfb1 de TFIIH. Par calorimĂ©trie Ă titrage isotherme, nous avons observĂ© que les segments de ces deux protĂ©ines contenant ce motif interagissent avec une grande affinitĂ© au domaine PH de Tfb1. Le site de liaison de ces segments sur Tfb1PH est trĂšs semblable au site de liaison du domaine de transactivation de p53 et au domaine carboxy-terminal de TFIIEα avec Tfb1PH, tel que dĂ©montrĂ© par rĂ©sonance magnĂ©tique nuclĂ©aire (RMN). De plus, tous ces segments peuvent faire compĂ©tition les uns aux autres pour la liaison Ă Tfb1PH. Nous avons aussi dĂ©montrĂ© in vivo chez la levure quâune dĂ©lĂ©tion de Tfb1PH crĂ©e une sensibilitĂ© aux radiations UV. De plus, la dĂ©lĂ©tion de multiples segments de Rad2 et Rad4, dont les segments dâinteraction Ă Tfb1PH, est nĂ©cessaire pour voir une sensibilitĂ© aux rayons UV. Ainsi, de multiples interactions sont impliquĂ©es dans la liaison de Rad2 et Rad4 Ă TFIIH. Finalement, les structures des complexes Rad2-Tfb1PH et Rad4-Tfb1PH ont Ă©tĂ© rĂ©solues par RMN. Ces structures sont identiques entre elles et impliquent des rĂ©sidus hydrophobes interagissant avec des cavitĂ©s peu profondes de Tfb1PH. Ces structures sont trĂšs semblables Ă la structure de TFIIEα-p62PH.
Ces dĂ©couvertes fournissent ainsi un lien important entre la transcription et la rĂ©paration de lâADN. De plus, elles permettent dâĂ©mettre un modĂšle du mĂ©canisme de dĂ©placement de XPC/Rad4 par XPG/Rad2 au site de dommage Ă lâADN. Ces connaissances aident Ă mieux comprendre les mĂ©canismes de maintient de la stabilitĂ© gĂ©nomique et peuvent ainsi mener Ă dĂ©velopper de nouvelles thĂ©rapies contre le cancer.The nucleotide excision repair pathway (NER) is a mechanism capable of removing a wide variety of helix-distorting lesions, such as those caused by ultraviolet irradiation (UV). As all DNA repair pathways, NER contributes to the prevention of carcinogenesis by preventing DNA mutation. During this process, the lesion is first recognized by the protein XPC/Rad4 (human/yeast), which then recruits TFIIH. This complex unwinds the DNA with its helicase activity and then recruits the endonuclease XPG/Rad2 and other proteins necessary for DNA excision. Upon arrival at the lesion site, XPG/Rad2 displaces XPC/Rad4.
TFIIH also acts in DNA transcription, using its helicase activity. In addition to the similarity of the presence of TFIIH in transcription and DNA repair, it is possible to ask ourselves how the two pathways are similar. We were interested in the interactions involving TFIIH and the DNA repair machinery.
We have therefore undertaken a structural and functional characterization of these interactions. We have found that Rad2 and Rad4 have a motif of interaction based on other interactions of the Tfb1 subunit of TFIIH. Using isothermal titration calorimetry, we found that segments of these two proteins containing this motif interact with high affinity to the PH domain of Tfb1. The binding site of these segments is very similar to Tfb1PH binding site of transactivation domain of p53 and the carboxyl-terminal domain of TFIIEα with Tfb1PH, as demonstrated by nuclear magnetic resonance (NMR). In addition, these segments can compete with each other for binding to Tfb1PH. We also demonstrated in vivo that deletion of Tfb1PH in yeast creates a sensitivity to UV irradiation. In addition, the deletion of multiple segments of Rad2 and Rad4, including segments of interaction Tfb1PH, is required to observe a sensitivity to UV. Thus, multiple interactions are involved in the binding of TFIIH to Rad2 and Rad4. Finally, the structures of the Rad2-Tfb1PH and Rad4-Tfb1PH complexes were solved by NMR. These structures are identical to each other and involve hydrophobic residues interacting with shallow grooves on Tfb1PH. These structures are very similar to the structure of TFIIEα-p62PH.
These findings provide an important mechanistic link between transcription and DNA repair. In addition, they provide a model of the mechanism of the displacement of XPC/Rad4 by XPG/Rad2 at the damaged site. This knowledge helps to better understand the mechanisms of genomic stability and can lead to novel cancer therapies
Protein Design with Guided Discrete Diffusion
A popular approach to protein design is to combine a generative model with a
discriminative model for conditional sampling. The generative model samples
plausible sequences while the discriminative model guides a search for
sequences with high fitness. Given its broad success in conditional sampling,
classifier-guided diffusion modeling is a promising foundation for protein
design, leading many to develop guided diffusion models for structure with
inverse folding to recover sequences. In this work, we propose diffusioN
Optimized Sampling (NOS), a guidance method for discrete diffusion models that
follows gradients in the hidden states of the denoising network. NOS makes it
possible to perform design directly in sequence space, circumventing
significant limitations of structure-based methods, including scarce data and
challenging inverse design. Moreover, we use NOS to generalize LaMBO, a
Bayesian optimization procedure for sequence design that facilitates multiple
objectives and edit-based constraints. The resulting method, LaMBO-2, enables
discrete diffusions and stronger performance with limited edits through a novel
application of saliency maps. We apply LaMBO-2 to a real-world protein design
task, optimizing antibodies for higher expression yield and binding affinity to
several therapeutic targets under locality and developability constraints,
attaining a 99% expression rate and 40% binding rate in exploratory in vitro
experiments
AbDiffuser: Full-Atom Generation of in vitro Functioning Antibodies
We introduce AbDiffuser, an equivariant and physics-informed diffusion model
for the joint generation of antibody 3D structures and sequences. AbDiffuser is
built on top of a new representation of protein structure, relies on a novel
architecture for aligned proteins, and utilizes strong diffusion priors to
improve the denoising process. Our approach improves protein diffusion by
taking advantage of domain knowledge and physics-based constraints; handles
sequence-length changes; and reduces memory complexity by an order of
magnitude, enabling backbone and side chain generation. We validate AbDiffuser
in silico and in vitro. Numerical experiments showcase the ability of
AbDiffuser to generate antibodies that closely track the sequence and
structural properties of a reference set. Laboratory experiments confirm that
all 16 HER2 antibodies discovered were expressed at high levels and that 57.1%
of the selected designs were tight binders.Comment: NeurIPS 202
Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate
While other aldolases crystallize readily in the apo form, diffraction-quality crystals of B. henselae aldolase could only be obtained in the presence of the native substrate. The quaternary structure is tetrameric, as is typical of aldolases
Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi
The eukaryotic parasite E. cuniculi expresses a fructose bisphosphate aldolase that crystallizes readily in the presence of the partial substrate analog phosphate. This aldolaseâphosphate structure and that of the sugar-bound Schiff base are reported. E. cuniculi aldolase displays a dimeric structure rather than the expected tetrameric quaternary structure
The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress
The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81
Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2
The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3âČ-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2âTfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair
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