23 research outputs found
Ătude sur la reconnaissance de l'ubiquitine par les domaines de transactivation acides des activateurs de transcription
Les domaines de transactivation (TAD) acides sont prĂ©sents dans plusieurs protĂ©ines oncogĂ©niques, virales et dans des facteurs de diffĂ©renciation de cellules souches. Ces domaines acides contrĂŽlent la transcription Ă travers une myriade dâinteractions avec divers partenaires ce qui provoque lâactivation de la transcription ou leur propre Ă©limination. Cependant, dans la derniĂšre dĂ©cennie, de plus en plus de recherches ont dĂ©montrĂ© que les TAD possĂ©daient un sous-domaine activation/dĂ©gradation (DAD) responsable pour une fonction d'activation de la transcription dĂ©pendante de la dĂ©gradation de la protĂ©ine. Un tel phĂ©nomĂšne peut ĂȘtre accompli par plusieurs moyens tels que des modifications post-traductionnelles, lâassociation Ă des cofacteurs ou la formation dâun rĂ©seau dâinteraction complexe en chaĂźnes. Or, aucune preuve concrĂšte nâa pu clairement dĂ©montrer le fonctionnement de la dĂ©pendance paradoxale entre ces deux fonctions sur un activateur de transcription.
Le DAD, a Ă©tĂ© observĂ© dans plusieurs facteurs de transcription incluant la protĂ©ine suppresseur de tumeur p53 et le facteur de diffĂ©renciation Ă©rythrocyte EKLF. Un aspect particulier des DAD est que la composition de leur sĂ©quence dâacide aminĂ© est fortement similaire Ă celle des domaines de liaison Ă lâubiquitine (UBD) qui jouent un rĂŽle clĂ© dans le contrĂŽle de la transcription Ă travers leur interaction non-covalente avec lâubiquitine.
Ainsi, dans ce mĂ©moire, nous avons Ă©tudiĂ© la possibilitĂ© que les TAD acides soient capables dâagir comme UBD pour rĂ©guler leur fonction paradoxale Ă travers des interactions non-covalentes avec lâubiquitine. Lâanalyse est faite en utilisant la rĂ©sonnance magnĂ©tique nuclĂ©aire (RMN) ainsi quâavec des essais fonctionnels de dĂ©gradation. En somme, cette Ă©tude amĂšne une plus grande comprĂ©hension des protĂ©ines impliquĂ©es dans le contrĂŽle des TAD et caractĂ©rise le tout premier exemple de TAD capable dâinteragir avec lâubiquitine.Acidic transactivating domains have been shown to be potential targets for a number of different therapies but their dynamic nature and their ability to bind many interacting partners has made it difficult to fully understand their functioning mechanisms. What we do know about these domains is that they readily control transcription through a myriad of interactions capable of either activating specific aspects of their function or simply, signal for their own demise. Within the acidic TADs lies an unusual degradation/activation domain (DAD) capable of activating transcription at the cost of its degradation. In other words, DAD transcriptional activation is dependent on the degradation of the protein. Such a phenomenon could be explained by a wide variety of hypotheses like the play of post-translational modifications, co-factors, or maybe just a really sophisticated time scaled network of interactions. However, no concrete explanation of how this dual dependent functioning domain works has yet to surface.
The DAD has been observed within acidic TADs of several transcription factors including the tumor suppressor p53 and the red blood cell differentiation factor EKLF. Interestingly though, the amino acid sequence composition of DADs share a strong similarity with several types of sequences from domains that bind ubiquitin (UBDs). These domains have been shown in the past to, in addition to their role in degradation, play a key role in regulating transcription through non-covalent interaction with ubiquitin. Hence, in this project, we investigated weather acidic TADs had the ability to function as UBDs and form non-covalent interactions with ubiquitin and also to determine the functional significance of this interaction in regards to the dual function of acidic TADs
Large expert-curated database for benchmarking document similarity detection in biomedical literature search
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term FrequencyâInverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research
Large expert-curated database for benchmarking document similarity detection in biomedical literature search
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe
Ătudes structurales et fonctionnelles sur les mĂ©canismes de rĂ©gulation des interactions entre protĂ©ines SUMOs et les domaines SIMs.
La modification post-traductionnelle par les « Small Ubiquitin-Like MOdifyers (SUMOs) » est un processus majeur de rĂ©gulation qui influence plus dâune centaine de protĂ©ines. Cette modification (SUMOylation) touche plusieurs fonctions nuclĂ©aires telles que la rĂ©paration de lâADN, la rĂ©plication et la transcription. La SUMOylation affecte une protĂ©ine le plus souvent en permettant la formation de nouvelles interactions protĂ©ine-protĂ©ines avec des facteurs de rĂ©gulations qui possĂšdent un court segment hydrophobe dans leur sĂ©quence connu sous le nom de « SUMO interacting motif (SIM) ». Bien que les interactions SUMO-SIMs soient bien documentĂ©es, la description de leur rĂ©gulation nâest pas complĂšte. Cette thĂšse dĂ©crit des Ă©tudes fonctionnelles et structurales sur diffĂ©rents mĂ©canismes de rĂ©gulation des interactions SUMO-SIMs. Plus prĂ©cisĂ©ment, elle dĂ©crit les effets de lâacĂ©tylation et de la queue N-terminale des protĂ©ines SUMOs sur leur capacitĂ© Ă rĂ©guler les interactions entre SUMOs et les SIMs de trois protĂ©ines : le suppresseur de tumeur « promyelocytic leukemia (PML) », le corĂ©presseur de transcription « Death Domain Associated Protein 6 (Daxx) » et « Protein Inhibitor of Activated STAT (PIAS) », une ligase E3 pour SUMO.
La premiĂšre Ă©tude dĂ©crit lâeffet de lâacĂ©tylation de SUMO1 sur sa capacitĂ© Ă interagir avec les SIMs de PML et de Daxx. Ă partir dâexpĂ©riences de titrage calorimĂ©trique et dâĂ©tudes cristallographiques, nous avons dĂ©montrĂ© que lâacĂ©tylation prĂ©cise de certains rĂ©sidus conservĂ©s chez SUMO1 (K39 et K46) rĂ©duit fortement lâaffinitĂ© avec les deux SIMs testĂ©s. En contraste, nous dĂ©montrons que lâacĂ©tylation du rĂ©sidu K37 sur SUMO1 Ă un effet inhibiteur spĂ©cifique pour le SIM de Daxx. Les structures cristallographiques des complexes formĂ©s entre les variants acĂ©tylĂ©s de SUMO1 avec les SIMs concordent avec les donnĂ©es des titrages et suggĂšre une plasticitĂ© dans la formation des liens sur la surface dâinteraction. Partant de ce constat, nous postulons que la plasticitĂ© observĂ©e dans la structure des complexes acĂ©tylĂ©s dĂ©montre un mĂ©canisme de rĂ©gulation des interactions SUMO-SIMs par lâacĂ©tylation de rĂ©sidus conservĂ©s chez SUMO1.
Dans la deuxiĂšme Ă©tude, nous avons identifiĂ© un deuxiĂšme SIM Ă lâextrĂ©mitĂ© C-terminale de protĂ©ines de la famille (PIAS1-2-3). Nous dĂ©montrons que ce SIM est capable de lier SUMO1 et que structurellement la phosphorylation de rĂ©sidus clĂ©s dans ce domaine ainsi que lâacĂ©tylation de SUMO1 peut contrĂŽler cette interaction. Une comparaison avec le premier SIM des variants PIAS dĂ©montre que les deux SIMs sont affectĂ©s diffĂ©remment par la phosphorylation et lâacĂ©tylation. En outre, nous avons dĂ©terminĂ© que le nouveau SIM identifiĂ© joue un rĂŽle important dans la formation dâun complexe ternaire rĂ©presseur de la transcription, formĂ© des protĂ©ines PIAS, SUMO1 et de lâenzyme de conjugaison « UBiquitin Conjugating enzyme E2I (UBC9) ». Pris ensemble, ces rĂ©sultats donnent une description atomique de lâinteraction dâun nouveau SIM chez PIAS avec SUMO1 et dĂ©cris comment la phosphorylation et lâacĂ©tylation peuvent sĂ©lectivement rĂ©guler la spĂ©cificitĂ© des SIM trouvĂ©s chez les variants PIAS.
Finalement, dans la derniĂšre Ă©tude, nous avons explorĂ© le rĂŽle de la queue N-terminale des paralogues SUMO1 et 2 sur sa capacitĂ© Ă moduler les interactions SUMO-SIMs. Nous avons dĂ©montrĂ© que la queue N-terminale de SUMO1, mais pas SUMO2, avait un effet auto-inhibiteur sur les interactions SUMO-SIMs et que cet effet dĂ©pendait de la prĂ©sence de rĂ©sidus chargĂ©s nĂ©gativement prĂ©sent dans le SIM. Aussi, nous avons dĂ©montrĂ© que lâeffet auto-inhibiteur Ă©tait spĂ©cifique Ă la surface dâinteraction des SIMs sur SUMO1. De plus Ă partir dâĂ©tudes cristallographiques et de calorimĂ©trie, nous avons dĂ©montrĂ© que lâeffet auto-inhibiteur de la queue N-terminale de SUMO1 peut ĂȘtre neutralisĂ© par la prĂ©sence de zinc. La structure cristallographique du complexe entre SUMO1 et le SIM de PML dĂ©montre que le zinc stabilise la formation de liens entre des rĂ©sidus chargĂ©s nĂ©gativement du SIM et de la queue N-terminale de SUMO1. De plus, le zinc induit la formation dâune hĂ©lice α dans la queue N-terminale de SUMO1 qui est normalement intrinsĂšquement dĂ©sordonnĂ©e.
En rĂ©sumĂ©, cette Ă©tude donne une description atomique de lâeffet de lâacĂ©tylation sur les interactions SUMO-SIMs, dĂ©cris un nouveau SIM dans la famille de protĂ©ines PIAS et identifie un nouveau rĂŽle de la queue N-terminale de SUMO1 ainsi que comment cette rĂ©gion peut dĂ©finir la sĂ©lectivitĂ© des paralogues SUMOs.Post-translational modification with the « Small Ubiquitin-Like MOdifyer (SUMO) » is a major regulatory process (commonly referred to as SUMOylation) that regulates hundreds of proteins associated with a diverse array of biological activities including several nuclear functions such as DNA repair, replication and transcription. SUMOylation of a protein can impact its function in many ways most often by providing an additional binding surface for forming protein-protein interactions with regulatory factors through short hydrophobic regions on their binding partners known as « SUMO interacting motif (SIM) ». Although SUMO-SIM interactions are well documented, there are nevertheless outstanding questions that still need to be addressed regarding their controlling mechanisms. This thesis reports functional and structural studies on the regulatory mechanisms that govern SUMO-SIM interactions. More precisely, we studied how acetylation and the amino-terminal tail of SUMO proteins affects the interaction of SUMO with model SIMs from three proteins: the « promyelocytic leukemia (PML) » tumor suppressor, the transcriptional corepressor « Death Domain Associated Protein 6 (Daxx) » and the SUMO E3 ligase « Protein Inhibitor of Activated STAT (PIAS) ».
The first study reports the role that acetylation of SUMO1 plays on its binding to the SIMs of PML and Daxx. Isothermal Titration Calorimetry (ITC) experiments demonstrated that acetylation of SUMO1 at conserved residues (K39 and K46) dramatically reduces the binding to the SIMs of PML and Daxx. In contrast, SUMO1 acetylation at K37 dramatically reduced binding to the SIM of Daxx but only had minimal impact on binding to the SIM of PML. Crystal structures of the SUMO1 acetylated variants bound to the two SIMs support the ITC titrations and suggest that there is plasticity in SUMO-SIM interactions. The plasticity observed in the structures of these complexes would provide a robust mechanism for regulating SUMO-SIM interactions using a combination of signalling mechanisms that control post-translational modifications.
In the second study, we identified and characterized a novel SIM at the C-terminal extremity of three of the four known variants of the PIAS-family proteins (PIAS1-2-3). We demonstrated that this SIM binds to SUMO1 and structurally show that phosphorylation of the SIM or acetylation at select lysine residues of SUMO1 alters this interaction. In addition, we determined that it plays an important role in the formation of ternary complex made of SUMO1, PIAS1 and the « UBiquitin Conjugating enzyme E2I (UBC9) » in human cells. Together, these results provide an atomic description of the interaction between the C-terminal SIM of PIAS proteins and SUMO1 as well as important insight into how posttranslational modifications selectively regulate the specificity of the SIMs found in PIAS1-2-3.
Finally, our third study explores the intrinsically disordered N-terminal tail of SUMO paralogs and their ability to regulate SUMO-SIM interactions. We demonstrate that the N-terminal region of SUMO1, but not SUMO2, has an auto-inhibitory effect on the binding to SIMs and that this effect is dependent on the presence of acidic or phosphorylated residues that within the SIM. In addition, we also determined that this inhibition does not affect the interaction of SUMO1 with its E2 conjugating enzyme UBC9. Using titration calorimetry and crystallographic screening, we identified zinc as a negative regulator of this auto-inhibitory effect. The crystallographic structure of the complex between SUMO1 and the SIM of PML shows that zinc stabilises the formation of interactions with the negatively charged residues within the SIM and the N-terminal tail of SUMO1. Interestingly, zinc also appears to stabilize the formation of an α-helix within the N-terminal tail of SUMO1 which is normally intrinsically disordered.
In summary, this thesis describes the underlying atomic regulatory mechanisms of SUMO-SIM interactions by acetylation, reveals a novel SIM within the PIAS SUMO E3 ligase family and describes an unprecedented role of the N-terminal region of SUMO1 and provides important insight on how this region can define SUMO paralog specificity
Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region
Itch
is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases
involved in the control of inflammatory signaling pathways, several
transcription factors, and sorting of surface receptors to the degradative
pathway. In addition to these common domains, Itch also contains a
conserved proline-rich region (PRR) allowing its interaction with
Src homology 3 (SH3) domain-containing proteins. This region is composed
of 20 amino acids and contains one consensus class I and three class
II SH3-binding motifs. Several SH3 domain-containing partners have
been shown to recognize the Itch PRR, but their binding properties
have been poorly defined. Here we compare a subset of endocytic SH3
domain-containing proteins using bioluminescence resonance energy
transfer, isothermal titration calorimetry, and pull-down assays.
Results indicate that Endophilin is a high-affinity binding partner
of Itch both <i>in vivo</i> and <i>in vitro</i>, with a calculated <i>K</i><sub>D</sub> placing this complex
among the highest-affinity SH3 domain-mediated interactions reported
to date. All of the SH3 domains tested here bind to Itch with a 1:1
stoichiometry, except for ÎČ-PIX that binds with a 2:1 stoichiometry.
Together, these results indicate that Itch PRR is a versatile binding
module that can accommodate several different SH3 domain-containing
proteins but has a preference for Endophilin. Interestingly, the catalytic
activity of Itch toward different SH3 domain-containing proteins was
similar, except for ÎČ-PIX that was not readily ubiquitylated
even though it could interact with an affinity comparable to those
of other substrates tested
Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH
<div><p>Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431â487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448â471 (EBNA2<sub>448â471</sub>) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2<sub>448â471</sub> complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2<sub>448â471</sub> complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.</p></div
Tfb1PH and CBP KIX bind in a similar manner to EBNA2<sub>448â471</sub>.
<p>(<b>A</b>) Overlay of the <sup>1</sup>H-<sup>15</sup>N HSQC spectra for a 0.5 mM sample of <sup>15</sup>N-labeled EBNA2<sub>448â471</sub> in the absence (black) or presence (red) of 0.5 mM unlabeled Tfb1PH. (<b>B</b>) Overlay of the <sup>1</sup>H-<sup>15</sup>N HSQC spectra for a 0.5 mM sample of <sup>15</sup>N-labeled EBNA2<sub>448â471</sub> in the absence (black) or presence (red) of 0.5 mM unlabeled CBP KIX.</p
The hydrophobic residues of the ΊXXΊΊ motif from EBNA2 are important for transactivation.
<p>LexA-EBNA2<sub>431â487</sub> and mutant (W458T, I461 and F462S) fusion proteins were co-transformed in yeast with the reporter LexA operator-Lac-Z fusion plasmid pSH18â34. Results are presented as the percentage of the ÎČ-galactosidase units of the tested fusion proteins relative to that of the LexA-GAL4<sub>74â881</sub> positive control (100%). Error bars represent standard error about the mean of a minimum of three independent experiments.</p
Key interactions at the interface of the Tfb1PH-EBNA2<sub>448â471</sub> complex.
<p>(<b>A</b>) Ribbon representation of Tfb1PH (blue) and EBNA<sub>448â471</sub> (orange) highlighting the side chains (shown in sticks) of Tfb1PH (M59, M88 and R61) that interact with the aromatic ring of Phe462 (F462) of EBNA<sub>448â471</sub>. (<b>B</b>) Ribbon representation of Tfb1PH (blue) and EBNA<sub>448â471</sub> (orange) highlighting the side chains (shown in sticks) of Tfb1PH (M59, M88 and K57) that interact with the indole ring of Trp458 (W458) and the side chain of Ile461 (I461) of EBNA<sub>448â471</sub>.</p