Дисертації з теми "Protein interactions (PPI)"
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Weimann, Mareike. "A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactions." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16581.
Protein methylation on arginine and lysine residues is a largely unexplored posttranslational modification which regulates diverse cellular processes. The development of efficient proteome-wide approaches for detecting protein methylation is limited and technically challenging. We developed a novel workload reduced yeast-two hybrid (Y2H) approach to detect protein-protein interactions utilizing second generation sequencing. The novel Y2H-seq approach was systematically evaluated against our state of the art Y2H-matrix screening approach and used to screen 8 protein arginine methyltransferases, 17 protein lysine methyltransferases and 10 demethylases against a set of 14,268 proteins. Comparison of the two approaches revealed a higher sensitivity of the new Y2H-seq approach. The increased sampling rate of the Y2H-seq approach is advantageous when assaying transient interactions between substrates and methyltransferases. Overall 523 interactions between 22 bait proteins and 324 prey proteins were identified including 11 proteins known to be methylated. Network analysis revealed enrichment of transcription regulator activity, DNA- and RNA-binding function of proteins interacting with protein methyltransferases. The dataset represents the first proteome-wide interaction network of enzymes involved in methylation and provides a comprehensively annotated resource of potential new methylation substrates. An in vitro methylation assay coupled to mass spectrometry revealed amino acid methylation of candidate proteins. Seven of nine proteins tested were methylated including SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP and WDR42A indicating that the interaction network is likely to contain many putative methyltransferase substrate pairs. The presented protein-protein interaction network demonstrates that protein methylation is involved in diverse cellular processes and can inform hypothesis driven investigation into molecular mechanisms regulated through methylation.
Peri, C. "INVESTIGATING AND PREDICTING THE DETERMINANTS OF PROTEIN-PROTEIN INTERACTIONS THROUGH COMPUTATIONAL-STRUCTURAL BIOLOGY APPROACHES: IMPLICATIONS FOR STRUCTURAL VACCINOLOGY." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/243392.
Worseck, Josephine Maria. "Characterization of phosphorylation-dependent interactions involving neurofibromin 2 (NF2, merlin) isoforms and the Parkinson protein 7 (PARK7, DJ1)." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16533.
Alterations in phosphorylation-dependent signalling pathways, accumulation of aggregated proteins in the brain and neuronal apoptosis are common to neurodegeneration and implicate overlapping molecular mechanism. To gain insight into involved pathways, a modified yeast-two hybrid (Y2H) system was applied to screen 71 proteins associated with neurological disorders in a proteome-wide manner. For 21 of these proteins interactions were identified including 5 phosphorylation-dependent ones. In total, the network connected 79 proteins through 90 protein-protein interactions (PPIs). A fraction of these Y2H PPIs was tested in secondary interaction assays with a validation rate of 66 %. The described network-based approach successfully identified proteins associated with more than one disorder and cellular functions connected to specific disorders. In particular, the network revealed Ser/Thr kinase-dependent PPIs between the Parkinson protein 7 (PARK7, DJ1) and the E3 ligase components ASB3 and RNF31 (HOIP). The function of these proteins further substantiates the established connection between Parkinson’s disease (PD) and ubiquitination-mediated proteasome (dis)functions. Neurofibromin 2 (NF2, merlin) isoforms and PARK7 were identified as PI3K regulatory subunit p55-gamma (PIK3R3) interactors. These PPIs required Tyr kinase coexpression in the modified Y2H system and functional PIK3R3 pTyr-recognition modules (SH2 domains) in co-IP and Venus PCA experiments. This finding implicates the PI3K/AKT survival pathway in PD-associated neuronal apoptosis and Neurofibromatosis type 2-associated tumour formation. Investigation of PIK3R3, AOF2 (KDM1A, LSD1) and EMILIN1 PPIs on NF2 isoform level revealed preferential isoform 7 binding and cytoplasmic or membrane localisation of these PPIs for isoform 7 or 1, respectively. The generated modification-dependent and isoform-specific PPI network triggered many hypotheses on the molecular mechanisms implicated in neurological disorders.
Lima, Leandro de Araujo. "Uma abordagem integrativa usando dados de interação proteína-proteína e estudos genéticos para priorizar genes e funções biológicas em transtorno de déficit de atenção e hiperatividade." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-24082015-160400/.
Attention-Deficit/Hyperactivity Disorder (ADHD) is the most common neuro-developmental disorder in children, affecting 5.8% of children and adolescents in the world. Many studies have attempted to investigate the genetic susceptibility of ADHD without much success. The present study aimed to analyze rare and common variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios where the children were diagnosed with sporadic ADHD. We analyzed both single-nucleotide variants (SNVs) and copy-number variants (CNVs) in these trios and across multiple datasets, including a Brazilian sample of 503 children/adolescent controls from the High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of four CNV studies of ADHD involving children/adolescent Caucasian samples. The results from the Brazilian trios showed 3 major patterns: cases with inherited variations and de novo SNVs or de novo CNVs and cases with only inherited variations. Although the sample size is small, we could see that various comorbidities are more frequent in cases with only inherited variants. After exploring the rare variant composition in our 30 cases we selected genes with variations (SNVs or located in CNV regions) in our trio analysis that are recurrent in the families analyzed or in public data sets. Moreover, using only genes expressed in brain (post-mortem samples from Brain Atlas and The Genotype-Tissue Expression project), we constructed an in silico protein-protein interaction (PPI) network, with physical interactions confirmed by at least two sources. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.
Alleman, Cécile. "Accès synthétique au châssis [5-8-5] de la fusicoccine-A pour la synthèse d’analogues simplifiés en vue d'étudier les interactions protéine-protéine." Electronic Thesis or Diss., Université de Rennes (2023-....), 2023. http://www.theses.fr/2023URENS090.
In biological media, protein-protein interactions (PPI) are of huge importance, as they allow the regulation of many cellular events. PPI classically involve two partners: an adapter protein and its effector protein(s) regulated either in a positive or a negative manner. Inhibition of PPI has thus been considered as a solid therapeutic approach. On the other hand, stabilization of PPI remains scarcely investigated, but may lead to new promising approaches. This project focuses on the 14-3-3 family adapter protein which interacts with more than 200 protein partners. Among them, p53 protein is subjected to a lot of studies as this tumor suppressor protein regulates multiple biological processes (DNA repair, apoptosis). However, those major functions appear to be silenced in most cancer cases, thus allowing tumor cells proliferation. Some studies have shown that stabilization of the 14-3-3/p53 pair with the help of a molecular glue permitted to restore tumor suppressor activity of p53. Among the examined molecular glues, the fusicoccin-A (FC-A) natural product is shown to lodge in the valley formed by 14-3-3 and increases stabilization of the 14-3-3/p53 interaction. In this context, to enlarge the p53/14-3-3 molecular glue library, this project focuses on the access to simplified FC-A analogs through the synthesis of tricyclic scaffold. [6-8-5] analogs from an aromatic substrate are envisaged, as well as [5-8-5] analogs from a cyclopentane derivative, closer to the target structure. Various strategies have been explored in order to access these analogs
Gilker, Eva Adeline Gilker. "INTERACTIONS AND LOCALIZATION OF PROTEIN PHOSPHATASES, YWHA PROTEINS AND CELL CYCLE CONTROL PROTEINS IN MEIOSIS." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532699317257539.
Ravindranath, Velaga M. "Elucidating the role of mitoferrin (Mfrn), iron regulatory proteins (IRP1 and IRP2) and hephaestin (Heph) in iron metabolism by tagSNP and protein-protein interaction (PPI) analysis." Thesis, London Metropolitan University, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639414.
Johansson-Åkhe, Isak. "PePIP : a Pipeline for Peptide-Protein Interaction-site Prediction." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-138411.
Johansson, Joakim. "Modifying a Protein-Protein Interaction Identifier with a Topology and Sequence-Order Independent Structural Comparison Method." Thesis, Linköpings universitet, Bioinformatik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-147777.
Selim, Khaled [Verfasser]. "Structural and Functional Characterization of PII and PII-like Proteins and their Network of Interactions / Khaled Selim." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1233678507/34.
Simões, Sérgio Nery. "Uma abordagem de integração de dados de redes PPI e expressão gênica para priorizar genes relacionados a doenças complexas." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-17112015-172846/.
Complex diseases are characterized as being poligenic and multifactorial, so this poses a challenge regarding the search for genes related to them. With the advent of high-throughput technologies for genome sequencing and gene expression measurements (transcriptome), as well as the knowledge of protein-protein interactions, complex diseases have been sistematically investigated. Particularly, Protein-Protein Interaction (PPI) networks have been used to prioritize genes related to complex diseases according to its topological features. However, PPI networks are affected by ascertainment bias, in which the most studied proteins tend to have more connections, degrading the quality of the results. Additionally, methods using only PPI networks can provide just static and non-specific results, since the topologies of these networks are not specific of a given disease. In this work, we developed a methodology to prioritize genes and biological pathways related to a given complex disease, through an approach that integrates data from PPI networks, transcriptomics and genomics, aiming to increase replicability of different studies and to discover new genes associated to the disease. The methodology integrates PPI network and gene expression data, and then applies the Network Medicine Hypotheses to the resulting network in order to connect seed genes (obtained from association studies) through shortest paths possessing larger coexpression among their genes. Gene expression data in two conditions (control and disease) are used to obtain two networks, where each node (gene) in these networks is rated according to topological and coexpression aspects. Based on this rating, we developed two ranking scores: one that prioritizes genes with the largest alteration between their ratings in each condition, and another that favors genes with the greatest sum of these scores. The application of this method to three studies involving schizophrenia expression data successfully recovered differentially co-expressed gene in two conditions, while avoiding the ascertainment bias. Furthermore, when applied to the three studies, the method achieved a substantial improvement in replication of results, while other conventional methods did not reach a satisfactory replicability.
Dominguez, Palao Francisco. "Interferon induction by paramyxoviruses : investigations into specific RNA:protein interactions." Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/10750.
Aveiro, Susana Seabra. "The p22HBP heme binding protein: an NMR study of the dynamics and heme-protein interactions." Doctoral thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/14278.
The work presented in this Thesis investigates the dynamics and molecular interactions of p22HBP and the p22HBP-tetrapyrrole complex. Specifically, the key residues involved when a tetrapyrrole binds to p22HBP were sought. Previous molecular modelling studies identified three possible charged residues R56, K64 and K177 as possibly being important in tetrapyrrole binding via electrostatic interactions with the propionate groups of the tetrapyrrole. A number of variants of murine p22HBP were therefore prepared and fluorescence quenching and NMR used to verify the integrity of the variants and their interaction with tetrapyrrole. The same molecular modelling studies identified a mobile loop Y171-R180 in p22HBP that decreased in mobility on tetrapyrrole binding, therefore to confirm this mobility change dynamics studies based on NMR relaxation experiments were carried out. Finally in order to obtain a non heme-binding form of human p22HBP a chimeric p22HBP was designed and constructed. This construct, and the resulting protein, will be important for future siRNA knockdown studies where rescue or recovery of function experiments are required to prove the knockdown results. Chapter one discusses the current state of the art in terms of the biological, structural and functional aspects of p22HBP. The main objectives of the Thesis are also introduced here. Chapter two presents a detailed description of the different expression vectors (pNJ2 and pet28-a) and procedures used for overexpression and purification of murine p22HBP and its variants and human p22HBP. All expression and purification systems used gave good yields and allowed isotopic labeling to be carried out. The fluorescence quenching results for tetrapyrrole binding to murine p22HBP and variants are presented in chapter three along with the dissociation constants that were found to be in the nanomolar range for wild type murine and human p22HBP. The same studies were performed for murine p22HBP variants, with hydrophobic and polar changes being introduced at R56, K64 and K177. The dissociation constants were found to double in some cases but no significant changes in the strength of hemin-protein interactions were observed. The tetrapyrrole interaction with p22HBP was also followed by NMR spectroscopy, where chemical shift mapping was used to identify binding pocket location. All the variants and wild type human p22HBP were found to bind at the same location. Chapter 4 contains the data from 2D and 3D experiments carried out on 15N/13C labelled human p22HBP that was used to obtain backbone assignments. Comparison with wild type murine p22HBP assignments, PPIX titrations and theoretical calculations based on chemical shifts (Talos+) allowed 82% of the backbone resonances to be assigned. The results from the relaxation experiments used to probe the dynamics of the mobile loop in p22HBP on binding to tetrapyrrole are presented in chapter 5. The overall protein was found to tumble isotropically in the free and bound forms however the results to probe mobility changes in the 171-180 loop on tetrapyrrole binding proved inconclusive as only residue could be assigned and this did not seem to become significantly less mobile. The final chapter describes the design and construction of a chimeric p22HBP. For these purpose, the alfa1-helix sequence of human p22HBP in the phHBP1 plasmid was replaced by its homologous sequence in hSOUL, a non heme-binding protein with identical 3D structure. The results however indicated that either the incorrect sequence was introduced into the plasmid or the purification procedure was inadequate.
O trabalho apresentado nesta Tese focou-se na dinâmica e nas interações moleculares da p22HBP e do complexo p22HBP-tetrapirrol, nomeadamente nos resíduos chave envolvidos nesta interação. Estudos prévios de modelação molecular identificaram três possíveis resíduos chave R56, K64 e K177 como sendo importantes na interação com os tetrapirróis, através de interações eletrostáticas com os grupos propionato do tetrapirrol. Foram desenhados e construídos variantes da p22HBP murina e foram desenvolvidos estudos de extinção de fluorescência e RMN para avaliar a integridade dos variantes e a sua interação com os tetrapirróis. Os mesmos estudos de modelação molecular identificaram ainda uma zona flexível (Y171-R180) na p22HBP que diminui a mobilidade com a interação do tetrapirrol. Para confirmar esta alteração de mobilidade, foram realizados estudos de dinâmica, baseados em RMN. Por fim, com o intuito de obter uma versão não funcional da p22HBP humana, foi planeada e construída uma versão quimérica da p22HBP humana. No futuro, esta nova versão da p22HBP quimérica, será importante para os estudos de knockdown envolvendo siRNA. O capítulo um introduz uma revisão dos aspetos biológicos da p22HBP nomeadamente os estudos estruturais e as possíveis funções que foram identificadas. Os principais objetivos da tese são também apresentados neste capítulo. No capítulo dois é apresentada uma descrição detalhada dos diferentes vectores de sobreexpressão (pNJ2 e pet28-a) e dos métodos de sobreexpressão e purificação da p22HBP murina e respectivos variantes, bem como da p22HBP humana. Todos os sistemas de sobreexpressão e purificação utilizados obtiveram bons rendimentos e permitiram a marcação isotópica das proteínas. No capítulo 3 são apresentados os resultados de extinção de fluorescência para a interação da p22HBP murina e humana com hemina através das constantes de dissociação determinadas na ordem dos nanomolar. Os mesmos estudos foram realizados para os variantes da p22HBP murina, com alterações hidrofóbicas e de polaridade nos resíduos R56, K64 e K177. Em alguns casos, as constantes de dissociação determinadas são mais elevadas, embora não se tenham verificado alterações significativas na força da interação proteína-hemo. As interações tetrapirrólicas com a p22HBP foram também estudadas por espectroscopia de RMN, onde foram mapeadas as diferenças nos desvios químicos para identificar a localização da zona de interação. A localização da zona de interação dos variantes da p22HBP e a p2HBP humana mantém-se igual à p22HBP murina. No capítulo 4 encontram-se os resultados das experiências 2D e 3D realizadas na p22HBP humana, isotopicamente marcada com 15N/13C, para identificar as ressonâncias da cadeia principal. 82% dos sistemas de spin da cadeia principal foram identificados através da comparação com a p22HBP murina, das titulações com PPIX e de cálculos teóricos baseados nos desvios químicos (Talos+). No capítulo 5 são apresentados os resultados das experiências de relaxação, usados para comprovarem a dinâmica do loop na p22HBP aquando da interação com o tetrapirrol. A proteína no seu todo move-se de uma forma isotrópica na forma livre e ligada. No entanto os resultados para comprovar as alterações de mobilidade no loop 171-180 na presença de hemo, foram inconclusivos uma vez que só a um resíduo foi atribuído um sistema de spin, e não foi indicativo da perda significativa de mobilidade. O último capítulo descreve o planeamento e a construção da p22HBP quimérica. Para tal, a sequência que codifica a hélix alfa 1 da p22HBP humana, no plasmídeo phHBP1, foi substituída pela sequência homóloga da SOUL humana, uma proteína com uma estrutura 3D semelhante mas não liga ao hemo. Os resultados no entanto demonstraram que ou a sequência não foi introduzida corretamente no plasmídeo ou o sistema de purificação não foi adequado.
Ruiz-Gómez, Gloria, John C. Hawkins, Jenny Philipp, Georg Künze, Robert Wodtke, Reik Löser, Karim Fahmy, and M. Teresa Pisabarro. "Rational Structure-Based Rescaffolding Approach to De Novo Design of Interleukin 10 (IL-10) Receptor-1 Mimetics." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-215877.
Ruiz-Gómez, Gloria, John C. Hawkins, Jenny Philipp, Georg Künze, Robert Wodtke, Reik Löser, Karim Fahmy, and M. Teresa Pisabarro. "Rational Structure-Based Rescaffolding Approach to De Novo Design of Interleukin 10 (IL-10) Receptor-1 Mimetics." Public Library of Science, 2016. https://tud.qucosa.de/id/qucosa%3A30052.
Krieger, Natalie [Verfasser], and Klaus [Akademischer Betreuer] Harter. "Expression, localization, and interaction studies of the plastidic PII protein from Arabidopsis thaliana / Natalie Krieger ; Betreuer: Klaus Harter." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1212025156/34.
Nigavekar, Shraddha S. "Regulation of GLC7 encoded PP1 and analysis of synthetic lethal interactions with ade3 and leu2 in saccharomyces cerevisiae." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013007.
Heil, Katharina Friedlinde. "Systems biological approach to Parkinson's disease." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31043.
Wimble, Christopher. "Working Together: Using protein networks of bacterial species to compare essentiality, centrality, and conservation in Escherichia coli." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3878.
Gnangnon, Bénédicte. "Caractérisation moléculaire et fonctionnelle de la pseudo-tyrosine kinase-like (pTKL) de plasmodium." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S003/document.
Malaria is the first endemic parasitic disease in the world with nearly half million deaths in 2017 according to the WHO. This disease is the result of an infection by an agent belonging to the Plasmodium genus. This apicomplexan parasite infects two hosts over its complex life cycle: a definitive one – a mosquito belonging to the Anopheles genus – and a homoeothermic intermediate host. At least six Plasmodium species can infect humans. In its intermediate host, Plasmodium first replicates in hepatocytes before releasing erythrocyte-infectious stages in the bloodstream. Once there, parasites invade and replicate within erythrocytes, before lysing them to release other infectious stages. This triggers an exponential rise in the parasitemia, as well as malaria symptoms. Sexual stages, called gametocytes, are produced over this intra-erythrocytic cycle to be transmitted to the arthropod vector, thus allowing the completion of the parasite life cycle.Plasmodium co-evolved with its hosts and set up diverse gene expression regulation pathways accordingly. Phosphorylation is one of the major and fastest post-translational modifications used by the parasite to respond to environmental changes. Many of its kinases and phosphatases play key roles in host cell invasion, cellular growth and division, as well as motility of specific developmental stages. However, the role of the five pseudo-kinases expressed by Plasmodium has not been explored yet.During my PhD project, I have performed the characterization of the unique Plasmodium pseudo-Tyrosine Kinase-like (pTKL) and explored its role over the parasite intra-erythrocytic cycle.P. falciparum pTKL (PfpTKL) in silico annotation allowed the delineation of the protein domains. Notably, a SAM (Sterile Alpha Motif) domain, two RVxF motifs (known for their binding potential with the major protein phosphatase type 1, PP1) and a pseudo-kinase domain belonging to Tyrosine Kinase-like (TKL) family were found. This pseudo-kinase domain was found to be able to bind ATP in a cation-independent way although devoid of kinase activity. Two parasite protein partners of PfpTKL have been identified using in vitro protein-protein interaction studies together with heterologous models (yeast, Xenopus ovocytes). First, PfSERA5 (SErine Repeat Antigen 5) specifically and strongly interacts with PfpTKL SAM domain and second, PfPP1c binds the two RVxF-containing regions of PfpTKL. Interestingly, the second RVxF motif, which is located within the pseudo-kinase domain, directly binds PfPP1c and seems to be involved in the allosteric regulation of the phosphatase activity. The subcellular localization of P. berghei pTKL (PbpTKL) was studied by IFA as well as sequential lysis of erythrocytes followed by immunoprecipitation assays. PbpTKL was shown to be exported to the host cell cytosol at the trophozoite stage, but retained in the parasitophorous vacuole and the parasite cytosol at the schizont stage. Furthermore, our interactome analysis conducted at the trophozoite stage by IP/MS showed that PbpTKL binds many host cell proteins involved in erythrocyte cytoskeleton organization, as well as erythropoiesis and cell homeostasis. These data suggest that pTKL plays a role at the parasite/host interface, either directly or via its protein partners.Finally, in an attempt to understand the role of pTKL for the parasite development, we generated genetically modified P. berghei strains. The phenotypic study of PbpTKL KO and iKD strains did not show any difference between the defective parasites and the parental wild type ones during the intra-erythrocytic cycle, gametocyte expression and male gametocyte activation. These data suggest the dispensability of pTKL or the expression of redundant gene(s) with similar functions in these parasite stages. Whatever the explanation, it is still important to follow up this investigation in other parasite stages, from zygotes to hepatic stages
Martin, Gil. "Etude de la proteine p5 du bacteriophage phi 29 presentant une affinite pour le dna monocatenaire." Toulouse 3, 1987. http://www.theses.fr/1987TOU30258.
Haj, Hassan Maya. "Caractérisation de protéines bovines potentiellement impliquées dans la reproduction : GPA2, GPB5, PDI, PEBP et Ubiquitine." Thesis, Tours, 2011. http://www.theses.fr/2011TOUR4037/document.
We characterized five bovine proteins that are potentially involved in reproduction. We started with the cloning of gpa2 and gpb5 cDNAs in order to eventually purify recombinant and natural GPA2 and GPB5 to study their possible quaternary structure. GPA2 and GPB5 are the evolutionary ancestors of Glycoprotein hormones α and β subunits respectively. Meanwhile, we have shown the relative quaternary structure fragility of bovine FSH compared to human and sheep FSH. We also studied the effect of endocrine disruptors on PDI (Protein Disulfide Isomerase) before addressing GPA2/GPB5 PDI activity of GPA2/GPB5 once purified.We succeeded to purify the phosphatidyl-ethanolamine-binding protein (PEBP) and ubiquitin from bovine testis by hydrophobic interaction chromatography at very high ammonium sulfate concentration and we produced specific antibodies (anti-PEBP) in rabbits that allowed us to be the first to develop a reliable Elisa assay for this protein
Di, Antonio Veronica. "Towards the identification of small molecules inhibiting he dimerization of HCMV DNA polymerase processivity factor UL44." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3423139.
Cytomegalovirus (CMV) è un importante patogeno di interesse umano. Al momento gli antivirali disponibili ed utilizzati per la terapia contro l’infezione da CMV presentano una serie di controindicazioni dovute all’alto costo, bassa biodisponibilità, alta tossicità ed il presentarsi di ceppi virali resistenti. Inoltre non è disponibile un vaccine ed ancora non è ancora stato approvato l’uso di alcun farmaco per prevenire la trasmissione verticale durante la gravidanza. Per questi motivi, sono necessari nuovi ed efficaci farmaci antivirali. La proteina accessoria UL44 della DNA polimerasi di CMV, svolge un ruolo essenziale nella replicazione virale, conferendo processività alla subunità catalitica UL54 ancorando il complesso oloenzimatico al DNA. Il legame di UL44 al dsDNA avviene in assenza di ATP e dei clamp loaders, e dipende dalla omodimerizzazione di UL44. Infatti, il nostro gruppo di ricerca ha recentemente dimostrato che la proteina può dimerizzare in cellule e che mutazioni puntiformi in grado di inficiare tale dimerizzazione prevengono il legame con il DNA ed aboliscono la replicazione del DNA virale oriLyt-dipendente in saggi di transcomplementazione transiente. Perciò, la distruzione dell’omodimerizzazione UL44 rappresenta un potenziale allettante bersaglio per lo sviluppo di nuovi anti-virali. Partendo da queste osservazioni ed usando la struttura cristallografica recentemente pubblicata degli omodimeri UL44, il nostro gruppo di ricerca ha eseguito un virtual screening con il software Glide in combinazione con una libreria di 1.3 x 10^6 piccole molecole (SMs) per identificare SMs che potenzialmente potessero interferire con l’omodimerizzazione di UL44. Dopo tre rounds di screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), seguiti da un’analisi delle proprietà chimiche dei composti, sono state selezione 18 SM per ulteriori analisi. I composti selezionati sono stati acquistati presso un fornitore commerciale, per essere testati in diversi saggi per valutare le loro abilità di inibire l’omodimerizzazione di UL44, sia in cellule che in vitro. A questo scopo, abbiamo utilizzato diversi saggi in cellule e in vitro per monitorare l’effetto delle SMs sulla dimerizzazione di UL44. Nei saggi cellulari, le tecniche utilizzate includono Fluorescent Resonant Energy Transfer (FRET) e Bioluminescence Resonant Energy Transfer (BRET), mentre per saggi in vitro è stato utilizzato il saggio GST-pull down. I nostri dati indicano che la metodica FRET acceptor photobleaching è in grado di rilevare sia l’omodimerizzazione di UL44, sia il legame con il dominio C-terminale della subunità catalitica (residui 1125-1242). Queste interazioni sono sensibili all’introduzione di mutazioni puntiformi che alterano i due processi, evidenziando la specificità di questa tecnica. Siamo stati quindi in grado di confermare che UL44 forma dimeri in un contesto cellulare. Purtroppo, l’acquisizione dei dati e la loro analisi richiedono un lungo tempo e dipendono da alti livelli di espressione delle proteine di fusione. Per contro, il saggio BRET permette un rapido e preciso monitoraggio quantitativo dell’omodimerizzazione di UL44 e il legame con UL54 in cellule viventi. Inoltre, attraverso esperimenti di saturazione, che permettono di calcolare in modo preciso i valori di Bmax e B50 relative all’omodimerizzazione o al legame con UL54 per varianti di UL44 che contengono singole sostituzioni amminoacidiche che affliggono la dimerizzazione di UL44, il suo legame a UL54 o il DNA, nonché il trasporto al nucleo della proteina. I dati ottenuti possono aiutare a comprendere il processo di formazione dell’oloenzima della DNA polimerasi di CMV, suggerendo cambiamenti conformazionali nel complesso olenzimatico in seguito a legame con il DNA. Inoltre, il calcolo di Bmax e B50 è stato usato per sviluppare tre differenti sistemi cellulari esprimenti un’ideale quantità di UL44 da utilizzare come piattaforma per lo screening di SM, poiché sono in grado di generare valori di BRET ratio simili al 50% della Bmax .Questi includono un sistema di espressione completamente stabile, in cui sia RLuc-UL44 che YFP-UL44 sono stabilmente espresse in cellule derivate da HEK293 A, un sistema ibrido stabile/transiente, in cui YFP-UL44 è stabilmente espressa e RLuc-UL44 è espressa in transiente, o un sistema completamente transiente in cui entrambe le proteine sono espresse transientemente. Saggi di competizione eseguiti sovra-esprimendo quantità crescenti di UL44 fusa a un FLAG tag hanno evidenziato che nessun inibizione può essere ottenuta per il sistema completamente stabile, probabilmente per la difficoltà di distruggere un complesso proteico preformato piuttosto che prevenirne la formazione. Per questo motive ci siamo focalizzati su sistemi transienti di espressione piuttosto che sul sistema interamente stabile. Sulla base di questi dati, un primo screening su piccolo scala è stato eseguito per studiare l’effetto di 18 SMs sulla dimerizzazione di UL44 usando il sistema BRET stabile/transiente. Le 18 piccole molecole sono state risospese in DMSO e la loro tossicità è stata valutata in coltura cellulare, prima di essere aggiunte a concentrazioni subtossiche alla linea YFP-UL44, sei ore dopo la trasfezione per esprimere RLuc-UL44. La nostra analisi ha identificato solo composti che inibivano blandamente il BRET ratio relativo alla dimerizzazione di UL44. Per questo motivo abbiamo ri-valutato il set up del saggio BRET, diminuendo il tempo d’incubazione delle SM prima di testare i valori BRET da 42 a 18 ore, utilizzando un saggio completamente transiente e diminuendo la quantità di proteine espresse. Per quest’ultima è stato necessario cambiare il substrato utilizzato per generare il segnale bioluminescente da CTZ a hCTZ. È stato eseguito un nuovo screening, con risultati molto simili a quelli ottenuti utilizzando il setup originale. Inoltre, quando i candidati migliori sono stati rivalutati usando un controllo negativo, il loro effetto sul BRET ratio è risultato aspecifico. Ulteriormente, la BRET, come la FRET, non è riuscita a rilevare uno specifico effetto nell’interazione UL44/UL54 di una SM in grado di distruggere questa interazione. Possiamo quindi concludere che BRET e FRET non sono tecniche ideali per la ricerca di SM inibitrici dell’interazione tra le proteine. Ci siamo poi focalizzati su metodi in vitro, partendo dal saggio GST-pull down, il quale è stato effettato utilizzando il dominio C-terminale (residui 1-290) di UL44, fuso o con GST o con 6His-tag. I risultati ottenuti mostrano che la tecnica GST-pull down è in grado di rilevare differenze nel legame tra UL44 wild-type e i mutanti con mutazioni nel sito di dimerizzazione, suggerendo una possibile applicazione di GST-pull down per lo screening delle SMs. Questa tecnica è stata implementata per questo studio, e i dati preliminari suggeriscono che il numero di SMs testate potrebbe portare all’inibizione della dimerizzazione di UL44. In conclusione, abbiamo sviluppato diversi saggi per monitorare la dimerizzazione di UL44. I saggi cellulari basati su RET confermano che UL44 forma dimeri in cellule viventi, e dimostrano di essere subottimali per lo screening. D’altro canto, i metodi in vitro come GST-pull down dimostrerebbero un maggiore sensibilità e sono stati implementati per l’identificazione degli inibitori della dimerizzazione di UL44.
Park, Alex. "Characterization of a novel class of anti-HCV agents targeting protein-protein interactions." Thèse, 2016. http://hdl.handle.net/1866/18893.
Hepatitis C virus (HCV) is an important causative agent for liver diseases and is responsible for a worldwide pandemic affecting roughly 180 million individuals worldwide. Late diagnosis following the progression to fibrosis and to cirrhosis, in nearly 16% of chronic infections, is attributed to the absence of symptoms in the first years of infection. By exploiting membrane protein-protein interactions (PPI), live cell assays using bioluminescence resonance energy transfer (BRET) technology have previously been optimized to complete a comprehensive hepatitis C virus (HCV) protein interaction network. Using the groundwork laid by this network study, a high-throughput assay (HTS) cell-based assay was implemented to identify novel inhibitory compounds targeting an unreported NS3/4A-NS3/4A interaction. Approximately 110,000 compounds from a small-molecule collection were screened to monitor modulation of NS3/4A homodimerization and were discriminated based on specificity and potency. UM42811 was identified as a potential NS3/4A-NS3/4A interaction activator and found to have a promising antiviral activity boasting an excellent therapeutic window. Combined deep sequencing and mutation mapping have yielded a resistance profile based on statistical and functional probability pointing towards a novel inhibitory mechanism targeting the HCV NS3/4A independent from protease activity inhibition. Data from an HCV to host protein interaction network generated by our group was used to analyze alternative effects of UM42811 on interactions which potentially benefit viral persistence. NS3/4A-specific host interactors were heavily associated with nuclear and mitochondrial transport based on Gene Ontology (GO). Among these specific interactors, karyopherin subunit beta 1 (KPNB1) and heat shock protein 60 (HSP60) were selected for further study. Interestingly, co-immunoprecipitation experiments revealed that UM42811 was able to prevent both KPNB1-NS3/4A and HSP60-NS3/4A interactions. Moreover, functional and western analysis revealed the KPNB1-NS3/4A interaction to have deleterious effects on iv interferon stimulated gene (ISG) induction. Unexpectedly, analysis revealed a putative NS3/4A mediated cleavage of KPNB1. Overall, this study demonstrates the strength of cell-based PPI strategies in the identification of novel HCV antiviral compounds, characterizes a novel inhibitory mechanism for HCV and reveals a potentially novel viral immune evasion mechanism.
Su, Junjie. "Accurate and Reliable Cancer Classi cation Based on Pathway-Markers and Subnetwork-Markers." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8865.
Tsai, Pai-Chi, and 蔡百騏. "Characterization of the interaction between PP1 and its inhibitory protein by surface plasmon resonance biosensors." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/41900696493652747412.
國立中正大學
分子生物研究所
90
Inhobitor-2 (I2) is the regulatory subunit of the catalytic subunit of protein phosphatase-1 (PP1) and inhibits the enzyme activity via multiple sites. Our previous studies have demonstrated that the region encompassing residues 9-13 (PIKGI) plays critical role in inhibition of PP1. However, no obvious evidence indicated the sequence Lys144-Leu-His-Tyr147 corresponding to the BB(V/I)XF PP1-binding motif, involved in this action. In the presentation, we have characterized in detail the function of this sequence to test whether it has such activity. Biacore analysis demonstrated that deletion of C-terminal sequence after the residue 120 of I2 did not significantly affect the binding constant between PP1 and I2. In comparison with I2(WT), mutation of Tyr-147 【I2-(Y147A)】 also showed no significant difference on affinity with PP1. These findings suggested that this sequence Lys144-Leu-His-Tyr147 is probably not involved in the inhibition with PP1.
Tuan, Li-Fen, and 段麗芬. "The Identification and Characterization of the CellularLocalization of p53 and Its Interacting Protein, PIP." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/68783412382688676244.
國防醫學院
生物化學研究所
89
PIP is a novel p53-interacting protein. It has been well known that p53-binding proteins play important roles in either mediating or modulating the activity of p53, and consequently affect its tumor suppression function. In normal cells, p53 mainly localizes in nucleus, whereas the cytoplasmic distribution of p53 was found in certain type of cancers in which p53 is wild-type. The focus of this research is to identify the cellular localization of p53 and PIP in various cell lines, and to determine their distribution after exposing to DNA damage agents such as UV or IR. Our data indicate that the localization of singly transfected fluorescent p53 and PIP fusion proteins is primarily in nucleus and cytosol, respectively, and UV or IR did not affect their cellular localization. When cells were co-expressed the fluorescent p53 and PIP fusion proteins, three different results were obtained from a series of independent experiments in both MCF-7 and H1299: first, the prescence of p53-DsRed fusion protein was sufficient to translocate EGFP-PIP fusion protein from its original place, cytosol, to nucleus, second, both the expression of p53-DsRed and the stimulation of IR were require for changing the cellular localization of EGFP-PIP, and third, the localization of EGFP-PIP did not change upon the expression of p53-DsRed and IR stimulation. Besides the fluorescent fusion proteins, we also used immunohistochemistry to determine the localization of endogenous PIP and transfected PIP with different sizes. The results showed that the endogenous PIP and the transfected PIP with ring-finger motif are localized in nucleus, whereas the cytoplasmic localization was observed when cells were transfected with the other PIP constructs including the PIP fragments derived from the longest ORF and the usage of the fourth ATG as initiation codon. In consistent with the third observation mentioned above, the localization of endogenous and transfected PIP remains in the cytosol regardless the expression of p53 and/or the treatment of DNA damaging agents. Overall, the results in this thesis, although with certain conflicting observations, show that we are fail to demonstrate the colocalization of p53 and PIP, and indicate that these two proteins do not interact with each other in the conditions which we employed. However, this conclusion is contradictory to what we obtained from in vivo binding assay, in which the interaction between p53 and PIP can be measured in irradiated mouse tissues. The results in my thesis might provide some clues and information in pursuing the interaction of p53 and PIP in the future.
Weiß, Martin. "A protein in search of a function: The c-di-AMP-binding protein DarA of Bacillus subtilis." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C177-D.
Beiraghi, Salek Asma. "Spinophilin-Dependent Regulation of the Phosphorylation, Protein Interactions, and Function of the GluN2B Subunit of the NMDAR and its Implications in Neuronal Cell Death." Thesis, 2020. http://hdl.handle.net/1805/24770.
Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.
Winter, Sherry Lynn. "Genetic and functional characterization of the interaction of BRCA1 with the serine/threonine phosphatase, PP1, and the circadian clock proteins, Per1 and Per2 /." 2006. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=442638&T=F.
(9746078), Asma Beiraghi Salek. "Spinophilin-dependent regulation of the phosphorylation, protein interactions, and function of the GluN2B subunit of the NMDAR and its implications in neuronal cell death." Thesis, 2021.
Lussier-Price, Mathieu. "Étude sur la reconnaissance de l'ubiquitine par les domaines de transactivation acides des activateurs de transcription." Thèse, 2014. http://hdl.handle.net/1866/11225.
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.