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Tattersall, Daniel. "The identification of retromer partner proteins." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615241.
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Njunge, James Mwangi. "Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1013186.
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Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
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Krishnan, Kadalmani. "Characterisation of the G protein controlled tyrosine kinase, ACK1 and its interaction with nucleolar partner proteins." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610698.
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Chan, Pui Wai. "Fhit : a novel interacting partner of G[alpha][subscript q] proteins /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BICH%202008%20CHAN.
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Mohamad, Nada. "The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export." Doctoral thesis, Universitat Politècnica de València, 2017. http://hdl.handle.net/10251/90397.
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Nuclear export of messenger ribonucleic acid (mRNA) is a complex and essential process for a correct gene expression in all eukaryotic cells. The export of mRNA through the nuclear pore complex depends mostly on the crosstalk and coordination of several proteins forming what is known as mRNPs (messenger ribonucleoproteins) that play dynamic, interconnecting roles in the different mRNA biogenesis steps such as pre-mRNA processing, stability, and export.
One key protein in this process is Mex67, conserved from yeast to humans, is the major messenger RNA exporter also involved in ribosomal RNA export. Mex67 interacts with Mtr2 to form an evolutionary conserved heterodimer essential for proper mRNA export and subsequently the survival of the cell. Mex67 have been studied for many years, however due to the complexity and interconnectivity of the different processes in mRNA biogenesis, there is yet to uncover many details on the dynamics of the process and the crosstalk between Mex67 and its many partners.
In this study, using a combination of biochemical, biophysical, and structural analysis, we characterize the interaction between Mex67 and a novel partner protein called Mip6 (Mex67 interacting protein 6). We were able to reconstitute a stable complex in vitro, and extensively study the mechanism in which the two proteins interact. We also solved the crystal structure of the C-terminal region of Mex67 that interacts with Mip6 and identified the UBA domain of Mex67, known to bind FG nucleoporins and Hpr1 protein as also the site where Mip6 binds. However, little was known about the structure or function of Mip6 and its paralogue Pes4. Here we proved that Mip6 is an RNA binding protein with four RNA recognition motifs that binds RNA in vitro with high affinity. Additionally, its fourth RNA recognition motif was also the site of binding of Mex67. Furthermore, we showed that the Mex67 complex formation with Mip6 RRM4 compromises its ability to bind RNA or vice versa. We also designed a point mutation on Mip6 RRM4 that disrupts its interaction with Mex67 but not with RNA. Subsequent in vivo yeast assays led us to hypothesize a role of Mip6 as an adaptor protein for Mex67 in nuclear export especially upon stress. Additional function of Mip6 was the localization of its bound mRNA to cytoplasmic stress granules in cellular stress conditions.
Moreover, the crystal structures of Mip6 RRM3, Pes4 RRM3, Pes4 RRM4, and Pes4 RRM3/4 were also solved. All RRMs adopted a canonical RRM fold with conserved RNP1 and RNP2 sequences normally involved in RNA binding, except Mip6 RRM3 that was missing the aromatic ring in RNP2. In the structure of RNA-free Pes4 RRM3/4, the tandem RRM domains were connected with a flexible disordered linker and no inter-domain contact between them. Finally, although Pes4 RRM4 was binding RNA in vitro, it did not have the ability to interact with Mex67 thus suggesting a separate evolutionary function for Mip6 and Pes4.La exportación nuclear de ácido ribonucleico mensajero (ARNm) es un proceso complejo y esencial para una expresión correcta de los genes en todas las células eucariotas. La exportación de ARNm a través del complejo del poro nuclear depende principalmente de la interacción y coordinación de varias proteínas, que forman lo que se conoce como mRNPs (ribonucleoproteínas mensajeras), que tienen un papel dinámico e interconectado en las diferentes etapas de la biogénesis de ARNm, tales como el procesamiento del pre-ARNm, estabilidad, y exportación. Una proteína clave en este proceso es Mex67, conservada de levaduras a humanos, que es la principal exportadora de ARN mensajero y también está implicada en la exportación de ARN ribosomal. Mex67 interacciona con Mtr2 para formar un heterodímero conservado evolutivamente esencial para una exportación adecuada de ARNm y la consiguiente supervivencia de la célula. Se ha estudiado Mex67 durante muchos años, sin embargo, debido a la complejidad e interconectividad de los diferentes procesos de biogénesis de ARNm, todavía quedan por descubrir muchos detalles de la dinámica del proceso y las interacciones entre Mex67 y sus muchas proteínas asociadas. En este estudio, combinando un análisis bioquímico, biofísico y estructural, hemos caracterizado la interacción entre Mex67 y una nueva proteína asociada denominada Mip6 (proteína 6 que interacciona con Mex67). Hemos podido reconstituir un complejo estable in vitro y estudiar extensivamente el mecanismo por el cual interaccionan estas dos proteínas. También hemos resuelto la estructura cristalográfica de la región C-terminal de Mex67 que interacciona con Mip6 e identificado el dominio UBA de Mex67, conocido por unirse a nucleoporinas FG y a la proteína Hpr1, así como el sitio por el que se une Mip6. No obstante, se sabía muy poco sobre la estructura o la función de Mip6 y su parálogo Pes4. Hemos probado que Mip6 es una proteína de unión a ARN con cuatro motivos de reconocimiento de ARN que se unen a ARN in vitro con una afinidad alta. Además, su cuarto motivo de reconocimiento de ARN es también el sitio de unión a Mex67. Posteriormente, demostramos que la formación del complejo de Mex67 con el dominio RRM4 de Mip6 compromete su capacidad para unir ARN o viceversa. También diseñamos una mutación puntual en el RRM4 de Mip6 que rompe la interacción con Mex67 pero no con el ARN. Los ensayos posteriores in vivo en levaduras nos permitieron establecer una hipótesis sobre el papel de Mip6 como proteína adaptadora para Mex67 en la exportación nuclear, especialmente en condiciones de estrés. Una función adicional de Mip6 era la localización del ARNm que se unía a ella en gránulos de estrés en condiciones de estrés celular. Además, hemos resuelto las estructuras cristalográficas del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 y los RRM3 y 4 de Pes4. Todos los RRMs adoptaron una conformación canónica RRM con secuencias RNP1 y RNP2 conservadas generalmente implicadas en la unión a ARN, excepto el RRM3 de Mip6 que carecía del anillo aromático en RNP2. En la estructura sin ARN de los RRM3 y 4 de Pes4, los dominios RRM tándem estaban conectados por una región flexible desordenada y no había un contacto inter-dominio entre ellos. Finalmente, aunque el RRM4 de Pes4 se unía a ARN in vitro, no presentaba la capacidad de interaccionar con Mex67 lo cual sugiere una divergencia evolutiva de la función de Mip6 y Pes4.
L¿exportació nuclear d¿àcid ribonucleic missatger (mRNA) es un procés complex i essencial per a una correcta expresió gènica en totes cèl¿lules eucariotes. L¿exportació del mRNA a través del complex del porus nuclear depén principalment de la interacció i coordinació de diverses proteïnes, que formen el que es coneix com mRNPs (ribonucleoproteïnes missatgeres), que tenen un paper dinàmic i interconnectat en les diferents etapes de la biogènesi d¿ARNm, com el processament del pre-ARNm, estabilitat, localització i exportació. Una proteïna clau en aquest procés és MEX67, conservada de llevats fins a humans, que és la principal exportadora de ARN missatger i també està implicada en l¿exportació de ARN ribosomal. Mex67 interacciona amb Mtr2 per a formar un heterodímer conservat evolutivament essencial per a una exportació adequada d¿ARNm i la consegüent supervivència de la cèl¿lula. S¿ha estudiat Mex67 durant molts anys, però degut a la complexitat i interconectivitat dels diferents processos de biogènesi d¿ARNm, encara queden per descobrir molts detalls de la dinàmica del procés i les interaccions entre Mex67 i les seues moltes proteïnes associades. En aquest estudi, combinant l¿anàlisi bioquímic, biofísic i estructural, hem caracteritzat la interacció entre Mex67 i una nova proteïna associada anomenada Mip6 (proteïna 6 que interacciona amb Mex67). Hem pogut reconstituir un complex estable in vitro i estudiar extensivament el mecanisme pel qual interaccionen estes dos proteïnes. També hem resolt l¿estructura cristal¿logràfica de la regió C-terminal de Mex67 que interacciona amb Mip6 i identificat el domini UBA de Mex67, conegut per unir-se a nucleoporines FG i a la proteïna Hpr1, així com ser el lloc pel que s¿uneix Mip6. No obstant, se sabia molt poc sobre l¿estructura o la funció de Mip6 i el seu paràleg Pes4. Hem comprobat que Mip6 es una proteïna d¿unió a ARN amb quatre motius de reconeixement d¿ARN que s¿uneixen a ARN in vitro amb una afinitat alta. A més, el seu quart motiu de reconeixement d¿ARN és també el lloc d¿unió a Mex67. Posteriorment, demostràrem que la formació del complex de Mex67 amb el domini RRM4 de Mip6 compromet la seua capacitat per a unir ARN o viceversa. També vam dissenyar una mutació puntual en el RRM4 de Mip6 que trenca la interacció amb Mex67 però no amb l¿ARN. Els assajos posteriors in vivo en llevats ens van permetre establir una hipòtesi sobre el paper de Mip6 com a proteïna adaptadora per a Mex67 en l¿exportació nuclear, especialment en condicions d¿estrès. Una funció adicional de Mip6 era la localització de l¿ARNm que s¿unia a ella en grànuls d¿estrès en condicions d¿estrès cel¿lular. A més, hem resolt les estructures cristal¿logràfiques del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 i els RRM3 i 4 de Pes4. Tots els RRMs adoptaren una conformació canònica RRM amb seqüències RNP1 i RNP2 conservades generalment implicades en la unió a ARN, excepte el RRM3 de Mip6 que mancava del anell aromàtic en RNP2. En la estructura sense ARN dels RRM3 i 4 de Pes4, els dominis RRM tàndem estàven conectats per una regió flexible desordenada i no hi havia un contacte interdomini entre ells. Finalment, encara que el RRM4 de Pes4 es unia a ARN in vitro, no presentava la capacitat d¿interaccionar amb Mex67, la cual cosa sugerix una divergencia evolutiva de la funció de Mip6 y Pes4.
Mohamad, N. (2017). The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90397
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Ekiert, Robert. "Analysis of partner proteins of MeCP2 and their relevance to Rett syndrome." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9901.
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Methyl-CpG binding protein 2 (MeCP2) was discovered as a protein binding to methylated DNA more than 20 years ago. It is very abundant in the brain and was shown to be able to repress transcription. The mutations in MeCP2 cause Rett syndrome, an autism-spectrum neurological disorder affecting girls. Yet, the exact role of MeCP2 in Rett disease, its function and mechanism of action are not fully elucidated. In order to shed some light on its role in the disease the aim of this project was to identify proteins interacting with MeCP2. Affinity purification of MeCP2 from mouse brains and mass spectrometry analysis revealed new interactions between MeCP2 and protein complexes. Detailed analysis confirmed the findings and narrowed down the top interactions to distinct regions of MeCP2. One of the domains interacts with identified NCoR/SMRT co-repressor complex and is mutated in many patients with Rett syndrome. In vitro assays proved that these mutations abolish the putative transcriptional repressor function of MeCP2. We propose a model in which Rett syndrome is caused by two types of mutations: either disrupting the interaction with DNA or affecting the interaction with the identified complex, which has an effect on the global state of chromatin. The presented findings can help to develop new therapies for Rett syndrome in the future.
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Humphrey, Tania Vivienne. "Characterisation of a putative G-protein coupled receptor and its protein interacting partner in Arabidopsis /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16260.pdf.
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Cheung, Ngai. "Structural and functional characterization of EEN/EndophilinA2, a fusion partner in acute leukemia /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31540284.
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Cheung, Ngai, and 張毅. "Structural and functional characterization of EEN/EndophilinA2, a fusion partner in acute leukemia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45014760.
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O'Kane, Neil D. "TREK-1 investigation of a physiological role and identification of novel binding partner proteins /." abstract and full text PDF (UNR users only), 2009. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1472969.
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Bloor, Adrian John Clifton. "Identification of novel partner proteins for the basic helix-loop-helix transcription factor SCL." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619716.
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Bhattacharjee, Sonali. "The role of Fml1 and its partner proteins Mhf1 and Mhf2 in promoting genome stability." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711640.
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Müller, Marietta. "YIP1 family member 4 (YIPF4) is a novel cellular binding partner of the papillomavirus E5 proteins." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/6446/.
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Papillomaviruses (PVs) are capable of causing a broad spectrum of diseases with the human PVs (HPVs) being responsible for a great portion of cervical, anogenital and head and neck cancers worldwide. The PV oncoprotein E5 plays roles in host cell transformation, the PV life-cycle and viral immune evasion. However, the mechanisms by which E5 achieves this are unclear. A yeast two-hybrid screen identified a novel Golgi protein, YIPF4, as a potential interactor of 16E5. YIPF4 is a member of the integral membrane protein family YIP1 that is thought to mediate intracellular trafficking. Quantitative polymerase chain reaction, Western blot and immuno-histochemistry analysis confirmed that YIPF4 is expressed in host cells of HPV infection in cell culture systems and in clinical samples of HPV16 induced cervical lesions. This implies that YIPF4 could be a relevant in vivo binding partner of E5. Upon the differentiation of HPV18 positive keratinoctyes in semisolid medium, the YIPF4 expression levels were stabilised compared to control cells suggesting that YIPF4 might play a role during the productive viral life-cycle. A differential, detergent permeabilisation assay provided the first experimental evidence for a three trans-membrane domain model of YIPF4. Co-immuno-precipitation revealed a conserved interaction of YIPF4 with E5 proteins from clinically important PVs indicating a potentially invaluable role of this complex for the virus. A flow cytometry approach unexpectedly revealed that neither E5 nor YIPF4 proteins modulate the trafficking of human leukocyte antigen class I molecules to facilitate viral immune evasion. A preliminary cellular interactome of YIPF4 was determined in a label free mass spectrometry experiment to facilitate the search for the function of the highly conserved E5/YIPF4 protein complex. This knowledge might contribute to elucidating new targets for the development of therapeutic agents against the broad spectrum of PV associated diseases.
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Nipper, Rick William Jr 1978. "Molecular function of the cell polarity protein partner of inscuteable in Drosophila neuroblasts." Thesis, University of Oregon, 2007. http://hdl.handle.net/1794/6194.
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xiii, 48 p. : (col. ill.) A print copy of this title is available through the UO Libraries under the call number: SCIENCE QL537.D76 N57 2007Asymmetric cell division (ACD) is a unique mechanism employed during development to achieve cellular diversity from a small number of progenitor cells. Cells undergoing ACD distribute factors for self-renewal at the apical cortex and factors for differentiation at the basal cortex. It is critical for proper development that the mitotic spindle be tightly coupled to this axis of polarization such that both sets of proteins are exclusively segregated into the daughter cells. We use ACD in Drosophila neuroblasts as a model system for understanding the molecular mechanisms that govern spindle-cortical coupling. Neuroblasts polarize Partner of Inscuteable (Pins), Gαi and Mushroom Body Defect (Mud) at the apical cell cortex during mitosis. Gαi and Pins are required for establishing cortical polarity while Mud is essential for spindle-cortical alignment. Gαi and Mud interact through Pins GoLoco domains and tetratricopeptide repeats (TPR) respectively, however it is unclear how Mud activity is integrated with Pins and Gαi to link neuroblast cortical polarity to the mitotic spindle. This dissertation describes how Pins interactions with Gαi and Mud regulate Iwo fundamental aspects of neuroblast ACD: cortical polarity and alignment of the spindle with the resulting polarity axis. I demonstrate that Pins is a dynamic scaffolding protein that undergoes a GoLoco-TPR intramolecular interaction, resulting in a conformation of Pins with low Mud and reduced Gαi binding affinity. However, Pins TPR domains fail to completely repress Gαi binding, as a single GoLoco is unaffected by the intramolecular isomerization. Gαi present at the apical cortex specifies Pins localization through binding this "unregulated" GoLoco. Liberation of Pins intramolecularly coupled state occurs through cooperative binding of Gαi and Mud to the other GoLoco and TPR domains, creating a high-affinity Gαi-Pins-Mud complex. This autoregulatory mechanism spatially confines the Pins-Mud interaction to the apical cortex and facilitates proper apical-spindle orientation. In conclusion, these results suggest Gαi induces multiple Pins states to both properly localize Pins and ensure tight coupling between apical polarity and mitotic spindle alignment.
Adviser: Ken Prehoda
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Davies, Joanna Marie. "The identification of novel POZ domain zinc finger proteins : characterisation of a heterodimeric partner of BCL-6." Thesis, Institute of Cancer Research (University Of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264995.
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Rushing, Stacy Renée. "Proteins that interact with and mediate the activity of the aryl hydrocarbon receptor and its partner ARNT /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Geiger, Michael [Verfasser]. "Mutational analysis of the P. falciparum ARO protein, functional analysis of its predicted binding partner AIP and identification of AIP interacting proteins / Michael Geiger." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1227582420/34.
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Hardwick, Steven. "Structural and functional characterisation of partner switching proteins involved in the environmental stress response of gram-positive bacteria." Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438402.
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Luciakova, Dominika. "Characterisation of novel cytochrome P450-fusion enzymes." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-novel-cytochrome-p450fusion-enzymes(08d9f0eb-666c-4f0f-b3ad-1fbf52555a0e).html.
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This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partners with functions distinct from electron transfer. Understanding why evolution drove the ‘partner’ proteins to evolve into a single unit is often unclear, but provides an important challenge for the understanding of the breadth of biochemical reactions mediated by P450s. The first P450-fusion analysed (Chapter 3) is CYP116B1 from a soil bacterium, Cupriavidus metallidurans, that displays important environmental implications. The enzyme was characterised as a functional fusion, composed of three domains: a P450 from the CYP116B family, and a phthalate dioxygenase reductase (PDOR)-like reductase binding FMN and a 2Fe-2S cluster. CYP116B1 is a stable, cytosolic enzyme but can undergo FMN cofactor loss. Studies included redox potentiometry of the intact fusion and its individual domains using spectro-electrochemical and EPR methods to enable the determination of midpoint redox potentials for individual cofactors. The CYP116B1 EPR signature was shown to be typical of P450s, and changed upon binding heme-coordinating inhibitors of the azole class. Extensive compound library screening did not reveal a substrate-like physiological “hit”. However, catalytic activity was detected towards selected thiocarbamate herbicides. GC-MS data revealed the enzymatic mechanism of herbicide degradation. The second system studied (Chapter 4) is P450-CAD, an atypical fusion of an uncharacterised soluble P450 and a cinnamyl alcohol dehydrogenase (ADH) module from Streptomyces ghanaensis; a member of the major antibiotic producing genus of bacteria. The CAD module appears unlikely to be a redox partner, but instead possibly mediates substrate/product exchange with the P450. The intact fusion was shown to aggregate during extraction. Genetic dissection of domains revealed that this was due to the highly insoluble ADH moiety. The heme domain (HD) was soluble and was characterised extensively. The enzyme displays an unusual spectrum when in the FeII-CO complex (Amax = 445 nm). The P450-CAD HD catalytic activity is supported by heterologous redox partners (E. coli flavodoxin reductase [FldR] and flavodoxin [FldA], and spinach ferredoxin reductase [FdR] and ferredoxin [Fdx]). The CAD-HD binds fatty acid substrates of carbon chain length C8-14, with the highest affinity for 12-methylmyristic acid (12M14C acid), the C12 lauric acid, its aldehyde and alcohol, indicating that the terminal methyl group is important for binding to the enzyme. Unusually, the CAD-HD also binds a range of detergent compounds. Further analysis included SEC-MALLS, thermostability and structural studies. The final enzyme studied (Chapter 5) is the P450-BDOR (a P450 linked to a benzoate dioxygenase reductase) redox-partner fusion. The unconventional trait of this enzyme is the inclusion of an FCD (a fatty acid metabolism regulator protein [FadR] C-terminal DNA-binding domain). From the point of view of P450s, DNA interaction would represent an unprecedented function, suggesting novel functions for a P450 enzyme. Thus, this enzyme requires extensive research with the expectations that new information will contribute to an expansion of knowledge of P450 diversity. This study provides initial analytical insights into the P450-BDOR system, supported with functional and kinetic data on the P450 and its reductase domain.
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Crane, Jennine Marie. "Characterization of two modes of interaction between the chaperone SecB and its binding partners." Free to MU Campus, others may purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3144410.
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Koneni, Rupa. "The Biological Function of Interacting Partners of ZXD Family Proteins." Cleveland State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=csu1250261050.
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El, Osmani Nour. "Identification de corps nucléaires TET1-positifs dans les cellules tumorales coliques : comment la vitamine C et les herbicides pourraient avoir un impact sur la déméthylation de l'ADN dans les tumeurs solides." Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONT008.
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Les protéines Ten – Eleven Translocation (TET1-3) sont des régulateurs clés de la voie active de déméthylation de l'ADN. Ces enzymes déclenchent une série de réactions d’oxydation qui jouent un rôle essentiel dans le contrôle de l’expression des gènes et, de fait, dans le contrôle de multiple mécanismes cellulaires y compris la prolifération et la différenciation cellulaire. Dans de nombreux cancers y compris dans certains cancers solides tels que le cancer colorectal (CRC), l’expression et l’activité des TETs sont fréquemment diminuées. Certains facteurs alimentaires, métaboliques et environnementaux ont été identifiés comme capables d’influencer l'activité et/ou l'expression des TETs. La VitC est bien reconnue pour son rôle stimulant de la déméthylation de l’ADN mais les mécanismes moléculaires impliqués ne sont pas clairement élucidés. D’autre part, le Linuron, un herbicide à base de phénylurée, est connu pour sa génotoxicité, mais ses effets sur les TETs n'ont pas été étudiés.L'objectif principal de cette thèse a été d’étudier la distribution subcellulaire des TETs et en particulier de TET1 dans les cellules coliques non tumorales et tumorales et d'évaluer l'impact de la VitC et du Linuron sur cette distribution subcellulaire. Une étude par immunofluorescence a mis en évidence une nouvelle compartimentation de TET1 et le marqueur de son activité de déméthylation, la 5hydroxyméthylcytosine (5hmC), dans des corps nucléaires des cellules de CRC. En étudiant l'interaction potentielle entre TET1/5hmC et les protéines formant des corps nucléaires dans les cellules CRC, nous avons observé leur colocalisation avec les corps de Cajal (CB) mais pas avec ceux marqués positivement pour la protéine de leucémie promyélocytaire (PML). De plus, les cellules CRC traitées à VitC ont révélé une augmentation du nombre et de la taille des corps nucléaires de 5hmC, PML et CBs. Fait intéressant, l'imagerie confocale a révélé que la VitC potentialisait l'interaction des corps de 5hmC avec les corps de PML et de Cajal. A l’inverse, l'analyse confocale de TET1 a révélé que l'exposition au Linuron induisait une diminution significative du nombre de corps nucléaires de TET1 ainsi qu’une augmentation de la prolifération et de l'invasivité des cellules tumorales coliques.Ce travail a permis d’identifier de nouveaux corps nucléaires concentrant TET1 et 5hmC dans les cellules tumorales coliques et de démontrer que le nombre, la taille et les propriétés dynamiques de ces corps nucléaires sont inversement modulées par la VitC et le Linuron. Ces résultats mettent en évidence de nouvelles fonctions biologiques pour TET1 et ouvrent de nouvelles perspectives de recherche en oncologie digestiveTen-eleven translocation proteins (TET1-3) are key regulators of the active DNA demethylation pathway via triggering a series of oxidation reactions. TET proteins play an essential role in controlling gene expression and in modulating multiple cellular mechanisms. These proteins are frequently downregulated in many cancers, including colorectal cancer (CRC).Moreover, some dietary and environmental factors have been identified as capable of influencing the activity and/or expression of TETs. Vitamin C (VitC), a therapeutic adjuvant, is well known to actively induce demethylation, but the molecular mechanisms involved are not clearly elucidated. On the other hand, Linuron, a phenyl-urea herbicide, is known for its genotoxicity, yet its effects on TETs have not been studied.The main objective of this thesis was to study the subcellular distribution of TETs, in particular of TET1, in non-tumoral and tumoral colonic cells and to evaluate the impact of VitC and Linuron on this subcellular distribution. Immunofluorescence assays, detected novel compartmentalization of TET1 and its demethylation mark, 5hydroxymethylcytosine (5hmC), characterized by their recruitment into coarse nuclear bodies (NBs) in the nucleoplasm of CRC cells. While studying the potential cross-talk between TET1/5hmC-NBs and nuclear-body forming proteins in CRC cells, we observed their colocalization with Cajal bodies (CBs) but not with those positively stained for the promyelocytic leukemia protein (PML). CRC cells treated with VitC revealed stimulation in the number and size of 5hmC-NBs, PML-NBs, and CBs. Interestingly, confocal imaging, revealed that VitC enhanced the interaction of 5hmC-NBs with both PML- and Cajal bodies. Conversely, confocal analysis of TET1 revealed that exposure to Linuron induced a significant decrease in the number of TET1-NBs in CRC associated with an increase in cellular proliferation and invasiveness.This work identified new nuclear bodies concentrating TET1 and 5hmC in CRC cells and demonstrated that the number, size, and dynamic properties of these nuclear bodies are inversely modulated by VitC and Linuron. These results highlight new biological functions for TET1 and open new research perspectives in digestive oncology
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Mikołajka, Aleksandra. "Functional and structural studies of the FGFR1 oncogene partner protein and biochemical investigations of the retinoblastoma protein and its binding partners." [S.l.] : [s.n.], 2007. http://mediatum2.ub.tum.de/doc/614708/document.pdf.
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Hamlet, Isla. "GATA1 protein partners in megakaryocytes." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442463.
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Cheng, Chi-wai, and 鄭智威. "Identification and characterization of PIN1 binding partners." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45199656.
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Glenn, Steve Ehren. "Investigations of HP1 and insulator partner protein 1 (HIPP1)." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6583.
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Drosophila HP1 and Insulator Partner Protein 1 (HIPP1) is the homologue of the human co-repressor Chromodomain Y family of proteins that repress neuronal gene expression in mammals. HIPP1 was identified by its extensive co-localization with Heterochromatin Protein 1a (HP1a) in heterochromatic regions of the genome and insulator binding proteins in euchromatic regions. The majority of HIPP1 binding to euchromatin is at binding sites for Drosophila Suppressor of Hairy-wing [Su(Hw)]. Su(Hw) is a zinc finger DNA binding protein that functions as an insulator, activator, and repressor. Transcriptional regulation by Su(Hw) is essential in the ovary and testis, where Su(Hw) functions primarily as a repressor of neuronal genes. However, the mechanism of Su(Hw) dependent repression is not clear. The focus of my thesis work has been defining the role of HIPP1 in development and its contribution to Su(Hw) function and heterochromatin formation. As part of this work, CRISPR was used to generate multiple Hipp1 null alleles and a tagged derivative of the endogenous gene (Hipp1GFP). Hipp1 null flies were found to be viable. Study of HIPP1 expression revealed it is present in most tissues and restricted to the nucleus. HIPP1 showed limited colocalization with HP1a, and tests of repression of transgenes in heterochromatin suggested that HIPP1 is not required for heterochromatin formation. Investigations of HIPP1 binding revealed that Su(Hw) is responsible for the majority of HIPP1 recruitment to euchromatin. Despite this, HIPP1 was found to be dispensable for the transcriptional and insulator functions of Su(Hw) as well as for female and male fertility. These data indicate that HIPP1 is not a critical Su(Hw) cofactor. Further studies are needed to clarify the role of HIPP1 in Drosophila development.
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de, Laurentiis Evelina Ines, and University of Lethbridge Faculty of Arts and Science. "Two partners of the ribosome, EF-Tu and LepA." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, c2009, 2009. http://hdl.handle.net/10133/2515.
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The translational GTPases elongation factor Tu (EF-Tu) and LepA modulate the dynamics of tRNA on the ribosome. EF-Tu facilitates the delivery of aminoacyl-tRNA (aa-tRNA) to the translating ribosome and LepA catalyzes the retro-translocation of tRNA•mRNA from the E- and P-sites of the ribosome back to the P- and A-sites. Although an increasing body of structural and biochemical information is available, little is known about the functional cycle of LepA during retro-translocation, the kinetics of EF-Tu dissociation from the ribosome and the rate of EF-Tu conformational change during aa-tRNA delivery. This thesis reports the successful construction and biochemical characterisation of a mutant form of EF-Tu from Escherichia coli ideal for the specific incorporation of fluorescent labels, enabling measurements pivotal for uncovering the rate of EF-Tu conformational change and dissociation from the ribosome. Furthermore, to determine structural components critical for LepA’s function, mutant versions of the protein were constructed and biochemically characterised.xii, 127 leaves : ill. (some col.) ; 29 cm
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Durgan, Joanne. "Protein Kinase C-binding partners & phosphorylation." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444645/.
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Protein Kinase C (PKC) comprises a family of phospholipid-dependent Ser/Thr kinases, implicated in a broad array of cellular processes. PKC activity is subject to a complex network of regulatory inputs, including co-factor binding, phosphorylation and protein-protein interaction. In addition, the chronic activation of PKC frequently leads to its down-regulation this process may be intrinsic to the tumour promoting activity of the phorbol esters. The aim of this work was to investigate the regulation of the novel PKC-e isoform by binding partners and phosphorylation, with a particular focus on the process of agonist-induced degradation. A yeast 2-hybrid screen was performed using a PKC-e bait and two novel binding partners were identified, both with associations to the ubiquitin/proteasome system VHL Binding Protein 1 (VBP1) and F-box WD40 protein 7 (Fbw7). Interactions were verified in mammalian cells and mapped to the catalytic domain of PKC-e. Extensive studies revealed that neither partner influenced the process of PKC-e down-regulation. However, Fbw7a was demonstrated to represent an in vitro PKC substrate. The site of phosphorylation was mapped to Ser-18 and, using phospho-specific antibodies, was shown to be phosphorylated in the cell. PKC-e activity is required for its agonist-induced degradation. Studies were therefore undertaken to investigate autophosphorylation, which may be implicated in this process. Serine residues 234, 316 and 368 were identified as novel PKC-e autophosphorylation sites. Using phospho- specific antibodies, all three sites were shown to be occupied in response to PKC-e activation. Phosphorylation at these sites was found not to influence agonist-induced PKC-e down-regulation. However, a critical role was established for phosphorylated Ser-368, in the recruitment of the PKC-e binding partner, 14-3-3(3. Together these findings provide insight into the mechanisms controlling PKC-e activity and demonstrate a relationship between regulation through phosphorylation and protein-protein interaction.
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Perrody, Elsa. "The viral protein Rki : an atypical cochaperone partner of Hsp70." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1870/.
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Les chaperons moléculaires de la famille Hsp70 sont des protéines ubiquitaires qui interviennent dans de nombreux processus cellulaires liés au repliement des protéines. Les fonctions d'Hsp70 sont indissociables de la présence des cochaperons JDP (J-domain protein). Ces protéines, grâce à leur J-domaine caractéristique, stimulent l'activité ATPasique de Hsp70, facilitent leur prise en charge des substrats et leur confèrent leur localisation cellulaire. L'analyse de la séquence du génome de l'entérobactriophage RB43, membre de la famille du bacteriophage T4, a révélé la présence d'un gène codant une JDP putative (l'orfan 057w). Dans ce travail, nous avons montré que le J-domaine de cette JDP, nommée Rki, est fonctionnel in vivo et que Rki interagit spécifiquement avec le chaperon multifonctionnel Hsp70/DnaK de l'hôte Escherichia coli. Cependant, à la différence des trois cochaperons JDP de DnaK présents chez E. Coli, la protéine Rki ne fonctionne pas comme un cochaperon de type général de DnaK in vivo et in vitro. Au contraire, l'expression de Rki est fortement toxique dans une souche sauvage d'E. Coli. De façon remarquable, cette toxicité est totalement abolie en l'absence de DnaK endogène ou quand le J-domaine de Rki est inactivé. D'autres expériences in vivo ont ensuite révélé que Rki est exprimée précocement durant l'infection par RB43 et que la délétion du gène rki diminue significativement la prolifération du bactériophage. De plus, nous avons trouvé que des mutations dans le gène dnaK de l'hôte suppriment efficacement le phénotype de retard de croissance du phage muté pour le gène rki, indiquant que Rki interfère spécifiquement avec les fonctions cellulaires de DnaK. Enfin, nous avons montré que l'interaction de Rki avec le chaperon DnaK de l'hôte stabilise rapidement le facteur s32 de réponse au choc thermique, qui est normalement adressé à la dégradation par DnaKThe highly conserved molecular chaperone Hsp70 is involved in a plethora of cellular processes associated with protein folding. To function as a molecular chaperone, Hsp70 requires the presence of its obligate J-domain cochaperone partners (JDP). These proteins, thanks to their J-domain signature, stimulate Hsp70's ATPase activity, facilitate substrate delivery and confer specific cellular localization to Hsp70. Genome analysis of the T4-like enterobacteriophage RB43, revealed a gene encoding a putative JDP (orfan 057w). In this work, we first show that the J-domain of this JDP, named Rki, is functional in vivo. Moreover, we show that Rki specifically interacts with the E. Coli host multifunctional Hsp70/DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. Coli, Rki full-length does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. Coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor s32, which is normally targeted for degradation by DnaK
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Hudson, Jaylen Braxton. "Identifying Endogenous Binding Partners of Btf and TRAP150." Wright State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=wright1591182283366217.
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Chung, Jo-Lan. "Identifying protein-protein binding sites and binding partners using sequence and structure information /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3244170.
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Gray, Amy Jetaun. "Novel Phosducin-Like Protein Binding Partners: Exploring Chaperone and Tumor Suppressor Protein Interactions." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3408.
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Many proteins cannot fold into their native state without the assistance of one or more molecular chaperones. Chaperonins are an essential class of chaperones that provide an isolated chamber for proteins to fold. CCT, a group II chaperonin found in eukaryotes assists in the folding of actins, tubulins, and many other cellular proteins. PhLP1 is a member of the phosducin protein family that assists CCT in the folding of Gβ and its subsequent assembly with Gγ. However, previous studies have not addressed the scope of PhLP1 and CCT-mediated Gβγ; assembly. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of all Gγ subunits that form dimers with Gβ2 and the assembly of Gγ2 with Gβ1-4, without affecting the specificity of the Gβγ interactions. These findings suggest that PhLP1 has a general role for the assembly of all Gβγ combinations. Although the role of PhLP1 as a co-chaperone for Gβγ assembly has been established, other possible functions for PhLP1 either as a co-chaperone or otherwise are yet to be investigated. A known tumor suppressor protein, PDCD5, was found to interact with PhLP1 in a co-immunoprecipitation proteomics screen. The data presented in Chapter 3 show that PDCD5 binds PhLP1 indirectly through a ternary complex with CCT. Our results signify that the apoptotic function of PDCD5 is cytosolic, is phosphorylation dependent, and most likely involves CCT. Moreover, structural analysis suggests that over-expressed PDCD5 blocks β-actin from entering the CCT folding cavity, suggesting a co-chaperone role for PDCD5 in inhibiting or enhancing folding of yet-to-be determined CCT substrates. Compared to PhLP1, the functions of other members of the phosducin family, PhLP2A, PhLP2B, and PhLP3, are poorly understood. They have no role in G-protein signaling, but appear to assist CCT in the folding of actin, tubulin and proteins involved in cell cycle progression. Chapter 4 investigates the possibility of PhLP2 and/or PhLP3 acting as co-chaperones in the folding and assembly of actins and tubulins. In addition, another mediator of cellular signaling, 14-3-3ε, was found to interact with PhLP2A in a phosphorylation dependent manner and relieve the inhibition of β-actin folding caused by PhLP2A over-expression.
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Collier, Miranda. "Small heat shock protein interactions with in vivo partners." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:24cf8041-c82d-4bc4-87a7-0ae7e38f1879.
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Small heat-shock proteins (sHsps) are part of a broad cellular sys- tem that functions to maintain a stable proteome under stress. They also perform a variety of regulatory roles at physiological conditions. Despite the multitude of sHsp targets, their interactions with partners are not well understood due to highly dynamical structures. In this thesis, I apply a variety of biophysical and structural approaches to examine distinct interactions made by the abundant human sHsps αβ-crystallin and Hsp27. First, I find that αβ-crystallin binds a cardiac-specific domain of the muscle sarcomere protein titin. A cardiomyopathy-causative variant of αβ-crystallin is shown to disrupt this interaction, with demonstrated implications for tissue biomechanics. Next, I investigate the conformation and unfolding behaviour of another sarcomere-associated protein, filamin C, finding support for the hypothesis that it is mechanosensitive. This leads into an interrogation of the interaction between filamin C and Hsp27, which we find is modulated by phosphorylation of Hsp27. This modulation only manifests during filamin C unfolding, pointing toward a protective chaperoning mode against over-extension during mechanical stress. This finding is bolstered by up-regulation and interaction of both proteins in a mouse model of heart failure. I establish a system for similar studies of a third sHsp, cvHsp, which is muscle-specific and implicated in various myopathies but scantly understood at the molecular level compared to αβ-crystallin and Hsp27. Finally, I probe the stoichiometries and kinetics of complexes formed between αβ-crystallin and Hsp27 themselves, which co-assemble into a highly polydisperse ensemble. This involved the development of a high-resolution native mass spectrometry method for disentangling heterogeneous systems. Together these findings add to our understanding of the roles and mechanisms of ATP-independent molecular chaperones.
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Chiu, Peng-hang Raymond, and 趙炳铿. "Identification of protein-interacting partners of testis-specific protein y-encoded like 2 (TSPYL2)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41508725.
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Chiu, Peng-hang Raymond. "Identification of protein-interacting partners of testis-specific protein y-encoded like 2 (TSPYL2)." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508725.
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Nicewonger, John David. "Potential interaction of cysteine rich intestinal protein with a protein partner and their regulation in immune cells." [Gainesville, Fla.] : University of Florida, 2003. http://purl.fcla.edu/fcla/etd/UFE0000757.
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Ho, Joseph Tsung-yo. "Bridging cell growth and proliferation : identification and characterization of binding partners for pescadillo, a novel nucleolar protein involved in tumorigenesis and DNA damage /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/10659.
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Dawson, Linda Fiona. "FLJ22318 : a novel binding partner of the NKX3-1 homeodomain protein in prostate cancer cells /." Access via Murdoch University Digital Theses Project, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070622.92421.
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Hall, Anita. "The protein tyrosine phosphatase-PEST interactome with novel interaction partners." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96685.
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Cellular regulation and signalling can occur at numerous levels and post-translational modification by phosphorylation is a method of regulating these pathways. This reversible phenomenon requires a strict balance between kinases and phosphatases at various steps along a signalling pathway. One phosphatase that has gained increasing interest in cytoskeleton regulation and cellular signalling is the protein tyrosine phosphatase PTP-PEST. Much of these investigations have uncovered numerous binding partners and key interactors of PTP-PEST that were found to be regulated in part by their phosphorylation status. Our goal was to examine and expand the role of PTP-PEST in applying the technique of affinity-purification coupled with mass spectrometry (AP-MS) and constructing an interactive network. Our previous work involved identifying putative substrates in utilizing a modified yeast two-hybrid method with PTP-PEST. Several of which were determined to be true interactors by this method, including p130Cas, paxillin, and a novel substrate termed Skap-Hom. We applied the AP-MS technique to screen for novel PTP-PEST interactions. Herein, we've identified two new interactors of PTP-PEST, Arhgap26 and PPM1A. Both may be involved in regulating migration or cytoskeletal changes which may correlate with PTP-PEST function. AP-MS is proving itself to be a useful method and other potential targets will be further confirmed. In addition, we applied a bioinformatics approach using the Cytoscape visualization software to generate a first PTP protein-protein interaction map; the PTP-PEST interactome. An understanding of the substrates that are regulated by PTP-PEST and their associated roles in the cell allows us to elucidate mechanisms and develop potential therapeutic targets for various malignancies, especially that of cancer.La signalisation cellulaire et sa régulation prennent place à plusieurs niveaux dans la cellule. Ici nous avons étudié le mécanisme des modifications post-transcriptionnelles par phosphorylation. Ce phénomène réversible nécessite un équilibre entre les kinases et les phosphatases à plusieurs niveaux d'une voie de signalisation. PTP-PEST est une phosphatase qui suscite de plus en plus d'intérêt dans la régulation du cytosquelette et la signalisation cellulaire. Plusieurs études ont identifié de nombreuses protéines d'interactions et de liaisons de PTP-PEST qui sont régulées par leur statut de phosphorylation. Notre but est d'examiner et d'élargir nos connaissances sur le rôle de PTP-PEST en utilisant des techniques de purification par affinité ainsi que la spectrophotométrie de masse (PA-SM) afin de construire un réseau d'interaction des protéines.Nous avons précédemment identifié des substrats putatifs de PTP-PEST avec la méthode modifiée de double-hybride dans la levure. Plusieurs substrats identifiés sont des protéines de liaison de PTP-PEST, incluant p130Cas, paxillin et un nouveau substrat appelé Skap-Hom. Grâce à la méthode PA-SM, pour le criblage de nouvelles protéines d'interactions, nous avons identifié 2 nouveaux partenaires de liaison de PTP-PEST : Arhgap26 et PPM1A. Ces deux protéines pourraient être impliquées dans la régulation de la migration ou dans les changements cytosqueletiques avec PTP-PEST. La méthode PA-SM a prouvé son utilité et d'autres cibles de PEST seront analysées de cette façon. Avec le logiciel bioinformatique Cytoscape visualization, nous avons généré la première carte d'interactions protéine-protéine : l'intéractome de PTP-PEST. Comme les protéines d'intéractions de PEST identifiées ici sont impliquées dans la migration cellulaire, il y aura une possibilité d'une corrélation avec le cancer. Ceci nous permettrons de l'expansion sur le développement de cibles thérapeutiques potentielles pour le cancer.
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Czub, B. "Identification of interacting protein partners of TOPORS in the retina." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1469146/.
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Retinitis pigmentosa (RP, MIM#268000) is a heterogeneous disease characterised by loss of rod photoreceptors and pigment deposits in the retina. Historically, genes linked to RP were associated with rod-specific functions. Recently, a novel class of ubiquitously expressed causative genes has emerged including splicing factor genes and TOPORS (NM_005802). To date, studies show TOPORS is expressed in all tested human tissues, including the retina. However, mutations in this ubiquitously expressed gene only cause RP without any systemic symptoms. The purpose of this work was to understand why mutations in TOPORS, which encodes a multifunctional protein, cause a retina-only disease by identifying protein interacting partner(s) of TOPORS, using a yeast-two hybrid (Y2H) screen. In case the interacting partner(s) turn out to be retina specific, it may explain the retina-only phenotype. Human retinal cDNA library was constructed from total retinal cDNA directly in the Y187 Saccharomyces cerevisiae yeast strain. Retina-specificity of the cDNA library was validated by sequencing, leading to identification of several retina-specific genes, including rhodopsin (RHO; NM_000539). The library was screened for protein interacting partners of TOPORS, using MatchmakerTM Gold Yeast Two-Hybrid System (Clontech, CA, USA). Over 10^7 cDNA clones were screened, leading to isolation of 53 potential interactions. The identified interacting partners were prioritised for further evaluation, based on literature and database searches, and re-tested in yeast leading to identification of three candidates for further functional studies: a soluble fragment of integral membrane protein 2B (ITM2B; NM_0219999), previously linked to neurodegenerative disorders, and more recently associated with an inherited retinal dystrophy; a brain prostaglandin D2 synthase (PTGDS; NM_000954), highly expressed in the retina, previously suggested to play a role in retinal homeostasis; a regulatory subunit 4 of the 26 S protease (PSMC1; NM_002802), conferring substrate specificity to the proteasome complex during degradation of ubiquitinated proteins. The outcomes suggest several scenarios for why mutations in TOPORS result only in RP; however, further studies are essential to elucidate the role of TOPORS and its interacting partners in the aetiology of this debilitating disease.
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Nipper, Rick William. "Molecular function of the cell polarity protein Partner of Inscuteable in Drosophila neuroblasts /." Connect to title online (Scholars' Bank) Connect to title online (ProQuest), 2007. http://hdl.handle.net/1794/6194.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 45-48). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.
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Williams, Sandile Elroy Johnathan. "Identification of a BK channel protein-partner and its involvement in Oâ‚‚ sensing." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411134.
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Lee, C. "Redox binding partners of the hBCAT proteins and their distribution and expression in control and disease human brains : implications in protein folding in neurodegeneration." Thesis, University of the West of England, Bristol, 2017. http://eprints.uwe.ac.uk/30019/.
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INTRODUCTION: Novel binding partners of the branched chain aminotransferase proteins (hBCAT) proteins have been identified and indicate that these metabolic proteins may have alternative functions to their housekeeping role. In particular, the molecular chaperone, protein disulphide isomerase (PDI) was shown to co-localise with mitochondrial hBCAT, where a role for hBCAT in protein folding was demonstrated. Moreover, the cytosolic isoform, hBCATc, showed redox-specific interactions with key regulators of cytoskeletal metabolism, implicating hBCATc in protein trafficking. These associations were influenced by changes in the redox environment, where specific interactions were lost on oxidation. Knowledge of these and other redox-dependent and independent associations are important, in particular as hBCAT are significantly increased in the brains of Alzheimer’s disease. If these cellular associations are dysregulated they could contribute to the pathological changes observed in Alzheimer’s disease brain. We hypothesise that hBCAT is a multifunctional enzyme and its direction of metabolism is directed by its response to changes in the redox environment, dictated through modification of its CXXC redox switch. Moreover, identification of these proteins in neurodegenerative conditions may highlight new markers of disease association. AIMS: The overall aim of this thesis is to identify new binding partners for the hBCAT proteins (both redox-dependent and –independent), which will offer insight into their multifunctional role within the cell. Within this aim, novel indicators of pathology in neurodegenerative conditions, associated with hBCAT function will be identified. These will be examined for their potential role as biomarkers in disease conditions. METHODS: Using nano LC-MS/MS, specific binding partners were identified following affinity hBCAT-tagged chromatography. Under specific redox conditions, proteins extracted from the neuronal cell lines: IMR32 and SH- SY-5Y, and endothelial cells, hCMEC/D3 were shown to differentially bind to affinity columns tagged with either wild-type hBCAT or mutant hBCAT. Key proteins identified in these studies were further mapped in human brain, using immunohistochemistry, to understand their association with hBCAT. Finally, to evaluate their expression in Alzheimer’s disease and Parkinson’s disease, we used immunohistochemistry and Western blot analysis. RESULTS AND DISCUSSION: Here, we have shown for the first time that the hBCAT proteins differentially bind to specific proteins involved in protein folding, cytoskeletal regulation, alternative splicing and metabolic energy metabolism. These interactions were both redox-dependent and redox-independent, indicating that this protein can function in the cell even if oxidised to its inactive metabolic form. Chronic oxidation, as suggested to feature in neurodegenerative conditions, is thought to contribute to protein misfolding,cytoskeletal dysfunction in addition to disrupting other processes such as mitochondrial function, which all contribute to cellular death. Here, oxidation prevented hBCAT interacting with cytoskeleton and key metabolic proteins but did not affect interactions with chaperones or the proteasome, and even evoked new interactions with the enzymes of ubiquitinylation. These and other associations offer exciting new insights into the role of hBCAT in cells. Several of these chaperones that were involved with protein folding, including, the chaperones BiP, BAG1 and TCP1, were mapped to the human brain for the first time. Distribution was ubiquitous throughout the brain in all cell types except for BAG1, which was not present within granular cells of the hippocampus and the cerebellum. Co-expression with hBCATc was evident in several neuronal cell types of the temporal lobe, frontal lobe and the cerebellum, and hBCATm showed co-expression in endothelial cells and some neuronal cells of the same brain regions. Confirmation that these proteins are localised to the same brain cells and regions indicates that these interactions that were identified through these in vitro associations may have relevance in the human brain. To evaluate if these expression patterns are modified in neurodegenerative conditions, expression of hBCAT was examined in the hippocampus of Alzheimer's disease and expression of hBCAT, BiP and TCP1 was examined in the midbrain of Parkinson's disease. Expression of hBCAT was elevated in Alzheimer's disease however, expression of hBCAT, BiP and TCP1 were all decreased in Parkinson's disease and therefore hBCAT may represent a potential biomarker with good differentiation between diseases. Overall, these studies indicate that proteins involved in protein folding/chaperone activity are modified differentially in certain brain areas of Alzheimer's disease and Parkinson's disease. CONCLUSION: Overall, data generated in this thesis supports the role of hBCAT as a multi-functional cellular protein. Future studies of their functional role with these partners will offer exciting insight into these moonlighting roles. Moreover, their co-localisation in the human brain and differential expression may point to new areas of biomarker development and therapeutic targeting.
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Langston, Kelsey Murphey. "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/3822.
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Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.
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Yip, Kit-yan. "Identification of the protein interacting partners of testis specific protein, Y-encoded like-2 (TSPYL2)." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557844.
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Yip, Kit-yan, and 葉潔茵. "Identification of the protein interacting partners of testis specific protein, Y-encoded like-2 (TSPYL2)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557844.
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au, L. Dawson@murdoch edu, and Linda Dawson. "FLJ22318: A Novel Binding Partner of the NKX3-1 Homeodomain Protein in Prostate Cancer Cells." Murdoch University, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070622.92421.
Full textAbstract:
Prostate cancer is a frequently diagnosed malignancy which ranges from an indolent asymptomatic tumour to an aggressive, rapidly lethal systemic disease. Determination of chromosomal, genetic and epigenetic alterations associated with prostate carcinogenesis have led to the characterisation of functional consequences of these alterations, thereby elucidating pathways contributing to malignant growth that can be utilised clinically and therapeutically.
FLJ22318 is a novel hypothetical protein that was identified by yeast two-hybrid analysis to interact with the prostatic homeodomain protein, NKX3-1. Expression of NKX3-1 is largely restricted to epithelial cells of the adult prostate where it is involved in maintaining the prostatic phenotype, while NKX3-1 expression is reduced or absent in prostate tumours. In contrast, FLJ22318 expression is documented in cDNA libraries derived from a variety of human adult and foetal tissues including the prostate, suggesting that it may be ubiquitously expressed and that it potentially interacts with a number of proteins in addition to NKX3-1. FLJ22318 expression is undocumented in human prostate tumours.
Bioinformatic analyses have postulated multiple FLJ22318 mRNA transcripts however the proposed open reading frames encoded by these transcripts predict the FLJ22318 protein to contain three strong protein-protein interaction domains, a Lissencephaly type-1-like homology (LisH), a C-terminal to LisH (CTLH) and a CT11-RanBPM (CRA) domain. Of the 44 single nucleotide polymorphisms identified within the FLJ22318 gene, none are located within the protein coding region suggesting that FLJ22318 may be critical for cell survival and/or function. Comparison of the amino acid sequence between human FLJ22318 and its orthologues in a diverse range of mammalian species identified >97% sequence homology, providing further strong evidence of the critical cellular function of FLJ22318.
To characterise the biological activity of FLJ22318 in prostate cancer cells, the FLJ22318 coding region was amplified by polymerase chain reaction (PCR) and ligated into mammalian and bacterial expression vectors to encode V5-, myc-, GFP-, HA-, and GST-FLJ22318 fusion proteins. Interaction between FLJ22318 and NKX3-1 was confirmed using (reverse) yeast two-hybrid, GST pull-down and co-immunoprecipitation assays. These data were supported by confocal microscopy which demonstrated the perinuclear and nuclear co-localisation of FLJ22318 and NKX3-1 in prostate cancer cells. Northern blotting identified expression of ~2Kb and ~4Kb FLJ22318 mRNAs in prostate cancer cell lines, which was consistent with bioinformatic analyses of mRNA species. Transfection of prostate cancer cells to overexpress FLJ22318 did not alter endogenous NKX3-1 levels, however FLJ22318 exhibited transcriptional repressor function on an NKX3-1 responsive element and increased NKX3-1 transcriptional repressor activity on this element.
To further investigate FLJ22318 function, additional yeast two-hybrid analyses were performed in prostate cancer cells to identify potential FLJ22318 binding proteins. These studies isolated cDNAs encoding 33 different proteins involved in cell metabolism and apoptosis as well as transcriptional regulators associated with control of cellular proliferation. One of the candidate FLJ22318 interactors, protein kinase, interferon-inducible double stranded RNA dependent activator (PRKRA/PACT) was shown using confocal microscopy to extensively co-localise with FLJ22318 in the cytoplasm and perinuclear regions of prostate cancer cells. Preliminary co-immunoprecipitation and GST pull-down assays have provided additional evidence of the interaction of PRKRA and FLJ22318.
Results of this thesis have generated important information characterising the structure of the FLJ22318 gene and protein, the interaction between FLJ22318 and NKX3-1 and the potential functions of FLJ22318 in prostate cancer cells. As the FLJ22318 gene is located on 5q35, a chromosomal region frequently disrupted in a variety of tumours, future studies of the biological activity of FLJ22318 will clarify its normal cellular functions and its contribution to tumorigenesis or malignant progression in the prostate and in other tissues.
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Müller, Judith. "Protein-Protein-Interaktionen zwischen Homöodomänenproteinen der Gerste Identifizierung und Charakterisierung interagierender Partner von BKN3, dem Genprodukt des Hooded-Lokus /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960354999.
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Jolly, Carrie E. "Localization of a Microsporidia ADAM (A Disintegrin and Metalloprotease Domain) Protein and Identification of Potential Binding Partners." Digital Commons @ East Tennessee State University, 2007. https://dc.etsu.edu/etd/2052.
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Microsporidia are spore-forming, obligate intracellular pathogens typically associated with opportunistic infections in immunocompromised individuals. Treatment options for microsporidia infections in humans are limited and additional research is necessary to create better therapeutic agents. For many pathogenic organisms, adhesion to the host cell surface is a prerequisite for tissue colonization and invasion. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. In an effort to better understand adherence, we have turned our attention to determining what proteins may be involved in this process. Examination of the Encephalitozoon cuniculi genome database revealed a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. The microsporidia ADAM (MADAM) protein is of interest because ADAMs are known to be involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. The objectives for this study were to examine the localization of MADAM, analyze its potential involvement during adherence and/or host cell infection, and to identify potential binding partners or substrates. Through the use of immunoelectron transmission microscopy, we demonstrated that MADAM is localized to the surface exposed exospore, plasma membrane, and the polar sac-anchoring disk complex (a bell-shaped structure at the spore apex involved in the infection process). Location of MADAM within the exospore and polar sac-anchoring disk suggests that MADAM is in a position to facilitate spore adherence or host cell infection. Thus far, we have been unable to conclusively demonstrate that MADAM is involved in either event. Through the use of a yeast two-hybrid system, we were able to identify polar tube protein 3 (PTP3) as a potential binding partner or substrate for the MADAM protein. The interaction between MADAM and PTP3 was confirmed by in vitro co-immunoprecipitation. PTP3 is hypothesized to be involved in the process of polar tube extrusion by stabilizing the interaction between PTP1-PTP2 polymers. Further analysis of the interaction between MADAM and PTP3 may lead to a better understanding of the events that occur during polar tube extrusion.
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Quan, Xueping. "The molecular recognition and partner prediction for transient protein complexes : CDK-cyclin homologue interactions." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/15661.
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This thesis describes a novel computational strategy that combines multiple bioinformatic program components to predict specific transient protein-protein interactions. This protocol was developed focusing especially on the transient interactions between cyclin-dependent kinases (CDK) and cyclins. We adopted a comparative modelling strategy to build 3-D models of cyclins and CDKs using known human CDK and cyclin structures as templates. These modelled structures were then subjected to a large scale docking experiment with the program ZDOCK in which all cyclin-CDK combinations were considered. In the following steps of the procedure, additional selection criteria were applied to select the most compelling complexes from the ZDOCK result list. The two principal selection criteria used were the relative CDK-cyclin subunit orientation in the complex, and interface surface property correlation. Calibration of interface surface property correlation coefficients as computed by the program MOLSURFER was based on a positive reference dataset consisting of 104 true, non-homologous, transient heterodimeric protein-protein complexes, and a negative reference dataset consisting of 70 false protein-protein complexes. Prediction accuracies achieved using this approach are expected to be around 80% based on cross-validation of the interface selection criteria. The entire modelling and prediction approach has been applied to the well-characterised set of human CDKs and cyclins. Of the resulting positive predictions, 80% were in agreement with complex formation according to HPRD and Swiss-Prot annotation. Finally, when the approach was applied to 33 CDK homologues and 35 cyclin-homologues in Arabidopsis thaliana it yielded 19 mostly likely interacting CDK-cyclin pairs. The prediction strategy developed and applied in this work should be transferable to other transient heterodimer protein-protein interactions.