We are using the yeast two-hybrid (Y2H) system to identify novel protein-protein interactions (PPI) relevant to wood formation. Bait proteins for Y2H binary assays and screening against a xylem cDNA prey library were selected from approximately 400 Populus trichocarpa genes that are at least 8-fold more highly expressed in differentiating secondary xylem versus phloem-cambium, and designated here as poplar biomass (PB) genes. Here we report some of the interactions involving selected PB proteins and efforts to characterize their functions in Populus and Arabidopsis. Members of the ROP GTPase family, PB15 in poplar and ROP11 in Arabidopsis, interact with the domain of unknown function (DUF) 620 (DUF620) proteins (e.g., PB129 in poplar). Ectopic co-expression of PB15 and PB129 in Arabidopsis caused outgrowths at the base of flower pedicels and altered leaf morphology. Interestingly, the co-expression phenotype could not be observed in transgenic plants that are only expressing either one of the interacting partners separately. Transgenics altered in expression of PB15 and/or PB129 were prepared in Populus and characterization of transgenic trees will be performed in greenhouse and field. In addition to DUF620 family proteins, ROP11 also interacts with the COP9 subunit CSN5A in Arabidopsis. We confirmed the interaction of ROP11 and CSN5A in Y2H and employed available mutants for ROP11 and CSN5A in Arabidopsis to genetically characterize this interaction. Surprisingly, loss of ROP11 was found to rescue the csn5a-2 pleiotropic phenotype. Ectopic expression of a ROP11 dominant negative mutant in the csn5a-2 background also complemented the stunted growth phenotype. Transcript analysis and gel blot assays showed that CSN5A transcript levels remained unchanged in all rescue lines, whereas CSN5A protein levels increased relative to WT. Taken together, we concluded that ROP11 negatively regulate CSN5A protein level in plant by some as yet unknown mechanism. / Ph. D.

The project goals for this dissertation were to manipulate Populus trichocarpa source-sink relationships to optimize this woody crop species for specific agricultural traits such as increased growth rate, stress tolerance and/or improvements in overall biomass accumulation. We targeted specific tissues such as xylem, where alterations in the relationship of source and sink tissues can lead to the control of xylem cell deposition or of various wood properties. This led to the characterization of 165 protein-protein interactions and 20 protein-DNA interaction which constitute numerous woody tissue related subnetworks. One such network, centered on the DIVARACATA and RADIALIS INTERACTING FACTOR (PtrDRIF), identified PtrWOX13c as an interacting protein. Characterization of PtrWOX13c shows that it displays the ability to control promoters related to lignin biosynthesis genes and overexpression phenotypes show alterations in axillary branch activity. Genes which control the differentiation and specialization of cells such as members of the WOX family are also highly responsive to abiotic stress which can force major changes in plant metabolism and nutrient mobilization. ABA, a prominent plant phytohormone with known roles in the adaptation to stress has shown novel connections in the regulation of growth promoting complexes such as TOR through antagonistic regulatory actions of the SnRK2 protein kinase in Arabidopsis. Characterization of the core ABA signaling in P. trichocarpa has identified a regulatory clade A protein phosphatase which interacts with numerous PtrSnRK2 proteins and when overexpressed in hybrid poplar results in increased height and node production potentially by indirect control of growth promoting complexes like TOR through SnRK2 inhibition. This work has also demonstrated that in addition to the involvement of phytohormones in the regulation of plant development, sugar phosphates such as T6P can exert significant control of plant architecture. Together, these studies comprise the discovery and subsequent characterization of novel wood associated networks, hormone pathways and sugar signaling in the manipulation of P. trichocarpa source-sink relationships for the promotion of biomass accumulation. / PHD / Detailed analyses of gene activity in different tissues or under the influence of various environmental conditions have identified numerous genes that control desirable traits and plant characteristics. However, the activities and functions of the proteins produced from these genes is less understood. One of the ways proteins work is through the formation of complexes with other proteins. Using the commercially valuable tree Populus trichocarpa (poplar) as our research model, we have identified novel complexes of interacting proteins with the potential to sense and respond to the environment and to promote plant growth. We tested the function of some of the members of these newly discovered protein complexes using transgenic poplar. As a result, we revealed previously unknown functions for two poplar proteins: PtrWOX13c promoted increased branching and PtrHAB2 promoted an increase in tree height. Independent of these functional analyses of poplar proteins, we also tested the ability of a sugar phosphate, trehalose6-phosphate, known from previous work to regulate plant growth, for its ability to promote poplar growth. We found that reducing levels of trehalose-6-phosphate resulted in increased branch growth, similar to the impact of the PtrWOX13c protein. In summary, identification of new protein complexes is a valuable strategy for the discovery of proteins that can increase tree growth. Additionally, combining targeted changes in both proteins and regulatory sugars may be a promising path toward future crop improvement and tree domestication.

No description available.

Die dynamische Regulation von Protein-Protein Interaktionen (PPIs) ist wichtig für den Ablauf von biologischen Prozessen. Die circadiane Uhr, die einen ~24 Stunden Rhythmus generiert und eine Vielzahl von physiologischen Parametern steuert kann auch die Dynamik von PPIs regulieren. Um neue Erkenntnisse über regulatorische Mechanismen innerhalb des molekularen Oszillators zu gewinnen, habe ich zunächst alle möglichen PPIs zwischen 46 circadianen Komponenten mittels eines systematischen yeast-two-hybid (Y2H) Screens bestimmt. Dabei habe ich 109 bis dahin noch unbekannte PPIs identifiziert und einen repräsentativen Anteil mittels Co-Immunopräzipitationsexperimenten in humanen Zellen validiert. Unter den neuen PPIs habe ich bis dahin unbekannte Modulatoren der CLOCK/BMAL1 Transaktivierung identifiziert und dabei die Rolle der Proteinphosphatase 1 (PP1) als dynamischen Regulator der BMAL1 Stabilität funktionell charakterisiert. Das experimentelle PPI Netzwerk wurde mit bereits aus der Literatur bekannten PPIs und Interaktionspartnern ergänzt. Eine systematische RNAi Studie belegte außerdem die Relevanz der aus der Literatur stammenden Interaktoren für die ~24 Stunden Periodizität. Um eine Aussage über die Dynamik der PPIs im Netzwerk treffen zu können, wurden circadiane mRNA Expressionsdaten in das PPI Netzwerk integriert. Systematische Perturbationsstudien, in denen alle Komponenten des experimentellen Netzwerkes mittels RNAi herunterreguliert oder überexprimiert wurden, zeigten eine essentielle Bedeutung für die dynamischen PPIs innerhalb des circadianen Oszillators auf. Desweiteren wurden im circadianen PPI Netzwerk funktionelle Module identifiziert, welche dynamisch organsiert sind. Durch eine systemweite Analyse des humanen Proteoms wurden viele dynamische PPIs identifiziert, die biologische Prozesse wie z.B. Signaltransduktion und Zellzyklus miteinander verbinden. Rhythmische PPIs sind daher von Bedeutung für die zeitliche Organisation zellulärer Physiologie. / Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24 hrs) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes PPI dynamics on this timescale are largely unknown. To elucidate so far unknown regulatory mechanisms within the circadian clockwork, I have systematically mapped PPIs among 46 circadian components using high-throughput yeast-two-hybrid (Y2H) interaction experiments. I have identified 109 so far uncharacterized interactions and successfully validated a sub-fraction via co-immunoprecipitation experiments in human cells. Among the novel PPIs, I have identified modulators of CLOCK/BMAL1 function and further characterized the role of protein phosphatase 1 (PP1) in the dynamic regulation of BMAL1 abundance. Furthermore, to generate a more comprehensive circadian PPI network, the experimental network was enriched and extended with additional interactions and interaction partners from literature, some of which turned out to be essential for normal circadian dynamics. The integration of circadian mRNA expression profiles allowed us to determine the interaction dynamics within our network. Systematic genetic perturbation studies (RNAi and overexpression in oscillating human cells) revealed a crucial role of dynamic regulation (via rhythmic PPIs) for the molecular clockwork. Furthermore, dynamic modular organization as a pervasive circadian network feature likely contributes to time-of-day dependent control of many cellular processes. Global analysis of the proteome regarding circadian regulation of biological processes via rhythmic PPIs revealed time-of-day dependent organization of the human interactome. Circadian PPIs dynamically connect many important cellular processes like signal transduction and cell cycle, which contribute to temporal organization of cellular physiology.

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2009. / The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortex. Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders.

Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína &#945-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a &#945-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 &#181M) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da &#945-sinucleína por que a interação aconteceria com maior afinidade quando a &#945-sinucleína está na presença de SDS. / Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and &#945-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with &#945-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 &#181M) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the &#945-synuclein conformation, because the affinity of SEPT4 and &#945-synuclein seemed to be higher in the presence of SDS.

Src homology 3 (SH3) domains are one family of the peptide recognition modules (PRMs), which bind peptides rich in proline or positively charged residues in the target proteins, and play important assembly or regulatory functions in dynamic eukaryotic cellular processes, especially in signal transduction and endocytosis. SH3 domains are conserved from yeast to human, and improper SH3 domain mediated protein-protein interaction (PPI) leads to defects in cellular function and may even result in disease states. Since commonly used large-scale PPI mapping strategies employed full-length proteins or random protein fragments as screening probes and did not identify the particular PPIs mediated by the SH3 domains, I employed a combined experimental and computational strategy to address this problem. I used yeast two-hybrid (Y2H) as my major experimental tool, as well as individual SH3 domains as baits, to map SH3 domain mediated PPI networks, “SH3 domain interactomes”. One of my important contributions has been the improvement for Y2H technology. First, I generated a pair of Y2H host strains that improved the efficiency of high-throughput Y2H screening and validated their usage. These strains were employed in my own research and also were adopted by other researchers in their large-scale PPI network mapping projects. Second, in collaboration with Nicolas Thierry-Mieg, I developed a novel smart-pooling method, Shifted Transversal Design (STD) pooling, and validated its application in large-scale Y2H. STD pooling was proven to be superior among currently available methods for obtaining large-scale PPI maps with higher coverage, high sensitivity and high specificity. I mapped the SH3 domain interactomes for both budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans, which contain 27 and 84 SH3 domains, respectively. Comparison of these two SH3 interactomes revealed that the role of the SH3 domain is conserved at a functional but not a structural level, playing a major role in the assembly of an endocytosis network from yeast to worm. Moreover, the worm SH3 domains are additionally involved in metazoan-specific functions such as neurogenesis and vulval development. These results provide valuable insights for our understanding of two important evolutionary processes from single cellular eukaryotes to animals: the functional expansion of the SH3 domains into new cellular modules, as well as the conservation and evolution of some cellular modules at the molecular level, particularly the endocytosis module.

Src homology 3 (SH3) domains are one family of the peptide recognition modules (PRMs), which bind peptides rich in proline or positively charged residues in the target proteins, and play important assembly or regulatory functions in dynamic eukaryotic cellular processes, especially in signal transduction and endocytosis. SH3 domains are conserved from yeast to human, and improper SH3 domain mediated protein-protein interaction (PPI) leads to defects in cellular function and may even result in disease states. Since commonly used large-scale PPI mapping strategies employed full-length proteins or random protein fragments as screening probes and did not identify the particular PPIs mediated by the SH3 domains, I employed a combined experimental and computational strategy to address this problem. I used yeast two-hybrid (Y2H) as my major experimental tool, as well as individual SH3 domains as baits, to map SH3 domain mediated PPI networks, “SH3 domain interactomes”. One of my important contributions has been the improvement for Y2H technology. First, I generated a pair of Y2H host strains that improved the efficiency of high-throughput Y2H screening and validated their usage. These strains were employed in my own research and also were adopted by other researchers in their large-scale PPI network mapping projects. Second, in collaboration with Nicolas Thierry-Mieg, I developed a novel smart-pooling method, Shifted Transversal Design (STD) pooling, and validated its application in large-scale Y2H. STD pooling was proven to be superior among currently available methods for obtaining large-scale PPI maps with higher coverage, high sensitivity and high specificity. I mapped the SH3 domain interactomes for both budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans, which contain 27 and 84 SH3 domains, respectively. Comparison of these two SH3 interactomes revealed that the role of the SH3 domain is conserved at a functional but not a structural level, playing a major role in the assembly of an endocytosis network from yeast to worm. Moreover, the worm SH3 domains are additionally involved in metazoan-specific functions such as neurogenesis and vulval development. These results provide valuable insights for our understanding of two important evolutionary processes from single cellular eukaryotes to animals: the functional expansion of the SH3 domains into new cellular modules, as well as the conservation and evolution of some cellular modules at the molecular level, particularly the endocytosis module.

Η μυο-ειδική πρωτεΐνη δεσμίνη, αποτελεί μέλος των πρωτεϊνών του κυτταροσκελετού των ενδιαμέσων ινιδίων και εκφράζεται στους λείους και τους γραμμωτούς μυς. Στους συσταλτούς μύες, το πλέγμα του κυτταροσκελετού της Δεσμίνης περιβάλλει τους Ζ-δίσκους διασυνδέοντάς τους, ενώ παράλληλα συνδέει μεταξύ τους τις συσταλτές περιοχές της μυικής ίνας με την σαρκοπλασματική μεμβράνη, με διάφορα οργανίδια και με τον πυρήνα. Για να προσδιορίσουμε τους ακριβείς μηχανισμούς δράσης της δεσμίνης χρησιμοποιήσαμε το σύστημα υβριδισμού των ζυμών – yeast two hybrid screen system – προκειμένου να ανιχνεύσουμε πρωτεΐνες που αλληλεπιδρούν με τη δεσμίνη. Χρησιμοποιήσαμε ως «δόλωμα» αλληλουχίες των άκρων του μορίου της δεσμίνης του αμινο-τελικού και το καρβόξυ-τελικού. Μελετώντας τις πρωτεΐνες που προέκυψαν, διαπιστώσαμε ότι το αμινο-τελικό άκρο της δεσμίνης αλληλεπιδρά με διάφορες μιτοχονδριακές πρωτεΐνες. Με το ίδιο σύστημα αποκαλύψαμε αλληλεπιδράσεις της δεσμίνης με λυοσωματικές πρωτεΐνες όπως η καθεψίνη D και η προσαποσίνη οι οποίες αλληλεπιδρούν με το αμινοτελικό άκρο της δεσμίνης. Η καθεψίνη D είναι μια λυοσωματική πρωτεάση, που οδηγείται και ωριμάζει πλήρως στα λυοσώματα ενώ η προσαποσίνη είναι ένα πρόδρομο λυοσωματικό μόριο με πρωτεόλυση του οποίου, εντός του λυοσώματος, προκύπτουν οι σαποσίνες Α έως D. Η καθεψίνη D αποτελεί δείκτη καταστάσεων αυτοφαγία και τελευταία φαίνεται ότι επεμβαίνει σε φαινόμενα απόπτωσης επάγωντάς την κατά περίπτωση. Η αλληλεπίδραση της δεσμίνης με την καθεψίνη D επιβεβαιώθηκε και με βιοχημικές τεχνικές (in vitro) όπως η συνεργιστική ανοσοκαθίζηση /ανοσοκατακρήμνιση (co-immuno-precipitation) και η τεχνική GST pull-down. Μετά και από αυτές τις in vitro αποδείξεις, φαίνεται πως μάλλον συμβαίνει ευθεία αλληλεπίδραση μεταξύ της δεσμίνης και της καθεψίνης D. Γι’ αυτό, και με βάση όσα είναι γνωστά για την καθεψίνη D, προτείνουμε μια νέα λειτουργία του κυτταροσκελετού της δεσμίνης πιθανόν στην μετακίνησης και τη δημιουργία των λυοσωμάτων αλλά και έναν νέο ρυθμιστικό ίσως ρόλο της, σε διαδικασίες αυτοφαγίας και απόπτωσης, μέσω της πρόσδεσής της με σημαντικά μόρια ρυθμιστές τέτοιων διαδικασιών.................... / Desmin is the muscle - specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In order to identify the exact mechanisms of desmin’s action, we performed a yeast two-hybrid screen for desmin-interacting proteins. For this purpose, we used as baits the two non helical terminal regions of the desmin molecule, the amino (head)- and the carboxy (tail)- terminal domain. We have found that the head domain of desmin potentially interacts with two new groups of proteins, mitochondria and lysosome related. Specifically, in the second category, we have revealed an association of the head domain of desmin with Cathepsin D (one of the lysosomal proteinases) and prosaposin (a single precursor which gives rise to Saposins A-D by proteolytic cleavage in lysosomes and is also referred to as sphigolipid activator proteins). In addition to its targeting to lysosomes, Cathepsin D is also involved in apoptosis and autophagy processes. This protein interaction result has been retested. The interaction between cathepsin D and desmin has also been further confirmed both with reverse yeast transformation as well as biochemical assays such as co-immunoprecipitation and GST pull down assay. The above described strong evidence of direct interaction between desmin and cathepsin D, has allowed us to propose a novel function of desmin IFs in lysosomal trafficking and/or as a new regulator of autophagy and apoptotic cell death.

No description available.