The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

Οι πρωτεϊνικές αλληλεπιδράσεις (PPI) αναφέρονται στην σύνδεση δύο ή περισσοτέρων πρωτεϊνών ώστε να εκτελεστεί μια βιολογική λειτουργία. Την τελευταία δεκαετία, νέες τεχνολογίες υψηλής απόδοσης για τον εντοπισμό αυτών των αλληλεπιδράσεων έχουν παράγει μεγάλης κλίμακας σύνολα δεδομένων τόσο του ανθρώπου όσο και των περισσοτέρων ειδών. Με την αναπαράσταση αυτών των δεδομένων σε δίκτυα, με τους κόμβους να αναπαριστούν τις πρωτεΐνες και τις ακμές τις αλληλεπιδράσεις, μπορούν να εξαχθούν χρήσιμες πληροφορίες σχετικά με τον προσδιορισμό της λειτουργίας των πρωτεϊνών/πρόβλεψη ή σχετικά με το πώς να σχεδιαστούν κατάλληλα φάρμακα που προσδιορίζουν τα νέα γονίδια-στόχους για τον καρκίνο ή τους μηχανισμούς που ελέγχουν (ή ρυθμίζουν) τις βιολογικές αλληλεπιδράσεις που είναι υπεύθυνες για την καλή ή την κακή λειτουργία ενός κυττάρου. Στα πλαίσια της παρούσας διπλωματικής, κληθήκαμε να κάνουμε λειτουργική πρόβλεψη των πρωτεϊνών στο δίκτυο πρωτεϊνικών αλληλεπιδράσεων του ανθρώπου εφαρμόζοντας μια μέθοδο δυναμικής επικάλυψης η οποία βασίζεται στον έλεγχο του πώς οι ταλαντωτές οργανώνονται σε ένα «αρθρωτό»(modular) δίκτυο σχηματίζοντας διεπαφές συγχρονισμού και κοινότητες επικάλυψης. Μελετήσαμε το δίκτυο πρωτεϊνικών αλληλεπιδράσεων του ανθρώπου και τις κλάσεις λειτουργιών θεωρώντας ένα σύνολο ταλαντωτών φάσης (phase oscillators) με μία τοπολογία συνδέσεων που ορίζεται από το δίκτυο πρωτεϊνικών αλληλεπιδράσεων του ανθρώπου. Συγκεκριμένα, αρχίσαμε με μία απλή ομαδοποίηση για κάθε πρωτεΐνη και έπειτα χρησιμοποιήσαμε την μέθοδο δυναμικής επικάλυψης για τον προσδιορισμό των λειτουργιών των πρωτεϊνών του PPI δικτύου. Στην συνέχεια, εντοπίσαμε εκείνες τις πρωτεΐνες οι οποίες δεν είχαν ομαδοποιηθεί σωστά καθώς και τις πρωτεϊνες που ήταν πιθανόν να «συμμετείχαν» σε περισσότερες από μία λειτουργικές κλάσεις (πολυλειτουργικές πρωτεΐνες). Με κατάλληλο έλεγχο των αλληλεπιδράσεων μεσαίας κλίμακας του δικτύου των δυναμικών συστημάτων που δημιουργήθηκε παρήχθησαν χρήσιμες πληροφορίες για τις μικρής και μεγάλης κλίμακας διαδικασίες μέσω των οποίων οι βιολογικές διεργασίες οργανώνονται σε ένα κύτταρο γεγονός που αποκαλύπτει ότι η μέθοδος είναι ικανή όχι μόνο να εντοπίσει τις μη σωστά ομαδοποιημένες πρωτεΐνες αλλά και να αποκαλύψει αυτές που έχουν διπλή λειτουργικότητα (2 λειτουργίες). / Protein-protein interactions (PPI) refer to the binding of two or more proteins to perform a biological function. In the last decade, novel high-throughput technologies for detecting those interactions have produced large-scale data sets across human and most model species. By embedding these data in networks, with nodes representing proteins and edges the detected PPIs, useful information can be extracted regarding protein functional annotation/prediction or on how to design proper drugs, identifying new targets on cancer, or mechanisms to control (or regulate) the biological interactions responsible for the functioning,or malfunctioning, of a cell. Under the framework of my master thesis, I had to apply a method of dynamical overlap based on the inspection of how oscillators organize in a modular network by forming synchronization interfaces and overlapping communities to the human PPI network. I studied the human protein interaction network (PIN) and its functional modules by considering an ensemble of phase oscillators with a topology of connections defined by the human PIN. In particular, I started with a single classification for each protein and I used the dynamical overlap method for identifying/predicting of the proteins function(s) in the PPI network. Then, I identified all those proteins that were misclassified and those proteins that were likely to be involved in more than one of the functional categories in our data(multifunctional proteins). A proper inspection on the meso-scale interactions of the generated network of dynamical systems provided useful information on the micro- and macro- scale processes through which biological processes are organized in a cell, that is, the method is not only able to identify the misclassified proteins but also to unveil those proteins that have double functionality.

published_or_final_version / Computer Science / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

<p>Individual proteins, and small collections of proteins, have been extensively studied for at least two hundred years. Today, more than 350 genomes have been completely sequenced and the proteomes of these genomes have been at least partially mapped. The inventory of protein coding genes is the first step toward understanding the cellular machinery. Recent studies have generated a comprehensive data set for the physical interactions between the proteins of <i>Saccharomyces cerevisiae</i>, in addition to some less extensive proteome interaction maps of higher eukaryotes. Hence, it is now becoming feasible to investigate important questions regarding the evolution of protein-protein networks. For instance, what is the evolutionary relationship between proteins that interact, directly or indirectly? Do interacting proteins co-evolve? Are they often derived from each other? In order to perform such proteome-wide investigations, a top-down view is necessary. This is provided by network (or graph) theory.</p><p>The proteins of the cell may be viewed as a community of individual molecules which together form a society of proteins (nodes), a network, where the proteins have various kinds of relationships (edges) to each other. There are several different types of protein networks, for instance the two networks studied here, namely metabolic networks and protein-protein interaction networks. The metabolic network is a representation of metabolism, which is defined as the sum of the reactions that take place inside the cell. These reactions often occur through the catalytic activity of enzymes, representing the nodes, connected to each other through substrate/product edges. The indirect interactions of metabolic enzymes are clearly different in nature from the direct physical interactions, which are fundamental to most biological processes, which constitute the edges in protein-protein interaction networks.</p><p>This thesis describes three investigations into the evolution of metabolic and protein-protein interaction networks. We present a comparative study of the importance of retrograde evolution, the scenario that pathways assemble backward compared to the direction of the pathway, and patchwork evolution, where enzymes evolve from a broad to narrow substrate specificity. Shifting focus toward network topology, a suggested mechanism for the evolution of biological networks, preferential attachment, is investigated in the context of metabolism. Early in the investigation of biological networks it seemed clear that the networks often display a particular, 'scale-free', topology. This topology is characterized by many nodes with few interaction partners and a few nodes (hubs) with a large number of interaction partners. While the second paper describes the evidence for preferential attachment in metabolic networks, the final paper describes the characteristics of the hubs in the physical interaction network of <i>S. cerevisiae</i>.</p>

The enzyme 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1) catalyses the intracellular biosynthesis of the active glucocorticoid cortisol. Tissue specific dysregulation of the enzyme has been implicated in the development of metabolic syndrome and other associated diseases. Experiments with transgenic mice and prototype inhibitors show that inhibition of 11β-HSD1 in visceral adipose tissue and liver leads to a resistance of diet-induced hyperglycemia and a favourable lipid and lipoprotein profile as compared to controls. 11β-HSD1 inhibition has thus been proposed as an effective strategy to decrease intracellular glucocorticoid levels without affecting circulating glucocorticoid levels that are essential for stress responses. The clinical development of selective and potent drugs has therefore become a priority. In this research, a process of virtual screening employing the novel algorithm UFSRAT (Ultra Fast Shape Recognition with Atom Types) was used to discover compounds which had specific physicochemical and spatial atomic parameters deemed essential for inhibition of 11β-HSD1. The top scoring compounds were assayed for inhibitory activity against recombinant human and mouse enzyme, using a fluorescence spectroscopy approach. In addition, HEK-293 cell based assays with either human, mouse or rat enzymes were carried out using a scintillation proximity assay (SPA). The most potent compound competitively inhibited human 11β-HSD1 with a Kiapp value of 51 nM. Recombinant mouse and human enzyme were expressed, purified and characterised and used in a series of ligand binding assays. Further to this, an X-ray crystal structure of mouse 11β-HSD1 in complex with a tight binding inhibitor – carbenoxolone was solved.

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signaling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. G protein-coupled receptor 50 (GPR50) is one such orphan, and its exact role is yet unknown. There is however emerging functional and genetic evidence suggesting a function for GPR50 in psychiatric illness and lipid metabolism. It was hypothesised that investigating GPR50’s protein-protein interactions would lead to a greater understanding of the role of GPR50 in normal brain functioning and in mental illness. Putative protein interactors were initially isolated by a yeast two-hybid study and were further tested here. To address GPR50’s links to mental illness, the GPR50∆502-505 deletion variant associated with mood disorders was also investigated. To test this hypothesis I sought to confirm some of the key yeast two-hybrid interactions. Using co-immunoprecipitation and immunocytochemistry the interaction of GPR50 with reticulon family members Nogo-A, Nogo-C and RTN3, and with cell-cell adhesion molecule CDH8 and lipid-associated protein ABCA2 were validated. In order to identify the location of interactions, subcellular fractionation of mouse brain and rt-PCR and immunohistochemistry in developing and adult mouse brain were performed. GPR50 and several interactors were found to be enriched at the synapse by subcellular fractionation of whole adult brain, and at embryonic day 18 (E18) and 5 weeks by rt-PCR. Colocalisation of GPR50 and interactors was found in the amygdala, hypothalamus, cortex and specific brain stem nuclei by immunohistochemistry. The discovery of GPR50 expression in noradrenergic, serotonergic and dopaminergic nuclei in the adult brain stem suggests a further role for GPR50 in neurotransmitter signaling and stress. To investigate the function of GPR50 two assays were performed that measure processes which are known to be affected by Nogo and RTN3: The first assay was a neurite outgrowth assay in Neuroscreen-1 cells, a PC12 cell clone. A significant increase in neurite length was detected after transient overexpression of GPR50 and this effect was increased in the GPR50∆502-505/T532A variant. Additionally GPR50-overexpression resulted in an increase in filopodia formation suggesting a role in actin dynamics. As a second functional assay in vitro BACE1 activity assays were performed in HEK293 cells. GPR50 but not GPR50∆502-505/T532A overexpression resulted in a significant increase in BACE1 activity. Lastly a final series of pilot experiments were performed to gain insight into the secondary structure of the C-terminal domain and the effects of the polymorphisms on structure. The 35kDa GPR50 C-terminal domain was purified and Circular Dichroism studies indicated a predominantly unstructured protein with increased a- helical content in the GPR50∆502-505 variant. The results in this thesis indicate a role for GPR50 in neuronal development and synaptic functioning. The results also strengthen an association with major mental illness, with links to several disease mechanisms.

<p>Eukaryotic Topoisomerase 2 is an essential enzyme that solves DNA topological problems such as DNA knotting, catenation, and supercoiling. It alters the DNA topology by introducing transient double strand break in one DNA duplex as a gate for the passage of another DNA duplex. Two different aspects of studies about eukaryotic Topoisomerase 2 will be covered in this thesis. In the first half of the thesis, we investigated conformational changes of human Topoisomerase 2&#61537; (hsTop2&#61537;) in the presence of cofactors and inhibitors. In the second half, we focused on an unknown regulatory function in the C-terminal domain (CTD) of Drosophila Topoisomerase 2 (Top2).</p><p>In the project of studying enzyme conformational changes, we adapted a previously developed methodology, Pulse-Alkylation Mass Spectrometry, with monobromobimane to study the protein dynamics of hsTop2&#61537;. Using this method, we captured the evidence of conformational changes in the presence of ATP and Mg2+ or the Top2 inhibitor, ICRF-193 which were not previously observed. Last, by using CTD truncated hsTop2&#61537;, the increasing reactivity of Cys427 suggested the CTD domain might be tethered adjacent to the core enzyme.</p><p>Following the study of enzyme conformational changes, we switched gear to examine an interaction between Drosophila Top2 and Mus101, homolog of human TopBP1. We first found that Mus101 interacts with CTD of Top2 in a phosphorylation-dependent manner. Next, in the co-immunoprecipitation and pull-down experiments using truncated or mutant Top2 with various Ser to Ala substitutions, we mapped the binding motif to the last amino acids of Top2 and identified that phosphorylation of Ser1428 and Ser1443 is important for Top2 to interact with the N-terminus of Mus101, which contains BRCT1/2 domains (BRCT, BRCA1 C-terminus). The binding affinity of the N-terminal Mus101 with a synthetic phosphorylated peptide covering the last 25 amino acids of Top2 (with pS1428 and pS1443) was determined by surface plasmon resonance with a Kd of 0.57 &#956;M. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response to Mus101. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2&#61508;20, truncation of 20 amino acids from the C-terminus of Top2, developed abnormally high chromosome numbers, which implies an infidelity in chromosome segregation during mitosis. Lastly, Top2-null flies rescued by Top2 with S1428A and S1443A were found to be viable but sterile. After investigating spermatogenesis, telophase of meiosis I was delayed, indicating Top2-Mus101 interaction is also important in segregating DNA in meiosis.</p> / Dissertation

Proteins are the functional units of cells. However, a major portion of the proteome does not have a known functional annotation. This dissertation explores protein -protein interactions, involving these uncharacterized or unknown function proteins. Initially, protein – protein interactions were tested and analyzed for paralogous proteins in Escherichia coli. To expand this concept further and to get an overview, protein – protein interactions were analyzed using ‘comparative interactomics’ for four pathogenic bacterial species including Escherichia coli, Yersinia pestis, Vibrio cholerae and Staphylococcus aureus. This approach was used to study unknown function protein pairs as well as to focus on uncharacterized hub proteins. The dissertation aims at using protein – protein interactions along with other research data about proteins as a possible approach to narrow down on functions of proteins.