The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Essential proteins affect the cellular life deeply, but it is hard to identify them. Protein-protein interaction is one of the ways to disclose whether a protein is essential or not. We notice that many researchers use the feature set composed of topology properties from protein-protein interaction to predict the essential proteins. However, the functionality of a protein is also a clue to determine its essentiality. In this thesis, to build SVM models for predicting the essential proteins, our feature set contains the sequence properties which can influence the protein function, topology properties and protein properties. In our experiments, we download Scere20070107, which contains 4873 proteins and 17166 interactions, from DIP database. The ratio of essential proteins to nonessential proteins is nearly 1:4, so it is imbalanced. In the imbalanced dataset, the best values of F-measure, MCC, AIC and BIC of our models are 0.5197, 0.4671, 0.2428 and 0.2543, respectively. We build another balanced dataset with ratio 1:1. For balanced dataset, the best values of F-measure, MCC, AIC and BIC of our models are 0.7742, 0.5484, 0.3603 and 0.3828, respectively. Our results are superior to all previous results with various measurements.

Finding reliable gene markers for accurate disease classification is very challenging due to a number of reasons, including the small sample size of typical clinical data, high noise in gene expression measurements, and the heterogeneity across patients. In fact, gene markers identified in independent studies often do not coincide with each other, suggesting that many of the predicted markers may have no biological significance and may be simply artifacts of the analyzed dataset. To nd more reliable and reproducible diagnostic markers, several studies proposed to analyze the gene expression data at the level of groups of functionally related genes, such as pathways. Given a set of known pathways, these methods estimate the activity level of each pathway by summarizing the expression values of its member genes and using the pathway activities for classification. One practical problem of the pathway-based approach is the limited coverage of genes by currently known pathways. As a result, potentially important genes that play critical roles in cancer development may be excluded. In this thesis, we first propose a probabilistic model to infer pathway/subnetwork activities. After that, we developed a novel method for identifying reliable subnetwork markers in a human protein-protein interaction (PPI) network based on probabilistic inference of subnetwork activities. We tested the proposed methods based on two independent breast cancer datasets. The proposed method can efficiently find reliable subnetwork markers that outperform the gene-based and pathway-based markers in terms of discriminative power, reproducibility and classification performance. The identified subnetwork markers are highly enriched in common GO terms, and they can more accurately classify breast cancer metastasis compared to markers found by a previous method.

Genome scale protein-protein interactions (PPIs) are interpreted as networks or graphs with thousands of nodes from the perspective of computer science. PPI networks represent various types of possible interactions among proteins or genes of a genome. PPI data is vital in protein function prediction since functions of the cells are performed by groups of proteins interacting with each other and main complexes of the cell are made of proteins interacting with each other. Recent increase in protein interaction prediction techniques have made great amount of protein-protein interaction data available for genomes. As a consequence, a systematic visualization and analysis technique has become crucial. To the best of our knowledge, no PPI visualization tool consider multi-resolution viewing of PPI network. In this thesis, we implemented a new approach for PPI network visualization which supports multi-resolution viewing of compound graphs. We construct compound nodes and label them by using gene set enrichment methods based on Gene Ontology annotations. This thesis further suggests new methods for PPI network visualization.

Protein-protein interactions are key to several biological processes that facilitate signal transduction and many other processes. These interactions are involved in pathways that are critical to many human diseases. Targeting specific protein-protein interactions is a challenging goal because protein-protein interactions are predominately through hydrophobic interactions. Antagonists of the protein-protein interactions need to be perfectly fit into the binding pockets to ensure the activity. The -helical domain of the proteins behaves as the recognition motifs for numerous protein-protein, and protein-nucleic acid interactions. Research has shown that pathways of many diseases contain protein-protein interactions involving -helical domains, e.g. neurological disorders, bacterial infections, HIV and cancer, etc. It is difficult yet very important to design small molecules to target the shallow binding areas of protein-protein interactions. So far the most successful one is Hamilton’s 1,4-terphenylene scaffold, which has been used to target the interactions between p53/MDM2, Bak/Bcl-xL etc. Inspired by this, we designed and synthesized three new scaffolds of non-pepditic -helical mimetics, mimicking the i, i + 4, i + 7 positions of an -helix. There are three basic principles that were leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an -helix. Second, our molecules should possess improved water solubility. Third, the molecules should be easy to synthesize to generate a focused library. Some of our molecules, including the ones whose molecular weight are as low as 294, started to show some inhibition against p53/MDM2 interactions.

←Within this dissertation the topic of virtual screening is discussed with regard to three different cancer targets and also a brief introduction of the tools used in virtual screening. In Chapter 1, the reader will be introduced to virtual screening and the programs that are used in virtual screening. In Chapter 2, the first of three projects are discussed. This project consists of the work that was done to find inhibitors of the P53 binding domain of MDMX. In this project the mobility of residues within the binding site of MDMX are discussed and the ways in which we attempted to model how drugs would bind two adjacent pockets within MDMX. In Chapter 3, the virtual screening and modeling work done for RING domain of MDM2 and MDMX is discussed. This work was done in conjunction with Moffitt Cancer Center in order to solve the 60 year old mystery of the mechanism of how thalidomide and possibly its analog lenalidomide caused children to be born limbless. Current thinking is that Cereblon through an unknown teratogenic mechanism activates an increase in FGF8. We suggest a mechanism that may happen in parallel that involves stabilization of MDM2 and the reduction of P63 levels. Chapter 4, the work that was done against the BH3 binding domain of MCL-1 is discussed in conjunction with collaboration with the Manetsch lab. In order to complete this screening the validation of IC50 values and then attempt to modify those products based upon the structure of MCL-1. Chapter 5 discusses the work done to find inhibitors of deoxycytidine kinase. All of these chapters taken together provide a brief overview of the computational work done produce inhibitors of Protein-Protein Interaction against three major cancer targets.

Staphylococcus aureus sortase A (SrtA) transpeptidase is a prominent membrane bound virulence factor in gram-positive bacteria, which organizes the peptidoglycan cell wall of the organism. Here, we report the first direct observation of the self-association behavior of SrtA. Formation of a SrtA dimer is highly selective in vitro in E. coli and in vivo on the S. aureus cell membrane. Quantitative analysis of protein binding affinity indicated a moderate association between two SrtA molecules with an apparent K[subscript d] of about 55 [micrometres] in vitro. Furthermore, to address the importance of dimerization for enzyme function, site-directed mutagenesis on potential target residues was performed to generate monomer only SrtA mutant proteins to completely disrupt dimer formation both in vitro and in vivo. Finally, an in vivo activity assay was performed to evaluate the function of SrtA wild type protein as well as its monomer only mutants. Our data demonstrated that S. aureus cells expressing mutant SrtA in a monomer only form are more successful at invading human epithelial cells than those expressing wild type SrtA in dimer-monomer equilibrium. It suggested that the monomeric form of SrtA is more active than the dimeric enzyme. We also demonstrated the uniqueness of SrtA dimerization by identifying that at least one other sortase family protein, SrtB only exists in monomer form. SrtA dimerization may have significant implications for understanding its biological function at both the cellular and molecular levels, which will lead to the development of new anti-infective therapies against gram-positive pathogens. / text

Kinetic target-guided synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of nine thio acids and nine sulfonyl azides leading to eighty one potential acylsulfonamides, the target protein, Bcl-XL selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-XL/BH3 interactions. To further investigate the Bcl-XL templation effect, control experiments were carried out using two mutants of Bcl-XL. In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, have been substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, has been replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual "hot spot" of Bcl-XL. These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs. Protein-protein interactions of the Bcl-2 family have been extensively investigated and the anti-apoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1) have been validated as crucial targets for the discovery of potential anti-cancer agents. At the outset, Bcl-2 and Bcl-XL were considered to play an important role in the regulation of apoptosis. Accordingly, several small molecule inhibitors targeting Bcl-2 and/or Bcl-XL proteins were primarily designed. A series of acylsulfonamides targeting these proteins were reported by Abbott laboratories, ABT-737 and ABT-263 being the most potent candidates. Remarkably, these molecules were found to exhibit weaker binding affinities against Mcl-1, another anti-apoptotic protein. Further experimental evidence suggests that, inhibitors targeting Mcl-1 selectively or in combination with other anti-apoptotic proteins would lead to desired therapeutic effect. As a result, numerous small molecules displaying activity against Mcl-1 have been identified so far. Specifically, acylsulfonamides derived from structure activity relationship by interligand nuclear overhauser effect (SAR by ILOEs), a fragment-based approach, have been recently reported with binding affinities in the nanomolar range. In the meantime, we have reported that the kinetic TGS approach can also be applied to identify acylsulfonamides as PPIMs targeting Bcl-XL. Taken together, structurally novel acylsulfonamides can be potentially discovered as Mcl-1 inhibitors using the kinetic TGS approach. Thus, a library of thirty one sulfonyl azides and ten thio acids providing three hundred and ten potential products was screened against Mcl-1 and the kinetic TGS hits were identified. Subsequently, control experiments involving Bim BH3 peptide were conducted to confirm that the fragments are assembled at the binding site of the protein. The kinetic TGS hits were then synthesized and subjected to the fluorescence polarization assay. Gratifyingly, activities in single digit micromolar range were detected, demonstrating that the sulfo-click kinetic TGS approach can also be used for screening and identification of acylsulfonamides as PPIMs targeting Mcl-1. The amide bond serves as one of nature's most fundamental functional group and is observed in a large number of organic and biological molecules. Traditionally, the amide functionality is introduced in a molecule through coupling of an amine and an activated carboxylic acid. Recently, various alternative methods have been reported wherein, the aldehydes or alcohols are oxidized using transition metal catalysts and are treated with amines to transform into the corresponding amides. These transformations however, require specially designed catalysts, long reaction times and high temperatures. We herein describe a practical and efficient amidation reaction involving aromatic aldehydes and various azides under mild basic conditions. A broad spectrum of functional groups was tolerated, demonstrating the scope of the reaction. Consequently, the amides were synthesized in moderate to excellent yields, presenting an attractive alternative to the currently available synthetic methods.

Η ακρίβεια και η πιστότητα της διαδικασίας διπλασιασμού του γονιδιώματος είναι μείζωνος σημασίας για την κυτταρική επιβίωση. Τυχόν ανωμαλίες όπως μεταλλάξεις ή χρωμοσωμικές ανωμαλίες είναι δυνατόν να οδηγήσουν στην εμφάνιση κακοήθειας ή σε πρόωρο κυτταρικό θάνατο. Τα παραπάνω συνηγορούν στη μεγάλη σημασία που έχει η άρτια λειτουργία και ρύθμιση του κυτταρικού κύκλου. Δύο πρωτεϊνες, οι geminin και cdt1, κατέχουν πολύ σημαντικό ρόλο κατά τη διαδικασία ρύθμισης του κυτταρικού κύκλου. Πιο συγκεκριμένα, η cdt1 αποτελεί έναν βασικό παράγοντα αδειοδότησης της αντιγραφής. Δρα συνεργατικά με την πρωτεϊνη Cdc6 προκειμένου να γίνει η πρόσδεση του εξαμερούς συμπλόκου MCM2-7 στο προ-αντιγραφικό σύμπλοκο (pre-RC), διασφαλίζοντας με τον τρόπο αυτό τις απαραίτητες συνθήκες για τη διαδικασία αδειοδότησης της αντιγραφής (Maiorano D. et al., 2000). Η geminin συνιστά τον φυσικό αναστολέα της cdt1 στα μετάζωα. Προσδένεται ισχυρά στην τελευταία μετά την έναρξη της σύνθεσης του DNA, παρεμποδίζοντας με αυτόν τον τρόπο την επαναπρόσδεσή της στο προ-αντιγραφικό σύμπλοκο (Tada S. et al., 2001; Wohlschlegel J.A. et al., 2000). Η διαδικασία αδειοδότησης της αντιγραφής παρουσιάζει ανωμαλίες σε περιπτώσεις καρκινικών κυττάρων. Πιο συγκεκριμένα, έχει δειχθεί σταθερή υπερέκφραση της cdt1 σε καρκινικές κυτταρικές σειρές, τόσο σε πρωτεϊνικό όσο και σε μεταγραφικό επίπεδο (Xouri G. et al., 2004). Παρομοίως, αυξημένη έκφραση της cdt1 έχει παρατηρηθεί και σε περιπτώσεις καρκίνου του παχέος εντέρου καθώς και πρώιμου καρκίνου του πνεύμονα, στον άνθρωπο (Bravou V. et al., 2005; Karakaidos P. et al., 2004). Τα παραπάνω αποτελέσματα έχουν προκύψει κατόπιν μελέτης, η οποία πραγματοποιήθηκε στο εργαστήριό μας. Αναφορικά με τη geminin, αυξημένα επίπεδα έκφρασής της έχουν συσχετιστεί με καρκινώματα παχέος εντέρου καθώς και με τη διαίρεση κακοηθών κυττάρων, γενικότερα (Gonzalez M.A. et al., 2005; Wohlschlegel J.A. et al., 2002). Επιπροσθέτως, η geminin βρίσκει εφαρμογή ως ανεξάρτητος δείκτης σε περιπτώσεις επιθετικού καρκίνου του μαστού (Gonzalez M.A. et al., 2004). Βασιζόμενοι στα παραπάνω, θεωρούμε ότι το σύμπλοκο geminin-cdt1 συνιστά έναν ιδανικό στόχο για το σχεδιασμό νέων αντικαρκινικών φαρμάκων. Το πρώτο βήμα προς αυτήν την κατεύθυνση αποτελεί ο μαζικός έλεγχος συνθετικών συστατικών, τα οποία να έχουν την ικανότητα διάσπασης του συμπλόκου. Ενδεχόμενη εύρεση τέτοιων συνθετικών συστατικών καθώς και μετέπειτα φυσικοχημική βελτιστοποίησή τους είναι δυνατόν να οδηγήσει στη δημιουργία ενός νέου αντικαρκινικού φαρμάκου. Η ολοκλήρωση της χαρτογράφησης του ανθρώπινου γονιδιώματος συνέβαλε στην ταυτοποίηση νέων πρωτεϊνικών μορίων στόχων, τα οποία εμπλέκονται στο μοριακό μηχανισμό διαφόρων ασθενειών. Με βάση τις εξελίξεις των τελευταίων χρόνων στον τομέα της φαρμακοβιομηχανίας, η αξιοποίηση αυτής της γνώσης συνδέεται με το μαζικό έλεγχο συνθετικών συστατικών έναντι αυτών των μορίων στόχων. Απώτερο στόχο αποτελεί η αναγνώριση κατάλληλων συνθετικών συστατικών τα οποία θα έχουν την ικανότητα να αλληλεπιδρούν με το μόριο-στόχος και κατ’επέκταση να μεταβάλλουν τον τρόπο λειτουργίας του, προκειμένου να έχουμε το επιθυμητό φαρμακολογικό αποτέλεσμα. Αποφασιστικής σημασίας στην παραπάνω διαδικασία, αποτελεί η σωστή επιλογή της κατάλληλης μεθόδου μαζικού ελέγχου των νέων υποψήφιων φαρμάκων – συνθετικών συστατικών – έναντι του μορίου στόχου. Στην παρούσα διπλωματική, επιλέχθηκε προς εφαρμογή η μέθοδος FRET. Ένα από τα βασικά της πλεονεκτήματα είναι η υψηλή αναλογία ‘σήματος-θορύβου’ που εμφανίζει καθώς και η υψηλή ποιότητα των δεδομένων που παρέχει. Αν και αποτελεί μία σχετικά καινούρια τεχνική, εντούτοις αποτελεί μία από τις πιο βασικές μεθόδους της σύγχρονης φαρμακοβιομηχανίας λόγω της αξιοπιστίας που την χαρακτηρίζει και επιπλέον της συμβατότητάς της με αυτοματοποιημένες τεχνικές. Ασφαλώς, απαραίτητη προϋπόθεση για την εφαρμογή της συγκεκριμένης μεθόδου αποτελεί η απομόνωση της υπό μελέτη πρωτεϊνης. Η έκφραση του πρωτεϊνικού συμπλόκου geminin-cdt1 πραγματοποιήθηκε με τη χρήση βακτηριακών συστημάτων έκφρασης ετερόλογων πρωτεϊνών. Επίσης, ο καθαρισμός του συμπλόκου υπήρξε επιτυχής και βασίστηκε στην εφαρμογή των τεχνικών της χρωματογραφίας συγγένειας και της χρωματογραφίας διήθησης σε πηκτή. Το επόμενο βήμα ήταν να διαπιστώσουμε εάν η μέθοδος FRET καθιστά δυνατή την ανίχνευση σχηματισμού του συμπλόκου. Πράγματι, κάτι τέτοιο ήταν εφικτό καθώς σε συγκέντρωση 60-80nM του συμπλόκου, παρατηρήθηκε αύξηση του σήματος σχεδόν κατά πέντε φορές υψηλότερα από το επίπεδο “θορύβου”. Το αποτέλεσμα αυτό είναι ιδιαίτερα σημαντικό καθώς συνεπάγεται ότι με τη συγκεκριμένη μέθοδο είναι εφικτός ο μαζικός έλεγχος συνθετικών συστατικών, τα οποία θα έχουν την ικανότητα διάσπασης του συμπλόκου. Οι λόγοι που συντελούν στην καταλληλότητα της μεθόδου για αυτό το σκοπό έγκεινται αφενός στην ευαισθησία την οποία εμφανίζει (αύξηση του σήματος μέχρι και πέντε φορές) και αφετέρου στην ειδικότητα και την αξιοπιστία της, όπως έχει δειχθεί και με τα αντίστοιχα πειράματα. Σημαντικό επίσης πλεονέκτημα αποτελεί και η μικρή σχετικά ποσότητα πρωτεϊνης (60-80nM), η οποία απαιτείται. Σύμφωνα με τα δεδομένα που έχουν προκύψει από την παρούσα διπλωματική , το FRET assay συνιστά μία ιδανική μέθοδο για την πραγματοποίηση μαζικού ελέγχου συνθετικών συστατικών, τα οποία θα έχουν την ικανότητα διάσπασης του συμπλόκου geminin-cdt1. Δεδομένης της πολύ ισχυρής αλληλεπίδρασης του συγκεκριμένου συμπλόκου, πραγματοποιήθηκαν μεταλλαγές σε τρία υψηλά συντηρημένα κατάλοιπα της cdt1, τα οποία κατέχουν κυρίαρχο ρόλο στην αλληλεπίδραση με τη geminin, σύμφωνα με κρυσταλλογραφικά δεδομένα (Lee C. et al., 2004). Οι μεταλλαγές αυτές ενδέχεται να συμβάλλουν σε ένα πολύ πιο εύκολα διασπάσιμο σύμπλοκο, γεγονός που μπορεί να οδηγήσει στην ευκολότερη και γρηγορότερη ταυτοποίηση υποψήφιων συνθετικών συστατικών. / The accurate and timely duplication of the genome is vital for cell survival. Mutations rearrangements or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing malignant cell growth or death. Origins of replication are licensed by a multi-subunit complex (pre-replicative complex: pre-RC) during G1 (Lei M & Tye B.K., 2001). Pre-RC assembly is an ordered, sequential process in which the Origin Recognition Complex (ORC) first binds to each replication origin and then recruits two other proteins: Cdc6 and Cdt1 (Bell S.P. & Dutta A., 2002). These two proteins function synergistically to load the six mini-chromosome maintenance helicase proteins (MCM2-7) onto the pre-RC, establishing the conditions for DNA licensing (Maiorano D. et al., 2000). The prevention of ectopically induced re-replication is accomplished by functionally redundant mechanisms, including sequestration of MCM by Crm1 (Yamaguchi R. & Newport J., 2003), inactivation and export from the nucleus of Cdc6 (Delmolino L.M. et al., 2001; Jiang W. et al., 1999; Pelizon C. et al., 2000) and degradation of Cdt1 (Nishitani H. et al., 2001). Metazoans, have evolved an additional system for preventing re-replication: Geminin, which was originally identified in Xenopus as essential factor to exit from mitosis (McGarry T.J. & Kirschner M.W., 1998), binds tightly to Cdt1 and prevents Cdt1 assembly onto pre-RC (Tada S. et al., 2001; Wohlschlegel J.A. et al., 2000). Licensing system members are misregulated in cancer cells and differential expression of licensing components could be used for the diagnosis and prognosis of cancer (Shreeram S. & Blow J.J., 2003). Over-expression of Cdt1 can predispose cells to a malignant transformation. It has been shown that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level (Xouri G. et al., 2004). Moreover, Cdt1 protein is highly expressed in cases of human colon cancer and primary human lung carcinomas (Bravou V. et al., 2005; Karakaidos P. et al., 2004). Similarly, Geminin is also over-expressed in colon carcinomas and its expression levels were directly related to the cellular proliferation index in proliferating malignant cells (Gonzalez M.A. et al., 2005; Wohlschlegel J.A. et al., 2002). Furthermore, expression of Geminin is considered as an independent indicator of adverse prognosis in cases of invasive breast cancer (Gonzalez M.A. et al., 2004). Given the crucial role of the Geminin-Cdt1 complex in cell cycle regulation and cancer, this complex could serve as target for the discovery and development of novel anti-cancer drugs. This requires the screening of compounds that are capable of disrupting the geminin-cdt1 complex. For that purpose we performed a HTP (high-throughput)-compatible assay, called FRET-assay. The acronym FRET stands for Fluorescence Resonance Energy transfer. The principle of the assay is based on the radiationless transfer of energy from an excited donor fluorophore (Europium Cryptate) to a suitable acceptor fluorophore (XL665). The first step was the expression and purification of the geminin-cdt1 complex. The complex was expressed by using E. coli bacterial cells and purified by metal affinity chromatography on a Ni+2 column. As soon as the complex was ready to use, we next tried to investigate whether the formation of the complex was detectable by using the FRET assay. Indeed, at the complex concentration of 60nM and 80nM, the signal was about 5 times above background. This was a first indication that the Geminin-Cdt1 complex can be used successfully for energy transfer based assays. Given the very high binding affinity of the two proteins (Lee C. et al., 2004), it could be quite unlikely to find a compound that can disrupt the complex. To overcome that obstacle, three single mutations were made at the highly conserved Geminin-contacting residues of hCdt1, Y170, F173 and L176. The mutation of these residues to alanine can possibly provide a more easily disruptable complex, which could be of importance concerning the faster and easier identification of any candidate compounds. Our data suggest that hGeminin-Cdt1 complex can be considered as a promising target for compound screening. Given the high importance of Geminin-Cdt1 balance for maintaining genomic stability integrity and that both proteins have been correlated with cases of cancer, that screening could hopefully lead to the discovery and development of lead compounds towards anti-cancer drugs.

A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.