Not available

Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.

Not available / text

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

Many protein-protein interactions, especially those involved in eukaryotic signalling, are mediated by PDZ domains through the recognition of hydrophobic C-termini. The availability of experimental PDZ interaction data sets have led to the construction of computational methods to predict PDZ domain-peptide interactions. Such predictors are ideally suited to predict interactions in single organisms or for limited subsets of PDZ domains. As a result, the goal of my thesis has been to build general predictors that can be used to scan the proteomes of multiple organisms for ligands for almost all PDZ domains from select model organisms. A framework consisting of four steps: data collection, feature encoding, predictor training and evaluation was developed and applied for all predictors built in this thesis. The first predictor utilized PDZ domain-peptide sequence information from two interaction data sets obtained from high throughput protein microarray and phage display experiments in mouse and human, respectively. The second predictor used PDZ domain structure and peptide sequence information. I showed that these predictors are complementary to each other, are capable of predicting unseen interactions and can be used for the purposes of proteome scanning in human, worm and fly. As both positive and negative interactions are required for building a successful predictor, a major obstacle I addressed was the generation of artificial negative interactions for training. In particular, I used position weight matrices to generate such negatives for the positive only phage display data and used a semi-supervised learning approach to overcome the problem of over-prediction (i.e. prediction of too many positives). These predictors are available as a community web resource: http://webservice.baderlab.org/domains/POW. Finally, a Bayesian integration method combining information from different biological evidence sources was used to filter the human proteome scanning predictions from both predictors. This resulted in the construction of a comprehensive physiologically relevant high confidence PDZ mediated protein-protein interaction network in human.

Many protein-protein interactions, especially those involved in eukaryotic signalling, are mediated by PDZ domains through the recognition of hydrophobic C-termini. The availability of experimental PDZ interaction data sets have led to the construction of computational methods to predict PDZ domain-peptide interactions. Such predictors are ideally suited to predict interactions in single organisms or for limited subsets of PDZ domains. As a result, the goal of my thesis has been to build general predictors that can be used to scan the proteomes of multiple organisms for ligands for almost all PDZ domains from select model organisms. A framework consisting of four steps: data collection, feature encoding, predictor training and evaluation was developed and applied for all predictors built in this thesis. The first predictor utilized PDZ domain-peptide sequence information from two interaction data sets obtained from high throughput protein microarray and phage display experiments in mouse and human, respectively. The second predictor used PDZ domain structure and peptide sequence information. I showed that these predictors are complementary to each other, are capable of predicting unseen interactions and can be used for the purposes of proteome scanning in human, worm and fly. As both positive and negative interactions are required for building a successful predictor, a major obstacle I addressed was the generation of artificial negative interactions for training. In particular, I used position weight matrices to generate such negatives for the positive only phage display data and used a semi-supervised learning approach to overcome the problem of over-prediction (i.e. prediction of too many positives). These predictors are available as a community web resource: http://webservice.baderlab.org/domains/POW. Finally, a Bayesian integration method combining information from different biological evidence sources was used to filter the human proteome scanning predictions from both predictors. This resulted in the construction of a comprehensive physiologically relevant high confidence PDZ mediated protein-protein interaction network in human.

The baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, thereby enhancing dissemination of progeny virions in nature. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH) is poorly understood and a mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not been determined. The cathepsins of Autographa californica multiple nucleoplyhedrovirus (AcMNPV) and most other group I alphabaculoviruses have well-conserved chymotrypsin cleavage (Y11) and myristoylation sites (G12) suggestive of proteolytic cleavage to generate proV-CATH, and subsequent acylation which could promote membrane anchoring in order to foster cellular retention of the protein. Proteolytic iii N-terminal processing of baculoviral procathepsin was determined by fusing HA epitope-coding tags to the 5’ and/or 3’ ends of v-cath, indicating that the gene is expressed as a pre-proenzyme. However no evidence for myristoylation of proV-CATH was found, suggesting that another mechanism is responsible for retaining proV-CATH in cells. Prior evidence suggested that CHIA is a proV-CATH folding chaperone and that lack of chiA expression causes proV-CATH to become insoluble and unable to mature into V-CATH enzyme. A putative CHIA chaperone activity for assisting in proV-CATH folding implies that proV-CATH and CHIA interact in the ER of infected cells. Fluorescence microscopy demonstrated co-localization of CHIA-GFP and proV-CATH-RFP fusion proteins in the ER. An mRFP-based bimolecular fluorescence complementation (BiFC) assay helped to determine not only that AcMNPV proV-CATH interacts directly with the full-length viral CHIA, but also that it independently binds to the N-terminal chitin-binding domain (CBD) and C-terminal active site domain (ASD) of CHIA, in the ER during virus replication. Moreover, reciprocal Ni/HIS pull-downs of HIS-tagged proteins confirmed the proV-CATH interactions with CHIA, or with the CBD and ASD biochemically. The reciprocal co-purification of proV-CATH with all three polypeptides (CHIA, CBD, ASD) suggests proV-CATH specifically interacts with each of them, and corroborates the BiFC data. Furthermore, CHIA KDEL deletion allowed for premature secretion of not only CHIA but also of proV-CATH, suggesting that the CHIA/proV-CATH interaction in the ER aids cellular retention of proV-CATH. In contrast to prior reports, it was also determined that CHIA is iv dispensable for correct folding of proV-CATH since proV-CATH produced by a chiA-deficient virus was soluble, prematurely secreted from cells and could mature into V-CATH enzyme. Taken together, these data indicate that the viral chitinase plays a major role in ensuring that proV-CATH is neither prematurely secreted nor activated to V-CATH enzyme.

The E2A proteins are transcription factors critical for B-lymphopoiesis. A chromosomal translocation involving the E2A gene promotes acute lymphoblastic leukemia (ALL) through expression of the oncoprotein E2A-PBX1. Two activation domains of E2A-PBX1, AD1 and AD2, have been implicated in transcription mediated by recruitment of the transcriptional co-activator CBP/p300. A motif has been identified within AD1 that is important for recruiting CBP/p300, known as PCET. This recruitment requires an interaction between the activation domains of E2A-PBX1 and the KIX domain of CBP/p300. The KIX domain recognizes a generic ΦXXΦΦ sequence (Φ corresponds to a hydrophobic residue) found in the activation domains of numerous transcription factors. Mutation of leucine 20 in PCET has been shown to abrogate ex vivo immortalization of murine bone marrow and oncogenesis in a murine bone marrow transplantation model. A similar sequence is also found in AD2 and is implicated in E2A transcriptional activity and recruitment of CBP/p300. The structural details of these interactions remain largely unknown. NMR spectroscopy was used to determine the solution structure of the PCET:KIX complex, and the functional consequences of the Leu20Ala mutation were structurally rationalized. Other PCET mutations informed by this structure were tested and correlations were found between in vitro binding affinities and both transcriptional activation and immortalization. The binding site of the ΦXXΦΦ-containing E2A AD2 peptide was mapped to the same site on the KIX domain used by the PCET motif. A model of this complex was generated and mutations were tested using a similar approach as was used for PCET. E2A AD2 binds the KIX domain with lower affinity than the PCET motif and is not required for immortalizing bone marrow. A mutation that increases the affinity of E2A AD2 for the KIX domain to levels approaching that seen for the PCET:KIX interaction restores transcriptional activation and immortalization, demonstrating that immortalization by E2A-PBX1 is an affinity dependent process involving the KIX domain of CBP/p300. These studies indicate that the activation domains of E2A-PBX1 serve to support the in vivo function of the oncoprotein and that the PCET:KIX complex is a potential target for novel therapeutics in E2A-PBX1+ leukemia. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-09-13 13:30:48.848

Sweet Corn Inhibitor (SCI), a small (11811Da.) protein from the seeds of opaque-2 corn is a potent and specific inhibitor of trypsin and the activated Hageman Factor (Factor βXIIa) of the human blood plasma coagulation system. With the eventual aim of obtaining insight into the structure- function relationships of the selective SCI-pXIIa interaction, a synthetic gene for SCI was cloned into Saccharomyces cerevisiae (yeast) and Escherichia coli (E.coli) expression systems in an attempt to obtain overexpression of the recombinant gene product. The establishment of functional expression, together with an isolation and purification procedure for SCI would provide a system for obtaining selected reactive-site mutants of SCI by cassette- and oligonucleotide-directed mutagenesis. A yeast secretion vector for a truncated form of SCI (tSCI) was constructed by cloning the gene for α-factor prepro-tSCI fusion, downstream to the α-mating factor (MFα1) promoter of yeast. Yeast transformants containing the expression vector failed to express and secrete the desired product. The synthetic gene encoding the complete SCI sequence was cloned into E.coli expression vectors that directed both cytoplasmic and periplasmic expression. In cytoplasmic expression, the SCI gene was cloned directly downstream to the powerful, inducible λ-phage PL- and trc-promoters. No expression was obtained with the latter. With the former, expression levels of up to 3% of the total bacterial protein were obtained. These levels were improved 3- to 4-fold on incorporation of the E.coli dnaY gene product. Solubilisation and refolding of the purified SCI inclusion bodies failed to yield the active, correctly folded product. Failure to obtain an N-terminal sequence indicated an incompletely processed N-terminal methionine. For periplasmic expression, SCI, fused in-frame to the signal sequence of OmpA, a major E.coli outer membrane protein, was cloned into the same λ-phage P_L promoter vector. High levels (=10%) of expression of insoluble SCI were obtained. The nearly homogeneous product was obtained by a two-step procedure, involving ion-exchange chromatography, followed by hydrophobic interaction chromatography. Characterisation by N-terminal sequencing, SDS-PAGE and electrospray mass spectrometry, confirmed the presence of correctly processed SCI in the form of covalently associated dimers. Refolding studies are at present in progress.

Interactions between plant and virus proteins play pivotal roles in many processes during the viral infection cycle. Analysis of protein-protein interactions is crucial for understanding virus and host protein functions and the molecular mechanisms underlying viral infection. Several interactions between virus-encoded proteins have been reported. However, few interactions between viral and plant proteins have been identified so far. To examine interactions between Turnip mosaic potyvirus (TuMV) proteins and plant proteins, recombinant proteins were produced and used in ELISA-type assays and in in vitro co-immunoprecipitation experiments. An interaction between TuMV P1 proteinase and wheat poly(A)-binding protein (PABP) was identified. An interaction between P1 protein and the plant Arabidopsis thaliana eukaryotic initiation factor (iso)4E [eIF(iso)4E] was also found. Finally, potential interactions between both TuMV CI and P1 proteins and between TuMV CI protein and eIF(iso)4E were identified.