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Functions of the viral chitinase (CHIA) in the processing, subcellular trafficking and cellular retention of proV-CATH from Autographa californica multiple nucleopolyhedrovirusHodgson, Jeffrey James 05 January 2012 (has links)
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.
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Structural and functional characterization of E2A:KIX interactions in leukemiaDenis, Christopher 15 September 2012 (has links)
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
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A study of the expression of a protein proteinase inhibitor from sweet cornDe Silva, H. A. Rohan January 1991 (has links)
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<sub>L</sub> 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.
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Protein-protein interactions in turnip mosaic potyvirus replication complexThivierge, Karine January 2003 (has links)
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.
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From developing protein-protein interaction strategies to identifying gene functions case studies for transcription factor complexes and ribosome biogenesis genes /Li, Zhihua, January 1900 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.
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A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae characterization of its interaction with a deubiquitinating enzyme /Kee, Younghoon, January 1900 (has links) (PDF)
Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.
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Site directed mutagenesis of lozenge a yeast two-hybrid analysis of transcription factor protein interaction /Boumaza, Lailla. January 2007 (has links)
Thesis (M.S.)--Duquesne University, 2007. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 76-80) and index.
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Regulation of the neuronal K⁺-Cl⁻ cotransporter KCC2 by protein associated with MycGarbarini, Nicole Jodela. January 2008 (has links)
Thesis (Ph. D. in Neuroscience)--Vanderbilt University, May 2008. / Title from title screen. Includes bibliographical references.
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Use of electrospray ionization mass spectrometry to study protein conformation and protein-protein interactionsWatt, Stephen J. January 2005 (has links)
Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. EMBARGOED-this thesis is subject to a six months embargo (07/09/06) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 159-194.
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Structure-function analysis of two Drosophila neuronal cell adhesion proteins fasciclin I and amalgam /Liu, Xiao-yu. January 2007 (has links)
Thesis (Ph. D.)--Ohio State University, 2007.
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