Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.

Universiẗat, Diss., 2005--Freiburg (Breisgau). / Erscheinungsjahr an der Haupttitelstelle: 2004.

Human papilloma virus (HPV) is a ubiquitous virus implicated in a growing list of cancers, particularly cervical cancer‐ the second most common cancer among women worldwide. Although persistent infection with high‐risk oncogenic HPVs such as types ‐16 or ‐18 is necessary, additional factors like co‐infection with other viruses can play a role in cancer progression. Protein‐protein interactions play a central role in the infection, survival and proliferation of the virus in the host. Although some interactions of HPV proteins are well characterised, it is essential to discover other key viral interactions to further improve our understanding of the virus and to use this knowledge for the development of newer biomarkers and therapeutics. The aim of this study was to systematically analyse the interactions of HPV‐16 proteins using yeast two‐hybrid (Y2H). To achieve this, a clone collection of the viral proteome was generated by recombinatorial cloning and three independent Y2H screens were performed: (i) Intra‐viral screen to identify interactions among the HPV‐16 proteins; (ii) Inter‐viral screen to identify interactions with proteins of Herpes Simplex Virus (HSV) which is suggested to be a co‐factor; and (iii) Virus‐host screen to identify novel cellular binding partners. The intra‐viral Y2H screen confirmed some of the previously known interactions and also identified binding of the E1 and E7 proteins. Deletion mutagenesis was performed to map the interaction domains to the amino‐terminal 92 amino acids of E1 and carboxy‐terminal CxxC domain of E7. Replication assays suggest a possible repression of E1‐mediated episomal replication by direct binding of E7. The inter‐viral Y2H screen identified interactions of HPV proteins with seventeen HSV‐1 proteins including transcriptional regulator ICP4 and neurovirulance factor ICP34.5. The biological relevance of these interactions in the context of co‐infection is discussed. The virus‐host screen performed against a human cDNA library identified 54 interactions, a subset of which was validated by biochemical pull‐down assays. The functional relevance of an interaction between E7 and a proto‐oncogene spermatogenic leucine zipper protein (SPZ1) was further investigated suggesting a role of SPZ1 in E7‐mediated cell proliferation. The work presented in this thesis identifies several novel interactions of HPV proteins. Future work will involve the in‐depth elucidation of biological relevance of these interactions. In particular, the interactions of E7 with E1 and SPZ1 are of great interest to improve our understanding of the life cycle and pathogenesis of the virus which can be applied for improved strategies of prevention and treatment of malignancies caused by HPV.

The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.

The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.