Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 104-139). Also available online through Digital Dissertations.

Universiẗat, Diss., 2001--München.

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, the intracellular localization of Arabidopsis GLYR1 was reappraised by conducting microscopy-based experiments that address some novel mechanisms by which proteins can be directed to peroxisomes. For instance, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, was not sufficient for peroxisomal targeting. Collectively, the results define the cytosol as the intracellular location of GLYR1 and provide a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol. / NSERC

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska instututet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.