The 26S proteasome is the principal degradation structure of intracellular proteins in eukaryotes. The proteasome is composed of two 19S regulatory particles and a 20S catalytic core. The lid region of the 19S proteasome comprises at least 8 subunits that recognizes proteasomal substrates, including misfolded, truncated, or erroneously posttranslated proteins, as well as other tightly controlled and short-lived proteins. Such substrates are typically tagged with a small peptide called ubiquitin to facilitate their rapid and efficient recognition and translocation to the proteasome. The objective of this study was to characterize the Schistosoma mansoni and human homologues of proteasomal lid subunit POH1/RPN11. Earlier reports had suggested that overexpression of the fission yeast padl homologue can influence the activity of an AP1-like transcription factor. We showed that SmPOH, the Schistosoma mansoni homologue, stabilizes AP1 transcription factor c-Jun both in vitro and in cultured mammalian cells. We also demonstrated a dose-dependent SmPOH-induced stabilization of c-Jun in vitro. In contrast, SmPOH did not stabilize another well-known proteasomal substrate, p53, in vitro. Others later demonstrated that yeast POH1/RPN11 is a divalent cation-dependent metalloprotease deubiquitinase and outlined a conserved catalytic motif termed JAMM/MPN+ responsible for this activity. To better understand the structure-function relationship of POH1/RPN11 with regard to c-Jun stabilization, we showed that the Cys-120 residue of human POH1 (hPOH1), embedded in the JAMM/MPN+ motif, is required for the stabilizing effect on c-Jun. Furthermore, we established that hPOH1 influences c-Jun stability by altering its state of ubiquitination and intracellular localization. Parallel experiments were conducted to test the effect of hPOH1 on another proteasomal substrate, p27Kip, but no changes could be seen either in its stability or its localization. RNAi experiments targeting SmPOH transcripts in cultured schistosomula resulted in a lethal phenotype, thus illustrating the critical role of SmPOH expression in the parasite. Expression levels of SmPOH were reduced by ∼ 90% following a 9-day incubation with siRNAs targeting the SmPOH sequence. We further performed a survey to examine the expression levels of multiple proteasome subunits in cercaria, schistosomula, and adult stages of Schistosoma mansoni. Our results suggest a pivotal role for the proteasome in the development of the parasite. A better understanding of the function of the proteasome in S. mansoni may lead to the identification of novel drug targets and newer approaches for chemotherapy.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111843 |
| Date | January 2006 |
| Creators | Nabhan, Joseph. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Institute of Parasitology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002612127, proquestno: AAINR32222, Theses scanned by UMI/ProQuest. |
Page generated in 0.002 seconds