As the first structural example of this class of proteins, crystals of Pro-RIP and MOD were grown and diffracted to 2.4 and 2.5 A respectively. The structures of the two proteins are solved and found to be highly similar, with main chain RMSD of 0.519. Each protein has two domains. The N-terminal domain consists of five alpha-helices and five-stranded mixed beta-sheet. The conserved active site residues Y94, Y130, E207, R210 and W241, similar to those of other RIPs, are located at the cleft between the N-terminal and C-terminal domains. In Pro-RIP, the 25-amino acid internal inactivation region is found on the surface of the N-terminal domain and consists of a flexible loop followed by a long alpha-helix. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure of maize RIP reveals that the active site is too small to accommodate two glutamate residues and suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. / Pull-down assay indicated that the internal inactivation region diminished the interaction of Pro-RIP with purified ribosomes and ribosomal proteins P0, P1 and P2. Surface plasmon resonance assays showed that Pro-RIP has a slower association rate and faster dissociation rate on intact ribosomes when compared to MOD, resulting 80-fold decrease in binding affinity. These evidences strongly suggested that the reduced ribosome-inactivating activity and cytotoxicity of Pro-RIP is the result of its diminished interaction with the ribosomes. The ribosome binding site of MOD is found to be different from TCS and saporin, which are located between the anti-parallel beta-sheet in the C-terminal domain. In MOD, the positive-charged residues K158, K159, K160 and K161 that were found to be important for ribosome binding are located in the N-terminal domain, underneath the internal inactivation region. / Ribosome-inactivating proteins (RIPs) are rRNA N-glycosidases, which hydrolyze the N-glycosidic bond of A-4324 in 28S rRNA of eukaryotic ribosomes. Based on the number of subunits, RIPs are grouped into three classes. Type I RIPs (e.g. trichosanthin and saporin) are monomeric polypeptide with molecular weights of 25-32 kDa. Type II RIPs (e.g. ricin and cinnamomin) are heterodimeric proteins whose subunits are linked by a disulphide bridge, with molecular weights of 60-65 kDa. Chain A of type II RIPs is the catalytic subunit, while chain B is the lectin subunit, which facilitates the cellular entry of the protein by interacting with carbohydrates on the cell surface. Maize ribosome-inactivating protein is classified as a type III RIP, or an atypical RNA N-glycosidase. It is synthesized and stored in the kernel as a 34 kDa inactive precursor (Pro-RIP). During germination, the precursor undergoes proteolysis to generate a two-chain active RIP (MOD). Previous study has found that the 25-amino acid residues at the acidic internal inactivation region, which are removed during activation of Pro-RIP, is the major control element to suppress its in vitro protein synthesis inhibition activity. / Since the internal inactivation region of Pro-RIP controls the ribosome-inactivating activity and cytotoxicity, it provides an opportunity to engineer an on/off switch forits activity by HIV-1 protease through engineering HIV-1 protease recognition sites into the internal inactivation region of Pro-RIP. A variant that contains two HIV-1 protease recognition sites incorporated to the 25-amino acid internal inactivation region was found to be activated by HIV-1 protease in vitro. This variant entered cells more efficiently than Pro-RIP and was as cytotoxic as MOD. This switch may be applied to other RIPs such as ricin A chain and other protease recognition sequences may be used for increasing the specificity of an RIP toward viral infected cells. / The internal inactivation region of Pro-RIP greatly decreases its cytotoxicity, but not cellular uptake through alpha-2 macroglobulin receptor. On the contrary, the acidic residues within the region hinder fluid-phase endocytosis. Moreover, it is found that the internal inactivation region does not affect sub-cellular localization of the protein - MOD and Pro-RIP locate in the same cellular compartment (nucleus in JAR or cytoplasm in J774A.1 and C8166). / Mak, Nga Sze Amanda. / "July 2007." / Adviser: Shaw Pang Chui. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 216-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344954 |
| Date | January 2009 |
| Contributors | Mak, Nga Sze Amanda., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (236 leaves : ill. (chiefly col.)) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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