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.

Ribosome-inactivating proteins (RIPs) are cytotoxins that remove a specific adenine from the sarcin-ricin loop (SRL) of large ribosomal RNA and in turn inhibit protein synthesis. Apart from N-glycosidase activity, some RIPs are found to possess antiviral activity and the suppression on human immunodeficiency virus (HIV) has been extensively studied. / Maize RIP stands out from other members for having an internal inactivation region and requires proteolytic removal to regain full activity. We have exploited the innate regulatory mechanism of maize RIP and increased its specificity towards HIV by adding the HIV protease recognition site to the inhibitory segment. Our results demonstrated the wild-type maize RIP is inhibitory on simian immunodeficiency virus (SIV) replication in rhesus macaque and showed the HIV sensitive variant undergoes specific proteolytic activation upon viral infection and exerts enhanced in vitro antiviral effects. Therapeutic applications of RIPs are often restricted by short in vivo half-life and strong allergic responses and we attempted to improve the therapeutic potential of maize RIP by coupling with polyethylene glycol (PEG). Two mutants were generated for PEGylation and the resultant MOD-PEG₂₀ₖ variant was shown to be less sensitive to antibody recognition and has a prolonged plasma half-life, suggesting it may have enhanced therapeutic values. Besides, the applicability of protease-activation system in RIPs without inactivation loop was tested using ricin A chain (RTA) as the test case and HIV recognition sites were introduced either within or at C-terminus of the protein. The C-terminal RTA variants were specifically processed and had the anti-HIV activity increased in HIV-infected cells. / The present work illustrates the potential development of maize RIP as an anti-HIV agent and shows PEGylation can serve to enhance the protein for in vivo applications. Besides, the engineering of RTA with HIV recognition site suggests the potential of the protease-activation strategy to other RIPs for activity control. / 核糖體失活蛋白（RIPs）是一種細胞毒素蛋白，能特異地水解核糖體sarcin-ricin環（SRL），通過脫嘌呤抑制核糖體的蛋白合成功能。除此功能以外，很多RIP還具有抗病毒的活性，如抗人免疫缺陷病毒（HIV）的活性。 / 玉米RIP與其他的RIP不同，它含有一段內部失活結構域，需通過蛋白水解作用移除該結構域才能成為活性體。我們利用玉米RIP的這一特性，通過對內部結構域的改造，獲得了兩個可被HIV蛋白酶特異識別的突變體。體外實驗証明HIV可以特異地識別突變體上的蛋白酶切割位點，從而將其啟動產生抗病毒活性。另外，我們以蓖麻毒素A鏈（RTA）為例，分別於蛋白質的內部和碳端插入HIV識別序列，驗證了蛋白酶啟動系統在沒有內部失活結構域的RIP中，也能通過HIV蛋白酶的切割而活化並取得抗病毒活性。我們還揭示玉米RIP活性體可以有效抑制猿免疫缺陷病毒（SIV）在感染恒河猴體內的複制。此外，我們嘗試通過與聚乙二醇（PEG）融合來優化玉米RIP的免疫原性和半衰期，成功製備了兩種融合突變體，MOD-PEG₂₀ₖ顯示較不容易被抗體識別且延長了血液半衰期。 / 綜上所述，我們的研究表明玉米RIP作為抗HIV抑制劑具有良好的研發前景，而RTA的改造展示蛋白酶啟動系統可應用於其他RIP，同時我們還證明了PEG修飾可以很好的應用於蛋白類藥物的研發。 / Au, Ka Yee. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Title from PDF title page (viewed on 17, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

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

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 205-212.

No description available.

Human Immunodeficiency Virus Type 1 (HIV-1), the virus that causes Acquired Immunodeficiency Syndrome (AIDS), attacks the immune system leaving patients susceptible to opportunistic infections that eventually cause death. Highly Active Antiretroviral Therapy, HAART, is the current drug strategy used to combat HIV. It is a combination therapy that includes HIV-1 Reverse Transcriptase and HIV-1 Protease inhibitors. Drug resistant strains arise that evade current HAART treatments; therefore novel drugs are needed. HIV-1 regulatory proteins such as Tat, Rev, Nef, Vpr, Vpu, and Vif are attractive new drug targets. Of particular interest is the HIV-1 Vif protein and its cellular binding partner APOBEC3G. In the absence of HIV-1 Vif, APOBEC3G, a cytidine deaminase, is able to mutate the viral cDNA and render the virus noninfectious. HIV-1 Vif binds to APOBEC3G and targets it for proteosomal degradation through an interaction with a Cullin-RING ligase complex. Blocking the HIV-1 Vif APOBEC3G interaction would allow APOBEC3G to perform its antiviral function. An attractive strategy to target the HIV-1 Vif APOBEC3G interaction would be a structure-based one. To apply structure-based drug design approaches to HIV-1 Vif and APOBEC3G, I attempted to collect high resolution structural data on HIV-1 Vif and APOBEC3G. My attempts were unsuccessful because the milligram quantities of soluble protein required were not obtained. Therefore, in Chapter III I used chemical cross-linking and mass spectrometry to probe the structural topology of HIV-1 Vif obtaining low resolution structural data. Chemical cross-linking formed HIV-1 Vif multimers including dimers, trimers, and tetramers. Analysis of the cross-linked monomer revealed that HIV-1 Vif’s N-terminal domain is a well-folded, compact, globular domain, where as the C-teriminal domain is predicted to be disordered. In addition, disorder prediction programs predicted the C-terminal domain of HIV-1 Vif to be disordered. Upon oligomerization the C-terminal domain undergoes a disorder-to-order transition that not only facilitates oligomerization but may facilitate other protein-protein interactions. In addition, HIV-1 Vif oligomerization bring Lys34 and Glu134 in close proximity to each other likely creating one molecular surface forming a “hot spot” of biological activity. In Chapter IV I confirmed my low resolution structural data via peptide competition experiments where I identified peptides that can be used as scaffolds for future drug design. HIV-1 Vif oligomerization is concentration dependent. The HIV-1 Vif peptides Vif(29-43) and Vif(125-139) were able to disrupt HIV-1 Vif oligomerization, which confirms the low resolution structural data. HIV-1 Vif peptides Vif(25-39) and Vif(29-43) reduced the amount of APOBEC3G immobilized on the Protein A beads, reduced the amount of HIV-1 Vif interacting with APOBEC3G, or degraded APOBEC3G itself. These peptides could be used as scaffolds to design novel drugs that disrupt the function of HIV-1 Vif and or APOBEC3G. Therefore, low resolution structural data and peptide competition experiments were successful in identifying structurally important domains in HIV-1 Vif. They also provided insight into a possible mechanism for HIV-1 Vif function where a disorder-to-order transition facilitates HIV-1 Vif’s ability to interact with a diverse set of macromolecules. These data advance our structural understanding of HIV-1 Vif and they will facilitate future highresolution studies and novel drug designs.

No description available.

Amorphous solid dispersion (ASD) is a popular method to increase drug solubility and consequently poor drug bioavailability. Cellulose ω-carboxyesters were designed and synthesized specifically for ASD preparations in Edgar lab that can meet the ASD expectations such as high Tg, recrystallization prevention and pH-triggered release due to the free -COOH groups. Rifampicin (Rif), Ritonavir (Rit), Efavirenz (Efa), Etravirine (Etra) and Quercetin (Que) cellulose ester ASDs were investigated in order to increase drug solubility, prevent release at low pH and controlled release of the drug at small intestine pH that can improve drug bioavailability, decrease needed drug content and medication price to make it affordable in third world countries, and extent pill efficiency period to improve patient quality of life and adherence to the treatment schedule. The studies were compared with cellulose based commercial polymers to prove the impact of the investigation and potential for the application. Furthermore, the in vitro results obtained were further supported by in vivo studies to prove the significant increase in bioavailability and show the extended release. The need of new cellulose derivatives for ASD applications extended the research area, the design and synthesis of a new class of polymers, alkyl cellulose ω-carboxyesters for ASD formulations investigated and the efficiency of the polymers were summarized to show that they have the anticipated properties. The polymers were synthesized by the reaction of commercial cellulose alkyl ethers with benzyl ester protected, monofunctional hydrocarbon chain acid chlorides, followed by removal of protecting group using palladium hydroxide catalyzed hydrogenolysis to form the alkyl cellulose wcarboxyalkanoate. Having been tested for ASD preparation, it was proven that the polymers were efficient in maintaining the drug in amorphous solid state, release the drug at neutral pH and prevent the recrystallization for hours, as predicted. / Ph. D.