Browsing by Subject "Prenylation"
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Item Exploitation of prenylation in biomolecules: cell-penetrating prenylated peptides and prenyalted proteins.(2009-05) Wollack, James W.Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and in vivo analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. These peptides were shown to have intrinsic cell-penetrating abilities and enter cells through a passive transport mechanism and localize to the endomembrane surrounding the nucleus. Their cell-penetrating properties were shown to be mostly due to their prenylation state and not their peptide sequence. Once discovered other derivatives of these peptides were used to study peptide prenylation and enzymatic processing in living cells. Also in this work other peptides and proteins were modified with non-natural prenyl diphosphates. This work aimed at honing in on the smallest alkyne or azide labeled prenyl diphosphate that is a substrate for PFTase. An alkyne containing PFTase substrate was identified that contained only 9 non-hydrogen atoms. The substrate was used to modify "Caax Box" containing proteins and peptides. Further proteolysis of the "Caax Box" allowed for alkyne modification of biomolecules with the addition of only a single modified cysteine. This result allows for the addition of alkyne functionality without the addition of a long hydrophobic chain that can hinder a biomolecules solubility or reactivity.Item Negative regulation of oncogenic Ras signaling by mitogen-activated protein kinase phosphatase-3.(2010-07) Zeliadt, Nicholette A.Activating mutations in the Ras oncogene are found in ~30% of all human cancers. Ras is a central regulator of intracellular signaling pathways, such as the Raf/MEK1/2/ERK1/2 protein kinase cascade, that modulate cell cycle progression, cell proliferation, and cell survival. Chronic activation of Ras-stimulated pathways is believed to play a key role in cellular transformation, although constitutive activation of Ras alone is not sufficient for tumor development. The goal of these studies is to further understand the biochemical events that occur during carcinogenesis in cells that express activated Ras that make cells susceptible to tumor promoting stimuli. This knowledge is important for the development of new strategies to diagnose, prevent and treat cancer. To date, attempts at developing small molecule inhibitors of oncogenic Ras have been unsuccessful. Therefore, efforts have instead focused on targeting downstream effectors of Ras, such as the Raf/MEK1/2/ERK/12 cascade, or preventing the posttranslational prenylation of Ras that is critical for its function. The aim of these studies is two-fold: 1) to elucidate the signaling events that occur in cells exposed to tumor-promoting stimuli; and 2) to develop tools for the study of prenylation in living cells.Item New Developments in Solid Phase Peptide Chemistry Facilitate the Study of the Unique Metalloprotease, Ste24(2023-03) Bader, TaysirProtein and peptide prenylation is an essential biological process involved in many signal transduction pathways. In its most prevalent form, prenylation involves three enzymatic steps; transfer of an isoprenoid moiety by FTase or GGTaseI to the cysteine of a C-terminal CaaX sequence (where C is cysteine, a is an aliphatic amino acid, and X is a variable amino acid dictating whether a farnesyl or longer geranylgeranyl chain is added), proteolytic removal of the aaX sequence by ZMPSTE24 or RCE1, and finally carboxymethylation of the newly exposed C-terminal cysteine by ICMT enzyme. ZMPSTE24 also catalyzes a second cleavage step upstream of the CaaX site in prelamin A, and mutations abolishing this step lead to progeroid diseases. Ste24 is the yeast homolog of ZMPSTE24 and is the founding member of a unique class of integral membrane metalloproteases. Its precise mechanism of action has yet to be explored fully at the molecular level, and it is unique in that it performs two separate cleavage reactions sequentially at distinct sites in the same substrate molecule. The system historically used for studying prenylation is the mating pheromone a-Factor, a dodecameric peptide with a methyl ester C-terminal farnesylated cysteine. Producing this and other prenylated peptides presents four key challenges: the C-terminal cysteine is prone to epimerization, the terminal methyl ester is not readily available through traditional SPPS, the terminal cysteine has to be chemoselectively modified with a hydrophobic prenyl chain on the C-terminal cysteine, and often there are several cysteines in the sequence which necessitates additional orthogonal protecting group chemistry. In this work, various synthetic methodologies were developed in order to overcome these challenges, and then utilized for the production of a myriad of peptide probes based on the structure of a-Factor. These probes were used to study both cleavage steps of ZMPSTE24, as well as the other enzymes involved in the prenylation pathway.Item Prenylated Chemically Self-Assembled Nanorings: A Versatile Platform for Macro-Chemical Biology(2022-02) Wang, YiaoMacro-chemical biology uses chemical methods and biomacromolecules to study and manipulate biological systems at the cellular level, and biomacromolecules capable of manipulating cell fates have been demonstrated exceedingly valuable for assorted fundamental research and therapeutic applications. Protein conjugates, as a type of chemically engineered hybrid macromolecules, are synthesized through conjugations of proteins with functional molecules of diverse types and have been exploited to manipulate cell functions in various aspects, including regulation of intercellular interactions, intervention in intracellular biological pathways, and termination of cell proliferation. Although effective in numerous pre-clinical studies, the clinical translation of therapeutic protein conjugates remains challenging and hindered by some limitations, which in turn underlines some ideal features for designing clinically desirable protein conjugates. Hence, to meet these challenges, our group has constructed a versatile macromolecular platform with self-assembling protein-lipid conjugates, namely, prenylated chemically self-assembled nanorings (CSANs), for macro-chemical biology studies. This multivalent system has exhibited exceptional stability, function reversibility, target cell selectivity, and broad utility, and was shown to effectively regulate cell fates for multiple biomedical applications. We first used the prenylated CSANs as a universal non-genetic system to stably modify cell surface and demonstrated they can mediate reversible cell-cell interactions for fundamental research and adoptive cell therapy. Thereafter, we discovered the CSANs can specifically transfer from the CSAN-modified cell to the target cell during cell-cell interactions, and such intercellular CSAN transfer is dependent on specific ligand-receptor engagement. Therefore, with fluorescent dyes conjugated to the farnesylated CSANs through click reactions, the CSAN assisted cell-cell cargo transfer (C4T) was utilized as a tool to record cell-cell interactions. Furthermore, by conjugating functional oligonucleotides and cancer drugs to the farnesylated CSANs, we were able to manipulate cell functions by the C4T approach. Finally, the cancer drug MMAE was conjugated to the CSANs to form a targeted drugdelivery system for the treatment of EGFR-positive cancer. The MMAE-loaded anti-EGFR CSANs manifested notable cytotoxicity and selectivity against EGFR-positive cancer cells and were also found to provoke immunogenic death of the cancer cells. Altogether, these promising results demonstrate that the prenylated CSANs are a versatile platform applicable to a variety of fundamental research and therapeutic purposes.Item Protein prenylation in the pathogenesis of Alzheimer’s Disease and its therapeutic potential(2021-06) Jeong, AngelaWith the unprecedented growth of senior population, Alzheimer’s disease (AD) has risen in prevalence to approximately 50 million people afflicted worldwide. While the molecular mechanisms underlying the AD pathogenesis have yet to be elucidated, emerging evidence suggests that a key posttranslational lipid modification of proteins, called prenylation, may play an important role in the pathogenesis of AD. Isoprenoids, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), are synthesized from mevalonate in the cholesterol biosynthesis pathway, and the attachment of these lipid prenyl groups is catalyzed by farnesyltransferase (FT) and geranylgeranyl transferases (GGTs), respectively. Once prenylated, proteins localize to cell membranes, where they interact with downstream effectors, and regulate various cellular processes. Multiple lines of evidence have shown that modulation of protein prenylation affects AD neuropathology. To investigate the contribution of each prenylation pathway to AD pathogenesis, our laboratory previously generated the transgenic AD model mice (APP/PS1) with systemic haplodeficiency of FT or GGT. While haplodeficiency of either FT or GGT reduced Aβ deposition and neuroinflammation, only FT haplodeficiency rescued cognitive deficits, suggesting distinct functions of the two prenylation pathways. To follow up on the previous work, my dissertation research is aimed to: 1) investigate the AD pathology-associated changes in prenylation; 2) elucidate the role of neuronal FT in the AD pathogenesis; 3) identify prenylation substrates involved in AD pathogenesis, which can be potentially targeted for therapeutic development. Studies with postmortem human brain tissue samples from two cohorts found that FT levels and the farnesylation of H-Ras were significantly elevated in the brains with AD. In line with this evidence from human samples, neuronal FT deletion attenuated memory impairment and mitigated the Aβ levels by reducing its production as well as normalizing the heightened mTORC1 activity in APP/PS1 mice. Finally, experiments employing a novel prenylomic profiling approach identified proteins whose prenylations were significantly upregulated in APP/PS1 mice compared to WT controls. Taken together, the findings from my dissertation research indicate that aberrant upregulation of protein farnesylation contributes to the pathogenic process of AD, and targeting protein farnesylation and/or downstream signaling pathways of farnesylated proteins may confer therapeutic benefits against AD.Item Solid-phase synthesis of C-Terminal peptide Libraries for studying the specificity of protein farnesyltransferase(2014-07) Wang, Yen-ChihProtein prenylation is a common post-translational modification of specific protein-derived cysteine residues in eukaryotic cells. To study the substrate specificity of these enzymes, the primary strategy employed to date has involved the synthesis, purification and assaying of individual peptides. As an improvement, here we describe the synthesis of peptides containing free C-termini on solid supports. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with R. norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results while the analogue synthesized from a C5 unit gave a different pattern of reactivity. Finally the substrate specificities of PFTases from three organisms (R. norvegicus, S. cerevisiae and C. albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.