Dissecting the different biological effects of oncogenic Ras isoforms in cancer cell lines: could stimulation of oxidative stress be the one more weapon of H-Ras? Regulation of oxidative stress and Ras biological effects

Ras proteins are small GTPase functioning as molecular switches that, in response to particular extracellular signalling, as growth factors, activate a diverse array of intracellular effector cascades regulating cell proliferation, differentiation and apoptosis. Human tumours frequently express Ras proteins (Ha-, Ki-, N-Ras) activated by point mutations which contribute to malignant phenotype, including invasiveness and angiogenesis. Despite the common signalling pathways leading to similar cellular responses, studies clearly demonstrate unique roles of the Ras family members in normal and pathological conditions and the lack of functional redundancy seems to be explainable, at least in part, by the ability of Ras isoforms to localize in different microdomains to plasma membrane and intracellular organelles. This different intracellular compartmentalization could help Ras isoforms to contact different downstream effectors finally leading to different biological outcomes. Interestingly, it has also been shown that Ha- and Ki-Ras exert an opposite role in regulating intracellular redox status. In this regard we suggest that H-Ras specific induction of ROS (reactive oxygen species) production could be one of the main determinants of the invasive phenotype which characterize cancer cells harbouring H-Ras mutations. In our hypothesis then, while K-Ras (not able to promote oxidative stress) could mainly contribute to cancer progression and invasiveness through activation of MAPK and PI3K, H-Ras-mediated oxidative stress could play a unique role in modulation of intercellular contacts leading to a loss of cell adhesion and eventually also to a metastatic spread

Site-selective incorporation and ligation of protein aldehydes

The incorporation of aldehyde handles into proteins, and subsequent chemical reactions thereof, is rapidly proving to be an effective way of generating homogeneous, covalently linked protein constructs that can display a vast array of functionality. In this review, we discuss methods for introducing aldehydes into target proteins, and summarise the ligation strategies for site-selective modification of proteins containing this class of functional handles

Organocatalysis in total synthesis

Peer Reviewe

Synthesis of C-5 and C-10 Vinyloxybenzene containing substrates for the enzyme Protein Farnesyltransferase

Protein Farnesyltransferase (PFTase) is an enzyme that incorporates farnesyl groups into proteins and peptides that end in a certain amino acid sequence. Previously, non-natural substrates that could have also been transferred by PFTase have undergone bioconjugation reactions via copper catalyzed click chemistry. Because of copper’s cytotoxicity, these substrates are not compatible with in vivo applications. Presented are two bioorthogonal substrates that do not require the use of a copper catalyst and they contain a vinyloxybenzene moeity for a photoreaction on a diaryl tetrazole. Both substrates are predicted to be PFTase substrates that will allow for the eventual incorporation of new properties such as fluorescence on targeted proteins. Because many prenylated proteins are involved in signaling processes, this has generated interest in protein prenyl transferases as possible anticancer targets

Host-Mediated Post-Translational Prenylation of Novel Dot/Icm-Translocated Effectors of Legionella Pneumophila

The Dot/Icm type IV translocated Ankyrin B (AnkB) effector of Legionella pneumophila is modified by the host prenylation machinery that anchors it into the outer leaflet of the Legionella-containing vacuole (LCV), which is essential for biological function of the effector in vitro and in vivo. Prenylation involves the covalent linkage of an isoprenoid lipid moiety to a C-terminal CaaX motif in eukaryotic proteins enabling their anchoring into membranes. We show here that the LCV harboring an ankB null mutant is decorated with prenylated proteins in a Dot/Icm-dependent manner, indicating that other LCV membrane-anchored proteins are prenylated. In silico analyses of four sequenced L. pneumophila genomes revealed the presence of eleven other genes that encode proteins with a C-terminal eukaryotic CaaX prenylation motif. Of these eleven designated Prenylated effectors of Legionella (Pel), seven are also found in L. pneumophila AA100. We show that six L. pneumophila AA100 Pel proteins exhibit distinct cellular localization when ectopically expressed in mammalian cells and this is dependent on action of the host prenylation machinery and the conserved cysteine residue of the CaaX motif. Although inhibition of the host prenylation machinery completely blocks intra-vacuolar proliferation of L. pneumophila, it only had a modest effect on intracellular trafficking of the LCV. Five of the Pel proteins are injected into human macrophages by the Dot/Icm type IV translocation system of L. pneumophila. Taken together, the Pel proteins are novel Dot/Icm-translocated effectors of L. pneumophila that are post-translationally modified by the host prenylation machinery, which enables their anchoring into cellular membranes, and the prenylated effectors contribute to evasion of lysosomal fusion by the LCV

Enzymatic Construction of DARPin-Based Targeted Delivery Systems Using Protein Farnesyltransferase and a Capture and Release Strategy

Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC$_{50}$ of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates

Dual modification of biomolecules

With the advent of novel bioorthogonal reactions and “click” chemistry, an increasing number of strategies for the single labelling of proteins and oligonucleotides have emerged. Whilst several methods exist for the site-selective introduction of a single chemical moiety, site-selective and bioorthogonal dual modification of biomolecules remains a challenge. The introduction of multiple modules enables a plethora of permutations and combinations and can generate a variety of bioconjuguates with many potential applications. From de novo approaches on oligomers to the post-translational functionalisation of proteins, this review will highlight the main strategies to dually modify biomolecules

Doctor of Philosophy

dissertationArchaea utilize the mevalonate (MVA) pathway for the biosynthesis of isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP), the two building blocks of all isoprenoids. The archaeal MVA pathway deviates from the classical eukaryotic MVA pathway in the two-step conversion of mevalonate phosphate to IPP by utilizing a putative phosphomevalonate decarboxylase and the recently discovered isopentenyl phosphate kinase (IPK). The latter catalyzes the ATP-dependent phosphoryl transfer reaction to isopentenyl phosphate (IP) to form IPP. Chen and Poulter recently characterized IPKs from Thermoplasma acidophilum (THA) and Methanothermobacter thermautotrophicus (MTH), using its substrate IP and other small molecule isoprenoid monophosphates. In this dissertation, we report the first crystal structures of THA and MTH IPKs in complex with their substrates and products. The structures reveal key active site residues involved in substrate binding and catalysis. We also describe the promiscuity of IPK towards fosfomycin, an antibiotic produced in Streptomyces and the substrate of the fosfomycin resistance enzyme, FomA. The structure of FomA is highly homologous to that of IPK, indicating a possible evolutionary relationship between the two enzymes. Finally, we describe four THA IPK mutants with kinase activities toward the longer chain isoprenoids geranyl phosphate (GP) and farnesyl phosphate (FP), achieved by mutations located in the IP binding site