In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

Three mammalian nuclear lamin proteins, lamin B1, lamin B2 and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility that farnesyl transferase inhibitors can represent effective treatment for HGPS. However, there are concerns about their use for this purpose given the potential for alternative prenylation pathways.

The discovery of mammalian protein prenylation was originally motivated by an effort to identify a nonsterol isoprenoid which indirect evidence suggested was a coregulator of isoprenoid biosynthesis and played a critical role in cellular proliferation. The first prenylated proteins to be identified were the nuclear lamin proteins-B lamins and prelamin A-which were subsequently shown to be farnesylated at a carboxyl-terminal CAAX motif. In both types of lamin, the farnesylation and carboxymethylation play a role in targeting these proteins to the nuclear envelope. The nucleus can be demonstrated to be a CAAX processing compartment for the lamins. In the case of prelamin A, there is removal of a carboxyl-terminal polypeptide which is specifically catalyzed by the enzyme Zmpste24. This processing event is necessary for assembly of lamin A into the lamina and may play a role in cell cycle control. Because the nucleus contains only one target membrane, lamin farnesylation and carboxymethylation may be sufficient to allow association with this membrane. This stands in contrast to farnesylated proteins expressed in the cytoplasm.

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. Following the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 as a candidate to carry out one or both of these proteolytic reactions. In this body of work, the CAAX endopeptidase activity of recombinant, membrane reconstituted, Zmpste24 is demonstrated using a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage a 33 kDa prelamin A carboxyl terminal tail of prelamin A was expressed in insect cells. This purified substrate possesses a fully processed CAAX box, and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. In vitro reactions with this substrate and insect cell membranes bearing recombinant Zmpste24 demonstrate that Zmpste24 may possess the ability to directly catalyze the second endoproteolytic cleavage. Previous studies on nuclear envelope fractions have ascribed this second activity to a chymotrypsin like protease. However, Zmpste24 contains the canonical HEXXH domain, a common characteristic of zinc metalloproteinases. Experiments on Zmpste24 in this work demonstrate that inactivating the HEXXH domain by site directed mutagenesis results in a loss of the first endoproteolysis reaction, while the second endoproteolytic activity is retained. Supporting these data is the observation that a truncated mutant of Zmpste24 (residues: Met1 - Pro230) that does not contain the HEXXH motif, loses the first endoproteolytic activity while retaining the second. Furthermore, this second activity is not sensitive to the metalloproteinase inhibitors EDTA and 1,10-orthophenanthroline, but is sensitive to the chymotrypsin inhibitor TPCK and its fluorescent analogue, FFCK. The fact that Zmpste24 can be affinity labeled with FFCK suggests that this second activity is directly caused by a second, yet unidentified, active site with a chymotrypsin-like catalytic mechanism.

Malaria kills at least one million people each year, mostly children - a death every 30 seconds. Almost one half of the world population is at risk from malaria. Antimalarial drugs are the only means for the treatment of about 500 million annual global malaria cases. Because of prevalent drug-resistance it is extremely urgent to identify new drug targets. Many proteins involved in eukaryotic signal transduction and cell cycle progression undergo post-translational lipid modification by a prenyl group. Protein prenyltransferases, which catalyze the post-translational prenyl modification, have been established as a target for anticancer therapy. Research done in our laboratory has demonstrated recently that prenyl modification of proteins could be a novel target for the development of antimalarial drugs.The goal of this study is to understand the molecular mechanism of protein prenylation in Plasmodium. The key to use of prenyltransferase inhibitors for the pharmacological intervention is a thorough understanding of the in vivo prenylation pathways in the malaria parasite. Knowledge of the physiological functions of the cellular protein substrates of malarial prenyltransferases is an important first step in the elucidation of the mechanism of antimalarial action of inhibitors of protein prenylation. The research described in this thesis revealed the evidence for the existence of farnesylated and geranylgeranylated malaria parasite proteins. The study shows that the dynamics of protein prenylation changes with the intraerythrocytic development cycle of the parasite. We detected that prenylated proteins in the 50 kDa range were mostly farnesylated and that the proteins in the 22-25 kDa range were mostly geranylgeranylated. The prenylation of P. falciparum proteins is inhibited by prenyltransferase inhibitors. We have also demonstrated unique features of protein prenylation in P. falciparum compared to the human host such as farnesylation of proteins are sensitive to inhibition by geranylgeranyltransferase inhibitors.. In-silico search of the malarial genome sequence identified potential protein prenyltransferase substrates. One of these substrates is a SNARE protein Ykt6 homologue. The malarial Ykt6 was recombinantly expressed and subjected to an in-vitro prenylation assay. We showed that the recombinant Ykt6 was indeed a substrate for the malarial prenyltransferase.

O desenvolvimento de antimaláricos é necessario pois, há linhagens de Plasmodium resistentes às drogas em uso e um alvo importante é a via de isoprenoides. Importantes alvos derivados desta via são: heme O já que, há antimaláricos relacionados ao heme; e a giberilina pois, há inibidores desta que não são prejudiciais ao homem. Inabenfide (INA) e uniconazol-P (UNP) inibem a biossíntese de giberilina em plantas e o crescimento de P. falciparum. Inicialmente, identificamos no parasita genes homólogos para a síntese de heme O e A, cox10 e 15, que codificam as enzimas HOS e HAS. Parasitas transgênicos com HOS e HAS marcados com GFP permitiram identificar a localização de cox10 no núcleo e de cox15 na mitocôndria. Identificamos heme O por marcações metabólicas e espectrometria de massa. Entretanto, não identificamos heme A e giberelina. INA e UNP diminuem a biossíntese de heme O e a parasitemia, observado por oxido-redução e marcação metabólica, o que torna a sintese de heme O um interessante alvo para antimaláricos. / The development of antimalarials is necessary because there are Plasmodium strains resistant to the drugs in use and an important target is the isoprenoid pathway. Targets derived from the isoprenoid pathway are: heme O as there are antimalarial drugs related to heme; And gibberillin, because there are inhibitors which are not harmful to man. Inabenfide (INA) and uniconazole-P (UNP) inhibit biosynthesis of gibberillin in plants and of growth the P. falciparum. Initially, we identified in P. falciparum genes homologous to cox10 and 15 encoding the enzymes (HOS and HAS) for synthesis of heme O and A. We created transgenic parasite lines which had HOS and HAS tagged to GFP. These revealed that the subcellular location of cox10 is in the nucleus and of cox15 in mitochondria. We identified heme O by metabolic labeling and mass spectrometry. However, no heme A or gibberellin was detected. INA and UNP decreased heme O biosynthesis and parasitemia as observed by oxido-reduction and metabolic labeling. Our data point to heme O as an important target for antimalarials.

Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LAΔ50/progerin). These modifications cause an abnormal association of LAΔ507 progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LAΔ50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.

No description available.