Orientadores: Anibal Eugênio Vercesi, Karina Gottardelo Zecchin / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T16:40:04Z (GMT). No. of bitstreams: 1 Rossato_FrancoAparecido_D.pdf: 7526681 bytes, checksum: 3768c017da7bde02ddd95e8e41881438 (MD5) Previous issue date: 2014 / Resumo: Recentemente mostramos que os inibidores da enzima ácido graxo sintase (FASN - EC 2.3.1.85), cerulenina e orlistat, reduzem a proliferação e induzem apoptose em células B16-F10 de melanoma murino via mecanismos mitocondriais. Neste presente estudo investigamos os efeitos desses inibidores de FASN em linhagem celular não-tumorigênica derivada de melanoblastos de camundongos (melan-a). O tratamento in vitro de células melan-a com 5 µg/mL de cerulenina ou com 30 µM de orlistat inibiu a proliferação celular, com acúmulo da proteína supressora de tumor p21WAF1/Cip1, assim como induziu a via intrínseca da apoptose com liberação de citocromo c e ativação de caspases-3 e -9, sem ativação da caspase-8. Os inibidores de FASN não alteram o conteúdo de ácidos graxos livres nas células melan-a, verificados por espectrometria de massas, sugerindo que o tratamento com cerulenina ou orlistat induz apoptose independente da inibição desta enzima. Análise das funções da bioenergética mitocondrial das células melan-a mostraram inibição da respiração, seguido por aumento da produção de superóxido. A inibição da respiração, promovida pelo tratamento com cerulenina ou orlistat, foi restrita à oxidação de substratos ligados a NADH (39,9% DMSO x cerulenina; ou 60,8% EtOH x orlistat) e succinato (45,8% DMSO x cerulenina; ou 51,8% EtOH x orlistat), e não foi significativa quando as mitocôndrias estavam respirando com substrato do complexo IV, N,N,N',N'-tetrametil-p-fenilenodiamina. A proteção conferida pelo sequestrador de radicais livres N-acetil cisteína (NAC) sugere que a disfunção mitocondrial provocada por estes compostos está associada a estresse oxidativo e é provável que seja mediada pela ação de superóxido na cadeia respiratória nos níveis de complexos de I e II. Análise proteômica de mitocôndria dessas células também mostra alterações ligadas ao estresse oxidativo. Nossos dados em conjunto sugerem que cerulenina e orlistat induzem apoptose em células não tumorais como resultado de uma disfunção mitocondrial e de maneira independente de FASN / Abstract: We have previously reported that the fatty acid synthase (FASN) inhibitors, cerulenin or orlistat, induce apoptosis in B16-F10 mouse melanoma cells mediated by mitochondria. Here we investigate the effects of these inhibitors on the non-tumorigenic mouse cell line melan-a. Cerulenin or orlistat treatment decreased cells proliferation, accompanied by increased amounts of the tumor suppressor protein p21WAF1/Cip1, as well as induced apoptosis, but not necrosis, in melan-a cell line. Mitochondrial cytochrome c release and activation of caspases-9 and -3 were detected in melan-a-treated cells. siRNAi for FASN did not culminate in apoptosis, and FASN inhibitors treatment did not alter free fatty acids content in the non-tumorigenic cells, as verified by mass spectrometry, suggesting that cerulenin or orlistat induces apoptosis independent on FASN inhibition. Analysis of energy-linked functions of melan-a mitochondria showed inhibition of respiration followed by large stimulation of superoxide production. Respiratory inhibition after cerulenin or orlistat treatment, respectively, was restricted to the oxidation of NADH-linked substrates (39.9 or 60,8%) and succinate (45.8 or 51.8%) and was not significant when mitochondria were respiring on the complex IV substrate, N,N,N?,N?-tetramethyl-p-phenylendiamine. The protection conferred by the free radical scavenger NAC suggests that the mitochondrial dysfunction caused by these compounds is associated with oxidative stress and is mediated by the action of superoxide on the respiratory chain at the levels of complexes-I and II. Proteomic analysis of mitochondria melan-a cells also indicate major changes linked to oxidative stress. Taken together, the present results show that cerulenin or orlistat induces apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent on FASN inhibition / Doutorado / Fisiopatologia Médica / Doutor em Ciências

Malaria, caused by Plasmodium, is one of the most devastating infectious diseases of the world in terms of mortality as well as morbidity (WHO, 2002). The development of resistance in the Plasmodium falciparum against the present antimalarials has made the situation very alarming (Trape et al., 2000). To combat this situation, new antimalarials as well as identification of new drug targets are urgently required. The discovery of the presence of type II fatty acid biosynthesis system in the malarial parasite has offered several promising new targets for this mission. This thesis describes the successful cloning of fabZ from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and most importantly discovery of two small molecule inhibitors (Sharma et al., 2003). The study was executed to gain insights into the structure and function of PfFabZ to get better understanding of the interactions with its substrate analogs, novel inhibitors and also acyl carrier protein (PfACP). The molecular details of the interactions of the two novel inhibitors were also determined. Lastly, the residues of PfFabZ important for the interaction with PfACP were successfully elucidated. Chapter 1 presents a brief review of the literature about the disease as well as the life cycle, biology and the metabolic pathways operational in malarial parasite, Plasmodium falciaparum. The discovery of type II FAS in P. falciparum and the aims and the scope of the thesis are also discussed. The quest of developing new antimalarials, study of the mechanism of actions of antimalarials such as quinine and its derivatives along with the major metabolic pathways (Purine, pyrimidine, phospholipids, carbohydrate metabolism, folate and heme biosynthesis pathways etc.) existing in P. falciparum are described in detail in this chapter. Origin and importance of apicoplast in P. falciaprum is also described in brief. For long, it was believed that Plasmodium spp. are incapable of de novo fatty acid synthesis but this view has undergone substantial revision due to the recent discovery of plant and bacterial type of fatty acid biosynthesis pathway in them (Surolia and Surolia, 2001). As this pathway is distinct from that of the human host it has accelerated the momentum for the discovery of new antimalarials (Surolia and Surolia, 2001). The Chapter also surveys the details of type II FAS in bacteria, particularly that of E. coli (Rock and Cronan, 1996). The dehydratase step which is the third step of fatty acid elongation cycle has been covered in considerable detail. Lastly, it focuses on the recent advancement in the understanding of fatty acid biosynthesis system in Plasmodium falciparum along with some inhibitors targeting the malarial FAS. As each enzyme of the Plasmodium FAS can serve as good antimalarial targets, my work focuses on the dehydratase step catalyzed by β-hydroxyacyl-ACP dehydratase (PfFabZ). Cloning, expression and kinetic characterization of PfFabZ forms the major content of Chapter 2. The PlasmoDB data base was searched for this gene and the mined out open reading frame contained sequence of the putative FabZ together with the bipartite leader polypeptide. Our aim was to clone the mature PfFabZ without the bipartite leader sequence. Amplification of the mature pffabZ using Plasmodium falciparum genomic DNA revealed the presence of an intron in the ORF and the gene was finally cloned by RT-PCR in pET-28a(+) vector. It was expressed with an N-terminal hexahistidine tag in BL-21(DE3) cells and purified to near homogeneity but the protein was insoluble and unstable. Truncation of 12 residues from the N-terminal end improved the stability and solubility of the protein by 3-5 fold. Truncated PfFabZ was used for all future experiments. FabZs from other sources are reported to be hexamer in solution but PfFabZ showed homodimeric arrangement in the conditions used for gel filtration as well as dynamic light scattering studies. Kinetics of PfFabZ was characterized using substrate analogs, β-hydroxybutryl-CoA (forward substrate) and Crotonoyl-CoA (reverse substrate). Both the forward and reverse reaction were thoroughly characterized by spectrophotometry and HPLC and the reverse reaction was found to be 7 times faster than the forward reaction. Km οf crotonoyl-CoA was calculated to be 86 µM and kcat/Km of 220 M-1s-1 whereas the Kmfor β-hydroxybutryl-CoA was found to be 199 µM and kcat/Kmof 80.2 M-1s-1. The kinetic data clearly indicates the higher affinity of PfFabZ for the reverse substrate. Chapter 3 describes the discovery of two small molecules inhibitors, NAS-21 and NAS-91 for PfFabZ, their detailed inhibition kinetics and their effect on the growth of Plasmodium falciparum in culture. These inhibitors were the first inhibitors to be reported for FabZ class of enzymes with an IC50 ranging below 15 µM. Both of them inhibited PfFabZ following competitive kinetics with respect to the substrates utilized for both the forward and reverse reactions. The inhibition data were analyzed by Lineweaver-Burk and Dixon plots and both inhibitors showed competitive inhibition kinetics with dissociation constant in submicromolar range. Binding constants for both the inhibitors were also determined by fluorescence titration method and were calculated to be 1.6 (± 0.04) X 106 M-1 for NAS-91 and 1.2 (± 0.03) X 106 M-1 for NAS-21. These inhibitors were checked on Plasmodium falciparum culture and both inhibited parasite growth with IC50 values of 7 µM and 100 µM for NAS-21 and NAS-91, respectively. They also inhibited the incorporation of [1,2-14C]-acetate in the fatty acids of the P. falciparum conforming the inhibition of fatty acid biosynthesis. FabZ class of enzymes are thought to contain His-Glu as a catalytic dyad. Based on the disparity in the arrangement of residues at the active site of the dimeric (Swarnamukhi et al., 2006) and hexameric forms of PfFabZ in the crystal structures (Kosteriva et al., 2005), we set out to elucidate the active site residues in PfFabZ which is described in Chapter 4. The role of each of the presumed active site residues His-133 and Glu-147 along with Arg-99 and His-98 were analyzed by chemical modification studies and site directed mutagenesis. Single and double mutants were prepared and the activity of the mutants was monitored by spectrophotometry and isothermal titration calorimetry (ITC). It was concluded that in PfFabZ, His-133 and Glu-147 makes the catalytic dyad, His-98 might be important in directing the substrate in correct orientation while Arg-99 is involved in maintaining the active site loop in proper orientation rather than taking direct part in catalysis. Chapter 4 also concludes that dimeric form of PfFabZ is inactive species and turns into active hexameric form in the presence of substrate. Chapter 5 describes the molecular details of NAS-21 and NAS-91 interactions with PfFabZ. The fact that both these compounds inhibited PfFabZ in competitive manner, prompted me to examine their interaction with the residues in the active site tunnel. Apart from the His-133 and Glu-147 catalytic dyad the only polar residue is His-98 and chemical modification and site directed mutagenesis studies were done to elucidate the interactions of these residues with NAS-21 and NAS-91. Both the inhibitors were able to protect the modification of histidines by DEPC in wild type PfFabZ, His-98-Ala mutant and His-133-Ala mutant but with differential strength, indicating that they do interact with histidines. The interaction of these inhibitors was further confirmed by determining the dissociation constants of wPfFabZ, His-98-Ala, His-133-Ala, His-98-Ala/His-133-Ala double mutant, Glu-147-Ala mutant by fluorescence titration method. The results obtained from chemical modification and fluorescence titration studies confirmed that NAS-21 interacts strongly with histidines, His-98 and His-133 but not with Glu-147. On the other hand NAS-91 interacts loosely with His-98 and His-133 but strongly with Glu-147. Chapter 5 concludes with the observation that both the inhibitors (NAS-21 and NAS-91) interact with the active site residues of PfFabZ, preventing the substrate to enter the active site tunnel. Acyl carrier protein (ACP) is a small acidic protein to which the acyl chain intermediates are tethered and shuttled from one enzyme to another for the completion of fatty acid elongation cycle. Whenever acyl carrier proteins are expressed in E. coli, they are present in three forms apo, holo and acyl-ACPs. Chapter 6 describes a novel method for the expression of histidine tagged PfACP in pure holo form, protocol for the cleavage of his-tag from PfACP by thrombin preparation of homogenous singly enriched ie PfACP [15N]-labeled or [13C]-labeled PfACP as well as doubly enriched [15N]-[13C] PfACP samples for its structure elucidation by NMR (Sharma et al., 2005). These studies also constituted reporting of a holo-ACP structure from any of the sources for the first time (Sharma, et. al. 2006). The purified pure holo-PfACP was further used for the interaction studies with PfFabZ. Earlier studies have shown that ACP interacts with FAS enzymes via helix II with conserved set of residues but the molecular details of the interactions are poorly known (Zhang, et. al., 2003). We have recently solved the NMR structure (Sharma, et. al., 2006) of PfACP and crystal structure of PfFabZ (Swarnamukhi, et. al., 2006). So, both the structures were docked using Cluspro server. Chapter 7 elucidates the roles of important residues on PfFabZ surface near the active site entry which are responsible for interacting with PfACP. The residues lining the active site entry were identified and mutated. The residues lining the active site tunnel of PfFabZ are Arg102, Lys104, Lys105, Lys123, Leu94, Phe95, Ala96, Gly97, Ile128, Ile145, Phe150 and Ala151. Charged residues were mutated to alanine and also to oppositely charged residues while the neutral residues were changed to charged residues. The interaction of PfFabZ mutants with PfACP was studied by ACP independent enzymatic assay and surface plasmon resonance (SPR) spectroscopy. It was concluded that PfFabZ and PfACP interaction is mainly governed by electrostatic interaction made by the charged residues (Lys104 being the most important residue) and is fine tuned by hydrophobic interactions. Chapter 8 summarizes the findings of the thesis. FabZ from Plasmodium falciparum was cloned and biochemically characterized. Two inhibitors for this enzyme were discovered and their molecular details of binding to PfFabZ were elucidated. The presence of catalytic dyad was confirmed and finally the residues of PfFabZ important for interaction with PfACP were elucidated. Appendix I describes the inhibition of PfENR (enoyl ACP reductase), the rate limiting and the fourth enzyme of the fatty acid elongation pathway by green tea extracts. Three tea catechins (EGCG, EGC and ECG) and two plant polyphenols (quercetin and buteine) were selected for the inhibition study. All the catechins inhibited PfENR potently with Ki values in nanomolar range. Among the five compounds studied, EGCG was found to be the best inhibitor. All of them blocked the NADH binding site showing competitive kinetics with respect to NADH and uncompetitive kinetics with crotonoyl-CoA, the substrate analog. Most importantly, the catechins potentiated the inhibition of PfENR by triclosan, a well known PfENR inhibitor. We also report that in the presence of tea catechins triclosan behaves as a slow-tight binding inhibitor of PfENR. The overall inhibition constant of triclosan in the presence of EGCG was calculated to be 2pM which is 50 times better than the earlier reported values with NAD+ (Kapoor, et. al., 2004).

Sink limitations in plants reduce the potential for photosynthesis and yield, particularly under conditions that favour enhanced source activity such as elevated CO2 (EC). Dark respiration, considered catabolic, has rarely been exploited to enhance sink activity in plants. Arabidopsis thaliana L. lines with partially-suppressed mitochondrial pyruvate dehydrogenase (mtPDH) kinase (mtPDHK), a negative post-translational regulator of the mtPDH complex, was shown previously to have both elevated mtPDH complex activity and increased seed weight and oil content at ambient CO2 (AC), suggesting an enhancement of sink activity. The mtPDH links glycolysis with the tricarboxylic acid (TCA) cycle. It was hypothesized that Arabidopsis having suppressed mtPDHK will display their greatest plant productivity at EC through a combined enhancement of source and sink activities. Control and transgenic Arabidopsis having either constitutive or seed-specific expression of antisense mtPDHK were grown at either AC or EC. Expression of mtPDHK and mtPDH complex activity in rosette leaves and reproductive tissues were measured, which required the development of an assay to quantify mtPDH activity. Vegetative and reproductive growth over time, seed oil parameters, and leaf net C exchange were also quantified. A parabolic relationship was found between mtPDHK expression and mtPDH activity, reflecting a role for mtPDH in balancing photosynthetic and respiratory processes. A number of growth and seed oil parameters were improved in transgenic lines, particularly at EC; many of these parameters showed a significant linear or quadratic correlation with mtPDHK expression and mtPDH activity. The proportion of very long chain fatty acids was increased in transgenic lines. Leaf net C exchange was enhanced at AC and EC, and particularly in lines showing repression of mtPDHK. The greatest enhancement in total seed and oil productivity was found for the constitutive lines 104 and 31 at EC (up to 2.8 times). These two lines exhibited a significant increase in inflorescence size, an increase in leaf water use efficiency, the lowest rate of mtPDH complex inactivation by ATP, and an intermediary enhancement of mtPDH complex activity in seeds. Thus, it is concluded that the mtPDH plays a key role in regulating sink and source activities in plants. / Natural Sciences and Engineering Research Council (NSERC) through the Green Crop Networks Research Network; Ontario Graduate Scholarship; Syngenta Graduate Scholarship; Ball Farm Services and Agrico Canada Ltd. Scholarship; Mrs. Fred Ball Scholarship; Arthur D. Latornell Scholarship; Hoskins Scholarship; Robb Travel Grant; Registrars and the Deans Scholarship and travel awards and bursaries from the University of Guelph, and the Ontario Agricultural College.