Host cell cAMP-Epac-Rap1b pathway inhibition by hawthorn extract as a potential target against Trypanosoma cruzi infection.

Although the two drugs currently available for the treatment of Chagas disease, Benznidazole and Nifurtimox, have proven to be effective in the acute phase of the disease, the 60-90-day treatment leads to high toxicity and unwanted side effects, presenting, in addition, a low efficacy in the chronic phase of the disease. For this reason, new therapies that are more effective are needed. In this regard, we have recently shown that the inhibition of the Epac-Rap1b pathway suppressed the cAMP-mediated host cell invasion by Trypanosoma cruzi. Interestingly, it has been described that vitexin, a natural flavone that protects against ischemia-reperfusion damage, acts by inhibiting the expression of Epac and Rap1 proteins. Vitexin can be found in plants of the genus Crataegus spp., traditionally known as hawthorn, which are of great interest considering their highly documented use as cardio-protectors. Pre-treating cells with an extract of Crataegus oxyacantha produced levels of T. cruzi invasion comparable to the ones observed for the commercially available Epac1-specific inhibitor, ESI-09. In addition, extract-treated cells exhibited a decrease in the activation of Rap1b, suggesting that the effects of the extract would be mediated by the inhibition of the cAMP-Epac-Rap1 signaling pathway. Using HPLC-HRMS2, we could confirm the presence of vitexin, and other flavones that could act as inhibitors of Epac/Rap1b, in the extracts of C. oxyacantha. Most significantly, when cells were treated with the extract of C. oxyacantha in conjunction with Nifurtimox, an increased modulation of invasion was observed.

Host Cell Rap1b mediates cAMP-dependent invasion by Trypanosoma cruzi.

Trypanosoma cruzi cAMP-mediated invasion has long been described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we gathered evidence indicating that the cAMP/Epac pathway is activated in different cells lines. In accordance, data collected from pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP), and infection assays using cells transfected with a constitutively active mutant of Rap1b (Rap1b-G12V), strongly suggest the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.

Lipase-Catalyzed Synthesis and Biological Evaluation of N-Picolineamides as Trypanosoma cruzi Antiproliferative Agents.

In our search for new safe antiparasitic agents, an enzymatic pathway was applied to synthesize a series of N-pyridinylmethyl amides derived from structurally different carboxylic acids. Thirty derivatives, including 11 new compounds, were prepared through lipase-catalyzed acylation in excellent yields. In order to optimize the synthetic methodology, the impact of different reaction parameters was analyzed. Some compounds were evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible for American trypanosomiasis (Chagas' disease). Some of them showed significant activity as parasite proliferation inhibitors. Amides derived from 2-aminopicoline and stearic and elaidic acids were as potent as nifurtimox against the amastigote form of T. cruzi, the clinically relevant form of the parasite. Even more, a powerful synergism between nifurtimox and N-(pyridin-2-ylmethyl)stereamide was observed, almost completely inhibiting the proliferation of the parasite. Besides, the obtained compounds showed no toxicity in Vero cells, making them excellent potential candidates as lead drugs.

Screening of Protein-Protein Interaction Modulators Using BRET-Based Technology.

Protein-protein interactions (PPIs) play central roles in most molecular mechanisms underlying cellular and biological processes. Within the methods developed to study PPIs is bioluminescence resonance energy transfer (BRET). Taking advantage of this technique, we have set a BRET-based assay that enables the screening of modulators of essential PPIs for Trypanosoma cruzi survival. Considering the complexity of the evaluated mixture, pure chemical compounds or natural extracts, two approaches are described, BRET in living cells or from lysates.

Sialic acid removal by trans-sialidase modulates MMP-2 activity during Trypanosoma cruzi infection.

Matrix metalloproteinases (MMPs) not only play a relevant role in homeostatic processes but are also involved in several pathological mechanisms associated with infectious diseases. As their clinical relevance in Chagas disease has recently been highlighted, we studied the modulation of circulating MMPs by Trypanosoma cruzi infection. We found that virulent parasites from Discrete Typing Units (DTU) VI induced higher proMMP-2 and MMP-2 activity in blood, whereas both low (DTU I) and high virulence parasites induced a significant decrease in proMMP-9 plasma activity. Moreover, trans-sialidase, a relevant T. cruzi virulence factor, is involved in MMP-2 activity modulation both in vivo and in vitro. It removes α2,3-linked sialyl residues from cell surface glycoconjugates, which then triggers the PKC/MEK/ERK signaling pathway. Additionally, bacterial sialidases specific for this sialyl residue linkage displayed similar MMP modulation profiles and triggered the same signaling pathways. This novel pathogenic mechanism, dependent on sialic acid removal by the neuraminidase activity of trans-sialidase, can be exploited by different pathogens expressing sialidases with similar specificity. Thus, here we present a new pathogen strategy through the regulation of the MMP network.

All Roads Lead to Cytosol: Trypanosoma cruzi Multi-Strategic Approach to Invasion.

T. cruzi has a complex life cycle involving four developmental stages namely, epimastigotes, metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. Although trypomastigotes are the infective forms, extracellular amastigotes have also shown the ability to invade host cells. Both stages can invade a broad spectrum of host tissues, in fact, almost any nucleated cell can be the target of infection. To add complexity, the parasite presents high genetic variability with differential characteristics such as infectivity. In this review, we address the several strategies T. cruzi has developed to subvert the host cell signaling machinery in order to gain access to the host cell cytoplasm. Special attention is made to the numerous parasite/host protein interactions and to the set of signaling cascades activated during the formation of a parasite-containing vesicle, the parasitophorous vacuole, from which the parasite escapes to the cytosol, where differentiation and replication take place.

Hybrids of Cinchona Alkaloids and Bile Acids as Antiparasitic Agents Against Trypanosoma cruzi.

The current chemotherapy of Chagas disease needs to be urgently improved. With this aim, a series of 16 hybrids of Cinchona alkaloids and bile acids were prepared by functionalization at position C-2 of the quinoline nucleus by a radical attack of a norcholane substituent via a Barton-Zard decarboxylation reaction. The antitrypanosomal activity of the hybrids was tested on different stages and strains of T. cruzi. In particular, eight out of 16 hybrids presented an IC50 ≤1 µg/mL against trypomastigotes of the CL Brener strain and/or a selectivity index higher than 10. These promising hybrids yielded similar results when tested on trypomastigotes from the RA strain of T. cruzi (discrete typing unit-DTU-VI). Surprisingly, trypomastigotes of the Y strain (DTU II) were more resistant to benznidazole and to most of the hybrids than those of the CL Brener and RA strains. However, the peracetylated and non-acetylated forms of the cinchonine/chenodeoxycholic bile acid conjugate 4f and 5f were the most trypanocidal hybrids against Y strain trypomastigotes, with IC50 values of 0.5 and 0.65 µg/mL, respectively. More importantly, promising results were observed in invasion assays using the Y strain, where hybrids 5f and 4f induced a significant reduction in intracellular amastigotes and on the release of trypomastigotes from infected cells.

Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase.

Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic ß-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 ß-sheet domain. The binding was stabilized by the lipid insertion into the ß-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors.

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein-protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein-protein interactions were set in living cells and lysates.

Host Epac1 is required for cAMP-mediated invasion by Trypanosoma cruzi.

Mechanistic details of the modulation by cAMP of Trypanosoma cruzi host cell invasion remain ill-defined. Here we report that activation of host's Epac1 stimulated invasion, whereas specific pharmacological inhibition or maneuvers that alter Epac1 subcellular localization significantly reduced invasion. Furthermore, while specific activation of host cell PKA showed no effect, its inhibition resulted in an increased invasion, revealing a crosstalk between the PKA and Epac signaling pathways during the process of invasion. Therefore, our data suggests that subcellular localization of Epac might be playing an important role during invasion and that specific activation of the host cell cAMP/Epac1 pathway is required for cAMP-mediated invasion.

Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi.

Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite's life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMP's mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi.

Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes?

OBJECTIVE: Chronic exercise and obesity both increase intramyocellular triglycerides (IMTGs) despite having opposing effects on insulin sensitivity. We hypothesized that chronically exercise-trained muscle would be characterized by lower skeletal muscle diacylglycerols (DAGs) and ceramides despite higher IMTGs and would account for its higher insulin sensitivity. We also hypothesized that the expression of key skeletal muscle proteins involved in lipid droplet hydrolysis, DAG formation, and fatty-acid partitioning and oxidation would be associated with the lipotoxic phenotype. RESEARCH DESIGN AND METHODS: A total of 14 normal-weight, endurance-trained athletes (NWA group) and 7 normal-weight sedentary (NWS group) and 21 obese sedentary (OBS group) volunteers were studied. Insulin sensitivity was assessed by glucose clamps. IMTGs, DAGs, ceramides, and protein expression were measured in muscle biopsies. RESULTS: DAG content in the NWA group was approximately twofold higher than in the OBS group and ~50% higher than in the NWS group, corresponding to higher insulin sensitivity. While certain DAG moieties clearly were associated with better insulin sensitivity, other species were not. Ceramide content was higher in insulin-resistant obese muscle. The expression of OXPAT/perilipin-5, adipose triglyceride lipase, and stearoyl-CoA desaturase protein was higher in the NWA group, corresponding to a higher mitochondrial content, proportion of type 1 myocytes, IMTGs, DAGs, and insulin sensitivity. CONCLUSIONS: Total myocellular DAGs were markedly higher in highly trained athletes, corresponding with higher insulin sensitivity, and suggest a more complex role for DAGs in insulin action. Our data also provide additional evidence in humans linking ceramides to insulin resistance. Finally, this study provides novel evidence supporting a role for specific skeletal muscle proteins involved in intramyocellular lipids, mitochondrial oxidative capacity, and insulin resistance.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2.

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1ß, TcP2α, TcP2ß and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1ß-TcP2α and TcP1ß-TcP2ß. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2ß were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

Downregulation of Rap1GAP through epigenetic silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors.

Thyroid cancer is the most common type of endocrine malignancy, encompassing tumors with various levels of invasive growth and aggressiveness. Rap1GAP, a Rap1 GTPase-activating protein, inhibits the RAS superfamily protein Rap1 by facilitating hydrolysis of GTP to GDP. In this study, we analyzed 197 thyroid tumor samples and showed that Rap1GAP was frequently lost or downregulated in various types of tumors, particularly in the most invasive and aggressive forms of thyroid cancer. The downregulation was due to promoter hypermethylation and/or loss of heterozygosity, found in the majority of thyroid tumors. Treatment with demethylating agent 5-aza-deoxycytidine and/or histone deacetylation inhibitor trichostatin A induced gene reexpression in thyroid cells. A genetic polymorphism, Y609C, was seen in 7% of thyroid tumors but was not related to gene downregulation. Loss of Rap1GAP expression correlated with tumor invasiveness but not with specific mutations activating the mitogen-activated protein kinase pathway. Rap1GAP downregulation was required in vitro for cell migration and Matrigel invasion. Recovery of Rap1GAP expression inhibited thyroid cell proliferation and colony formation. Overall, our findings indicate that epigenetic or genetic loss of Rap1GAP is very common in thyroid cancer, where these events are sufficient to promote cell proliferation and invasion.

Nitro-fatty acids reduce atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: Inflammatory processes and foam cell formation are key determinants in the initiation and progression of atherosclerosis. Electrophilic nitro-fatty acids, byproducts of nitric oxide- and nitrite-dependent redox reactions of unsaturated fatty acids, exhibit antiinflammatory signaling actions in inflammatory and vascular cell model systems. The in vivo action of nitro-fatty acids in chronic inflammatory processes such as atherosclerosis remains to be elucidated. METHODS AND RESULTS: Herein, we demonstrate that subcutaneously administered 9- and 10-nitro-octadecenoic acid (nitro-oleic acid) potently reduced atherosclerotic lesion formation in apolipoprotein E-deficient mice. Nitro-fatty acids did not modulate serum lipoprotein profiles. Immunostaining and gene expression analyses revealed that nitro-oleic acid attenuated lesion formation by suppressing tissue oxidant generation, inhibiting adhesion molecule expression, and decreasing vessel wall infiltration of inflammatory cells. In addition, nitro-oleic acid reduced foam cell formation by attenuating oxidized low-density lipoprotein-induced phosphorylation of signal transducer and activator of transcription-1, a transcription factor linked to foam cell formation in atherosclerotic plaques. Atherosclerotic lesions of nitro-oleic acid-treated animals also showed an increased content of collagen and alpha-smooth muscle actin, suggesting conferral of higher plaque stability. CONCLUSION: These results reveal the antiatherogenic actions of electrophilic nitro-fatty acids in a murine model of atherosclerosis.

Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop.

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity.

The influence of radioiodination made through prosthetic group N-succinimidyl-3-[131I]iodo-benzoate ([131I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC50 almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se.

Iodide benzamides for the in-vivo detection of melanoma and metastases.

The aim of this work was to synthesize, label and evaluate in vivo [I]N-(2-diethylaminoethyl)-3-iodo-4-methoxybenzamide ([I]IMBA) as a radiotracer for B16-F0 melanoma cells, in C57 mice bearing a subcutaneous melanoma tumour and experimentally induced lung metastases. The average radio-iodination yield achieved, after labelling and Sep-Pak purification, was 65%, with a radiochemical purity of > or = 96%. Biodistribution studies using [I]IMBA (2.2 GBq/micromol) showed high specific tumour uptake, with low non-target tissue background, due to a rapid renal clearance from the animal body (corporal retention was 19.7 +/- 7.1% of the injected dose at 6 h and 4.00 +/- 2.4% of the injected dose at 24 h). The very high targeting efficiency of this radiopharmaceutical was also confirmed by images in which primary subcutaneous tumour and induced lung metastases were clearly visualized. In addition, a clear correlation was found between the uptake of radioactivity in the lungs (percentage of the injected dose per gram of tissue) and the number of metastases carried by them.