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Trivella D.B.B.,University of California at San Diego | Trivella D.B.B.,University of Campinas | Trivella D.B.B.,National Center for Research in Energy and Materials | Pereira A.R.,University of California at San Diego | And 8 more authors.
Chemistry and Biology | Year: 2014

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,β-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy. © 2014 Elsevier Ltd.


De Giuseppe P.O.,National Center for Research in Energy and Materials | Souza T.D.A.C.B.,National Center for Research in Energy and Materials | Souza T.D.A.C.B.,Carlos Chagas Institute | Zanphorlin L.M.,University of Campinas | And 3 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification. © 2014 International Union of Crystallography.


Vargas G.,Federal University of Rio de Janeiro | Rocha J.D.B.,Federal University of Rio de Janeiro | Oliveira D.L.,Federal University of Rio de Janeiro | Albuquerque P.C.,Federal University of Rio de Janeiro | And 15 more authors.
Cellular Microbiology | Year: 2015

Summary: The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C.albicansEV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850nm. Two predominant seroreactive proteins (27kDa and 37kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C.albicansEV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-β) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G.mellonella larvae with EV followed by challenge with C.albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C.albicansEV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis. © 2014 John Wiley & Sons Ltd.


Nakayasu E.S.,University of Texas at El Paso | Nakayasu E.S.,Pacific Northwest National Laboratory | Sobreira T.J.P.,National Center for Research in Energy and Materials | Torres R.,University of Texas at El Paso | And 4 more authors.
Journal of Proteome Research | Year: 2012

Chagas disease, caused by Trypanosoma cruzi, is a devastating parasitic infection affecting millions of people. Although many efforts have been made for the development of immunotherapies, there is no available vaccine against this deadly infection. One major hurdle for the rational approach to develop a T. cruzi vaccine is the limited information about the proteins produced by different phylogenetic lineages, strains, and stages of the parasite. Here, we have adapted a 1D nanoHPLC system to perform online 2D LC-MS/MS, using the autosampler to inject the eluting salt solutions in the first dimension separation. The application of this methodology for the proteomic analysis of the infective trypomastigote stage of T. cruzi led to the identification of 1448 nonredundant proteins. Furthermore, about 14% of the identified sequences comprise surface proteins, most of them glycosylphosphatidylinositol (GPI)-anchored and related to parasite pathogenesis. Immunoinformatic analysis revealed thousands of potential peptides with predicted high-binding affinity for major histocompatibility complex (MHC) class I and II molecules. The high diversity of proteins expressed on the trypomastigote surface may have many implications for host-cell invasion and immunoevasion mechanisms triggered by the parasite. Finally, we performed a rational approach to filter potential T-cell epitopes that could be further tested and validated for development of a Chagas disease vaccine. © 2011 American Chemical Society.


Vallejo M.C.,University of Sao Paulo | Nakayasu E.S.,University of Texas at El Paso | Matsuo A.L.,University of Sao Paulo | Sobreira T.J.P.,National Center for Research in Energy and Materials | And 4 more authors.
Journal of Proteome Research | Year: 2012

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes. © 2012 American Chemical Society.


Santos C.R.,National Center for Research in Energy and Materials | Tonoli C.C.C.,National Center for Research in Energy and Materials | Trindade D.M.,National Center for Research in Energy and Materials | Betzel C.,University of Hamburg | And 6 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2011

Branching enzymes (BEs) catalyze the formation of branch points in glycogen and amylopectin by cleavage of α-1,4 glycosidic bonds and subsequent transfer to a new α-1,6 position. BEs generally belong to glycoside hydrolase family 13 (GH13); however TK1436, isolated from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, is the first GH57 member, which possesses BE activity. To date, the only BE structure that had been determined is a GH13-type from Escherichia coli. Herein, we have determined the crystal structure of TK1436 in the native state and in complex with glucose and substrate mimetics that permitted mapping of the substrate-binding channel and identification of key residues for glucanotransferase activity. Its structure encompasses a distorted (β/α) 7-barrel juxtaposed to a C-terminal α-helical domain, which also participates in the formation of the active-site cleft. The active site comprises two acidic catalytic residues (Glu183 and Asp354), the polarizer His10, aromatic gate-keepers (Trp28, Trp270, Trp407, and Trp416) and the residue Tyr233, which is fully conserved among GH13- and GH57-type BEs. Despite TK1436 displaying a completely different fold and domain organization when compared to E. coli BE, they share the same structural determinants for BE activity. Structural comparison with AmyC, a GH57 α-amylase devoid of BE activity, revealed that the catalytic loop involved in substrate recognition and binding, is shortened in AmyC structure and it has been addressed as a key feature for its inability for glucanotransferase activity. The oligomerization has also been pointed out as a possible determinant for functional differentiation among GH57 members. © 2010 Wiley-Liss, Inc.


Georgieva D.,University of Hamburg | Georgieva D.,Bulgarian Academy of Science | Murakami M.,National Center for Research in Energy and Materials | Perband M.,University of Hamburg | And 2 more authors.
Molecular BioSystems | Year: 2011

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn2+ ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed. © The Royal Society of Chemistry.


De Oliveira R.A.,University of Campinas | Filho R.M.,University of Campinas | Rossell C.E.V.,National Center for Research in Energy and Materials
Chemical Engineering Transactions | Year: 2016

In this work is presented a process with high lactic acid production as well as high sugar conversion and low by-products formation. The microorganism Lactobacillus plantarum uses preferably 6-carbon sugars. However, the xylose consumption occurs since the hexose concentration is not high. Lb. plantarum was shown to have a capacity for lactic acid production from cellulosic and hemicellulosic hydrolysates without detoxification. The results were similar to that using glucose, which is the source of carbon currently used by the lactic acid industry. Lactic acid is an organic acid with a hydroxyl and an acid functional group. It has an asymmetric carbon and it naturally occurs as two optical isomers, D and L lactic acid. Crystallinity and many other important physical properties such as rate of degradation, melting point and boiling point are controlled by the ratio of enantiomers used. Lactic acid production by fermentation has several advantages when compared to chemical synthesis, such as low temperatures, low energy consumption, better environmental concerns and high purity. Furthermore, by fermentation it is possible to obtain the optically pure lactic acid, while by chemical synthesis only the racemic mixture can be produced. Despite the wide variety of feedstock tested for the lactic acid production most of them have problems with price, seasonality, continuous availability for largescale production, fermentation rate, the amount of contaminants present, yields of lactic acid, formation of byproducts, location of lactic acid production plant due to both availability as logistics of transportation and use of it. Trying to find substrates that overcome some of these barriers, in this work is proposed the use of molasses from sugar industry, pentose and hexose from enzymatic hydrolysis of sugarcane bagasse aiming to produce lactic acid. Copyright © 2016, AIDIC Servizi S.r.l.,.


PubMed | Laboratory of Molecular Investigation of Cancer LIMC, University of Guelph, São Paulo State University and National Center for Research in Energy and Materials
Type: | Journal: Journal of mammary gland biology and neoplasia | Year: 2017

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mices lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-1 silencing and their effect on EMT.


PubMed | National Center for Research in Energy and Materials
Type: Journal Article | Journal: ACS applied materials & interfaces | Year: 2016

Vaccines and therapies are not available for several diseases caused by viruses, thus viral infections result in morbidity and mortality of millions of people every year. Nanoparticles are considered to be potentially effective in inhibiting viral infections. However, critical issues related to their use include their toxicity and their mechanisms of antiviral action, which are not yet completely elucidated. To tackle these problems, we synthesized silica nanoparticles with distinct surface properties and evaluated their biocompatibility and antiviral efficacy. We show that nanoparticles exhibited no significant toxicity to mammalian cells, while declines up to 50% in the viral transduction ability of two distinct recombinant viruses were observed. We designed experiments to address the mechanism of antiviral action of our nanoparticles and found that their hydrophobic/hydrophilic characters play a crucial role. Our results reveal that the use of functionalized silica particles is a promising approach for controlling viral infection and offer promising strategies for viral control.

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