RESUMO
Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.
Assuntos
Infecções por Mycobacterium , Mycobacterium bovis , Mycobacterium tuberculosis , Humanos , Mycobacterium bovis/fisiologia , Cromatografia Líquida , Espectrometria de Massas em Tandem , Macrófagos/metabolismo , Autofagia , Infecções por Mycobacterium/metabolismo , Proteína Quinase C/metabolismoRESUMO
Nanoparticles (NPs) are novel platforms that can carry both cancer-targeting molecules and drugs to avoid severe side effects due to nonspecific drug delivery in standard chemotherapy treatments. Cancer cells are characterized by abnormal membranes, metabolic changes, the presence of lectin receptors, glucose transporters (GLUT) overexpression, and glycosylation of immune receptors of programmed death on cell surfaces. These characteristics have led to the development of several strategies for cancer therapy, including a large number of carbohydrate-modified NPs, which have become desirable for use in cell-selective drug delivery systems because they increase nanoparticle-cell interactions and uptake of carried drugs. Currently, the potential of NP glycosylation to enhance the safety and efficacy of carried therapeutic antitumor agents has been widely acknowledged, and much information is accumulating in this field. This review seeks to highlight recent advances in NP stabilization, toxicity reduction, and pharmacokinetic improvement and the promising potential of NP glycosylation from the perspective of molecular mechanisms described for drug delivery systems for cancer therapy. From preclinical proof-of-concept to demonstration of therapeutic value in the clinic, the challenges and opportunities presented by glycosylated NPs, with a focus on their applicability in the development of nanodrugs, are discussed in this review.
RESUMO
A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP-HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8â¯kDa, proteins from 12 to 16â¯kDa and proteins from 20 to 30â¯kDa. Two components were fully sequenced: one α-neurotoxic peptide of 7210â¯Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A2 (PLA2) with molecular weight 13517â¯Da and no effect on the nAChR. PLA2 activity was evaluated for all RP-HPLC components. In addition, N-terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA2's, six to three-finger toxins (3FTx) and one to Kunitz-type serine protease inhibitors. Two-dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70â¯kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA2's, L-amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC-MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C-type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake.
Assuntos
Cobras Corais , Venenos Elapídicos/química , Proteômica , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Venenos Elapídicos/enzimologia , Venenos Elapídicos/toxicidade , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Humanos , Camundongos , PeptídeosRESUMO
Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect. BIOLOGICAL SIGNIFICANCE: This manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.
Assuntos
Proteínas de Insetos/análise , Proteômica/métodos , Saliva/química , Transcriptoma , Triatoma/química , Animais , Anticoagulantes , Cromatografia Líquida , Insetos Vetores , Inibidores da Agregação Plaquetária , Proteínas e Peptídeos Salivares , Espectrometria de Massas em TandemRESUMO
This communication reports the results of proteomic, transcriptomic, biochemical and electrophysiological analysis of the soluble venom and venom glands of the Mexican centipede Scolopendra viridis Say (here thereafter abbreviated S. viridis). Separation of the soluble venom permitted to obtain 54 different fractions, from which a mass finger printing analysis permitted the identification of at least 86 components, where 70% of the molecules have low molecular masses. Two-dimensional electrophoretic separation of this venom revealed the presence of about forty proteins with molecular weights ranging from 17 to 58kDa. The novo sequencing of 149 peptides obtained by LC-MS/MS from the 2D-gels showed the presence of proteins with amino acid sequences similar to several enzymes and venom allergens type 3. Furthermore, a total of 180 sequences were obtained from a cDNA library prepared with two venomous glands. From this, 155 sequences correspond to complete genes containing more than 200 base pairs each. Comparative sequence analyses of these sequences indicated the presence of different types of enzymes and toxin-like genes. Two proteins with molecular weights around 37,000 and 42,000Da were shown to contain hyaluronidase activity. Electrophysiological assays performed with soluble venom show that it decreases mammalian sodium channel currents. BIOLOGICAL SIGNIFICANCE: Animal venoms of Scolopendra species have been scarcely studied, although they have been reported to contain several bioactive compounds, some of which with potential therapeutic interest. The Mexican centipede S. viridis contains a powerful venom, capable of inflicting immediate effects on their preys. This communication is focused on the identification and description of a proteomic and transcriptomic analysis of the protein components of this venom. Several amino acid sequences similar to reported enzymes are the principal components in the S. viridis venom, but also a low number of toxins were identified. This knowledge should contribute to the understanding of the pharmacological effects caused by bites of this centipede species.
Assuntos
Venenos de Artrópodes/química , Artrópodes/química , Proteômica , Transcriptoma , Alérgenos , Animais , Astacoidea , Células CHO , Cromatografia Líquida , Biologia Computacional , Cricetulus , DNA Complementar/metabolismo , Eletroforese em Gel Bidimensional , Etiquetas de Sequências Expressas , Biblioteca Gênica , Gryllidae , Células HEK293 , Humanos , Hialuronoglucosaminidase/metabolismo , Peso Molecular , Peptídeos/química , Venenos de Escorpião/química , Espectrometria de Massas em TandemRESUMO
Scorpion beta-toxins (beta-ScTxs) modify the activity of voltage-gated sodium (Nav) channels, thereby producing neurotoxic effects in diverse organisms. For this reason, beta-ScTxs are essential tools not only for discriminating among different channel sub-types but also for studying the mechanisms of channel gating and the structure-function relationship involved in this process. This review considers both the structural and the functional implications of the beta-ScTxs after they bind to their receptor sites, in accord with their classification into a) anti-mammalian beta-ScTxs, b) anti-insect selective excitatory beta-ScTxs, c) anti-insect selective depressant beta-ScTxs and d) beta-ScTxs active on both insect and mammals Nav channels. Additionally, the molecular mechanism of toxin action by the "voltage sensor trapping" model is discussed, and the systemic effects produced by these toxins are reviewed.
Assuntos
Venenos de Escorpião/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/fisiologia , Sequência de Aminoácidos , Animais , Ácido Glutâmico/fisiologia , Humanos , Insetos/efeitos dos fármacos , Mamíferos , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/farmacologia , Venenos de Escorpião/química , Escorpiões/metabolismo , Alinhamento de SequênciaRESUMO
The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fabâ«scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Venenos de Escorpião/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Testes de Neutralização , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , TermodinâmicaRESUMO
Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.
Assuntos
Alérgenos/química , Peptídeos Catiônicos Antimicrobianos/química , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Hipersensibilidade ao Látex/imunologia , Lectinas de Plantas/química , Alérgenos/imunologia , Sequência de Aminoácidos , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina E/sangue , Dados de Sequência Molecular , Lectinas de Plantas/imunologia , Conformação Proteica , Alinhamento de SequênciaRESUMO
Decreased immune reactivity of isoforms of major allergens has been reported. However, such claims have always been based on experiments with recombinant proteins. This work describes the molecular and physicochemical characterization of a hevein (Hev b 6.0201) natural isoform (Hev b 6.0202), which is present in rubber latex from Hevea brasiliensis. The isoallergen has a single substitution Asn14Asp, which gives rise to local differences in the surface potential, as observed from the crystal structure presented here. Besides, ELISA inhibition using serum pools of adult and pediatric patients showed reduced IgE-binding capacity ( approximately 27%) with the isoallergen. Overall, these results are relevant to delineate crucial residues involved in this dominant discontinuous epitope.
Assuntos
Substituição de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Imunoglobulina E/metabolismo , Lectinas de Plantas/química , Isoformas de Proteínas/química , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Hevein (Hev b 6.02) is a major IgE-binding allergen in natural rubber latex and manufactured products. Both tryptophans (Trp(21) and Trp(23)) of the hevein molecule were chemically modified with BNPS-skatole (2-nitrophenylsulfenyl-3-methyl-3(')-bromoindolenine); derivatized allergen failed to significantly inhibit binding of serum IgE in ELISA assays. Similarly, skin prick tests showed that hevein-positive patients gave no response with the modified allergen. Dot blot experiments carried out with anti-hevein mono- and polyclonal antibodies confirmed the importance of Trp(21) and Trp(23) for antibody-recognition, and demonstrated the specific cross-reactivity of other molecules containing hevein-like domains. We also report the structure of Hev b 6.02 at an extended resolution (1.5A) and compare its surface properties around Trp residues with those of similar regions in other allergens. Overall our results indicate that the central part of the protein, which comprises three aromatic and other acidic and polar residues, constitutes a conformational epitope.