RESUMO
Purpose: Limbal epithelial stem cells (LSCs), located in the basal layer of the corneal epithelium in the corneal limbus, are vital for maintaining the corneal epithelium. LSCs have a high capacity of self-renewal with increased potential for error-free proliferation and poor differentiation. To date, limited research has focused on unveiling the composition of the limbal stem cell niche, and, more important, on the role the specific stem cell niche may have in LSC differentiation and function. Our work investigates the composition of the extracellular matrix in the LSC niche and how it regulates LSC differentiation and function. Methods: Hyaluronan (HA) is naturally synthesized by hyaluronan synthases (HASs), and vertebrates have the following three types: HAS1, HAS2, and HAS3. Wild-type and HAS and TSG-6 knockout mice-HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, TSG-6-/--were used to determine the importance of the HA niche in LSC differentiation and specification. Results: Our data demonstrate that the LSC niche is composed of a HA rich extracellular matrix. HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, and TSG-6-/- mice have delayed wound healing and increased inflammation after injury. Interestingly, upon insult the HAS knock-out mice up-regulate HA throughout the cornea through a compensatory mechanism, and in turn this alters LSC and epithelial cell specification. Conclusions: The LSC niche is composed of a specialized HA matrix that differs from that present in the rest of the corneal epithelium, and the disruption of this specific HA matrix within the LSC niche leads to compromised corneal epithelial regeneration. Finally, our findings suggest that HA has a major role in maintaining the LSC phenotype.
Assuntos
Diferenciação Celular/fisiologia , Microambiente Celular/fisiologia , Epitélio Corneano/metabolismo , Ácido Hialurônico/metabolismo , Limbo da Córnea/citologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Animais , Queimaduras Químicas/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/induzido quimicamente , Glucuronosiltransferase/metabolismo , Hialuronan Sintases , Ácido Hialurônico/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio , Cicatrização/fisiologiaRESUMO
BACKGROUND: Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. METHODOLOGY/PRINCIPAL FINDINGS: THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i) it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold), and also decrease in k 3 (3.5-fold). The large values of ΔG â=â 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25))-S(-)/(His(163))-Im(+) H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. CONCLUSIONS/SIGNIFICANCE: Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.
Assuntos
Catepsina L/metabolismo , Heparina/farmacologia , Leishmania mexicana/enzimologia , Animais , Sequência de Bases , Catepsina L/genética , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , Cinética , Reação em Cadeia da Polimerase , Espectrometria de FluorescênciaRESUMO
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.
Assuntos
Simulação de Dinâmica Molecular , Sulfotransferases/química , Substituição de Aminoácidos , Domínio Catalítico , Dissacarídeos/química , Heparitina Sulfato/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/química , Fosfoadenosina Fosfossulfato/química , Ligação Proteica , Sulfotransferases/genéticaRESUMO
The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/biossíntese , Sulfato de Queratano/biossíntese , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/deficiência , Humanos , Integrina beta1/metabolismo , Sulfato de Queratano/deficiência , Lumicana , Masculino , Camundongos , Camundongos Knockout , Neoplasias da Próstata/patologia , Regulação para CimaRESUMO
Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, α-neoendorphin, ß-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects.
Assuntos
Cocaína/administração & dosagem , Corpo Estriado/enzimologia , Encefalinas/metabolismo , Metaloendopeptidases/biossíntese , Precursores de Proteínas/metabolismo , Animais , Encefalinas/genética , Expressão Gênica/efeitos dos fármacos , Masculino , Metaloendopeptidases/genética , Precursores de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
Generic active pharmaceutical ingredients (APIs) have been commonly used in Brazil, since 1999, but most of them are synthetic and small molecules. Recently, a large number of generic enoxaparins were introduced into the market raising concerns related to product-to-product interchangeability, efficiency, and drug counterfeiting. These drugs are produced from biological sources and their production involves complex procedures and purification processes. The present article evaluates several generic enoxaparins, structurally and pharmacologically, and compares them with the branded products. Structural analysis showed that the generic products are, indeed, quite similar to the branded products, however, this similarity cannot be extended to their pharmacological activities. The results showed that generic products must go through extensive structural, pharmacological, and clinical evaluation in order to assess their quality, efficacy and, ultimately, avoid drug counterfeiting before clinical use. Variation was also observed between the branded products, showing that such drugs must be at constant surveillance.
Assuntos
Medicamentos Genéricos/análise , Medicamentos Genéricos/farmacocinética , Enoxaparina/análise , Enoxaparina/farmacocinética , Brasil , Medicamentos Genéricos/uso terapêutico , Enoxaparina/uso terapêutico , Feminino , Humanos , MasculinoRESUMO
During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.
Assuntos
Colágeno/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação para Cima , Idoso , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno/genética , Neoplasias Colorretais/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteoglicanas/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.
Assuntos
Enzimas/análise , Proteoglicanas/biossíntese , Proteoglicanas/química , Proteômica/métodos , Caramujos/metabolismo , Animais , Enzimas/química , Enzimas/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Modelos Animais , Proteoglicanas/análise , Proteoma/análise , Caramujos/química , Caramujos/genética , Caramujos/ultraestrutura , Distribuição Tecidual , Vertebrados/metabolismoRESUMO
Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2â=â21±1 s⻹) was much higher than the HNE deacylation step (k3â=â0.57±0.05 s⻹). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing kâ1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.
Assuntos
Heparina/farmacologia , Elastase de Leucócito/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Cinética , Elastase de Leucócito/farmacologia , Modelos Moleculares , Peptídeos/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.