Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.196
Filtrar
1.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502207

RESUMO

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.


Assuntos
Hidrolases Anidrido Ácido/fisiologia , Condrócitos/patologia , Anormalidades Craniofaciais/patologia , Nanismo/patologia , Glicosaminoglicanos/metabolismo , Instabilidade Articular/patologia , Ossificação Heterotópica/patologia , Fenótipo , Polidactilia/patologia , Animais , Linhagem Celular Transformada , Condrócitos/metabolismo , Anormalidades Craniofaciais/etiologia , Anormalidades Craniofaciais/metabolismo , Nanismo/etiologia , Nanismo/metabolismo , Instabilidade Articular/etiologia , Instabilidade Articular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ossificação Heterotópica/etiologia , Ossificação Heterotópica/metabolismo , Polidactilia/etiologia , Polidactilia/metabolismo
2.
FEBS Lett ; 595(18): 2341-2349, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34375459

RESUMO

Heparan sulfate (HS), a sulfated glycosaminoglycan (GAG), was reported to be a necessary host attachment factor that promotes SARS-CoV-2 infection. In this study, we developed GAG microarrays based on fluorescence detection for high-sensitivity screening of the GAG-binding specificity of proteins and applied it for the analysis of SARS-CoV-2 spike (S) protein. Among the 20 distinct GAGs, the S protein bound not only to heparin (HEP)/HS but also to chondroitin sulfate E (CSE) in a concentration-dependent manner. We then analyzed the specificity of each subunit of the S protein. While the S1 subunit showed exclusive binding to HEP, the S2 subunit also bound to CSE and HEP/HS. CSE might act as an alternative attachment factor for HS in SARS-CoV-2 infection.


Assuntos
Sulfatos de Condroitina/metabolismo , Glicosaminoglicanos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Humanos , Análise em Microsséries , Ligação Proteica , Espectrometria de Fluorescência/métodos
3.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
4.
Molecules ; 26(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209220

RESUMO

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.


Assuntos
Blástula/embriologia , Epitopos/metabolismo , Glicosaminoglicanos/metabolismo , Lytechinus/embriologia , Animais , Blástula/citologia , Adesão Celular/fisiologia , Lytechinus/citologia
5.
J Virol ; 95(19): e0058721, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34232726

RESUMO

Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake, and transduction, but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to integrin ß1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryoelectron microscopy at 2.49-Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. IMPORTANCE Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines, and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the three-dimensional structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.


Assuntos
Dependovirus/genética , Dependovirus/metabolismo , Evolução Molecular Direcionada , Receptores Virais/metabolismo , Motivos de Aminoácidos , Sistemas CRISPR-Cas , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Dependovirus/química , Dependovirus/ultraestrutura , Variação Genética , Glicosaminoglicanos/metabolismo , Humanos , Integrina beta1/química , Integrina beta1/metabolismo , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/química , Internalização do Vírus
6.
FASEB J ; 35(7): e21647, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165206

RESUMO

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfócitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/genética
7.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071909

RESUMO

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.


Assuntos
Queimaduras Químicas/metabolismo , Doenças da Córnea/induzido quimicamente , Doenças da Córnea/metabolismo , Matriz Extracelular/metabolismo , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/metabolismo , Álcalis/efeitos adversos , Animais , Biomarcadores , Queimaduras Químicas/diagnóstico , Doenças da Córnea/diagnóstico , Dermatan Sulfato/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/diagnóstico , Imunofluorescência , Expressão Gênica , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Sulfato de Ceratano/metabolismo , Camundongos , Proteoglicanas/metabolismo
8.
Nat Commun ; 12(1): 3543, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112803

RESUMO

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.


Assuntos
Neoplasias da Mama/sangue , Quimiocina CCL2/metabolismo , Exossomos/metabolismo , Receptores CCR2/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/patologia , Citocinas/metabolismo , Exossomos/imunologia , Exossomos/patologia , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteoglicanas/metabolismo , Receptores de Citocinas/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
9.
Biomolecules ; 11(5)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063530

RESUMO

Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing out the "bitter" and "sweet" aspects of this field of research. Furthermore, we attempt to bridge the gap between bioinformatics and glycobiology, which have so far been kept apart by conceptual and technical differences. For this purpose, we provide computational scientists and glycobiologists with the fundamentals of these two fields of research, with the aim of creating opportunities for their combined exploitation, and thereby contributing to a substantial improvement in scientific knowledge.


Assuntos
Membrana Celular/metabolismo , Biologia Computacional/métodos , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Animais , Glicosaminoglicanos/química , Humanos , Proteoglicanas/química , Relação Estrutura-Atividade
10.
Int J Biol Macromol ; 182: 1704-1712, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34052269

RESUMO

Designing clinical applicable polymeric composite scaffolds for auricular cartilage tissue engineering requires appropriate mechanical strength and biological characteristics. In this study, silk fiber-based scaffolds co-reinforced with poly-L-lactic acid porous microspheres (PLLA PMs) combined with either Bombyx mori (Bm) or Antheraea pernyi (Ap) silk fibers were fabricated as inspired by the "steel bars reinforced concrete" structure in architecture and their chondrogenic functions were also investigated. We found that the Ap silk fiber-based scaffolds reinforced by PLLA PMs (MAF) exhibited superior physical properties (the mechanical properties in particular) as compared to the Bm silk fiber-based scaffolds reinforced by PLLA PMs (MBF). Furthermore, in vitro evaluation of chondrogenic potential showed that the MAF provided better cell adhesion, viability, proliferation and GAG secretion than the MBF. Therefore, the MAF are promising in auricular cartilage tissue engineering and relevant plastic surgery-related applications.


Assuntos
Cartilagem da Orelha/fisiologia , Microesferas , Morus/química , Poliésteres/química , Seda/química , Tecidos Suporte/química , Animais , Bombyx , Proliferação de Células , Forma Celular , Sobrevivência Celular , Condrócitos/citologia , Condrócitos/metabolismo , Força Compressiva , DNA/metabolismo , Regulação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Porosidade , Coelhos , Seda/ultraestrutura , Fator de Necrose Tumoral alfa/metabolismo
11.
Viruses ; 13(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946963

RESUMO

Viruses are infectious agents that hijack the host cell machinery in order to replicate and generate progeny. Viral infection is initiated by attachment to host cell receptors, and typical viral receptors are cell-surface-borne molecules such as proteins or glycan structures. Sialylated glycans (glycans bearing sialic acids) and glycosaminoglycans (GAGs) represent major classes of carbohydrate receptors and have been implicated in facilitating viral entry for many viruses. As interactions between viruses and sialic acids have been extensively reviewed in the past, this review provides an overview of the current state of structural knowledge about interactions between non-enveloped human viruses and GAGs. We focus here on adeno-associated viruses, human papilloma viruses (HPVs), and polyomaviruses, as at least some structural information about the interactions of these viruses with GAGs is available. We also discuss the multivalent potential for GAG binding, highlighting the importance of charged interactions and positively charged amino acids at the binding sites, and point out challenges that remain in the field.


Assuntos
Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Fenômenos Fisiológicos Virais , Animais , Humanos , Conformação Molecular , Relação Estrutura-Atividade , Internalização do Vírus , Vírus/classificação , Vírus/metabolismo
12.
Am J Physiol Heart Circ Physiol ; 320(5): H2044-H2057, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33834865

RESUMO

Major depressive disorder (MDD) is an independent risk factor for cardiovascular disease (CVD) and its complications; however, causal mechanisms remain unclear. In the present study, we investigate cardiac structural and functional alterations and associated changes in myocardial glycosaminoglycans (GAGs) disaccharide profile in mice that exhibit depression-like behavior. Mice were assigned to the chronic mild stress (CMS) group and nonstress control group (CT). The CMS group was exposed to a series of mild, unpredictable stressors for 7 wk. Mice in the CMS group show a significant decrease in protein expression of hippocampal brain-derived neurotrophic factor (BDNF) and exhibit depression-like behavioral changes, such as learned helplessness and decreased exploration behavior, as compared with the control group. Although cardiac function remained unchanged between the groups, echocardiography analysis showed slightly increased left ventricular wall thickness in the CMS group. Furthermore, the CMS group shows an increase in cardiomyocyte cross-sectional area and an associated decrease in BDNF protein expression and increase in IL-6 mRNA expression, when compared with control mice. GAG disaccharide analysis of the left ventricles of the CMS and CT mice revealed an elevation in heparan (HS) and chondroitin sulfate (CS) content in the CMS hearts (35.3% and 17.9%, respectively, vs. control group). Furthermore, we also observed that unsulfated or monosulfated disaccharides were the most abundant units; however, we did not find any significant difference in mole percent or sulfation pattern of HS/CS disaccharides between the groups. The current investigation highlights a need for further research to explore the relationship between cardiac GAGs biology and myocardial remodeling as a causal mechanism that underlie cardiovascular complications in patients with MDD.NEW & NOTEWORTHY Comorbidity between depression and CVD is well established, whereas its etiology, especially the role of nonfibrous components (proteoglycans/GAGs) of the extracellular matrix, is unexplored. To the best of our knowledge, this is the first study to characterize cardiac proteoglycan/glycosaminoglycan profile in response to depression-like behavioral changes in mice. We observed that chronic mild stress (CMS)-induced depression-like behavior and alterations in glycosaminoglycan profile were associated with structural changes in the heart.


Assuntos
Depressão/metabolismo , Glicosaminoglicanos/metabolismo , Miocárdio/metabolismo , Estresse Psicológico/metabolismo , Animais , Comportamento Animal/fisiologia , Glicemia/metabolismo , Peso Corporal/fisiologia , Depressão/patologia , Ingestão de Alimentos/fisiologia , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Miocárdio/patologia , Estresse Psicológico/patologia
13.
Immunity ; 54(5): 962-975.e8, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33857420

RESUMO

Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.


Assuntos
Glicosaminoglicanos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Camundongos , Polimerização , Transdução de Sinais/fisiologia , Transportadores de Sulfato/metabolismo , Vaccinia/metabolismo , Vírus Vaccinia/patogenicidade
14.
Biomolecules ; 11(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800172

RESUMO

The tumor microenvironment (TME) is composed of cancerous, non-cancerous, stromal, and immune cells that are surrounded by the components of the extracellular matrix (ECM). Glycosaminoglycans (GAGs), natural biomacromolecules, essential ECM, and cell membrane components are extensively altered in cancer tissues. During disease progression, the GAG fine structure changes in a manner associated with disease evolution. Thus, changes in the GAG sulfation pattern are immediately correlated to malignant transformation. Their molecular weight, distribution, composition, and fine modifications, including sulfation, exhibit distinct alterations during cancer development. GAGs and GAG-based molecules, due to their unique properties, are suggested as promising effectors for anticancer therapy. Considering their participation in tumorigenesis, their utilization in drug development has been the focus of both industry and academic research efforts. These efforts have been developing in two main directions; (i) utilizing GAGs as targets of therapeutic strategies and (ii) employing GAGs specificity and excellent physicochemical properties for targeted delivery of cancer therapeutics. This review will comprehensively discuss recent developments and the broad potential of GAG utilization for cancer therapy.


Assuntos
Glicosaminoglicanos/metabolismo , Animais , Protocolos Antineoplásicos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Heparina/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Nanoestruturas/química
15.
Molecules ; 26(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799537

RESUMO

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund's adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.


Assuntos
Artrite/tratamento farmacológico , Extratos Vegetais/farmacologia , Zingiberaceae/metabolismo , Animais , Apoptose/efeitos dos fármacos , Artrite/genética , Artrite/imunologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , NF-kappa B/metabolismo , Cultura Primária de Células , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Rizoma/metabolismo , Suínos , Fator de Transcrição RelA/metabolismo
16.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921767

RESUMO

Cell surface proteoglycans are known to be important regulators of many aspects of cell behavior. The principal family of transmembrane proteoglycans is the syndecans, of which there are four in mammals. Syndecan-1 is mostly restricted to epithelia, and bears heparan sulfate chains that are capable of interacting with a large array of polypeptides, including extracellular matrix components and potent mediators of proliferation, adhesion and migration. For this reason, it has been studied extensively with respect to carcinomas and tumor progression. Frequently, but not always, syndecan-1 levels decrease as tumor grade, stage and invasiveness and dedifferentiation increase. This parallels experiments that show depletion of syndecan-1 can be accompanied by loss of cadherin-mediated adhesion. However, in some tumors, levels of syndecan-1 increase, but the characterization of its distribution is relevant. There can be loss of membrane staining, but acquisition of cytoplasmic and/or nuclear staining that is abnormal. Moreover, the appearance of syndecan-1 in the tumor stroma, either associated with its cellular component or the collagenous matrix, is nearly always a sign of poor prognosis. Given its relevance to myeloma progression, syndecan-1-directed antibody-toxin conjugates are being tested in clinical and preclinical trials, and may have future relevance to some carcinomas.


Assuntos
Carcinoma/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Sindecana-1/metabolismo , Animais , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Proteoglicanas/metabolismo
17.
Invest Ophthalmol Vis Sci ; 62(3): 28, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33749719

RESUMO

Purpose: This study characterized the role of glycosaminoglycans (GAGs) in the hydration, thickness, and biomechanical properties of posterior and anterior porcine sclera. Methods: The scleral discs and strips were obtained from the anterior and posterior parts of porcine eyes, and their initial hydration and thickness were measured. The anterior and posterior scleral discs were used to show the efficacy of the GAG removal protocol by quantifying their GAG content. The strips were divided into three groups of PBS treatment, buffer treatment, and enzyme treatment in order to assess the effects of different treatment procedures on the thickness, hydration, and viscoelastic properties of the samples. The mechanical properties of the strips were determined by performing uniaxial tensile stress relaxation experiments. Results: It was found that the control and buffer groups had insignificant differences in all measured quantities. The samples from the posterior region had a significantly larger GAG content and thickness in comparison with those from anterior region; however, there was an insignificant difference in their hydration. The GAG depletion process decreased the hydration of both anterior and posterior samples significantly (P < 0.05). Furthermore, the mechanical tests showed that the removal of GAGs resulted in stiffer mechanical behavior in both anterior and posterior samples (P < 0.05). In particular, the peak stress and equilibrium stress were significantly larger for the strips in the enzyme treatment group. Conclusions: GAGs and their interaction with the collagen network are important in defining the hydration and mechanical properties of both posterior and anterior sclera.


Assuntos
Fenômenos Biomecânicos/fisiologia , Água Corporal/metabolismo , Glicosaminoglicanos/metabolismo , Esclera/metabolismo , Animais , Elasticidade , Suínos , Resistência à Tração
18.
J Med Chem ; 64(6): 2937-2952, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33719441

RESUMO

There are currently no approved disease-modifying osteoarthritis (OA) drugs (DMOADs). The aggrecanase ADAMTS-5 is key in the degradation of human aggrecan (AGC), a component of cartilage. Therefore, ADAMTS-5 is a promising target for the identification of DMOADs. We describe the discovery of GLPG1972/S201086, a potent and selective ADAMTS-5 inhibitor obtained by optimization of a promising hydantoin series following an HTS. Biochemical activity against rat and human ADAMTS-5 was assessed via a fluorescence-based assay. ADAMTS-5 inhibitory activity was confirmed with human aggrecan using an AGC ELISA. The most promising compounds were selected based on reduction of glycosaminoglycan release after interleukin-1 stimulation in mouse cartilage explants and led to the discovery of GLPG1972/S201086. The anticatabolic activity was confirmed in mouse cartilage explants (IC50 < 1.5 µM). The cocrystal structure of GLPG1972/S201086 with human recombinant ADAMTS-5 is discussed. GLPG1972/S201086 has been investigated in a phase 2 clinical study in patients with knee OA (NCT03595618).


Assuntos
Proteína ADAMTS5/antagonistas & inibidores , Osteoartrite/tratamento farmacológico , Proteína ADAMTS5/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cães , Glicosaminoglicanos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Osteoartrite/metabolismo , Ratos
19.
Biomolecules ; 11(2)2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572941

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease, caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA). IDUA catalyzes the degradation of the glycosaminoglycans dermatan and heparan sulfate (DS and HS, respectively). Lack of the enzyme leads to pathologic accumulation of undegraded HS and DS with subsequent disease manifestations in multiple organs. The disease can be divided into severe (Hurler syndrome) and attenuated (Hurler-Scheie, Scheie) forms. Currently approved treatments consist of enzyme replacement therapy (ERT) and/or hematopoietic stem cell transplantation (HSCT). Patients with attenuated disease are often treated with ERT alone, while the recommended therapy for patients with Hurler syndrome consists of HSCT. While these treatments significantly improve disease manifestations and prolong life, a considerable burden of disease remains. Notably, treatment can partially prevent, but not significantly improve, clinical manifestations, necessitating early diagnosis of disease and commencement of treatment. This review discusses these standard therapies and their impact on common disease manifestations in patients with MPS I. Where relevant, results of animal models of MPS I will be included. Finally, we highlight alternative and emerging treatments for the most common disease manifestations.


Assuntos
Terapia de Reposição de Enzimas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Iduronidase/biossíntese , Mucopolissacaridose I/fisiopatologia , Mucopolissacaridose I/terapia , Animais , Doenças Ósseas/complicações , Doenças Ósseas/terapia , Transtornos Cognitivos/complicações , Transtornos Cognitivos/terapia , Feminino , Glicosaminoglicanos/metabolismo , Perda Auditiva/complicações , Perda Auditiva/terapia , Cardiopatias/complicações , Cardiopatias/terapia , Humanos , Masculino , Amplitude de Movimento Articular , Transplante de Células-Tronco/métodos , Transplante Homólogo
20.
Biomolecules ; 11(2)2021 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573119

RESUMO

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.


Assuntos
Glicosaminoglicanos/metabolismo , Glipicanas/metabolismo , Cabelo/crescimento & desenvolvimento , Proteoglicanas de Heparan Sulfato/metabolismo , Algoritmos , Alopecia/diagnóstico , Alopecia/prevenção & controle , Biópsia , Western Blotting , Análise por Conglomerados , Dermatologia , Proteínas da Matriz Extracelular , Cabelo/metabolismo , Folículo Piloso/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Análise de Componente Principal , Espectrofotometria Infravermelho/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...