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1.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674659

RESUMO

Heparan sulfate is a ubiquitous, variably sulfated interactive glycosaminoglycan that consists of repeating disaccharides of glucuronic acid and glucosamine that are subject to a number of modifications (acetylation, de-acetylation, epimerization, sulfation). Variable heparan sulfate chain lengths and sequences within the heparan sulfate chains provide structural diversity generating interactive oligosaccharide binding motifs with a diverse range of extracellular ligands and cellular receptors providing instructional cues over cellular behaviour and tissue homeostasis through the regulation of essential physiological processes in development, health, and disease. heparan sulfate and heparan sulfate-PGs are integral components of the specialized glycocalyx surrounding cells. Heparan sulfate is the most heterogeneous glycosaminoglycan, in terms of its sequence and biosynthetic modifications making it a difficult molecule to fully characterize, multiple ligands also make an elucidation of heparan sulfate functional properties complicated. Spatio-temporal presentation of heparan sulfate sulfate groups is an important functional determinant in tissue development and in cellular control of wound healing and extracellular remodelling in pathological tissues. The regulatory properties of heparan sulfate are mediated via interactions with chemokines, chemokine receptors, growth factors and morphogens in cell proliferation, differentiation, development, tissue remodelling, wound healing, immune regulation, inflammation, and tumour development. A greater understanding of these HS interactive processes will improve therapeutic procedures and prognoses. Advances in glycosaminoglycan synthesis and sequencing, computational analytical carbohydrate algorithms and advanced software for the evaluation of molecular docking of heparan sulfate with its molecular partners are now available. These advanced analytic techniques and artificial intelligence offer predictive capability in the elucidation of heparan sulfate conformational effects on heparan sulfate-ligand interactions significantly aiding heparan sulfate therapeutics development.


Assuntos
Glicosaminoglicanos , Proteoglicanas , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Simulação de Acoplamento Molecular , Inteligência Artificial , Ligantes , Heparitina Sulfato/metabolismo
2.
Methods Mol Biol ; 2619: 25-38, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662459

RESUMO

Chondroitin sulfate proteoglycans (CSPGs) are polyanionic extra/pericellular matrix macromolecules that surround almost all cell types and create microenvironmental niches to support miscellaneous cellular events. In general, the multifunctional properties of CSPGs are attributable to the structural divergence of the CS glycosaminoglycan (GAG) moieties. Because the expression profiles of the GAG chains of CSPGs change with developmental stage, aging, and disease progression, characterization of the GAG chains is essential to understand the functional roles of CSPGs. This chapter describes the basic protocols for GAG moiety-based immunochemical detection of CSPGs in biological samples in conjunction with CS disaccharide composition analysis.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Glicosaminoglicanos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Glicosaminoglicanos/metabolismo , Dissacarídeos , Sulfatos de Condroitina/química , Condroitina
3.
Methods Mol Biol ; 2619: 211-224, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662472

RESUMO

This chapter describes how to generate, visualize, and analyze interaction networks of glycosaminoglycans (GAGs), which are linear polyanionic polysaccharides mostly located at the cell surface and in the extracellular matrix. The protocol is divided into three major steps: (1) the collection of GAG-mediated interaction data, (2) the visualization of GAG interaction networks, and (3) the computational enrichment analyses of these networks to identify their overrepresented features (e.g., protein domains, location, molecular functions, and biological pathways) compared to a reference proteome. These analyses are critical to interpret GAG interactomic datasets, decipher their specificities and functions, and ultimately identify GAG-protein interactions to target for therapeutic purpose.


Assuntos
Glicosaminoglicanos , Mapas de Interação de Proteínas , Glicosaminoglicanos/metabolismo , Matriz Extracelular/metabolismo , Proteoma/metabolismo
4.
J Med Chem ; 66(2): 1321-1338, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36634271

RESUMO

Sulfated glycosaminoglycans (GAGs), or synthetic mimetics thereof, are not favorably viewed as orally bioavailable drugs owing to their high number of anionic sulfate groups. Devising an approach for oral delivery of such highly sulfated molecules would be very useful. This work presents the concept that conjugating cholesterol to synthetic sulfated GAG mimetics enables oral delivery. A focused library of sulfated GAG mimetics was synthesized and found to inhibit the growth of a colorectal cancer cell line under spheroid conditions with a wide range of potencies ( 0.8 to 46 µM). Specific analogues containing cholesterol, either alone or in combination with clinical utilized drugs, exhibited pronounced in vivo anticancer potential with intraperitoneal as well as oral administration, as assessed by ex vivo tertiary and quaternary spheroid growth, cancer stem cell (CSC) markers, and/or self-renewal factors. Overall, cholesterol derivatization of highly sulfated GAG mimetics affords an excellent approach for engineering oral activity.


Assuntos
Glicosaminoglicanos , Sulfatos , Glicosaminoglicanos/farmacologia , Glicosaminoglicanos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Biomimética
5.
Hum Gene Ther ; 34(1-2): 8-18, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36541357

RESUMO

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.


Assuntos
Disfunção Cognitiva , Iduronato Sulfatase , Mucopolissacaridose II , Mucopolissacaridose I , Doenças do Sistema Nervoso , Humanos , Animais , Camundongos , Idoso , Qualidade de Vida , Mucopolissacaridose II/genética , Mucopolissacaridose II/terapia , Mucopolissacaridose I/genética , Mucopolissacaridose I/terapia , Sistema Nervoso Central/metabolismo , Iduronidase/genética , Iduronidase/metabolismo , Iduronato Sulfatase/genética , Disfunção Cognitiva/metabolismo , Glicosaminoglicanos/metabolismo , Modelos Animais de Doenças
6.
Arterioscler Thromb Vasc Biol ; 43(2): 253-266, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36519467

RESUMO

BACKGROUND: Inappropriate activation and aggregation of platelets can lead to arterial thrombosis. Thrombin is the most potent platelet agonist that activates human platelets via two PARs (proteinase-activated receptors), PAR1 and PAR4. The aim is to study the activity and mechanism of an oligosaccharide HS-11 (the undecasaccharide, derived from sea cucumber Holothuria fuscopunctata) in inhibiting thrombin-mediated platelet activation and aggregation and to evaluate its antithrombotic activity. METHODS: Platelet activation was analyzed by detecting CD62P/P-selectin expression using flow cytometry. The HS-11-thrombin interaction and the binding site were studied by biolayer interferometry. Intracellular Ca2+ mobilization of platelets was measured by FLIPR Tetra System using Fluo-4 AM (Fluo-4 acetoxymethyl). Platelet aggregation, thrombus formation, and bleeding Assay were assessed. RESULTS: An oligosaccharide HS-11, depolymerized from fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata blocks the interaction of thrombin with PAR1 and PAR4 complex by directly binding to thrombin exosite II, and completely inhibits platelet signal transduction, including intracellular Ca2+ mobilization and protein phosphorylation. Furthermore, HS-11 potently inhibits thrombin-PARs-mediated platelet aggregation and reduces thrombus formation in a model of ex vivo thrombosis. CONCLUSIONS: The study firstly report that the fucosylated glycosaminoglycan oligosaccharide has antiplatelet activity by binding to thrombin exosite II, and demonstrates that thrombin exosite II plays an important role in the simultaneous activation of PAR1 and PAR4, which may be a potential antithrombotic target for effective treatment of arterial thrombosis.


Assuntos
Receptor PAR-1 , Trombose , Humanos , Plaquetas/metabolismo , Fibrinolíticos/farmacologia , Glicosaminoglicanos/metabolismo , Oligossacarídeos/farmacologia , Ativação Plaquetária , Agregação Plaquetária , Receptores de Trombina , Trombina/metabolismo , Trombose/prevenção & controle , Trombose/metabolismo
7.
Methods Mol Biol ; 2557: 709-720, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512246

RESUMO

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.


Assuntos
Fosfoadenosina Fosfossulfato , Proteoglicanas , Animais , Fosfoadenosina Fosfossulfato/metabolismo , Proteoglicanas/metabolismo , Complexo de Golgi/metabolismo , Glicosaminoglicanos/metabolismo , Sulfatos/metabolismo , Mamíferos/metabolismo
8.
Int J Mol Sci ; 23(24)2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-36555315

RESUMO

Glucocorticoids are steroid hormones that play diverse roles in numerous normal and pathological processes. They are actively used to treat a wide variety of diseases, including neurodegenerative and inflammatory diseases, cancers, and COVID-19, among others. However, the long-term use of glucocorticoids is associated with numerous side effects. Molecular mechanisms of these negative side effects are not completely understood. Recently, arguments have been made that one such mechanisms may be related to the influence of glucocorticoids on O-glycosylated components of the cell surface and extracellular matrix, in particular on proteoglycans and glycosaminoglycans. The potential toxic effects of glucocorticoids on these glycosylated macromolecules are particularly meaningful for brain physiology because proteoglycans/glycosaminoglycans are the main extracellular components of brain tissue. Here, we aim to review the known effects of glucocorticoids on proteoglycan expression and glycosaminoglycan content in different tissues, with a specific focus on the brain.


Assuntos
Glucocorticoides , Glicosaminoglicanos , Proteoglicanas , Humanos , Glucocorticoides/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo
9.
Appl Environ Microbiol ; 88(22): e0154622, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36342199

RESUMO

The degradation of glycosaminoglycans (GAGs) by intestinal bacteria is critical for their colonization in the human gut and the health of the host. Both colonic Bacteroides and Firmicutes have been reported to degrade GAGs; however, the enzymatic details of the latter remain largely unknown. Our bioinformatic analyses of fecal Firmicutes revealed that their genomes, especially Hungatella hathewayi strains, are an abundant source of putative GAG-specific catabolic enzymes. Subsequently, we isolated a Firmicutes strain, H. hathewayi N2-326, that can catabolize various GAGs. While H. hathewayi N2-326 was as efficient in utilizing chondroitin sulfate A (CSA) and dermatan sulfate as Bacteroides thetaiotaomicron, a well-characterized GAG degrader, it outperformed B. thetaiotaomicron in assimilating hyaluronic acid. Unlike B. thetaiotaomicron, H. hathewayi N2-326 could not utilize heparin. The chondroitin lyase activity of H. hathewayi N2-326 was found to be present predominantly in the culture supernatant. Genome sequence analysis revealed three putative GAG lyases, but only the HH-chondroitin ABC lyase was upregulated in the presence of CSA. In addition, five CAZyme gene clusters containing GAG metabolism genes were significantly upregulated when grown on CSA. Further characterization of the recombinant HH-chondroitin ABC lyase revealed that it cleaves GAGs predominantly in an exo-mode to produce unsaturated disaccharides as the primary hydrolytic product while exhibiting a higher specific activity than reported chondroitin ABC lyases. HH-chondroitin ABC lyase represents the first characterized chondroitin lyase from intestinal Firmicutes and offers a viable commercial option for the production of chondroitin, dermatan, and hyaluronan oligosaccharides and also for potential medical applications. IMPORTANCE An increased understanding of GAG metabolism by intestinal bacteria is critical in identifying the driving factors for the composition, modulation, and homeostasis of the human gut microbiota. In addition, GAG-depolymerizing polysaccharide lyases are highly desired enzymes for the production of GAG oligosaccharides and as therapeutics. At present, the dissection of the enzymatic machinery for GAG degradation is highly skewed toward Bacteroides. In this study, we have isolated an efficient GAG-degrading Firmicutes bacterium from human feces and characterized the first chondroitin ABC lyase from a Firmicutes, which complements the fundamental knowledge of GAG utilization in the human colon. The genomic and transcriptomic analysis of the bacterium shows that Firmicutes might use a distinct approach to catabolize GAGs from that used by Bacteroides. The high specific activity of the characterized chondroitin ABC lyase aids future attempts to develop a commercial chondroitinase for industrial and medicinal applications.


Assuntos
Condroitina ABC Liase , Glicosaminoglicanos , Humanos , Bacteroides/genética , Bacteroides/metabolismo , Condroitina ABC Liase/química , Condroitina ABC Liase/genética , Condroitina ABC Liase/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Firmicutes/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Oligossacarídeos/química , Especificidade por Substrato , Intestinos/metabolismo
10.
ACS Appl Bio Mater ; 5(11): 5457-5464, 2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36228282

RESUMO

The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases and is believed to be an autocatalytic process with a very high kinetic barrier. Intensive studies have focused on overcoming the kinetic barriers under extremely nonphysiological in vitro conditions by altering the pH of PrP solution on solid surfaces, such as gold, mica, and a lipid bilayer. Importantly, sulfated glycosaminoglycans (GAGs), including heparin, were found to be associated with PrP misfolding and aggregation, suggesting GAGs have catalytic roles in PrP aggregation processes. However, the exact role and details of GAGs in the PrP aggregation are not clear and need a thorough perusal. Here, we investigate the PrP aggregation process on a heparin functionalized gold surface by in situ, real-time monitoring of the atomic scale details of the whole aggregation process by single molecule atomic force microscopy (AFM), combining simultaneous topographic and recognition (TREC) imaging and single molecule force spectroscopy (SMFS). We observed the whole aggregation process for full-length human recombinant PrP (23-231) aggregation on the heparin modified gold surface, from the formation of oligomers, to the assembly of protofibrils and short fibers, and the formation of elongated mature fibers. Heparin is found to promote the PrP aggregation by facilitating the formation of oligomers during the early nucleation stage.


Assuntos
Doenças Priônicas , Príons , Humanos , Príons/química , Proteínas Priônicas/química , Heparina/química , Ouro , Doenças Priônicas/metabolismo , Glicosaminoglicanos/metabolismo
11.
Cells ; 11(19)2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36230907

RESUMO

Esophageal reconstruction through bio-engineered allografts that highly resemble the peculiar properties of the tissue extracellular matrix (ECM) is a prospective strategy to overcome the limitations of current surgical approaches. In this work, human esophagus was decellularized for the first time in the literature by comparing three detergent-enzymatic protocols. After decellularization, residual DNA quantification and histological analyses showed that all protocols efficiently removed cells, DNA (<50 ng/mg of tissue) and muscle fibers, preserving collagen/elastin components. The glycosaminoglycan fraction was maintained (70-98%) in the decellularized versus native tissues, while immunohistochemistry showed unchanged expression of specific ECM markers (collagen IV, laminin). The proteomic signature of acellular esophagi corroborated the retention of structural collagens, basement membrane and matrix-cell interaction proteins. Conversely, decellularization led to the loss of HLA-DR expression, producing non-immunogenic allografts. According to hydroxyproline quantification, matrix collagen was preserved (2-6 µg/mg of tissue) after decellularization, while Second-Harmonic Generation imaging highlighted a decrease in collagen intensity. Based on uniaxial tensile tests, decellularization affected tissue stiffness, but sample integrity/manipulability was still maintained. Finally, the cytotoxicity test revealed that no harmful remnants/contaminants were present on acellular esophageal matrices, suggesting allograft biosafety. Despite the different outcomes showed by the three decellularization methods (regarding, for example, tissue manipulability, DNA removal, and glycosaminoglycans/hydroxyproline contents) the ultimate validation should be provided by future repopulation tests and in vivo orthotopic implant of esophageal scaffolds.


Assuntos
Detergentes , Elastina , Colágeno , DNA/metabolismo , Esôfago/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Hidroxiprolina , Laminina , Proteômica
12.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232390

RESUMO

The dense neuropil of the central nervous system leaves only limited space for extracellular substances free. The advent of immunohistochemistry, soon followed by advanced diagnostic tools, enabled us to explore the biochemical heterogeneity and compartmentalization of the brain extracellular matrix in exploratory and clinical research alike. The composition of the extracellular matrix is critical to shape neuronal function; changes in its assembly trigger or reflect brain/spinal cord malfunction. In this study, we focus on extracellular matrix changes in neurodegenerative disorders. We summarize its phenotypic appearance and biochemical characteristics, as well as the major enzymes which regulate and remodel matrix establishment in disease. The specifically built basement membrane of the central nervous system, perineuronal nets and perisynaptic axonal coats can protect neurons from toxic agents, and biochemical analysis revealed how the individual glycosaminoglycan and proteoglycan components interact with these molecules. Depending on the site, type and progress of the disease, select matrix components can either proactively trigger the formation of disease-specific harmful products, or reactively accumulate, likely to reduce tissue breakdown and neuronal loss. We review the diagnostic use and the increasing importance of medical screening of extracellular matrix components, especially enzymes, which informs us about disease status and, better yet, allows us to forecast illness.


Assuntos
Doenças Neurodegenerativas , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Proteoglicanas/metabolismo
13.
Int J Mol Sci ; 23(19)2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36232472

RESUMO

Mucopolysaccharidosis type I (MPSI) (OMIM #252800) is an autosomal recessive disorder caused by pathogenic variants in the IDUA gene encoding for the lysosomal alpha-L-iduronidase enzyme. The deficiency of this enzyme causes systemic accumulation of glycosaminoglycans (GAGs). Although disease manifestations are typically not apparent at birth, they can present early in life, are progressive, and include a wide spectrum of phenotypic findings. Among these, the storage of GAGs within the lysosomes disrupts cell function and metabolism in the cartilage, thus impairing normal bone development and ossification. Skeletal manifestations of MPSI are often refractory to treatment and severely affect patients' quality of life. This review discusses the pathological and molecular processes leading to impaired endochondral ossification in MPSI patients and the limitations of current therapeutic approaches. Understanding the underlying mechanisms responsible for the skeletal phenotype in MPSI patients is crucial, as it could lead to the development of new therapeutic strategies targeting the skeletal abnormalities of MPSI in the early stages of the disease.


Assuntos
Iduronidase , Mucopolissacaridose I , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/genética , Mucopolissacaridose I/genética , Fenótipo , Qualidade de Vida
14.
Bone ; 165: 116548, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36122648

RESUMO

Bone material / compositional properties are significant determinants of bone quality, thus strength. Raman spectroscopic analysis provides information on the quantity and quality of all three bone tissue components (mineral, organic matrix, and tissue water). The overwhelming majority of the published reports on the subject concern adults. We have previously reported on these properties in growing children and young adults, in the cancellous compartment. The purpose of the present study was to create normative reference data of bone material / compositional properties for children and young adults, in the cortical compartment. We performed Raman (Senterra (Bruker Optik GmbH), 50× objective, with an excitation of 785 nm (100 mW) and a lateral resolution of ~0.6 µm) microspectroscopic analysis of transiliac bone samples from 54 individuals between 1.5 and 23 years of age, with no known metabolic bone disease, and which have been previously used to establish histomorphometric, bone mineralization density distribution, and cancellous bone quality reference values. The bone quality indices that were determined were: mineral/matrix ratio (MM) from the integrated areas of the v2PO4 (410-460 cm-1) and the amide III (1215-1300 cm-1) bands, tissue water in nanopores approximated by the ratio of the integrated spectral area ~ 494-509 cm-1 to Amide III band, the glycosaminoglycan (GAG) content (ratio of integrated area 1365-1390 cm-1 to the Amide III band, the sulfated proteoglycan (sPG) content as the ratio of the integrated peaks ~1062 cm-1 and 1365-1390 cm-1, the pyridinoline (Pyd) content estimated from the ratio of the absorbance height at 1660 cm-1 / area of the amide I (1620-1700 cm-1) band, and the mineral maturity / crystallinity (MMC) estimated from the inverse of the full width at half height of the v1PO4 (930-980 cm-1) band. Analyses were performed at the three distinct cortical surfaces (endosteal, osteonal, periosteal) at specific anatomical microlocations, namely the osteoid, and the three precisely known tissue ages based on the presence of fluorescence double labels. Measurements were also taken in interstitial bone, a much older tissue that has undergone extensive secondary mineralization. Overall, significant dependencies of the measured parameters on tissue age were observed, while at any given tissue age, sex and subject age were minimal confounders. The established Raman database in the cortical compartments complements the previously published one in cancellous bone, and provides healthy baseline bone quality indices that may serve as a valuable tool to identify alterations due to pediatric disease.


Assuntos
Osso e Ossos , Osso Cortical , Criança , Humanos , Adulto Jovem , Amidas , Osso e Ossos/metabolismo , Densidade Óssea , Osso Cortical/metabolismo , Glicosaminoglicanos/metabolismo , Minerais/metabolismo , Proteoglicanas , Água , Lactente , Pré-Escolar , Adolescente
15.
Molecules ; 27(18)2022 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-36144634

RESUMO

Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG-MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29-heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.


Assuntos
Sulfatos de Condroitina , Vírus da Varíola dos Macacos , Dermatan Sulfato , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Vírus da Varíola dos Macacos/metabolismo , Poliéster Sulfúrico de Pentosana , Ressonância de Plasmônio de Superfície , Vírus Vaccinia
16.
Int J Mol Sci ; 23(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36142595

RESUMO

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.


Assuntos
Gangliosidoses GM2 , Doença de Tay-Sachs , Desoxirribonuclease I/metabolismo , Fibroblastos/metabolismo , Proteína Ativadora de G(M2) , Gangliosídeo G(M2)/genética , Gangliosídeo G(M2)/metabolismo , Gangliosidoses GM2/genética , Gangliosidoses GM2/metabolismo , Gangliosidoses GM2/terapia , Edição de Genes , Globosídeos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidase A/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Doença de Tay-Sachs/genética , Doença de Tay-Sachs/metabolismo , Doença de Tay-Sachs/terapia , beta-N-Acetil-Hexosaminidases/metabolismo
17.
Molecules ; 27(17)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36080234

RESUMO

Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen-glucose deprivation (OGD)-induced human umbilical endothelial cells' (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the levels of the glutamate-cysteine ligase catalytic subunit (GCLc) and the glutathione synthetase (GSS) proteins, a key enzyme for de novo GSH synthesis, were determined using enzyme-linked immunoassay (ELISA). We demonstrated that the SDX treatment in OGD conditions significantly elevated the intracellular GSH, enhanced the GSH:GSSG ratio, shifting the redox potential to a more pro-reducing status. Furthermore, SDX increased the levels of both GCLc and GSS. The results show that SDX protects the human endothelial cells against ischemic stress by affecting the GSH levels and cellular redox state. These changes suggest that the reduction in the ischemia-induced vascular endothelial cell injury through repressing apoptosis and oxidative stress associated with SDX treatment may be due to an increase in GSH synthesis and modulation of the GSH redox system.


Assuntos
Células Endoteliais , Glucose , Endotélio/metabolismo , Glucose/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacologia , Humanos , Isquemia/metabolismo , Oxirredução , Estresse Oxidativo , Oxigênio/metabolismo
18.
Biomaterials ; 289: 121786, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36116171

RESUMO

Extracellular matrix is rich in biomolecules including structural proteins, glycosaminoglycans, and small molecules that are important for the maintenance and repair of tissue. Decellularized extracellular matrix (dECM) is expected to retain these key biomolecules and makes it a promising biomaterial candidate for regenerative medicine applications. To date, dECM-particle based biomaterials have been developed to engineer over 15 tissue types or organs, with the ultimate goal of mimicking specific biological and physical properties of the native tissue. The most common scaffold types are injectable hydrogels, electrospun scaffolds and bioprinted scaffolds. The purpose of this review paper is to highlight key challenges, fabrication methods and progress made for each tissue type, along with the discussion of other elements that are integral to push dECM biomaterials towards effective and specialized tissue repair.


Assuntos
Materiais Biocompatíveis , Medicina Regenerativa , Materiais Biocompatíveis/química , Matriz Extracelular Descelularizada , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Hidrogéis/metabolismo , Hidrogéis/uso terapêutico , Engenharia Tecidual , Tecidos Suporte/química
19.
Fluids Barriers CNS ; 19(1): 76, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36117162

RESUMO

Mucopolysaccharidoses comprise a set of genetic diseases marked by an enzymatic dysfunction in the degradation of glycosaminoglycans in lysosomes. There are eight clinically distinct types of mucopolysaccharidosis, some with various subtypes, based on which lysosomal enzyme is deficient and symptom severity. Patients with mucopolysaccharidosis can present with a variety of symptoms, including cognitive dysfunction, hepatosplenomegaly, skeletal abnormalities, and cardiopulmonary issues. Additionally, the onset and severity of symptoms can vary depending on the specific disorder, with symptoms typically arising during early childhood. While there is currently no cure for mucopolysaccharidosis, there are clinically approved therapies for the management of clinical symptoms, such as enzyme replacement therapy. Enzyme replacement therapy is typically administered intravenously, which allows for the systemic delivery of the deficient enzymes to peripheral organ sites. However, crossing the blood-brain barrier (BBB) to ameliorate the neurological symptoms of mucopolysaccharidosis continues to remain a challenge for these large macromolecules. In this review, we discuss the transport mechanisms for the delivery of lysosomal enzymes across the BBB. Additionally, we discuss the several therapeutic approaches, both preclinical and clinical, for the treatment of mucopolysaccharidoses.


Assuntos
Barreira Hematoencefálica , Mucopolissacaridoses , Barreira Hematoencefálica/metabolismo , Pré-Escolar , Glicosaminoglicanos/metabolismo , Humanos , Lisossomos/metabolismo , Mucopolissacaridoses/terapia
20.
Front Immunol ; 13: 915963, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36131938

RESUMO

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.


Assuntos
Antígenos CD28 , Linfócitos T CD4-Positivos , Colesterol/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transcrição Genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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