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1.
Invest Ophthalmol Vis Sci ; 62(14): 5, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34730792

RESUMO

Purpose: The arrangement of lens cells is regulated by ocular growth factors. Although the effects of these inductive molecules on lens cell behavior (proliferation, survival, and fiber differentiation) are well-characterized, the precise mechanisms underlying the regulation of growth factor-mediated signaling in lens remains elusive. Increasing evidence highlights the importance of heparan sulfate proteoglycans (HSPGs) for the signaling regulation of growth factors; however, the identity of the different lens HSPGs and the specific roles they play in lens biology are still unknown. Methods: Semiquantitative real-time (RT)-PCR and immunolabeling were used to characterize the spatial distribution of all known HSPG core proteins and their associated glycosaminoglycans (heparan and chondroitin sulfate) in the postnatal rat lens. Fibroblast growth factor (FGF)-2-treated lens epithelial explants, cultured in the presence of Surfen (an inhibitor of heparan sulfate [HS]-growth factor binding interactions) were used to investigate the requirement for HS in FGF-2-induced proliferation, fiber differentiation, and ERK1/2-signaling. Results: The lens expresses all HSPGs. These HSPGs are differentially localized to distinct functional regions of the lens. In vitro, inhibition of HS-sulfation with Surfen blocked FGF-2-mediated ERK1/2-signaling associated with lens epithelial cell proliferation and fiber differentiation, highlighting that these cellular processes are dependent on HS. Conclusions: These findings support a requirement for HSPGs in FGF-2 driven lens cell proliferation and fiber differentiation. The identification of specific HSPG core proteins in key functional lens regions, and the divergent expression patterns of closely related HSPGs, suggests that different HSPGs may differentially regulate growth factor signaling networks leading to specific biological events involved in lens growth and maintenance.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteoglicanas de Heparan Sulfato/genética , Cristalino/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Sulfatos de Condroitina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/metabolismo , Cristalino/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Ureia/análogos & derivados , Ureia/farmacologia
2.
Front Immunol ; 12: 676662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489931

RESUMO

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/sangue , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Células Cultivadas , Ativação do Complemento , Células Endoteliais/metabolismo , Heparina/análogos & derivados , Heparina/farmacologia , Humanos , Glomérulos Renais/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
3.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34413211

RESUMO

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.


Assuntos
Antivirais/farmacologia , Fatores Imunológicos/farmacologia , Lactoferrina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Células Epiteliais , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/imunologia , Heparitina Sulfato/metabolismo , Hepatócitos , Ensaios de Triagem em Larga Escala , Humanos , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , Células Vero
4.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34342561

RESUMO

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Dengue/virologia , Genótipo , Heparitina Sulfato/metabolismo , Proteínas do Envelope Viral/química , Animais , Sítios de Ligação , Encéfalo/patologia , Comunicação Celular , Linhagem Celular , Dengue/patologia , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Feminino , Heparina , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Fenótipo , Filogenia , Conformação Proteica , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética , Virulência , Ligação Viral
5.
J Virol ; 95(21): e0135721, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406867

RESUMO

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Assuntos
Evolução Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Biológica , Animais , Sítios de Ligação , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar , Furina/metabolismo , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral , Ligação Viral
6.
Exp Cell Res ; 407(1): 112785, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34411609

RESUMO

Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.


Assuntos
Acetilglucosaminidase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucopolissacaridose III/metabolismo , Células-Tronco Neurais/metabolismo , Acetilglucosaminidase/genética , Diferenciação Celular/fisiologia , Heparitina Sulfato/metabolismo , Humanos , Lisossomos/metabolismo , Mucopolissacaridose III/genética , Neurônios/metabolismo , Fenótipo
7.
Cell Rep ; 36(2): 109364, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34214467

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.


Assuntos
COVID-19/imunologia , COVID-19/metabolismo , Receptores Virais , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteômica , Receptores Virais/metabolismo , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Internalização do Vírus , Replicação Viral
8.
J Exp Med ; 218(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34106206

RESUMO

As indicated by its name, V-domain Ig suppressor of T cell activation (VISTA) is thought to serve primarily as an inhibitory protein that limits immune responses. VISTA antibodies can dampen the effects of several concomitantly elicited activation signals, including TCR and TLR activation, but it is currently unclear if VISTA agonism could singly affect immune cell biology. In this study, we discovered two novel VISTA antibodies and characterized their effects on human peripheral blood mononuclear cells by scRNA/CITE-seq. Both antibodies appeared to agonize VISTA in an Fc-functional manner to elicit transcriptional and functional changes in monocytes consistent with activation. We also used pentameric VISTA to identify Syndecan-2 and several heparan sulfate proteoglycan synthesis genes as novel regulators of VISTA interactions with monocytic cells, adding further evidence of bidirectional signaling. Together, our study highlights several novel aspects of VISTA biology that have yet to be uncovered in myeloid cells and serves as a foundation for future research.


Assuntos
Antígenos B7/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/imunologia , Sistemas CRISPR-Cas/genética , Heparitina Sulfato/metabolismo , Humanos , Ligação Proteica , Receptores Fc/metabolismo , Sindecana-2/metabolismo , Transcrição Genética , Transcriptoma/genética
9.
Nat Commun ; 12(1): 3495, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108486

RESUMO

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.


Assuntos
Transtorno do Espectro Autista/metabolismo , Dopamina/metabolismo , Heparitina Sulfato/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/patologia , Benzazepinas/uso terapêutico , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Antagonistas de Dopamina/uso terapêutico , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Heparitina Sulfato/farmacologia , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/patologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Mesencéfalo/patologia , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismo
10.
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
11.
PLoS One ; 16(6): e0252607, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086738

RESUMO

Heparan sulfate proteoglycans (HSPGs) consist of a core protein with side chains of the glycosaminoglycan heparan sulfate (HS). We have previously identified (i) the HSPGs syndecan-1 (SDC1), and collagen type XVIII (COL18) inside mouse and human islet beta cells, and (ii) a critical role for HS in beta cell survival and protection from reactive oxygen species (ROS). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress contributes to oxidative stress and type 2 diabetes (T2D) by depleting beta cell HSPGs/HS. A rapid loss of intra-islet/beta cell HSPGs, HS and heparanase (HPSE, an HS-degrading enzyme) accompanied upregulation of islet ER stress gene expression in both young T2D-prone db/db and Akita Ins2WT/C96Y mice. In MIN6 beta cells, HSPGs, HS and HPSE were reduced following treatment with pharmacological inducers of ER stress (thapsigargin or tunicamycin). Treatment of young db/db mice with Tauroursodeoxycholic acid (TUDCA), a chemical protein folding chaperone that relieves ER stress, improved glycemic control and increased intra-islet HSPG/HS. In vitro, HS replacement with heparin (a highly sulfated HS analogue) significantly increased the survival of wild-type and db/db beta cells and restored their resistance to hydrogen peroxide-induced death. We conclude that ER stress inhibits the synthesis/maturation of HSPG core proteins which are essential for HS assembly, thereby exacerbating oxidative stress and promoting beta cell failure. Diminished intracellular HSPGs/HS represent a previously unrecognized critical link bridging ER stress, oxidative stress and beta cell failure in T2D.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Estresse do Retículo Endoplasmático , Proteoglicanas de Heparan Sulfato/metabolismo , Estresse Oxidativo , Fatores Ativadores da Transcrição/genética , Fatores Ativadores da Transcrição/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glucuronidase/genética , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Lactonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Sesquiterpenos/farmacologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
PLoS One ; 16(6): e0246610, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086711

RESUMO

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.


Assuntos
Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Rim/metabolismo , Rim/fisiologia , Receptores de Superfície Celular/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Células CHO , Técnicas de Cultura de Células , Linhagem Celular , Cricetinae , Cricetulus , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/patogenicidade , Perfilação da Expressão Gênica/métodos , Heparitina Sulfato/metabolismo , Integrinas/metabolismo
13.
Emerg Microbes Infect ; 10(1): 1169-1179, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34013833

RESUMO

Zika virus (ZIKV) is a flavivirus transmitted by mosquitoes of the genus Aedes, but unlike other flaviviruses, ZIKV can be sexually transmitted by vaginal intercourse. The healthy vaginal pH ranges from 4.0 to 6.0, reaching values of 6.0-7.0 after semen deposition. Here, we report that low extracellular pH values (range 6.2-6.6) dramatically increase ZIKV infection on cell lines of different origin including some derived from the female genital tract and monocyte-derived macrophages. Furthermore, low pH significantly increased ZIKV infection of human ectocervix and endocervix cultured ex-vivo. Enhancement of infection by low pH was also observed using different ZIKV strains and distinct methods to evaluate viral infection, i.e. plaque assays, RT-PCR, flow cytometry, and fluorescence microscopy. Analysis of the mechanisms involved revealed that the enhancement of ZIKV infection induced by low pH was associated with increased binding of the viral particles to the heparan sulphate expressed on the target cell surface. Acidosis represents a critical but generally overlooked feature of the female genital tract, with major implications for sexual transmission diseases. Our results suggest that low vaginal pH might promote male-to-female transmission of ZIKV infection.


Assuntos
Colo do Útero/química , Vagina/química , Infecção por Zika virus/transmissão , Zika virus/patogenicidade , Acidose , Animais , Linhagem Celular , Colo do Útero/virologia , Chlorocebus aethiops , Feminino , Heparitina Sulfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Vagina/virologia , Células Vero , Zika virus/genética
14.
J Virol ; 95(16): e0001021, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037420

RESUMO

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.


Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Receptores Virais/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Animais , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Receptor 1 de Quimiocina CX3C/antagonistas & inibidores , Receptor 1 de Quimiocina CX3C/química , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/virologia , Heparitina Sulfato/metabolismo , Humanos , Mutação , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Sigmodontinae , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/genética
16.
Emerg Microbes Infect ; 10(1): 1065-1076, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34013835

RESUMO

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Hipóxia Celular/fisiologia , Heparitina Sulfato/metabolismo , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/fisiologia , Sindecana-1/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Heparitina Sulfato/genética , Humanos , Neuropilina-1/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sindecana-1/genética , Células Vero , Ligação Viral/efeitos dos fármacos
17.
Cells ; 10(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923753

RESUMO

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA-VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.


Assuntos
Heparitina Sulfato/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Neuropilina-2/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose , Humanos , Lipoproteínas VLDL/metabolismo , Sulfatases/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Cells ; 10(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33918094

RESUMO

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.


Assuntos
Heparitina Sulfato/metabolismo , Mucopolissacaridose III/metabolismo , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Fenômenos Biofísicos , Líquido da Lavagem Broncoalveolar , Colesterol/metabolismo , Cromatografia Líquida , Gangliosídeo G(M3)/metabolismo , Regulação da Expressão Gênica , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Monoglicerídeos/metabolismo , Fosfolipídeos/metabolismo , Padrões de Referência , Espectrometria de Massas em Tandem
19.
Nat Chem Biol ; 17(6): 684-692, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33846619

RESUMO

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.


Assuntos
Proteínas F-Box/antagonistas & inibidores , Estudo de Associação Genômica Ampla , Heparitina Sulfato/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Algoritmos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Matriz Extracelular/genética , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica/genética , RNA-Seq , Sulfotransferases/antagonistas & inibidores
20.
Semin Thromb Hemost ; 47(3): 240-253, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33794549

RESUMO

Cell surface proteoglycans are important constituents of the glycocalyx and participate in cell-cell and cell-extracellular matrix (ECM) interactions, enzyme activation and inhibition, and multiple signaling routes, thereby regulating cell proliferation, survival, adhesion, migration, and differentiation. Heparanase, the sole mammalian heparan sulfate degrading endoglycosidase, acts as an "activator" of HS proteoglycans, thus regulating tissue hemostasis. Heparanase is a multifaceted enzyme that together with heparan sulfate, primarily syndecan-1, drives signal transduction, immune cell activation, exosome formation, autophagy, and gene transcription via enzymatic and nonenzymatic activities. An important feature is the ability of heparanase to stimulate syndecan-1 shedding, thereby impacting cell behavior both locally and distally from its cell of origin. Heparanase releases a myriad of HS-bound growth factors, cytokines, and chemokines that are sequestered by heparan sulfate in the glycocalyx and ECM. Collectively, the heparan sulfate-heparanase axis plays pivotal roles in creating a permissive environment for cell proliferation, differentiation, and function, often resulting in the pathogenesis of diseases such as cancer, inflammation, endotheliitis, kidney dysfunction, tissue fibrosis, and viral infection.


Assuntos
Doença , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos
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