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1.
Cells ; 13(5)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38474382

RESUMO

Glypicans (Glps) are a family of heparan sulphate proteoglycans that are attached to the outer plasma membrane leaflet of the producing cell by a glycosylphosphatidylinositol anchor. Glps are involved in the regulation of many signalling pathways, including those that regulate the activities of Wnts, Hedgehog (Hh), Fibroblast Growth Factors (FGFs), and Bone Morphogenetic Proteins (BMPs), among others. In the Hh-signalling pathway, Glps have been shown to be essential for ligand transport and the formation of Hh gradients over long distances, for the maintenance of Hh levels in the extracellular matrix, and for unimpaired ligand reception in distant recipient cells. Recently, two mechanistic models have been proposed to explain how Hh can form the signalling gradient and how Glps may contribute to it. In this review, we describe the structure, biochemistry, and metabolism of Glps and their interactions with different components of the Hh-signalling pathway that are important for the release, transport, and reception of Hh.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Glipicanas/metabolismo , Proteínas de Drosophila/metabolismo , Ligantes , Proteínas Hedgehog/metabolismo , Proteoglicanas de Heparan Sulfato
2.
Viruses ; 16(2)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38400072

RESUMO

To identify host factors that affect Bovine Herpes Virus Type 1 (BoHV-1) infection we previously applied a genome wide CRISPR knockout screen targeting all bovine protein coding genes. By doing so we compiled a list of both pro-viral and anti-viral proteins involved in BoHV-1 replication. Here we provide further analysis of those that are potentially involved in viral entry into the host cell. We first generated single cell knockout clones deficient in some of the candidate genes for validation. We provide evidence that Polio Virus Receptor-related protein (PVRL2) serves as a receptor for BoHV-1, mediating more efficient entry than the previously identified Polio Virus Receptor (PVR). By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we further demonstrate the significance of HSPG in BoHV-1 entry. Another intriguing cluster of candidate genes, COG1, COG2 and COG4-7 encode six subunits of the Conserved Oligomeric Golgi (COG) complex. MDBK cells lacking COG6 produced fewer but bigger plaques compared to control cells, suggesting more efficient release of newly produced virions from these COG6 knockout cells, due to impaired HSPG biosynthesis. We further observed that viruses produced by the COG6 knockout cells consist of protein(s) with reduced N-glycosylation, potentially explaining their lower infectivity. To facilitate candidate validation, we also detailed a one-step multiplex CRISPR interference (CRISPRi) system, an orthogonal method to KO that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified eight candidates that have been implicated in the synthesis of surface heparan sulfate proteoglycans (HSPGs). In summary, our experiments confirmed the two receptors PVR and PVRL2 for BoHV-1 entry into the host cell and other factors that affect this process, likely through the direct or indirect roles they play during HSPG synthesis and glycosylation of viral proteins.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Poliomielite , Humanos , Proteoglicanas de Heparan Sulfato , Internalização do Vírus , Receptores Virais/genética , Proteínas de Transporte
3.
J Biol Chem ; 300(2): 105660, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38242322

RESUMO

Persistent high-risk HPV infection is closely associated with cervical cancer development, and there is no drug targeting HPV on the market at present, so it is particularly important to understand the interaction mechanism between HPV and the host which may provide the novel strategies for treating HPV diseases. HPV can hijack cell surface heparan sulfate proteoglycans (HSPGs) as primary receptors. However, the secondary entry receptors for HPV remain elusive. We identify myosin-9 (NMHC-IIA) as a host factor that interacts with HPV L1 protein and mediates HPV internalization. Efficient HPV entry required myosin-9 redistribution to the cell surface regulated by HPV-hijacked MEK-MLCK signaling. Myosin-9 maldistribution by ML-7 or ML-9 significantly inhibited HPV pseudoviruses infection in vitro and in vivo. Meanwhile, N-glycans, especially the galactose chains, may act as the decoy receptors for HPV, which can block the interaction of HPV to myosin-9 and influence the way of HPV infection. Taken together, we identify myosin-9 as a novel functional entry receptor for high-risk HPV both in vitro and in vivo, and unravel the new roles of myosin-9 and N-glycans in HPV entry, which provides the possibilities for host targets of antiviral drugs.


Assuntos
Papillomavirus Humano , Infecções por Papillomavirus , Internalização do Vírus , Humanos , Proteínas do Citoesqueleto , Proteoglicanas de Heparan Sulfato/metabolismo , Miosinas , Linhagem Celular , Animais , Cricetinae , Cricetulus , Polissacarídeos/metabolismo
4.
Food Funct ; 15(4): 1948-1962, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38270052

RESUMO

Resveratrol, renowned as an antioxidant, also exhibits significant potential in combatting severe respiratory infections, particularly the respiratory syncytial virus (RSV). Nevertheless, the specific mechanism underlying its inhibition of RSV replication remains unexplored. Heparan sulfate proteoglycans (HSPGs) play a pivotal role as attachment factors for numerous viruses, offering a promising avenue for countering viral infections. Our research has unveiled that resveratrol effectively curbs RSV infection in a dose-dependent manner. Remarkably, resveratrol disrupts the early stages of RSV infection by engaging with HSPGs, rather than interacting with RSV surface proteins like fusion (F) protein and glycoprotein (G). Resveratrol's affinity appears to be predominantly directed towards the negatively charged sites on HSPGs, thus impeding the binding of viral receptors. In an in vivo study involving RSV-infected mice, resveratrol demonstrates its potential by ameliorating pulmonary pathology. This improvement is attributed to the inhibition of pro-inflammatory cytokine expression and a reduction in viral load within the lungs. Notably, resveratrol specifically alleviates inflammation characterized by an abundance of neutrophils in RSV-infected mice. In summation, our data first shows how resveratrol combats RSV infection through interactions with HSPGs, positioning it as a promising candidate for innovative drug development targeting RSV infections. Our study provides insight into the mechanism of resveratrol antiviral infection.


Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sinciciais Respiratórios , Animais , Camundongos , Vírus Sinciciais Respiratórios/fisiologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/patologia , Proteoglicanas de Heparan Sulfato/farmacologia , Resveratrol/farmacologia , Pulmão/patologia
5.
Cell Rep ; 43(1): 113668, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38198277

RESUMO

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.


Assuntos
Agrina , Proteoglicanas de Heparan Sulfato , Humanos , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Agrina/metabolismo , Miócitos Cardíacos/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo
6.
Elife ; 122024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38265865

RESUMO

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.


Assuntos
Proteínas de Drosophila , Drosophila , Proteoglicanas de Heparan Sulfato , Glicoproteínas de Membrana , Proteoglicanas , Animais , Membrana Celular , Drosophila/crescimento & desenvolvimento , Glipicanas , Heparitina Sulfato
7.
Proc Natl Acad Sci U S A ; 121(3): e2316733121, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38215181

RESUMO

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Fator de Transcrição STAT3/metabolismo , Linhagem Celular , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Movimento Celular
8.
Am J Physiol Cell Physiol ; 326(3): C810-C828, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38223931

RESUMO

This review examined how Hippo cell signaling and heparan sulfate (HS)-proteoglycans (HSPGs) regulate tissue form and function. Despite being a nonweight-bearing tissue, the brain is regulated by Hippo mechanoresponsive cell signaling pathways during embryonic development. HS-proteoglycans interact with growth factors, morphogens, and extracellular matrix components to regulate development and pathology. Pikachurin and Eyes shut (Eys) interact with dystroglycan to stabilize the photoreceptor axoneme primary cilium and ribbon synapse facilitating phototransduction and neurotransduction with bipolar retinal neuronal networks in ocular vision, the primary human sense. Another HSPG, Neurexin interacts with structural and adaptor proteins to stabilize synapses and ensure specificity of neural interactions, and aids in synaptic potentiation and plasticity in neurotransduction. HSPGs also stabilize the blood-brain barrier and motor neuron basal structures in the neuromuscular junction. Agrin and perlecan localize acetylcholinesterase and its receptors in the neuromuscular junction essential for neuromuscular control. The primary cilium is a mechanosensory hub on neurons, utilized by YES associated protein (YAP)-transcriptional coactivator with PDZ-binding motif (TAZ) Hippo, Hh, Wnt, transforming growth factor (TGF)-ß/bone matrix protein (BMP) receptor tyrosine kinase cell signaling. Members of the glypican HSPG proteoglycan family interact with Smoothened and Patched G-protein coupled receptors on the cilium to regulate Hh and Wnt signaling during neuronal development. Control of glycosyl sulfotransferases and endogenous protease expression by Hippo TAZ YAP represents a mechanism whereby the fine structure of HS-proteoglycans can be potentially modulated spatiotemporally to regulate tissue morphogenesis in a similar manner to how Hippo signaling controls sialyltransferase expression and mediation of cell-cell recognition, dysfunctional sialic acid expression is a feature of many tumors.


Assuntos
Proteoglicanas de Heparan Sulfato , Via de Sinalização Hippo , Feminino , Gravidez , Humanos , Acetilcolinesterase , Matriz Extracelular , Proteínas da Matriz Extracelular , Via de Sinalização Wnt , Receptores Proteína Tirosina Quinases
9.
Protein Sci ; 33(1): e4843, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37996967

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global health crisis with significant clinical morbidity and mortality. While angiotensin-converting enzyme 2 (ACE2) is the primary receptor for viral entry, other cell surface and extracellular matrix proteins may also bind to the viral receptor binding domain (RBD) within the SARS-CoV-2 spike protein. Recent studies have implicated heparan sulfate proteoglycans, specifically perlecan LG3, in facilitating SARS-CoV-2 binding to ACE2. However, the role of perlecan LG3 in SARS-CoV-2 pathophysiology is not well understood. In this study, we investigated the binding interactions between the SARS-CoV-2 spike protein RBD and perlecan LG3 through molecular modeling simulations and surface plasmon resonance (SPR) experiments. Our results indicate stable binding between LG3 and SARS-CoV-2 spike protein RBD, which may potentially enhance RBD-ACE2 interactions. These findings shed light on the role of perlecan LG3 in SARS-CoV-2 infection and provide insight into SARS-CoV-2 pathophysiology and potential therapeutic strategy for COVID-19.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/química , SARS-CoV-2/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Ligação Proteica
10.
J Control Release ; 365: 43-59, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37935257

RESUMO

Imprecise targeting of chemotherapeutic drugs often leads to severe toxicity during breast cancer therapy. To address this issue, we have devised a strategy to load dacarbazine (DC) into fucose-based carbon quantum dots (CQDs), which are subsequently coated with exosomes (Ex-DC@CQDs) derived from breast cancer cells. Nanoparticle tracking analysis and western blotting revealed that Ex-DC@CQDs retained the structural and functional characteristics of exosomes. We found that exosomes facilitated the transport of DC@CQDs to cancer cells via heparan sulfate proteoglycan (HSPG) receptors, followed by an augmented depolarization of the mitochondrial membrane potential, ROS generation, and induction of apoptosis leading to cell death. In vivo imaging and pharmacokinetic studies demonstrated enhanced antitumor targeting and efficacy compared to free DC which we attribute to an improved pharmacokinetic profile, a greater tumor accumulation via exosome-mediated- HSPG receptor-driven cell uptake, and sustained release of the Ex-DC@CQDs. Our findings may pave the way for the further development of biologically sourced nanocarriers for breast cancer targeting.


Assuntos
Neoplasias da Mama , Exossomos , Pontos Quânticos , Humanos , Feminino , Pontos Quânticos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Exossomos/metabolismo , Dacarbazina , Proteoglicanas de Heparan Sulfato/metabolismo , Carbono/química
11.
Alzheimers Dement ; 20(2): 819-836, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37791598

RESUMO

INTRODUCTION: We discovered that the APOE3 Christchurch (APOE3Ch) variant may provide resistance to Alzheimer's disease (AD). This resistance may be due to reduced pathological interactions between ApoE3Ch and heparan sulfate proteoglycans (HSPGs). METHODS: We developed and characterized the binding, structure, and preclinical efficacy of novel antibodies targeting human ApoE-HSPG interactions. RESULTS: We found that one of these antibodies, called 7C11, preferentially bound ApoE4, a major risk factor for sporadic AD, and disrupts heparin-ApoE4 interactions. We also determined the crystal structure of a Fab fragment of 7C11 and used computer modeling to predict how it would bind to ApoE. When we tested 7C11 in mouse models, we found that it reduced recombinant ApoE-induced tau pathology in the retina of MAPT*P301S mice and curbed pTau S396 phosphorylation in brains of systemically treated APOE4 knock-in mice. Targeting ApoE-HSPG interactions using 7C11 antibody may be a promising approach to developing new therapies for AD.


Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Camundongos , Humanos , Animais , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Fosforilação , Apolipoproteínas E/metabolismo , Doença de Alzheimer/patologia , Fatores Imunológicos , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo
12.
J Biol Chem ; 300(1): 105544, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38072044

RESUMO

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Glipicanas , Heparitina Sulfato , Animais , Proteínas de Drosophila/metabolismo , Glipicanas/genética , Glipicanas/química , Glipicanas/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo
13.
Anal Methods ; 16(2): 152-160, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-38108085

RESUMO

Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI), widely expressed in human embryos, and is undetectable in healthy adult liver but overexpressed in human hepatocellular carcinoma (HCC). Therefore, accurate and sensitive detection of GPC3 is critical for disease diagnosis. In recent years, a series of methods have been developed for the highly sensitive detection of GPC3, but there is a lack of reviews on recent advances in GPC3-related assays. In this review, we provide the recent advances in GPC3 detection and GPC3 concentration detection, mainly in terms of various optical sensor-based assays and electrochemical assays, and also provide new insights into the challenges and future directions of the field.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Adulto , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glipicanas/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteoglicanas de Heparan Sulfato
14.
J Toxicol Sci ; 48(12): 655-663, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38044127

RESUMO

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway-the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead-as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.


Assuntos
Células Endoteliais , Proteoglicanas de Heparan Sulfato , Animais , Bovinos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/metabolismo , Sistema de Sinalização das MAP Quinases , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Cultivadas , Receptores ErbB/metabolismo
15.
Molecules ; 28(23)2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38067582

RESUMO

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.


Assuntos
Transdução de Sinais , Sindecana-4 , Sindecana-4/metabolismo , Citoplasma/metabolismo , Transdução de Sinais/fisiologia , Proteoglicanas de Heparan Sulfato/metabolismo , Espectroscopia de Ressonância Magnética
16.
Sci Rep ; 13(1): 22263, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097644

RESUMO

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.


Assuntos
Glicosaminoglicanos , Sulfatases , Humanos , Microscopia de Força Atômica , Proteoglicanas de Heparan Sulfato , Sulfatos de Condroitina/metabolismo , Espectrometria de Massas
17.
Immun Inflamm Dis ; 11(10): e1034, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37904703

RESUMO

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.


Assuntos
Células Endoteliais , Golpe de Calor , Humanos , Células Endoteliais/metabolismo , Glicocálix/metabolismo , Lipopolissacarídeos/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Fator de von Willebrand/metabolismo , Fator de von Willebrand/farmacologia , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/farmacologia , Ocludina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Molécula 1 de Adesão de Célula Vascular/farmacologia , Inflamação/metabolismo , Interleucina-6/farmacologia , Golpe de Calor/metabolismo , Resposta ao Choque Térmico
18.
Sci Rep ; 13(1): 14556, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37666868

RESUMO

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.


Assuntos
Diferenciação Celular , Proteoglicanas de Heparan Sulfato , Células-Tronco Pluripotentes Induzidas , Peptídeos e Proteínas de Sinalização Intercelular , Laminina , Fragmentos de Peptídeos , Domínios Proteicos , Pele , Humanos , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteoglicanas de Heparan Sulfato/química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Laminina/química , Laminina/farmacologia , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Pele/citologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia
19.
Hepatol Commun ; 7(9)2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37655967

RESUMO

BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.


Assuntos
Hepatite B , Lipase , Animais , Camundongos , Proteoglicanas de Heparan Sulfato/genética , Heparina , Hepatite B/genética , Vírus da Hepatite B , Lipase/genética
20.
Int J Mol Sci ; 24(17)2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37686158

RESUMO

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2ß1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbß3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.


Assuntos
Proteoglicanas de Heparan Sulfato , Trombose , Humanos , Proteínas da Matriz Extracelular , Adesividade Plaquetária
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