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1.
Nat Commun ; 12(1): 1072, 2021 02 16.
Article in English | MEDLINE | ID: mdl-33594057

ABSTRACT

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.


Subject(s)
DNA Demethylation , Nucleosomes/metabolism , Transcription Initiation, Genetic , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Chromatin/chemistry , Chromatin/metabolism , HEK293 Cells , HMGA2 Protein/metabolism , Histones/metabolism , Humans , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/pathology , Mice , Phosphorylation , Phosphoserine/metabolism , RNA Polymerase II/metabolism , Transcription Initiation Site , Transcriptional Activation/genetics , Transforming Growth Factor beta1/metabolism
2.
PLoS One ; 14(1): e0209573, 2019.
Article in English | MEDLINE | ID: mdl-30608949

ABSTRACT

Glycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer's disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer's disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer's disease brain.


Subject(s)
Alzheimer Disease/metabolism , Glycosaminoglycans/metabolism , tau Proteins/physiology , Aged , Aged, 80 and over , Brain/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Brazil , Chondroitin Sulfates/metabolism , Extracellular Matrix/metabolism , Female , Fibroblast Growth Factor 2/metabolism , Heparin/metabolism , Heparitin Sulfate/metabolism , Hippocampus/metabolism , Humans , Male , Middle Aged , Protein Binding , Temporal Lobe/metabolism , Vascular Endothelial Growth Factor A/metabolism
3.
FEBS Lett ; 592(23): 3806-3818, 2018 12.
Article in English | MEDLINE | ID: mdl-29729013

ABSTRACT

Neurodegenerative disorders, such as Alzheimer's, Parkinson's, and prion diseases, are directly linked to the formation and accumulation of protein aggregates in the brain. These aggregates, principally made of proteins or peptides that clamp together after acquisition of ß-folded structures, also contain heparan sulfates. Several lines of evidence suggest that heparan sulfates centrally participate in the protein aggregation process. In vitro, they trigger misfolding, oligomerization, and fibrillation of amyloidogenic proteins, such as Aß, tau, α-synuclein, prion protein, etc. They participate in the stabilization of protein aggregates, protect them from proteolysis, and act as cell-surface receptors for the cellular uptake of proteopathic seeds during their spreading. This review focuses attention on the importance of heparan sulfates in protein aggregation in brain disorders including Alzheimer's, Parkinson's, and prion diseases. The presence of these sulfated polysaccharides in protein inclusions in vivo and their capacity to trigger protein aggregation in vitro strongly suggest that they might play critical roles in the neurodegenerative process. Further advances in glyco-neurobiology will improve our understanding of the molecular and cellular mechanisms leading to protein aggregation and neurodegeneration.


Subject(s)
Alzheimer Disease/metabolism , Heparitin Sulfate/metabolism , Parkinson Disease/metabolism , Prion Diseases/metabolism , Protein Aggregates , Amyloid/chemistry , Amyloid/metabolism , Heparitin Sulfate/chemistry , Humans , Models, Chemical , Prion Proteins/chemistry , Prion Proteins/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism
4.
Brain ; 138(Pt 5): 1339-54, 2015 May.
Article in English | MEDLINE | ID: mdl-25842390

ABSTRACT

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau(P301L), and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau(P301L), that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.


Subject(s)
Alzheimer Disease/metabolism , Neurons/metabolism , Sulfotransferases/metabolism , tau Proteins/metabolism , Animals , Behavior, Animal , Cells, Cultured , Humans , NF-kappa B/metabolism , Phosphorylation , Tauopathies/metabolism
5.
J Biol Chem ; 287(14): 11363-73, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22298772

ABSTRACT

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.


Subject(s)
Aging/metabolism , Glycosaminoglycans/metabolism , Heparitin Sulfate/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Myocardium/metabolism , Aging/physiology , Animals , Disaccharides/metabolism , Fibroblast Growth Factor 2/metabolism , Glycosaminoglycans/isolation & purification , Heart Ventricles/metabolism , Heart Ventricles/physiopathology , Male , Rats , Rats, Wistar , Vascular Endothelial Growth Factor A/metabolism
6.
Stem Cell Res ; 8(2): 180-92, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22265738

ABSTRACT

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR(4120)] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR(4120)] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.


Subject(s)
Cell Differentiation/drug effects , Cell Movement/drug effects , Glycosaminoglycans/pharmacology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Adipogenesis/drug effects , Animals , Biomarkers/metabolism , Cattle , Cell Proliferation/drug effects , Clone Cells , Fibroblast Growth Factor 2/pharmacology , Flow Cytometry , Heparin/pharmacology , Humans , Male , Osteogenesis/drug effects , Phenotype , Rats , Rats, Wistar
7.
J Orthop Res ; 30(1): 61-71, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21688311

ABSTRACT

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations.


Subject(s)
Carrier Proteins/genetics , Cumulative Trauma Disorders/pathology , Cumulative Trauma Disorders/physiopathology , Cytokines/genetics , Glycosaminoglycans/metabolism , Rotator Cuff , Animals , Carrier Proteins/metabolism , Chondrogenesis/physiology , Cytokines/metabolism , Disease Progression , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression/physiology , Male , Rats , Rats, Sprague-Dawley , Rotator Cuff/pathology , Rotator Cuff/physiology , Rotator Cuff Injuries
8.
Neurobiol Aging ; 33(5): 1005.e11-22, 2012 May.
Article in English | MEDLINE | ID: mdl-22035591

ABSTRACT

Glycosaminoglycans (GAGs) are major extracellular matrix components known to tightly regulate cell behavior by interacting with tissue effectors as trophic factors and other heparin binding proteins. Alterations of GAGs structures might thus modify the nature and extent of these interactions and alter tissue integrity. Here, we studied levels and composition of GAGs isolated from adult and aged human hippocampus and investigated if their changes can influence the function of important trophic factors and the Aß42 peptide toxicity. Biochemical analyses showed that heparan sulfates are increased in the aged hippocampus. Moreover, GAGs from aged hippocampus showed altered capacities to regulate trophic factor activities without changing their capacities to protect cells from Aß42 toxicity, compared to adult hippocampus GAGs. Structural alterations in GAGs from elderly were suggested by differential transcripts levels of key biosynthetic enzymes. C5-epimerase and 2-OST expressions were decreased while NDST-2 and 3-OST-4 were increased; in contrast, heparanase expression was unchanged. Results suggest that alteration of GAGs in hippocampus of aged subjects could participate to tissue impairment during aging.


Subject(s)
Aging/pathology , Aging/physiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/physiology , Amyloid beta-Peptides/toxicity , Glycosaminoglycans/physiology , Hippocampus/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Peptide Fragments/physiology , Peptide Fragments/toxicity , Adolescent , Adult , Alzheimer Disease/pathology , Female , Hippocampus/pathology , Humans , Male , Middle Aged , Young Adult
9.
Curr Protein Pept Sci ; 12(3): 258-68, 2011 May.
Article in English | MEDLINE | ID: mdl-21348835

ABSTRACT

A number of neurodegenerative diseases, as Parkinson, prion, and Alzheimer's diseases, has been directly associated with altered conformations of certain peptides or proteins that assemble to form highly organized aggregates, also called amyloid fibers. Glycosaminoglycans have shown to play important roles on fibrils formation, stability and resistance to proteolysis. This manuscript reviews from basic concepts on the biochemistry and biology of glycosaminoglycans to their implications in neurodegeneration with particular emphasis in pathologic protein aggregation. Prion protein, Aß42, Tau, and α-synuclein, are all proteins that can interact with glycosaminoglycans. We document here how these interactions may modify protein conformation, aggregation kinetics, and fibers stabilization with important consequences in disease. We also raise questions which answers may make advance the understanding of the implication of GAGs in neurodegeneration.


Subject(s)
Glycosaminoglycans/metabolism , Neurodegenerative Diseases/physiopathology , Proteins/chemistry , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Glycosaminoglycans/chemistry , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Neurodegenerative Diseases/immunology , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Prion Diseases , Prions/chemistry , Prions/metabolism , Protein Conformation , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism
10.
BMC Genomics ; 10: 514, 2009 Nov 06.
Article in English | MEDLINE | ID: mdl-19895685

ABSTRACT

BACKGROUND: There are thousands of very diverse ciliate species from which only a handful mitochondrial genomes have been studied so far. These genomes are rather similar because the ciliates analysed (Tetrahymena spp. and Paramecium aurelia) are closely related. Here we study the mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus. These ciliates are only distantly related to Tetrahymena spp. and Paramecium aurelia, but more closely related to Nyctotherus ovalis, which possesses a hydrogenosomal (mitochondrial) genome. RESULTS: The linear mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus were sequenced and compared with the mitochondrial genomes of several Tetrahymena species, Paramecium aurelia and the partially sequenced mitochondrial genome of the anaerobic ciliate Nyctotherus ovalis. This study reports new features such as long 5'gene extensions of several mitochondrial genes, extremely long cox1 and cox2 open reading frames and a large repeat in the middle of the linear mitochondrial genome. The repeat separates the open reading frames into two blocks, each having a single direction of transcription, from the repeat towards the ends of the chromosome. Although the Euplotes mitochondrial gene content is almost identical to that of Paramecium and Tetrahymena, the order of the genes is completely different. In contrast, the 33273 bp (excluding the repeat region) piece of the mitochondrial genome that has been sequenced in both Euplotes species exhibits no difference in gene order. Unexpectedly, many of the mitochondrial genes of E. minuta encoding ribosomal proteins possess N-terminal extensions that are similar to mitochondrial targeting signals. CONCLUSION: The mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus are rather different from the previously studied genomes. Many genes are extended in size compared to mitochondrial genes from other sources.


Subject(s)
Euplotes/genetics , Genome, Mitochondrial/genetics , Electron Transport , Electron Transport Complex IV/genetics , Euplotes/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Molecular Sequence Data , Open Reading Frames , Phylogeny , RNA, Transfer/genetics , Repetitive Sequences, Nucleic Acid , Ribosomal Proteins/genetics
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