RESUMO
Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are microfibril-associated proteins essential for anchoring TGFß in the extracellular matrix (ECM) as well as for correct assembly of ECM components. Variants in LTBP2, LTBP3, and LTBP4 have been identified in several autosomal recessive Mendelian disorders with skeletal abnormalities with or without impaired development of elastin-rich tissues. Thus far, the human phenotype associated with LTBP1 deficiency has remained enigmatic. In this study, we report homozygous premature truncating LTBP1 variants in eight affected individuals from four unrelated consanguineous families. Affected individuals present with connective tissue features (cutis laxa and inguinal hernia), craniofacial dysmorphology, variable heart defects, and prominent skeletal features (craniosynostosis, short stature, brachydactyly, and syndactyly). In vitro studies on proband-derived dermal fibroblasts indicate distinct molecular mechanisms depending on the position of the variant in LTBP1. C-terminal variants lead to an altered LTBP1 loosely anchored in the microfibrillar network and cause increased ECM deposition in cultured fibroblasts associated with excessive TGFß growth factor activation and signaling. In contrast, N-terminal truncation results in a loss of LTBP1 that does not alter TGFß levels or ECM assembly. In vivo validation with two independent zebrafish lines carrying mutations in ltbp1 induce abnormal collagen fibrillogenesis in skin and intervertebral ligaments and ectopic bone formation on the vertebrae. In addition, one of the mutant zebrafish lines shows voluminous and hypo-mineralized vertebrae. Overall, our findings in humans and zebrafish show that LTBP1 function is crucial for skin and bone ECM assembly and homeostasis.
Assuntos
Colágeno/metabolismo , Cútis Laxa/etiologia , Variação Genética , Proteínas de Ligação a TGF-beta Latente/genética , Adolescente , Alelos , Animais , Células Cultivadas , Criança , Pré-Escolar , Cútis Laxa/patologia , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Masculino , Linhagem , Pele/metabolismo , Pele/patologia , Peixe-ZebraRESUMO
Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17â nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.
Assuntos
Fibronectinas , Microscopia , Epitopos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Peptídeos/metabolismoRESUMO
Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.
Assuntos
Mecanotransdução Celular , Miostatina , Animais , Camundongos , Fenômenos Biomecânicos , Colágeno/metabolismo , Miostatina/metabolismo , Proteômica , Tendões/metabolismoRESUMO
BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.
Assuntos
Escherichia coli , Tauopatias , Animais , Ratos , Escherichia coli/genética , Tauopatias/genética , Citoesqueleto , Emaranhados Neurofibrilares , MicrotúbulosRESUMO
O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.
Assuntos
Lisina , Proteômica , Animais , Glicosilação , Lisina/metabolismo , Colágeno Tipo I/metabolismo , Colágeno/metabolismoRESUMO
Amyloid formation continues to be a widely studied area because of its association with numerous diseases, such as Alzheimer's and Parkinson's diseases. Despite a large body of work on protein aggregation and fibril formation, there are still significant gaps in our understanding of the factors that differentiate toxic amyloid formation in vivo from alternative misfolding pathways. In addition to proteins, amyloid fibrils are often associated in their cellular context with several types of molecule, including carbohydrates, polyanions, and lipids. This review focuses in particular on evidence for the presence of lipids in amyloid fibrils and the routes by which those lipids may become incorporated. Chemical analyses of fibril composition, combined with studies to probe the lipid distribution around fibrils, provide evidence that in some cases, lipids have a strong association with fibrils. In addition, amyloid fibrils formed in the presence of lipids have distinct morphologies and material properties. It is argued that lipids are an integral part of many amyloid deposits in vivo, where their presence has the potential to influence the nucleation, morphology, and mechanical properties of fibrils. The role of lipids in these structures is therefore worthy of further study.
Assuntos
Amiloide , Amiloidose , Lipídeos , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Lipídeos/química , Agregados ProteicosRESUMO
Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-ß signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.This article has an associated First Person interview with the first author of the paper.
Assuntos
Processamento Alternativo , Fibronectinas , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , HumanosRESUMO
Collagen is the most ubiquitous biomacromolecule found in the animal kingdom and is commonly used as a biomaterial in regenerative medicine therapies and biomedical research. The collagens used in these applications are typically derived from mammalian sources which poses sociological issues due to widespread religious constraints, rising ethical concern over animal rights and the continuous risk of zoonotic disease transmission. These issues have led to increasing research into alternative collagen sources, of which marine collagens, in particular from jellyfish, have emerged as a promising resource. This study provides a characterization of the biophysical properties and cell adhesion interactions of collagen derived from the jellyfish Rhizostoma pulmo (JCol). Circular dichroism spectroscopy and atomic force microscopy were used to observe the triple-helical conformation and fibrillar morphology of JCol. Heparin-affinity chromatography was also used to demonstrate the ability of JCol to bind to immobilized heparin. Cell adhesion assays using integrin blocking antibodies and HT-1080 human fibrosarcoma cells revealed that adhesion to JCol is primarily performed via ß1 integrins, with the exception of α2ß1 integrin. It was also shown that heparan sulfate binding plays a much greater role in fibroblast and mesenchymal stromal cell adhesion to JCol than for type I mammalian collagen (rat tail collagen). Overall, this study highlights the similarities and differences between collagens from mammalian and jellyfish origins, which should be considered when utilizing alternative collagen sources for biomedical research.
Assuntos
Cnidários , Colágeno , Cifozoários , Animais , Humanos , Ratos , Adesão Celular , Cnidários/metabolismo , Colágeno/química , Integrinas/metabolismo , Cifozoários/químicaRESUMO
Alzheimer's disease (AD) is the most common neurodegenerative pathology among progressive dementias, and it is characterized by the accumulation in the brain of extracellular aggregates of beta-amyloid proteins and neurofibrillary intracellular tangles consisting of τ-hyperphosphorylated proteins. Under normal conditions, beta-amyloid peptides exert important trophic and antioxidant roles, while their massive presence leads to a cascade of events culminating in the onset of AD. The fibrils of beta-amyloid proteins are formed by the process of fibrillogenesis that, starting from individual monomers of beta-amyloid, can generate polymers of this protein, constituting the hypothesis of the "amyloid cascade". To date, due to the lack of pharmacological treatment for AD without toxic side effects, chemical research is directed towards the realization of hybrid compounds that can act as an adjuvant in the treatment of this neurodegenerative pathology. The hybrid compounds used in this work include moieties of a hydroxytyrosol, a nitrohydroxytyrosol, a tyrosol, and a homovanillyl alcohol bound to the N-benzylpiperidine moiety of donepezil, the main drug used in AD. Previous experiments have shown different properties of these hybrids, including low toxicity and antioxidant and chelating activities. The purpose of this work was to test the effects of hybrid compounds mixed with Aß1-40 to induce fibrillogenesis and mimic AD pathogenesis. This condition has been studied both in test tubes and by an in vitro model of neuronal differentiated human SH-SY5Y neuroblastoma cells. The results obtained from test tube experiments showed that some hybrids inhibit the activity of the enzymes AChE, BuChE, and BACE-1. Cell experiments suggested that hybrids could inhibit fibrillogenesis, negatively modulating caspase-3. They were also shown to exert antioxidant effects, and the acetylated hybrids were found to be more functional and efficient than nonacetylated forms.
Assuntos
Doença de Alzheimer , Neuroblastoma , Humanos , Doença de Alzheimer/tratamento farmacológico , Donepezila/farmacologia , Antioxidantes/farmacologia , Neuroblastoma/tratamento farmacológico , Proteínas tauRESUMO
A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.
Assuntos
Lesões da Córnea/metabolismo , Decorina/fisiologia , Colágenos Fibrilares/metabolismo , Organogênese/fisiologia , Cicatrização/fisiologia , Animais , Queimaduras Químicas/metabolismo , Lesões da Córnea/patologia , Proteínas da Matriz Extracelular/metabolismo , Queimaduras Oculares/induzido quimicamente , Colágenos Fibrilares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia com Lâmpada de Fenda , Hidróxido de SódioRESUMO
Aggregation and fibrillation of ß-amyloid peptides (Aß) as well as accumulation of toxic metal ions have been believed to be the central events to cause Alzheimer's disease (AD). Thus, an attractive therapeutic tactic for AD is to design and synthesize inhibitors and metal chelators to prevent Aß aggregation and chelate toxic metal ions. In this study, the polypeptide functionalized gold nanoparticles (PFGNP) were obtained by modifying polypeptides Cys-Gly-Gly-Gly-Leu-Pro-Phe-Phe-Asp (CGGGLPFFD) and Cys-Gly-Gly-Gly-Gly-Gly-His (CGGGGGH) onto gold nanoparticles through gold-sulfur bond. The inhibitory properties of PFGNP toward Aß1-42 fibril formation was assessed by thioflavin T (ThT) fluorescence method and corroborated by atomic force microscopy analysis. The ability of PFGNP to complex copper ions was studied by electrochemical method. The experimental results reveal that PFGNP can effectively chelate copper ions and significantly inhibit the fibrillation of Aß1-42 . Moreover, PFGNP exhibits significantly protective effect on Aß-induced cytotoxicity toward human neuroblastoma SH-SY5Y cells.
Assuntos
Doença de Alzheimer , Nanopartículas Metálicas , Neuroblastoma , Humanos , Doença de Alzheimer/tratamento farmacológico , Ouro/farmacologia , Cobre/farmacologia , Peptídeos beta-Amiloides , Fragmentos de Peptídeos/química , ÍonsRESUMO
Life is recognized as a sophisticated self-assembling material system. Cancer involves the overexpression and improper self-assembly of proteins, such as cytoskeleton protein vimentin, an emerging target related to tumor metastasis. Herein, we design a binding-induced fibrillogenesis (BIF) peptide that in situ forms fibrous networks, blocking the improper self-assembly of vimentin against cancer. The BIF peptide can bind to vimentin and subsequently perform fibrillogenesis to form fibers on vimentin. The resultant peptide fibrous network blocks vimentin skeletonization and inhibits the migration and invasion of tumor cells. In mouse models of tumor metastasis, the volume of tumor and the number of lung metastases are markedly decreased. Moreover, the efficacy of BIF peptide (5 mg/kg) is much higher than small molecular antimetastasis drug withaferin A (5 mg/kg) as a standard, indicating that the BIF peptide shows advantages over small molecular inhibitors in blocking the intracellular protein self-assembly.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Camundongos , Peptídeos , Vimentina/genéticaRESUMO
Fibrillar fibronectin (FFN), an active form of fibronectin (FN), plays important roles in various cellular processes. Our goal is to investigate effect of FFN morphology on cellular behaviors. Plasma FN at two concentrations was cross-linked into FFN by dialysis against 2 M urea followed by morphological analysis under Scanning Electron Microscopy. To evaluate effect of FFN morphology, fibroblasts were cultured on FN or different FFNs. Fibroblast behaviors including adhesion, spreading, and migration were evaluated. Our data showed that FN fibrillogenesis was dependent on FN concentration. At high concentrations (0.75 mg/mL), large FFN approximately 2.167 + 0.875 µm in diameter were formed with attached nodular structures and rough surface. In contrast, smooth surface FFN fibrils with diameter of 1.886 + 0.412 µm were formed from FN at 0.25 mg/mL. Cellular assays revealed morphological dependent biological effects of different FFNs. Fibroblast separately adhered to native FN and remained spherical while on FFN, cells attached with higher quantity and showed spreading morphology. A synergistic ligand interaction of integrin α5ß1 and αvß3 was observed in cell adhering on FFN. Cell migration results showed that large FFN decreased migration rate while small FFN did not. Taken together, our data draws new attention towards controlling biological function of FN by its fibrillar structure.
Assuntos
Fibronectinas/metabolismo , Substâncias Macromoleculares/metabolismo , Animais , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Fibronectinas/sangue , Substâncias Macromoleculares/sangue , Camundongos , Ureia/sangue , Ureia/metabolismoRESUMO
Proteoglycans consist of protein cores to which at least one glycosaminoglycan chain is attached. They play important roles in the physiology and biomechanical function of tendons, ligaments, cardiovascular system, and other systems through their involvement in regulation of assembly and maintenance of extracellular matrix, and through their participation in cell proliferation together with growth factors. They can be divided into two main groups, small and large proteoglycans. The small proteoglycans are also known as small leucine-rich proteoglycans (SLRPs) which are encoded by 18 genes and are further subclassified into Classes I-V. Several members of Class I and II, such as decorin and biglycan from Class I, and Class II fibromodulin and lumican, are known to regulate collagen fibrillogenesis. Decorin limits the diameter of collagen fibrils during fibrillogenesis. The function of biglycan in fibrillogenesis is similar to that of decorin. Though biomechanical function of tendon is compromised in decorin-deficient mice, decorin can substitute for lack of biglycan in biglycan-deficient mice. New data also indicate an important role for biglycan in disorders of the cardiovascular system, including aortic valve stenosis and aortic dissection. Two members of the Class II of SLRPs, fibromodulin and lumican bind to the same site within the collagen molecule and can substitute for each other in fibromodulin- or lumican-deficient mice.Aggrecan and versican are the major representatives of the large proteoglycans. Though they are mainly found in the cartilage where they provide resilience and toughness, they are present also in tensile portions of tendons and, in slightly different biochemical form in fibrocartilage. Degradation by aggrecanase is responsible for the appearance of different forms of aggrecan and versican in different parts of the tendon where these cleaved forms play different roles. In addition, they are important components of the ventricularis of cardiac valves. Mutations in the gene for versican or in the gene for elastin (which binds to versican ) lead to severe disruptions of normal developmental of the heart at least in mice.
Assuntos
Matriz Extracelular , Sulfato de Queratano , Animais , Colágeno , Decorina/genética , Camundongos , Versicanas/genéticaRESUMO
The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a "reversible" form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its "molecular chaperone" activity) and the formation of inactive fibrillary deposits (its "fibrillogenic" activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail.
Assuntos
Ácidos/farmacologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196-Leu2197 We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.
Assuntos
Proteína ADAMTS9/metabolismo , Fibroblastos/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Agregados Proteicos , Proteína ADAMTS9/genética , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibronectinas/genética , Humanos , Camundongos , Proteólise , Epitélio Pigmentado da Retina/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Here we show that Gas7 inhibits phosphorylated tau fibrillogenesis by binding to phosphorylated tau at its non-WW domain, presumably F-BAR domain. We revealed that Gas7 binds to the third repeat domain of tau, the core element of tau oligomerization and the C-terminal domain of tau and alters the conformation not to form fibrils. These results suggest that Gas7 may serve to protect against Alzheimer's disease and other tauopathies by preventing tau fibrillogenesis.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo , Células HEK293 , Humanos , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Tauopatias/metabolismo , Domínios WW , Proteínas tau/químicaRESUMO
This article presents 20 combinations of histochemical stainings for the determination of mast cell co-localization with the fibrous component of the connective tissue in the fibrillogenesis course. Best results were obtained using metachromatic detection of mast cells in combination with silver or picro-fuchsin impregnation, staining with brilliant green using van Gieson staining, and a combination of aniline blue staining with neutral red. Proposed variants of histochemical protocols open up new opportunities to analyze the participation of mast cells in extracellular matrix remodeling of the tissue microenvironment in the course of adaptive and pathological processes. Results obtained expand the current theoretical views of the process of fibrillogenesis in the extracellular matrix. They also shed new light on the participation of mast cell secretion components in the molecular mechanisms of fiber formation.
Assuntos
Colágeno/química , Matriz Extracelular/química , Mastócitos/química , Músculos do Pescoço/química , Animais , Corantes/química , Mastócitos/citologia , Ratos , Ratos Wistar , Prata/química , Coloração e Rotulagem , Cloreto de Tolônio/químicaRESUMO
Alzheimer's disease pathogenesis is measured by two key hallmarks viz extracellular senile plaques composed of insoluble amyloid beta (Aß) and neurofibrillary tangles composed of hyperphosphorylated tau, resulting in microtubule destabilization, synaptic damage and neurodegeneration. Accumulation of Aß is an introducing pathological incident in Alzheimer's disease; hence, the effect of dimethyl fumarate (DMF) on Aß1-42-induced alterations in phosphorylated tau, related protein kinases, fibrillogenesis and microtubule assembly in neuroblastoma SH-SY5Y cells was determined. DMF attenuated Aß1-42-induced neuronal apoptosis by down-regulating protein levels of Bcl-2/Bax, cleaved caspase-3 and caspase-9. Aß1-42-induced upsurge in tau phosphorylation at Ser396 and Thr231 epitopes was found to be declined by DMF pretreatment. The upregulated activity of glycogen synthase kinase-3 beta (GSK-3ß) by Aß142 treatment was blocked by DMF pretreatment. PI3K substrate Akt (at Ser473) as well as Wnt dependent ß-catenin and cyclin D1 activity was found to be upregulated by DMF pretreatment in Aß1-42 treated cells. ThT fluorescence and MTT assay showed that DMF reduces Aß fibrillogenesis and inhibit related cytotoxicity. Also, DMF exerts a protective effect on Aß1-42-induced microtubule disassembly caused due to a reduction in polymerized ß3-and α-tubulin. These results indicate that down-regulation of GSK-3ß activity and subsequent activation of PI3K/Akt and Wnt/ß-catenin signaling pathways are closely involved in the shielding effect of DMF against Aß1-42-induced tau hyperphosphorylation. Modulating cellular events related to Aß1-42-induced tau hyperphosphorylation, aggregation and microtubule stabilization offers new molecular insights into the defensive outcome of DMF towards appropriate management for Alzheimer's disease.
Assuntos
Fumarato de Dimetilo/farmacologia , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Tauopatias/tratamento farmacológico , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.