RESUMO
The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.
Assuntos
Caseína Quinase I/química , Caseína Quinase I/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Sequência de Aminoácidos , Proteínas Sanguíneas/metabolismo , Caseína Quinase I/genética , Adesão Celular , Movimento Celular , Proteínas do Líquido Cefalorraquidiano/metabolismo , Proteínas da Matriz Extracelular/genética , Técnicas de Inativação de Genes , Ontologia Genética , Humanos , Dados de Sequência Molecular , Fosfoproteínas/análise , Via Secretória , Especificidade por SubstratoRESUMO
Extracellular matrix (ECM) proteins constitute >1% of the proteome and interact with many modifiers and growth factors to affect most aspects of cellular behaviour during development and normal physiology, as well as in diseases such as fibroses, cancer and many genetic disorders. In addition to biochemical signals provided to cells by ECM proteins, important cellECM interactions involve bidirectional mechanotransduction influences, which are dependent on the physical structure and organization of the ECM. These are beginning to be understood using twenty-first-century approaches, including biophysics, nanotechnology, biological engineering and modern microscopy. Articles in this issue of Nature Reviews Molecular Cell Biology review progress in our understanding of the ECM.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Animais , Biologia Celular , Proteínas da Matriz Extracelular/química , Mecanotransdução Celular , PatologiaRESUMO
The biochemical and biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topographical features that both reflect and facilitate the functional requirements of the tissue.
Assuntos
Matriz Extracelular/química , Matriz Extracelular/metabolismo , Animais , Membrana Basal/química , Membrana Basal/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Humanos , Neurônios/citologia , Proteoglicanas/metabolismoRESUMO
Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.
Assuntos
Sulfatos de Condroitina , Proteínas da Matriz Extracelular , Heparitina Sulfato , Animais , Humanos , Camundongos , Biomarcadores Tumorais/química , Proteínas de Ligação ao Cálcio/química , Sulfatos de Condroitina/análise , Heparitina Sulfato/análise , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Triptases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genéticaRESUMO
Dorsal axial formation during vertebrate embryogenesis exhibits robust resistance to perturbations in patterning signals. However, how such stability is supported at the molecular level remains largely elusive. Here we show that Xenopus ONT1, an Olfactomedin-class secreted protein, stabilizes axial formation by restricting Chordin activity on the dorsal side. When ONT1 function is attenuated, the embryo becomes hyperdorsalized by a normally subeffective dose of Chordin. ONT1 binds Chordin and BMP1/Tolloid-class proteinases (B1TP) via distinct domains and acts as a secreted scaffold that enhances B1TP-mediated Chordin degradation by facilitating enzyme-substrate association. ONT1 is indispensable for fine-tuning BMP signaling in the axial tissue, and a similar role has been suggested for dorsally expressed BMPs such as ADMP. Simultaneous inhibition of ONT1 and dorsally expressed BMPs (ADMP and BMP2) synergistically caused drastic dorsalization. These results indicate that stable axial formation depends on two compensatory regulatory pathways involving ONT1/B1TP and dorsally expressed BMPs.
Assuntos
Padronização Corporal , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Proteína Morfogenética Óssea 1 , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Galinhas , Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Humanos , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Estrutura Terciária de Proteína , Metaloproteases Semelhantes a Toloide , Proteínas de Xenopus/químicaRESUMO
The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.
Assuntos
Caseína Quinase I/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Caseína Quinase I/química , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/química , Homeostase , Humanos , Miocárdio/metabolismo , Fosforilação , Via Secretória , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Numerous human disorders arise due to the inability of a particular protein to adopt its correct three-dimensional structure in the context of the cell, leading to aggregation. A new addition to the list of such protein conformational disorders is the inherited subtype of glaucoma. Different and rare coding mutations in myocilin, found in families throughout the world, are causal for early onset ocular hypertension, a key glaucoma risk factor. Myocilin is expressed at high levels in the trabecular meshwork (TM) extracellular matrix. The TM is the anatomical region of the eye that regulates intraocular pressure, and its dysfunction is associated with most forms of glaucoma. Disease variants, distributed across the 30 kDa olfactomedin domain (mOLF), cause myocilin to be sequestered intracellularly instead of being secreted to the TM extracellular matrix. The working hypothesis is that the intracellular aggregates cause a toxic gain of function: TM cell death is thought to lead to TM matrix dysfunction, hastening elevated intraocular pressure and subsequent vision loss.Our lab has provided molecular underpinnings for myocilin structure and misfolding, placing myocilin-associated glaucoma within the context of amyloid diseases like Alzheimer and diabetes. We have dissected complexities of the modular wild-type (WT) myocilin structure and associated misfolded states. Our data support the model that full-length WT myocilin adopts a Y-shaped dimer-of-dimers conferred by two different coiled-coil regions, generating new hypotheses regarding its mysterious function. The mOLF ß-propellers are paired at each tip of the Y. Disease-associated variants aggregate because mOLFs are less stable, leading to facile aggregation under physiological conditions (37 °C, pH 7.2). Mutant myocilin aggregates exhibit numerous characteristics of amyloid in vitro and in cells, and aggregation proceeds from a partially folded state accessed preferentially by disease variants at physiological conditions. Interestingly, destabilization is not a universal consequence of mutation. We identified counterintuitive, stabilizing point variants that adopt a non-native structure and do not aggregate; however, these variants have not been identified in glaucoma patients. An ongoing effort is predicting the consequence of any given mutation. This effort is relevant to interpreting data from large-scale sequencing projects where clinical and family history data are not available. Finally, our work suggests avenues to develop disease-modifying precision medicines for myocilin-associated glaucoma.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glaucoma/metabolismo , Glicoproteínas/metabolismo , Proteínas do Citoesqueleto/química , Proteínas da Matriz Extracelular/química , Proteínas do Olho/química , Glicoproteínas/química , Humanos , Modelos Moleculares , Dobramento de ProteínaRESUMO
The role of the extracellular matrix (ECM) remodeling in tumorigenesis and metastasis is becoming increasingly clear. Cancer development requires that tumor cells recruit a tumor microenvironment permissive for further tumor growth. This is a dynamic process that takes place by a cross-talk between tumor cells and ECM. As a consequence, molecules derived from the ECM changes associated to cancer are released into the bloodstream, representing potential biomarkers of tumor development. This article highlights the importance of developing and improving bioanalytical methods for the detection of ECM remodeling-derived components, as a step forward to translate the basic knowledge about cancer progression into the clinical practice.
Assuntos
Biomarcadores Tumorais , Proteínas da Matriz Extracelular/química , Neoplasias/diagnóstico , Proteínas da Matriz Extracelular/metabolismo , Humanos , Conformação ProteicaRESUMO
Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Mutação , Oligonucleotídeos Antissenso/administração & dosagem , Retinose Pigmentar/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Moleculares , Oligonucleotídeos Antissenso/farmacologia , Retina/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The extracellular matrix (ECM) is a complex and dynamic meshwork of cross-linked proteins that supports cell polarization and functions and tissue organization and homeostasis. Over the past few decades, mass-spectrometry-based proteomics has emerged as the method of choice to characterize the composition of the ECM of normal and diseased tissues. Here, we present a new release of MatrisomeDB, a searchable collection of curated proteomic data from 17 studies on the ECM of 15 different normal tissue types, six cancer types (different grades of breast cancers, colorectal cancer, melanoma, and insulinoma) and other diseases including vascular defects and lung and liver fibroses. MatrisomeDB (http://www.pepchem.org/matrisomedb) was built by retrieving raw mass spectrometry data files and reprocessing them using the same search parameters and criteria to allow for a more direct comparison between the different studies. The present release of MatrisomeDB includes 847 human and 791 mouse ECM proteoforms and over 350 000 human and 600 000 mouse ECM-derived peptide-to-spectrum matches. For each query, a hierarchically-clustered tissue distribution map, a peptide coverage map, and a list of post-translational modifications identified, are generated. MatrisomeDB is the most complete collection of ECM proteomic data to date and allows the building of a comprehensive ECM atlas.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteínas da Matriz Extracelular , Proteômica , Sequência de Aminoácidos , Proteínas da Matriz Extracelular/química , Humanos , Espectrometria de Massas , Peptídeos/química , Proteômica/métodos , NavegadorRESUMO
Competent elastic fibers are critical to the function of the lung and right circulation. Murine models of elastopathies can aid in understanding the functional roles of the elastin and elastin-associated glycoproteins that constitute elastic fibers. Here, we quantify together lung and pulmonary arterial structure, function, and mechanics with right heart function in a mouse model deficient in the elastin-associated glycoprotein fibulin-5. Differences emerged as a function of genotype, sex, and arterial region. Specifically, functional studies revealed increased lung compliance in fibulin-5 deficiency consistent with a histologically observed increased alveolar disruption. Biaxial mechanical tests revealed that the primary branch pulmonary arteries exhibit decreased elastic energy storage capacity and wall stress despite only modest differences in circumferential and axial material stiffness in the fibulin-5 deficient mice. Histological quantifications confirm a lower elastic fiber content in the fibulin-5 deficient pulmonary arteries, with fragmented elastic laminae in the outer part of the wall - likely the reason for reduced energy storage. Ultrasound measurements confirm sex differences in compromised right ventricular function in the fibulin-5 deficient mice. These results reveal compromised right heart function, but opposite effects of elastic fiber dysfunction on the lung parenchyma (significantly increased compliance) and pulmonary arteries (trend toward decreased distensibility), and call for further probing of ventilation-perfusion relationships in pulmonary pathologies. Amongst many other models, fibulin-5 deficient mice can contribute to our understanding of the complex roles of elastin in pulmonary health and disease.
Assuntos
Elastina , Proteínas da Matriz Extracelular/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Tecido Elástico , Elastina/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Feminino , Masculino , CamundongosRESUMO
Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.
Assuntos
Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Proteínas de Ligação a TGF-beta Latente/química , Proteínas de Ligação a TGF-beta Latente/metabolismo , Animais , Células Cultivadas , Elastina , Fibronectinas/metabolismo , Humanos , Camundongos , Ligação Proteica , Conformação Proteica , Tropoelastina/metabolismoRESUMO
Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.
Assuntos
Amelogênese Imperfeita/genética , Amelogenina/genética , Biomineralização/genética , Proteínas da Matriz Extracelular/genética , Adsorção/genética , Amelogênese Imperfeita/metabolismo , Amelogênese Imperfeita/patologia , Amelogenina/química , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Durapatita/química , Metabolismo Energético/genética , Proteínas da Matriz Extracelular/química , Humanos , Metaloproteinase 20 da Matriz/química , Metaloproteinase 20 da Matriz/genética , Camundongos , Microscopia de Força Atômica , Conformação Proteica , TermodinâmicaRESUMO
ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-ß binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose-modified TSRs are subsequently elongated to a glucose ß1-3-fucose (GlcFuc) disaccharide by ß1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2-/- but not in B3GLCT-/- cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.
Assuntos
Proteínas ADAMTS/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Proteínas da Matriz Extracelular/metabolismo , Deformidades Congênitas dos Membros/patologia , Proteínas ADAMTS/química , Proteínas ADAMTS/genética , Sequência de Aminoácidos , Animais , Doenças do Desenvolvimento Ósseo/genética , Sistemas CRISPR-Cas/genética , Dissacarídeos/química , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Edição de Genes , Glicosilação , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Células HEK293 , Humanos , Deformidades Congênitas dos Membros/genética , Manose/química , Camundongos , Mutagênese Sítio-Dirigida , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
Dentin matrix protein 1 (DMP1) is an acidic, extracellular matrix protein essential for biomineralization of calcium phosphate, in bone and dentin. It is proteolytically processed into two fragments, 44K and 56K. Recently, the presence of DMP1 was noticed in inner ear, specifically in otoconia, which are calcium carbonate biominerals involved in sensing of balance. In this study, the solution structure and biomineralization activity of otoconial 44K and 56K fragments toward calcium carbonate were investigated. The results of analytical ultracentrifugation, circular dichroism, and gel filtration indicated that DMP1 fragments are disordered in solution. Notably, 56K formed oligomers in the presence of calcium ions. It was also observed that both fragments influenced the crystal growth by in vitro biomineralization assay and scanning electron microscopy. In addition, they sequester the calcium ions during the calcite formation. Calcium carbonate crystals precipitated in vitro changed their size and shape in the presence of DMP1 fragments. Oligomerization propensity of 56K may significantly enhance this function. Our study indicates that intrinsically disordered DMP1 has a previously unknown regulatory function for biomineralization of otoconia.
Assuntos
Calcificação Fisiológica , Carbonato de Cálcio/química , Cristalização , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Humanos , Microscopia Eletrônica de Varredura , Conformação Proteica , Multimerização ProteicaRESUMO
Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.
Assuntos
Forma Celular , Proteínas da Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Linhagem Celular Tumoral , Colágeno/química , Simulação por Computador , Citocalasina D/farmacologia , Combinação de Medicamentos , Elasticidade , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Matriz Extracelular/química , Fibrina/química , Humanos , Laminina/química , Modelos Biológicos , Movimento (Física) , Pinças Ópticas , Proteoglicanas/química , Reologia/métodos , Estresse MecânicoRESUMO
The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Menisco/metabolismo , Osteoartrite/metabolismo , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Masculino , Proteoma/químicaRESUMO
Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1ß)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas da Matriz Extracelular/química , Receptores de Hialuronatos/química , Ácido Hialurônico/metabolismo , Interleucina-1beta/efeitos adversos , Células-Tronco Mesenquimais/citologia , Peptídeos/farmacologia , Animais , Anti-Inflamatórios/química , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Condrogênese , Ciclo-Oxigenase 2/genética , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Metaloproteases/genética , Camundongos , Peptídeos/químicaRESUMO
Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the ß-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.