Apatite reduces amelogenin proteolysis by MMP-20 and KLK4 in vitro.
Ontology highlight
ABSTRACT:
SUBMITTER: Sun Z
PROVIDER: S-EPMC3318039 | biostudies-literature | 2010 Apr
REPOSITORIES: biostudies-literature
Publications
Apatite reduces amelogenin proteolysis by MMP-20 and KLK4 in vitro.
Sun Z Z Carpiaux W W Fan D D Fan Y Y Lakshminarayanan R R Moradian-Oldak J J
Journal of dental research 20100216 4
Two enamel proteases, matrix metalloproteinase-20 (MMP-20) and kallikrein 4 (KLK4), are known to cleave amelogenin and are necessary for proper enamel formation. However, the effect of hydroxyapatite (HAP) on the proteolytic activity of these enzymes remains unclear. To investigate whether apatite affects normal amelogenin proteolysis, we used 2 different isoforms of amelogenin combined with the appropriate enzymes to analyze proteolytic processing rates in the presence or absence of synthetic h ...[more]
Similar Datasets
Project description:Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich amelogenin polypeptide (TRAP), the leucine-rich amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves amelogenin sequences after Pro(162), Ser(148), His(62), Ala(63), and Trp(45). These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) amelogenins. Mmp-20 cleaves LRAP after Pro(45) and Pro(40), producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His(62). We propose that Mmp-20 alone processes amelogenin during the secretory stage.
| S-EPMC2751868 | biostudies-literature
Project description:Amelogenin is cleaved by enamelysin (Mmp-20) soon after its secretion, and the cleavage products accumulate in specific locations during enamel formation, suggesting that parent amelogenin proteolysis is necessary for activating its functions. To investigate the precise roles of Mmp-20 and its influence on the assembly of amelogenin, an in vitro enzymatic digestion process mimicking the initial stages of amelogenin proteolysis was investigated at near-physiological conditions using recombinant porcine amelogenin (rP172) and enamelysin. Hierarchically organized nanorod structures formed during different digestion stages were detected by TEM. At the earliest stage, uniformly dispersed parent amelogenin spherical particles, mixed with some darker stained smaller spheres, and accompanying elongated chain-like nanostructures were observed. Cylindrical nanorods, which appeared to be the result of tight assembly of thin subunit cylindrical discs with thicknesses ranging from ?2.5 to ?6.0nm, were formed after an hour of proteolysis. These subunit building blocks stacked to form nanorods with maximum length of ?100nm. With the production of more cleavage products, additional morphologies spontaneously evolved from the cylindrical nanorods. Larger ball-like aggregates ultimately formed at the end of proteolysis. The uniform spherical particles, nanorods, morphological patterns evolved from nanorods, and globular aggregated microstructures were successively formed by means of co-assembly of amelogenin and its cleavage products during a comparatively slow proteolysis process. We propose that, following the C-terminal cleavage of amelogenin, co-assembly with its fragments leads to formation of nanorod structures whose properties eventually dictate the super-structural organization of enamel matrix, controlling the elongated growth of enamel apatite crystals.
| S-EPMC3185149 | biostudies-literature
Project description:Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.
| S-EPMC3513936 | biostudies-literature
Project description:Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.
| S-EPMC5119681 | biostudies-literature
Project description:Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.
| S-EPMC4037249 | biostudies-literature
Project description:The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.
| S-EPMC2707234 | biostudies-literature
Project description:Delayed removal of amelogenins, which are initially hydrolyzed by matrix metalloproteinase MMP-20, is a characteristic of enamel fluorosis. In this study, we investigated the regulation of MMP-20 and possible effects of fluoride on MMP-20 expression in human ameloblast lineage cells. Protein expression and signaling pathways of human ameloblast lineage cells, exposed to 10 muM fluoride, were compared to control cells without fluoride exposure. The role of activator protein-1 in MMP-20 regulation was analyzed by DNA-protein affinity precipitation and luciferase reporter gene assays. MMP-20 protein levels in human ameloblast lineage cells decreased in the presence of fluoride, while amelogenin and TIMP-2 were not altered. Fluoride also decreased the transcription of a luciferase reporter gene driven by the MMP-20 promoter. Down-regulation of MMP-20 by fluoride was related to suppression of JNK/c-Jun phosphorylation. In contrast, the JNK activator elevated the expression of MMP-20. Three c-Jun binding sites on the MMP-20 promoter were identified for the first time, and were occupied by c-Jun as MMP-20 was induced. Deletion of any one of AP-1 binding sites on the MMP-20 promoter significantly reduced the transcription of downstream luciferase reporter. These in vitro findings suggest that c-Jun is a key regulatory element for MMP-20 expression, and human ameloblast lineage cells can respond to fluoride by down-regulating MMP-20 transcription through the JNK/c-Jun signaling pathway.
| S-EPMC2194802 | biostudies-literature
Project description:Melanoma is a type of skin cancer that develops in pigment-producing melanocytes and often spreads to other parts of the body. Aberrant gene expression has been considered as a crucial step for increasing the risk of melanomagenesis, but how chromatin reorganization contributes to this pathogenic process is still not well understood. Here we report that matrix metalloproteinase 9 (MMP-9) localizes to the nucleus of melanoma cells and potentiates gene expression by proteolytically clipping histone H3 N-terminal tail (H3NT). From genome-wide studies, we discovered that growth regulatory genes are selectively targeted and activated by MMP-9-dependent H3NT proteolysis in melanoma cells. MMP-9 cooperates functionally with p300/CBP, because MMP-9 cleaves H3NT in a manner that is dependent on p300/CBP-mediated acetylation of H3K18. The functional significance of MMP-9-dependent H3NT proteolysis is further underscored by the fact that RNAi knockdown and small-molecule inhibition of MMP-9 and p300/CBP impede melanomagenic gene expression and melanoma tumor growth. Together, our data establish new functions and mechanisms for nuclear MMP-9 in promoting melanomagenesis and demonstrate how MMP-9-dependent H3NT proteolysis can be exploited to prevent and treat melanoma skin cancer.
2021-11-17 | GSE179367 | GEO
Project description:Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.
| S-EPMC3460178 | biostudies-literature
Project description:Amelogenin's capacity to regulate enamel formation is related to its conserved N- and C-terminal domains, its ability to self-assemble, and its ability to stabilize amorphous calcium phosphate (ACP) - a capacity enhanced by amelogenin phosphorylation. This in vitro study provides further insight into amelogenin function, using variations of the Leucine-Rich Amelogenin Peptide (LRAP), an alternative splice product comprised solely of amelogenin's N- and C-terminal domains. Peptide self-assembly was studied by dynamic light-scattering and transmission electron microscopy (TEM). TEM, selected area electron diffraction, and Fourier transform-infrared spectroscopy were also used to determine the effect of phosphorylated and non-phosphorylated LRAP on calcium phosphate formation. Results show that phosphorylated and non-phosphorylated LRAP can self-assemble into chain-like structures in a fashion dependent on the C-terminal domain. Notably, this capacity was enhanced by added calcium and to a much greater degree for phosphorylated LRAP. Furthermore, phosphorylated LRAP was found to stabilize ACP and prevent its transformation to hydroxyapatite (HA), while aligned HA crystals formed in the presence of non-phosphorylated LRAP. The N- and C-terminal amelogenin domains in non-phosphorylated LRAP are, therefore, sufficient to guide ACP transformation into ordered bundles of apatite crystals, making LRAP an excellent candidate for biomimetic approaches for enamel regeneration.
| S-EPMC3169881 | biostudies-other