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TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization

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ABSTRACT

Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 μM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization.

No MeSH data available.


rTRIP-1 binds to Type 1 Collagen.(6a) Binding of rTRIP-1 to immobilized collagen I was analyzed by Surface plasmon resonance (SPR) spectroscopy. SPR data analyses show the steady state fits used to calculate the binding constant between rTRIP-1 and immobilized Type 1 Collagen (KD = 48.59 μM). Values are the means from independent experiments that were performed in triplicates, and the error bars are the S.E.M. Figure 6b: SPR sensorgrams of a series of increasing concentrations of rTRIP-1 showing binding to immobilized type 1 Collagen.
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f6: rTRIP-1 binds to Type 1 Collagen.(6a) Binding of rTRIP-1 to immobilized collagen I was analyzed by Surface plasmon resonance (SPR) spectroscopy. SPR data analyses show the steady state fits used to calculate the binding constant between rTRIP-1 and immobilized Type 1 Collagen (KD = 48.59 μM). Values are the means from independent experiments that were performed in triplicates, and the error bars are the S.E.M. Figure 6b: SPR sensorgrams of a series of increasing concentrations of rTRIP-1 showing binding to immobilized type 1 Collagen.

Mentions: To assess the role of TRIP-1 in collagen biomineralization, we performed binding assay between rTRIP-1 (Fig. S6) as the analyte and type 1 collagen as the ligand using Surface Plasmon Resonance (SPR). Steady state analysis (Fig. 6a) demonstrated that TRIP-1 bound to Collagen in a concentration-dependent and saturated manner with an apparent KD of 48 μM. Figure 6b shows the sensorgrams of a series of increasing concentrations of rTRIP-1 flown on a Collagen 1-immobilized CM5 sensor surface clearly showing binding between TRIP-1 and type 1 Collagen.


TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization
rTRIP-1 binds to Type 1 Collagen.(6a) Binding of rTRIP-1 to immobilized collagen I was analyzed by Surface plasmon resonance (SPR) spectroscopy. SPR data analyses show the steady state fits used to calculate the binding constant between rTRIP-1 and immobilized Type 1 Collagen (KD = 48.59 μM). Values are the means from independent experiments that were performed in triplicates, and the error bars are the S.E.M. Figure 6b: SPR sensorgrams of a series of increasing concentrations of rTRIP-1 showing binding to immobilized type 1 Collagen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121659&req=5

f6: rTRIP-1 binds to Type 1 Collagen.(6a) Binding of rTRIP-1 to immobilized collagen I was analyzed by Surface plasmon resonance (SPR) spectroscopy. SPR data analyses show the steady state fits used to calculate the binding constant between rTRIP-1 and immobilized Type 1 Collagen (KD = 48.59 μM). Values are the means from independent experiments that were performed in triplicates, and the error bars are the S.E.M. Figure 6b: SPR sensorgrams of a series of increasing concentrations of rTRIP-1 showing binding to immobilized type 1 Collagen.
Mentions: To assess the role of TRIP-1 in collagen biomineralization, we performed binding assay between rTRIP-1 (Fig. S6) as the analyte and type 1 collagen as the ligand using Surface Plasmon Resonance (SPR). Steady state analysis (Fig. 6a) demonstrated that TRIP-1 bound to Collagen in a concentration-dependent and saturated manner with an apparent KD of 48 μM. Figure 6b shows the sensorgrams of a series of increasing concentrations of rTRIP-1 flown on a Collagen 1-immobilized CM5 sensor surface clearly showing binding between TRIP-1 and type 1 Collagen.

View Article: PubMed Central - PubMed

ABSTRACT

Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 μM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization.

No MeSH data available.