J Korean Acad Conserv Dent.  2007 Sep;32(5):459-468. 10.5395/JKACD.2007.32.5.459.

A study of APin-protein interactions using protein microarray

• Park JC
• Park SH
• Kim HJ
• Park JT
• Youn SH
• Kim JW
• Lee TY
• Son HH

• KMID: 2175939
• DOI: http://doi.org/10.5395/JKACD.2007.32.5.459

Abstract

Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interactions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR. The results were as follows: 1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively. 2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation. 3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the overexpression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expression of EF-hand calcium binding protein was markedly decreased by its inactivation. These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

Keyword

APin; Ameloblast; Protein microarray; Mineralization; Interaction; Differentiation

MeSH Terms

Ameloblasts
Animals
Aurora Kinase A
Calcium
Carrier Proteins
Clone Cells
DNA, Complementary
Gene Library
Odontoblasts
Protein Array Analysis*
Rats
RNA, Messenger
RNA, Small Interfering
Transfection
Calcium
Carrier Proteins
DNA, Complementary
RNA, Messenger
RNA, Small Interfering

Figure

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  Figure 1 Detection of the APin protein using protein microarray.

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  Figure 2 Relative affinity ranking with the APin protein.

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  Figure 3 RT-PCR amplification of the APin in ameloblast cell line after overexpression with CMV-APin plasmid and inactivation with U6-APin siRNA.

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  Figure 4 RT-PCR amplification of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein ameloblast cell line after over-expression with CMV-Apin protein plasmid and inactivation with U6-Apin protein siRNA.

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  Figure 5 The amplification for the APin reactions in control, over-expression, inactivation and mock groups. The relative amounts of MEF2 (A), Aurora kinase A (B), BMPR-IB (C) and EF-hand calcium binding (D) mRNA after normalization with the amounts of the GAPDH mRNA. The results were represented as means ± SD of three independent transfections. Con, normal ALC; Over, APin overexpression; Inact, APin inactivation; Mock, ALC expressing empty vector.

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