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CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

Ma Y, Tian S, Wang Z, Wang C, Chen X, Li W, Yang Y, He S - Mol Med Rep (2016)

Bottom Line: Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1).The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells.Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China.

ABSTRACT
Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

No MeSH data available.


Related in: MedlinePlus

Colocalization of FXR1P and CMAS in HEK293T and HeLa cells. (A) HEK293T and (B) HeLa cells were transiently transfected with pEGFP-N1-FXR1 and pDsRed-N1-CMAS, then visualized by fluorescence microscopy. FXR1P (green) is visible in the cytoplasm, CMAS (red) is visible mostly in the nucleus, with less in the cytoplasm. Positive colocalization (yellow) is visible in the cytoplasm and around the nucleus. Yellow indicated the colocalization of pEGFP-FXR1P with pDsRed-CMAS. The nuclei (blue) were stained by DAPI. Magnification, ×100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.
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f3-mmr-14-02-1501: Colocalization of FXR1P and CMAS in HEK293T and HeLa cells. (A) HEK293T and (B) HeLa cells were transiently transfected with pEGFP-N1-FXR1 and pDsRed-N1-CMAS, then visualized by fluorescence microscopy. FXR1P (green) is visible in the cytoplasm, CMAS (red) is visible mostly in the nucleus, with less in the cytoplasm. Positive colocalization (yellow) is visible in the cytoplasm and around the nucleus. Yellow indicated the colocalization of pEGFP-FXR1P with pDsRed-CMAS. The nuclei (blue) were stained by DAPI. Magnification, ×100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.

Mentions: To evaluate the colocalization of FXR1P and CMAS, pEGFP-N1-FXR1 and pDsRed-N1-CMAS plasmids were transiently cotransfected into HEK293T and HeLa cells (Fig. 3). Transfection with pEGFP-N1-FXR1 produced green fluorescence, predominantly in the cytoplasm, whereas pDsRed-N1-CMAS transfection predominantly produced red fluorescence in the nucleus and less in the cytoplasm. The two polypeptides (pEGFP-N1-FXR1 and pDsRed-N1-CMAS) maintained intracellular localization, with yellow fluorescence demonstrating FXR1P and CMAS colocalization in the cytoplasm and the edge of the nucleus (Fig. 3). Typically, these genes are expressed in different parts of the cell, with no fluorescence overlap, but co-overexpression leads to the formation of oligomers in the cytoplasmic and nucleus, producing yellow fluorescence.


CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

Ma Y, Tian S, Wang Z, Wang C, Chen X, Li W, Yang Y, He S - Mol Med Rep (2016)

Colocalization of FXR1P and CMAS in HEK293T and HeLa cells. (A) HEK293T and (B) HeLa cells were transiently transfected with pEGFP-N1-FXR1 and pDsRed-N1-CMAS, then visualized by fluorescence microscopy. FXR1P (green) is visible in the cytoplasm, CMAS (red) is visible mostly in the nucleus, with less in the cytoplasm. Positive colocalization (yellow) is visible in the cytoplasm and around the nucleus. Yellow indicated the colocalization of pEGFP-FXR1P with pDsRed-CMAS. The nuclei (blue) were stained by DAPI. Magnification, ×100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940058&req=5

f3-mmr-14-02-1501: Colocalization of FXR1P and CMAS in HEK293T and HeLa cells. (A) HEK293T and (B) HeLa cells were transiently transfected with pEGFP-N1-FXR1 and pDsRed-N1-CMAS, then visualized by fluorescence microscopy. FXR1P (green) is visible in the cytoplasm, CMAS (red) is visible mostly in the nucleus, with less in the cytoplasm. Positive colocalization (yellow) is visible in the cytoplasm and around the nucleus. Yellow indicated the colocalization of pEGFP-FXR1P with pDsRed-CMAS. The nuclei (blue) were stained by DAPI. Magnification, ×100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.
Mentions: To evaluate the colocalization of FXR1P and CMAS, pEGFP-N1-FXR1 and pDsRed-N1-CMAS plasmids were transiently cotransfected into HEK293T and HeLa cells (Fig. 3). Transfection with pEGFP-N1-FXR1 produced green fluorescence, predominantly in the cytoplasm, whereas pDsRed-N1-CMAS transfection predominantly produced red fluorescence in the nucleus and less in the cytoplasm. The two polypeptides (pEGFP-N1-FXR1 and pDsRed-N1-CMAS) maintained intracellular localization, with yellow fluorescence demonstrating FXR1P and CMAS colocalization in the cytoplasm and the edge of the nucleus (Fig. 3). Typically, these genes are expressed in different parts of the cell, with no fluorescence overlap, but co-overexpression leads to the formation of oligomers in the cytoplasmic and nucleus, producing yellow fluorescence.

Bottom Line: Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1).The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells.Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Biology, University of South China, Hengyang, Hunan 421001, P.R. China.

ABSTRACT
Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

No MeSH data available.


Related in: MedlinePlus