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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

Expression and localization of FXR1P and CMAS in HEK293T cells and HeLa cells. HEK293T cells respectively transfected with (A) pEGFP-N1-FXR1 and (B) pDsRed-N1-CMAS FXR1P is located in the cytoplasm. CMAS is located in the nucleus. HeLa cells respectively transfected with (C) pEGFP-N1-FXR1 and (D) pDsRed-N1-CMAS. Cells were visual-ized by fluorescence microscopy. FXR1P is also located in the cytoplasm and CMAS in the nucleus. The nuclei were stained with DAPI. Magnification, x100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.
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f2-mmr-14-02-1501: Expression and localization of FXR1P and CMAS in HEK293T cells and HeLa cells. HEK293T cells respectively transfected with (A) pEGFP-N1-FXR1 and (B) pDsRed-N1-CMAS FXR1P is located in the cytoplasm. CMAS is located in the nucleus. HeLa cells respectively transfected with (C) pEGFP-N1-FXR1 and (D) pDsRed-N1-CMAS. Cells were visual-ized by fluorescence microscopy. FXR1P is also located in the cytoplasm and CMAS in the nucleus. The nuclei were stained with DAPI. Magnification, x100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.

Mentions: HEK293T and HeLa cells transfected with pEGFP-N1-FXR1 exhibited strong cytoplasmic fluorescence, whereas transfection with pDsRed-N1-CMAS produced fluorescence in the nucleus (Fig. 2). Thus, the fusions to the N-terminus of EGFP or DsRed did not affect the fluorescence properties of the native protein, allowing the fusion protein to be correctly localized in vivo.


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)

Expression and localization of FXR1P and CMAS in HEK293T cells and HeLa cells. HEK293T cells respectively transfected with (A) pEGFP-N1-FXR1 and (B) pDsRed-N1-CMAS FXR1P is located in the cytoplasm. CMAS is located in the nucleus. HeLa cells respectively transfected with (C) pEGFP-N1-FXR1 and (D) pDsRed-N1-CMAS. Cells were visual-ized by fluorescence microscopy. FXR1P is also located in the cytoplasm and CMAS in the nucleus. The nuclei were stained with DAPI. Magnification, x100. 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

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

f2-mmr-14-02-1501: Expression and localization of FXR1P and CMAS in HEK293T cells and HeLa cells. HEK293T cells respectively transfected with (A) pEGFP-N1-FXR1 and (B) pDsRed-N1-CMAS FXR1P is located in the cytoplasm. CMAS is located in the nucleus. HeLa cells respectively transfected with (C) pEGFP-N1-FXR1 and (D) pDsRed-N1-CMAS. Cells were visual-ized by fluorescence microscopy. FXR1P is also located in the cytoplasm and CMAS in the nucleus. The nuclei were stained with DAPI. Magnification, x100. FXR1P, fragile X related 1; CMAS, CMP-N-acetylneuraminic acid synthetase; DAPI, 4′,6-diamidino-2-phenylindole.
Mentions: HEK293T and HeLa cells transfected with pEGFP-N1-FXR1 exhibited strong cytoplasmic fluorescence, whereas transfection with pDsRed-N1-CMAS produced fluorescence in the nucleus (Fig. 2). Thus, the fusions to the N-terminus of EGFP or DsRed did not affect the fluorescence properties of the native protein, allowing the fusion protein to be correctly localized in vivo.

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