An increasing number of proteins are being identified as multifunctional. A single protein may in fact perform different and unrelated functions in distinct cell compartments. Intriguing examples of moonlighting proteins are the glycolytic enzymes, which in addition to catalytic functions are involved in fully unrelated processes. These enzymes are extremely abundant in the cytoplasm and poorly expressed in other districts such as nuclei and plasma membranes, which makes difficult to discriminate among functional variants by standard biochemical and cellular approaches. The glycolytic enzyme alpha-enolase is a prototype of multifunctional protein. Lately, we focused on the role of plasminogen receptor exerted by surface alphaenolase in eukaryotic cells, function that contributes to the motility and invasiveness of cancer cells. To develop a method for the detection and quantification of surface alpha-enolase in living cells, and to set up experimental conditions, we used the HB2 mammary epithelial cell line, the MCF-7 low metastatic breast cancer cell line and the doxorubicin-resistant counterpart, MCF-7R. These cell lines express different levels of specific receptor proteins, which makes them suitable for a comparative analysis. Antibodies directed against extracellular epitopes of bona fide receptors or exclusively intracellular antigens were used for assessing the specificity of the detection. The method allows the quantitative analysis of surface proteins under different cell culture conditions, e.g. in cells subjected to different stimuli or drug treatments, without the interference of the cytoplasmic counterparts. We further show that this approach can be applied to other multifunctional proteins with characteristics similar to those of alpha-enolase.
A new experimental approach to study cell surface exposed multifunctional proteins
Giovanni Perconti;Agata Giallongo
2016
Abstract
An increasing number of proteins are being identified as multifunctional. A single protein may in fact perform different and unrelated functions in distinct cell compartments. Intriguing examples of moonlighting proteins are the glycolytic enzymes, which in addition to catalytic functions are involved in fully unrelated processes. These enzymes are extremely abundant in the cytoplasm and poorly expressed in other districts such as nuclei and plasma membranes, which makes difficult to discriminate among functional variants by standard biochemical and cellular approaches. The glycolytic enzyme alpha-enolase is a prototype of multifunctional protein. Lately, we focused on the role of plasminogen receptor exerted by surface alphaenolase in eukaryotic cells, function that contributes to the motility and invasiveness of cancer cells. To develop a method for the detection and quantification of surface alpha-enolase in living cells, and to set up experimental conditions, we used the HB2 mammary epithelial cell line, the MCF-7 low metastatic breast cancer cell line and the doxorubicin-resistant counterpart, MCF-7R. These cell lines express different levels of specific receptor proteins, which makes them suitable for a comparative analysis. Antibodies directed against extracellular epitopes of bona fide receptors or exclusively intracellular antigens were used for assessing the specificity of the detection. The method allows the quantitative analysis of surface proteins under different cell culture conditions, e.g. in cells subjected to different stimuli or drug treatments, without the interference of the cytoplasmic counterparts. We further show that this approach can be applied to other multifunctional proteins with characteristics similar to those of alpha-enolase.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
