The neuronal ceroid lipofuscinoses (NCLs) are rare forms of lysosomal storage diseases but the most common neurodegenerative disorders affecting children. All NCLs are characterized by dementia, epilepsy, and motor decline; in most NCL forms, children also manifest a progressive visual failure. Moreover, they share a common feature to accumulate lipopigments in lysosomes of neural cells and other cell types. However, they differ for the link to distinct genes and various mutations of the same gene that lead to a wide phenotypic variability of disease courses. The exact function of the most encoded proteins is still unclear and needs to be elucidated. Mutations of CLN8 gene lead to two distinct phenotypes: the progressive epilepsy and mental retardation (EPMR), and a variant of late-infantile NCL (vLINCL). CLN8 gene encodes an ER-resident transmembrane protein of 286 a.a.; despite its specific role is unknown, it has been tightly related to lipid homeostasis, specially to sphingolipids. In our laboratory, through a yeast-two-hybrid approach and a human cDNA library screening, it has been drawn a partial interactome. Of the identified CLN8 partners, the VAPA (vesicle-associated membrane protein/VAMP-associated-protein-A) protein is the docking site of ceramide transporter proteins in the ER, working in lipid physiology. EPMR patients and the murine model of vLINCL have impaired levels of ceramide metabolites. Thus, the interaction between VAPA and CLN8 could have a key role in regulating the traffic and physiology of ceramide. To gain insights on CLN8/VAPA interaction, we have prepared a "bicistronic" mammalian expression vector on which both CLN8 and VAPA were expressed under a CMV promoter and in fusion with two different Tags at N terminus. The "bicistronic" construct by overexpressing both proteins in the same cellular contest could help to investigate the functional role of CLN8/VAPA interaction in lipid homeostasis and, so its implication in CLN8 diseases-associated mechanisms.
A bicistronic vector to study the effect of CLN8/VAPA interaction on cell physiology and eventually the pathology of CLN8-associated NLC diseases
S Papasergi;P Saladino;I Deidda;D Russo;P Guarneri
2015
Abstract
The neuronal ceroid lipofuscinoses (NCLs) are rare forms of lysosomal storage diseases but the most common neurodegenerative disorders affecting children. All NCLs are characterized by dementia, epilepsy, and motor decline; in most NCL forms, children also manifest a progressive visual failure. Moreover, they share a common feature to accumulate lipopigments in lysosomes of neural cells and other cell types. However, they differ for the link to distinct genes and various mutations of the same gene that lead to a wide phenotypic variability of disease courses. The exact function of the most encoded proteins is still unclear and needs to be elucidated. Mutations of CLN8 gene lead to two distinct phenotypes: the progressive epilepsy and mental retardation (EPMR), and a variant of late-infantile NCL (vLINCL). CLN8 gene encodes an ER-resident transmembrane protein of 286 a.a.; despite its specific role is unknown, it has been tightly related to lipid homeostasis, specially to sphingolipids. In our laboratory, through a yeast-two-hybrid approach and a human cDNA library screening, it has been drawn a partial interactome. Of the identified CLN8 partners, the VAPA (vesicle-associated membrane protein/VAMP-associated-protein-A) protein is the docking site of ceramide transporter proteins in the ER, working in lipid physiology. EPMR patients and the murine model of vLINCL have impaired levels of ceramide metabolites. Thus, the interaction between VAPA and CLN8 could have a key role in regulating the traffic and physiology of ceramide. To gain insights on CLN8/VAPA interaction, we have prepared a "bicistronic" mammalian expression vector on which both CLN8 and VAPA were expressed under a CMV promoter and in fusion with two different Tags at N terminus. The "bicistronic" construct by overexpressing both proteins in the same cellular contest could help to investigate the functional role of CLN8/VAPA interaction in lipid homeostasis and, so its implication in CLN8 diseases-associated mechanisms.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
