title: Identifying protein partners of CLN8, an ER-resident protein involved in neuronal ceroid lipofuscinosis.
abstract: Neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of neurodegenerative diseases characterized by cognitive and motor decline, epilepsy, visual loss and by lysosomal autofluorescent inclusions. Two distinct clinical phenotypes, the progressive epilepsy with mental retardation (EPMR) and a late-infantile variant of NCLs (CLN8-vLINCL) are associated with mutations in the CLN8 gene that encodes a transmembrane protein predominantly located to the endoplasmic reticulum (ER). To gain insight into the function of CLN8 protein, we employed the split-ubiquitin membrane-based yeast two-hybrid (MYTH) system, which detects protein-protein interactions in a membrane environment, using the full-length human CLN8 as bait and a human brain cDNA library as prey. We identified several potential protein partners of CLN8 and especially referred to VAPA, c14orf1/hERG28, STX8, GATE16, BNIP3 and BNIP3L proteins that are associated with biologically relevant processes such as synthesis and transport of lipids, vesicular/membrane trafficking, autophagy/mitophagy and apoptosis. Interactions of CLN8 with VAPA and GATE16 were further validated by co-immunoprecipitation and co-localization assays in mammalian cells. Using a new C-terminal-oriented CLN8 antibody, CLN8-VAPA interaction was also confirmed by co-staining in close spatial proximity within different CNS tissues. The results of this study shed light on potential interactome networks of CLN8 and provide a powerful starting point for understanding protein function(s) and molecular aspects of diseases associated with CLN8 deficiency.
authors.affiliation: CNR Institute of Biomedicine and Molecular Immunology, 90146 Palermo, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Passantino
authors.forename: Caterina
authors.initials: C
authors.lastname: Cascio
authors.forename: Irene
authors.initials: I
authors.lastname: Deidda
authors.forename: Giacoma
authors.initials: G
authors.lastname: Galizzi
authors.forename: Domenica
authors.initials: D
authors.lastname: Russo
authors.forename: Gianpiero
authors.initials: G
authors.lastname: Spedale
authors.forename: Patrizia
authors.initials: P
authors.lastname: Guarneri
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D000917:Antibody Formation; D000071190:Autophagy-Related Protein 8 Family; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009472:Neuronal Ceroid-Lipofuscinoses / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011817:Rabbits; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; C090121:BNIP3 protein, human; C116838:BNIP3L protein, human; C121767:CLN8 protein, human; C121701:ERG28 protein, human; C498698:GABARAPL2 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; C114051:VAPA protein, human; D033921:Vesicular Transport Proteins