Drafting the CLN3 Protein Interactome in SH-SY5Y Human Neuroblastoma Cells: A Label-free Quantitative Proteomics Approach

Abstract

Neuronal ceroid lipofuscinoses (NCL) are the most common inherited progressive encephalopathies of childhood. One of the most prevalent forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3 disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3) remains elusive. In this study, we aimed to clarify the role of human CLN3 in the brain by identifying CLN3-associated proteins using a Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS) strategy combined with Significance Analysis of Interactome (SAINT). Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein complexes for subsequent TAP-MS. Bioinformatic analyses of isolated complexes yielded 58 CLN3 interacting partners (IP) including 42 novel CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP) previously identified in another high-throughput study by Behrends et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein 5 (CLN5) were identified (18 of which were in common with the CLN3 bait). Our findings support previously suggested involvement of CLN3 in transmembrane transport, lipid homeostasis and neuronal excitability, as well as link it to G-protein signaling and protein folding/sorting in the ER.

Advances in Biomarkers of Major Depressive Disorder

“…Protein interactomics often employ yeast two-hybrid screening (Y2H) [163], tandem affinity purification [164], and coimmunoprecipitation (coIP) [165].…”

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation

“…A lacuna in the field is that unlike other neurodegenerative diseases, notably Parkinson’s disease [25], it is not known if NCL genes function in common pathways. However, there is mounting evidence that these genes may interact, providing valuable insight into disease mechanisms [21, 28]. …”

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells

“…A more mature neuronal phenotype can be reached using growth medium supplemented with specific chemical factors, such as recombinant human brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), dibutyryl-cyclic AMP (db-cAMP), phorbol esters and others (Agholme et al 2010;Dwane et al 2013;Encinas et al 2000;Gimenez-Cassina et al 2006;Jamsa et al 2004;Kou et al 2008;Kume et al 2008;Sarkanen et al 2007). Thereafter, SH-SY5Y cells have been largely utilized to investigate the disease mechanisms associated with various neurodegenerative disorders (Agholme et al 2014;Ferreira et al 2013;Hadzhieva et al 2013;Jamsa et al 2004;Ke et al 2012;Krishna et al 2014;Lim et al 2015;Lopes et al 2010Lopes et al , 2012Palomo et al 2011;Scifo et al 2013Scifo et al , 2015.…”