Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells

J Proteomics. 2015 Jun 18;123:42-53. doi: 10.1016/j.jprot.2015.03.038. Epub 2015 Apr 10.

Abstract

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Biological significance: The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Keywords: Affinity purification; CLN1 disease; Interactome; Mass spectrometry; Neuronal ceroid lipofuscinoses; Palmitoyl protein thioesterase 1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Axons / metabolism
  • Brain / metabolism
  • Cell Line, Tumor
  • Cell Movement
  • Energy Metabolism
  • Gene Expression Regulation, Neoplastic*
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Lysosomes / metabolism
  • Mass Spectrometry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Mitochondria / physiology
  • Mutation
  • Neuroblastoma / metabolism*
  • Neuronal Ceroid-Lipofuscinoses / metabolism
  • Neurons / metabolism
  • Open Reading Frames
  • Proteomics / methods*
  • Signal Transduction
  • Thiolester Hydrolases

Substances

  • Membrane Proteins
  • Thiolester Hydrolases
  • PPT1 protein, human