Abstract
Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42–53).
Highlights
Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM256730)
The data shown here result from isolated protein complexes from PPT1expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS)
The terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network
Summary
Novel knowledge of the PPT1 interactome in SH-SY5Y cells Label-free quantitation of PPT1 IP to determine their relative abundances Identification of novel PPT1 IP and insight into novel roles of PPT1 Identification of putative PPT1 substrates Preliminary dataset for exploring functional relationships in the PPT1 network. In order to determine PPT1 IP, we first cloned full length human PPT1 into CTAP-Puro (a mammalian retroviral based expression vectors) and generated PPT1 expressing SH-SY5Y stable cells by retroviral infection. DNA of retroviral vectors was introduced into HEK 293T cells [2,3] simultaneously with two packaging plasmids: pCMV-Gag-Pol vector The three plasmids were mixed in proportions of 7.5:5:1, respectively and introduced into cells using the calcium phosphate method according to the manufacturer’s protocol (Life Technologies Europe BV). Infected cells were maintained in an undifferentiated state (.80% confluence) and constantly checked for consistent growth rates and morphological features
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