Proteomic analysis of the alteration of nuclear cytoplasmic distribution of intracellular proteins in motoneuron cell lines expressing mutant SOD1 G93A

Abstract

Background: The appropriate nuclear cytoplasmic localization of proteins is essential for eukaryotic cellular function and biological processes such as RNA processing, translation, protein-protein interaction and post-translational modification. Aberrant localization of proteins has been implicated in many human diseases, like cancer and neurodegenerative conditions. Evidence suggests that the cytoplasmic mislocalization of nuclear proteins including transactive response DNA-binding protein 43kDa (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis and frontotemporal lobar degeneration is associated with neurotoxicity. In this study, we investigated the proteome-wide alteration of nuclear cytoplasmic distribution in the motor neuron cell lines expressing mutant human SOD1 (G93A). Methods: The motor neuron-like cell line NSC34 transfected with wild or mutant human SOD1 (G93A) underwent for subcellular fractionation. The proteome in the nuclear and cytoplasm fractions were analyzed using liquid chromatography-tandem mass spectrometry. Abundance difference of proteins for genotype, subcellular fraction and their interaction were analyzed. Bioinformatics analysis using TargetMine and DAVID were used to understand the function of the identified proteins. Finally, immunoblots of the significant proteins that changed the subcellular distribution in mutant cells were performed for validation. Results: In total, 11,216 peptides and 1,925 proteins were identified, with 23% of the peptides and 32% of the proteins being found in both nuclear and cytoplasmic fractions. Using the intersection data set common to both fractions, we found that a considerable number of proteins (79%) were differentially distributed in the nuclear versus cytoplasmic compartments in the wild type. A total of 37 proteins showed a significant alteration in the nuclear cytoplasmic distribution in the mutant cells (p<0.05). The subcellular distribution in the mutant cells was shifted from the cytoplasm to the nucleus for the proteins of RNA transport and processing (Dhx9, Fmr1, Srsf3, Srsf6, Tra2b), whereas the opposite was the case for the pathways of protein folding(Cct5, Cct7, Cct8), the aminoacyl-tRNA biosynthesis (Farsb, Nars, Txnrd1), the synaptic vesicle cycle (Cltc, Nsf), the Wnt signaling (Cltc, Plcb3, Plec, Psmd3, Ruvbl1) and the Hippo signaling (Camk2d, Plcb3, Ruvbl1). Conclusions: This study allows a comprehensive understanding of the nuclear cytoplasmic distribution of intracellular proteins in motor neuron cell lines, and their alteration by the mutant SOD1 expression. Further studies are warranted, in order to elucidate the pathomechanistic implications of the aberrant localizations of the candidate proteins.