Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation

Abstract

A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosylinsoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

Increased 14-3-3 phosphorylation observed in Parkinson's disease reduces neuroprotective potential of 14-3-3 proteins

“…Our study shows some consistency with the findings of Kulanthingal et al who have demonstrated by proteomics alterations in 14-3-3 phosphorylation in tet-off BE-2-M17D neuroblastoma cell line that conditionally overexpresses αsyn (29). They observed increased phosphorylation of 14-3-3ε at 14 days after αsyn induction and increased phosphorylation of 14-3-3-3ζ at 28 days after αsyn induction (29).…”

“…Increased phosphorylation in response to αsyn overexpression has been described in a proteonomics study (29), but the site of phosphorylation is not known. We created a tetracycline-inducible M17 stable cell line that expresses wildtype αsyn upon doxycycline treatment.…”

“…Kulanthingal et al have previously demonstrated in a proteomics study that alterations in 14-3-3 phosphorylation are observed in neuroblastoma cells overexpressing αsyn (29). Which phosphorylation sites and which isoforms are involved have not been fully examined, nor the consequences of such phosphorylation changes in PD models.…”

Heat shock proteins: Cellular and molecular mechanisms in the central nervous system

“…HSP90 co-localizes with α-synuclein filaments of Lewy bodies in PD (Uryu et al , 2006), and reduced phosphorylation of HSP90 has been observed in the SN of PD brains and in response to α-synuclein accumulation (Kulathingal et al , 2009). The associations of reduced HSP90 phosphorylation, α-synuclein aggregation, and HSP90 interaction with aggregates are consistent with the need for HSP90 to be phosphorylated in order to release its substrate, and indicate that HSPC/HSP90 may be unable to dissociate from aggregated proteins.…”

Synthesis and Chromatographic Characteristics of Multidentate Ligand-Boned Silica Stationary Phases

“…1 Over the past three decades, IMAC has become an essential tool for the identification, detection, isolation and purification of proteins, especially been active in the areas such as proteomics, [2][3][4][5] refolding of recombinant proteins, [6][7][8][9] labeling and identification of proteins 5,[10][11][12] as well as cancer diagnosis [13][14][15][16] in recent few years. Ion exchangers using aminocarboxy chelating agent as ligand have been widely used in IMAC.…”