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Cell membrane proteome analysis in HEK293T cells challenged with α-Synuclein amyloids

ABSTRACT: Amyloids interact with plasma membranes. Extracellular amyloids cross the plasma membrane barrier. Internalized extracellular amyloids are reported to trigger amyloidogenesis of endogenous proteins in recipient cells. To what extent these extracellular and intracellular amyloids perturb the plasma membrane proteome is not investigated. Using α-Synuclein as a model amyloid protein, we performed membrane shaving followed by mass spectrometry experiment to identify the conformational changes in the cell surface proteins after extracellular amyloid challenge. We also performed membrane proteomics after the biogenesis of intracellular α-Synuclein amyloids. Our results suggest that promiscuous interaction with extracellular amyloids stochastically alter the conformation of plasma membrane proteins. This affects the biological process through the plasma membrane and result in loss in cell viability. Cells that survive the extracellular amyloid shock can grow normally and gradually develop intracellular amyloids which do not directly impact the plasma membrane proteome and associated biological processes. Thus, α-Synuclein amyloids can damage the plasma membrane and related processes only during cell to cell transfer and not during their intracellular biogenesis.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Cell Line Cell

SUBMITTER: Swasti Raychaudhuri

LAB HEAD: Swasti Raychaudhuri

PROVIDER: PXD046280 | Pride | 2024-07-09

REPOSITORIES: Pride

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			Action	DRS
	6days.allSpectra.HCD.FTMS.iso1.apl	Other
	6days.allSpectra.HCD.FTMS.iso10.apl	Other
	6days.allSpectra.HCD.FTMS.iso11.apl	Other
	6days.allSpectra.HCD.FTMS.iso12.apl	Other
	6days.allSpectra.HCD.FTMS.iso13.apl	Other

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Project description:Abnormal accumulation of aggregated proteins and sustained microglial activation are important contributors of neurodegenerative process in neurological diseases. Recent studies have shown that aggregation-prone proteins, such as a-synuclein, the protein implicated in Parkinson’s disease (PD), are released from neuronal cells and thus present in the extracellular fluid, pointing to the possible paracrine effects of these proteins on microglial immune responses. However, the mechanism underlying the disease-associated microglial activation and the role of neuronal proteins in this process remain unknown. Here, we show that extracellular a-synuclein released from neuronal cells is an endogenous ligand of toll-like receptor 2 (TLR2) and activates microglia, which in turn induces neurodegeneration. Interaction between neuron-released a-synuclein and TLR2 and subsequent activation of the TLR2 signaling were demonstrated comprehensively by using computational modeling of signaling network and by the experimental validation in TLR2-deficient microglia both in vitro and in vivo. In contrast to the neuron-released a-synuclein, recombinant a-synuclein proteins, including monomer, oligomer, fibril, or nitrated forms, were not able to interact or activate TLR2, suggesting that neuronal cells have a mechanism of enriching specific forms of a-synuclein capable of activating TLR2 during the process of releasing this protein. Taken together, the results suggest that both neuron-released extracellular a-synuclein and TLR2 might be novel therapeutic targets for modifying neuroinflammation in PD and related neurodegenerative diseases. We collected culture media from differentiated SH-SY5Y cells overexpressing either human a-synuclein (alpha-SCM) or beta-galactosidase (LZCM) and treat these media to primary rat microglia at the concentration of a-synuclein of 1.1M. Transcriptome analyses with microglial cells treated with either aSCM or LZCM at two different time points, 6 h and 24 h.

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Cell membrane proteome analysis in HEK293T cells challenged with α-Synuclein amyloids

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