Abstract
Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.
Highlights
Protein homeostasis, or proteostasis, refers to the cell’s inherent biological pathways and networks which ensure that proteins acquire their native and functional form with their levels being maintained within a strict regime to ensure optimal cell functionality [1,2].When proteostasis is compromised, proteins can partially unfold, misfold, and aggregate, leading to a plethora of deleterious consequences including cell death [3]
Cells, which possess neuronal‐like characteristics due to the presence of neurofilaments, vimentin and other proteins characteristic of a cell line with neuronal lineage [58], were treated with 1, 5, 10 and 20 μM of αBc:DAox (Figure 6A) or Hsp27:DAox (Figure 6B), there was no significant difference between cell viability in the presence of unmodified αBc or a phosphate‐buffered saline (PBS)
Protein aggregation is mitigated by sHsps such as αBc and Hsp27 which form the first line of defense against proteostatic dysregulation
Summary
Proteostasis, refers to the cell’s inherent biological pathways and networks which ensure that proteins acquire their native and functional form with their levels being maintained within a strict regime to ensure optimal cell functionality [1,2]. Small heat-shock proteins (sHsps) are a family of intracellular molecular chaperone proteins [5] They interact with and stabilize misfolded and partially unfolded proteins to prevent their aggregation [5]. The unstructured CTR provides solubility to the rather hydrophobic protein [15] and forms interactions between subunits, partially via its well-conserved IXI sequence [16]. Upon modification and cross-linking with DAox at an equivalent molar level, the sHsps were capable, to a degree comparable to that of the unmodified protein, of preventing amorphous and amyloid fibrillar protein aggregation, thereby corroborating the chaperones’ robust nature and their ability, in a cellular context, to mitigate the deleterious effects of protein unfolding and aggregation [5,6,7,42]
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