Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

ß-Synuclein: An Enigmatic Protein with Diverse Functionality.

α-Synuclein (αS) is a small, unstructured, presynaptic protein expressed in the brain. Its aggregated form is a major component of Lewy bodies, the large proteinaceous deposits in Parkinson's disease. The closely related protein, ß-Synuclein (ßS), is co-expressed with αS. In vitro, ßS acts as a molecular chaperone to inhibit αS aggregation. As a result of this assignation, ßS has been largely understudied in comparison to αS. However, recent reports suggest that ßS promotes neurotoxicity, implying that ßS is involved in other cellular pathways with functions independent of αS. Here, we review the current literature pertaining to human ßS in order to understand better the role of ßS in homeostasis and pathology. Firstly, the structure of ßS is discussed. Secondly, the ability of ßS to (i) act as a molecular chaperone; (ii) regulate synaptic function, lipid binding, and the nigrostriatal dopaminergic system; (iii) mediate apoptosis; (iv) participate in protein degradation pathways; (v) modulate intracellular metal levels; and (vi) promote cellular toxicity and protein aggregation is explored. Thirdly, the P123H and V70M mutations of ßS, which are associated with dementia with Lewy bodies, are discussed. Finally, the importance of post-translational modifications on the structure and function of ßS is reviewed. Overall, it is concluded that ßS has both synergistic and antagonistic interactions with αS, but it may also possess important cellular functions independent of αS.

The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27.

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.

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes.

Multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are α-synucleinopathies that exhibit widespread astrogliosis as a component of the neuroinflammatory response. Munc18, a protein critical to vesicle exocytosis, was previously found to strongly mark morphologically activated astrocytes in brain tissue of MSA patients. Immunofluorescence of MSA, DLB and normal brain tissue sections was combined with cell culture and co-culture experiments to investigate the relationship between extracellular α-synuclein and the transition to a secretory astrocyte phenotype. Increased Munc18-positive vesicles were resolved in activated astrocytes in MSA and DLB tissue compared to controls, and they were also significantly upregulated in the human 1321N1 astrocytoma cell line upon treatment with α-synuclein, with parallel increases in GFAP expression and IL-6 secretion. In co-culture experiments, rat primary astrocytes pretreated with α-synuclein inhibited the growth of neurites of co-cultured primary rat neurons and upregulated chondroitin sulphate proteoglycan. Taken together, these results indicate that the secretory machinery is significantly upregulated in the astrocyte response to extracellular α-synuclein and may participate in the release of neuroinhibitory and proinflammatory factors in α-synucleinopathies.

The multifaceted nature of αB-crystallin.

In vivo, small heat-shock proteins (sHsps) are key players in maintaining a healthy proteome. αB-crystallin (αB-c) or HspB5 is one of the most widespread and populous of the ten human sHsps. Intracellularly, αB-c acts via its molecular chaperone action as the first line of defence in preventing target protein unfolding and aggregation under conditions of cellular stress. In this review, we explore how the structure of αB-c confers its function and interactions within its oligomeric self, with other sHsps, and with aggregation-prone target proteins. Firstly, the interaction between the two highly conserved regions of αB-c, the central α-crystallin domain and the C-terminal IXI motif and how this regulates αB-c chaperone activity are explored. Secondly, subunit exchange is rationalised as an integral structural and functional feature of αB-c. Thirdly, it is argued that monomeric αB-c may be its most chaperone-species active, but at the cost of increased hydrophobicity and instability. Fourthly, the reasons why hetero-oligomerisation of αB-c with other sHsps is important in regulating cellular proteostasis are examined. Finally, the interaction of αB-c with aggregation-prone, partially folded target proteins is discussed. Overall, this paper highlights the remarkably diverse capabilities of αB-c as a caretaker of the cell.

Extracellular Interactions of Alpha-Synuclein in Multiple System Atrophy.

Multiple system atrophy, characterized by atypical Parkinsonism, results from central nervous system (CNS) cell loss and dysfunction linked to aggregates of the normally pre-synaptic α-synuclein protein. Mostly cytoplasmic pathological α-synuclein inclusion bodies occur predominantly in oligodendrocytes in affected brain regions and there is evidence that α-synuclein released by neurons is taken up preferentially by oligodendrocytes. However, extracellular α-synuclein has also been shown to interact with other neural cell types, including astrocytes and microglia, as well as extracellular factors, mediating neuroinflammation, cell-to-cell spread and other aspects of pathogenesis. Here, we review the current evidence for how α-synuclein present in the extracellular milieu may act at the cell surface to drive components of disease progression. A more detailed understanding of the important extracellular interactions of α-synuclein with neuronal and non-neuronal cell types both in the brain and periphery may provide new therapeutic targets to modulate the disease process.