Monomeric alpha-synuclein (alphaSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in alpha-helix structure. Here we show that at physiological pH and ionic strength, alphaSN incubated with different anionic lipid vesicles undergoes a marked increase in alpha-helical content at a temperature dictated either by the temperature of the lipid phase transition, or (in 1,2-DilauroylSN-Glycero-3-[Phospho-rac-(1-glycerol)] (DLPG), which is fluid down to 0 degrees C) by an intrinsic cold denaturation that occurs around 10-20 degrees C. This structure is subsequently lost in a thermal transition around 60 degrees C. Remarkably, this phenomenon is only observed for vesicles >100 nm in diameter and is sensitive to lipid chain length, longer chain lengths, and larger vesicles giving more cooperative unfolding transitions and a greater degree of structure. For both vesicle size and chain length, a higher degree of compressibility or permeability in the lipid thermal transition region is associated with a higher degree of alphaSN folding. Furthermore, the degree of structural change is strongly reduced by an increase in ionic strength or a decrease in the amount of anionic lipid. A simple binding-and-folding model that includes the lipid phase transition, exclusive binding of alphaSN to the liquid disordered phase, the thermodynamics of unfolding, and the electrostatics of binding of alphaSN to lipids is able to reproduce the two thermal transitions as well as the effect of ionic strength and anionic lipid. Thus the nature of alphaSN's binding to phospholipid membranes is intimately tied to the lipids' physico-chemical properties.

Cellulose is the most abundant biopolymer in the world, the main load-bearing element in plant cell walls, and represents a major sink for carbon fixed during photosynthesis. Previous work has shown that photosynthetic activity is partially regulated by carbohydrate sinks. However, the coordination of cellulose biosynthesis with carbohydrate metabolism and photosynthesis is not well understood. Here, we demonstrate that cellulose biosynthesis inhibition (CBI) leads to reductions in transcript levels of genes involved in photosynthesis, the Calvin cycle, and starch degradation in Arabidopsis (Arabidopsis thaliana) seedlings. In parallel, we show that CBI induces changes in carbohydrate distribution and influences Rubisco activase levels. We find that the effects of CBI on gene expression and carbohydrate metabolism can be neutralized by osmotic support in a concentrationdependent manner. However, osmotic support does not suppress CBI-induced metabolic changes in seedlings impaired in mechanoperception (mid1 complementing activity1 [mca1]) and osmoperception (cytokinin receptor1 [cre1]) or reactive oxygen species production (respiratory burst oxidase homolog DF [rbohDF]). These results show that carbohydrate metabolism is responsive to changes in cellulose biosynthesis activity and turgor pressure. The data suggest that MCA1, CRE1, and RBOHDFderived reactive oxygen species are involved in the regulation of osmosensitive metabolic changes. The evidence presented here supports the notion that cellulose and carbohydrate metabolism may be coordinated via an osmosensitive mechanism.

The ability of proteins to aggregate to form well-organized β-sheet rich amyloid fibrils is increasingly viewed as a general if regrettable property of the polypeptide chain. Aggregation leads to diseases such as amyloidosis and neurodegeneration in humans and various mammalian species but is also found in a functional variety in both animals and microbes. However, there are to our knowledge no reports of amyloid formation in plants. Plants are also the source of a large number of aggregation-inhibiting compounds. We reasoned that the two phenomena could be connected and that one of (many) preconditions for plant longevity is the ability to suppress unwanted protein aggregation. In support of this, we show that while protein extracts from the sugar maple tree Acer saccharum fibrillate readily on their own, this process is efficiently abolished by addition of small molecule extracts from the same plant. Further analysis of 44 plants showed a correlation between plant longevity and ability to inhibit protein aggregation. Extracts from the best performing plant, the sugar maple, were subjected to chromatographic fractionation, leading to the identification of a large number of compounds, many of which were shown to inhibit aggregation in vitro . One cautious interpretation is that it may have been advantageous for plants to maintain an efficient collection of aggregation-inhibiting metabolites as long as they do not impair metabolite function. From a practical perspective, our results indicate that long-lived plants may be particularly appropriate sources of new anti-aggregation compounds with therapeutic potential.

The intrinsically disordered protein α-synuclein (aSN) forms insoluble aggregates in the brains of Parkinson's disease (PD) patients. Cytotoxicity is attributed to a soluble aSN oligomeric species that permeabilizes membranes significantly more than monomers and fibrils. In humans, the A53T mutation induces early onset PD and increases the level of aSN oligomerization and fibrillation propensity, but Thr53 occurs naturally in aSNs of most animals. We compared aSNs from elephant, bowhead whale, and pig with human aSN. While all three animal aSNs showed significantly weakened fibrillation, elephant aSN formed much more oligomer, and pig aSN much less, than human aSN did. However, all animal aSN oligomers showed weakened permeabilization toward anionic lipid vesicles, indicative of decreased cytotoxicity. These animal aSNs share three substitutions compared to human aSN: A53T, G68E, and V95G. We analyzed aggregation and membrane binding of all eight mutants combining these three mutations. While the G68E mutation is particularly important in weakening fibrillation and possible toxicity, the strongest effect is seen when all three mutations are present. Thus, a small number of mutations can significantly decrease aSN toxicity.