Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-ß-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a ß-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or ß-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for ß-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between ß-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

Proposing a minimal set of metrics and methods to predict probabilities of amyloidosis disease and onset age in individuals.

Many amyloid-driven pathologies have both genetic and stochastic components where assessing risk of disease development requires a multifactorial assessment where many of the variables are poorly understood. Risk of transthyretin-mediated amyloidosis is enhanced by age and mutation of the transthyretin (TTR) gene, but amyloidosis is not directly initiated by mutated TTR proteins. Nearly all of the 150+ known mutations increase dissociation of the homotetrameric protein structure and increase the probability of an individual developing a TTR amyloid disease late in life. TTR amyloidosis is caused by dissociated monomers that are destabilized and refold into an amyloidogenic form. Therefore, monomer concentration, monomer proteolysis rate, and structural stability are key variables that may determine the rate of development of amyloidosis. Here we develop a unifying biophysical model that quantifies the relationships among these variables in plasma and suggest the probability of an individual developing a TTR amyloid disease can be estimated. This may allow quantification of risk for amyloidosis and provide the information necessary for development of methods for early diagnosis and prevention. Given the similar observation of genetic and sporadic amyloidoses for other diseases, this model and the measurements to assess risk may be applicable to more proteins than just TTR.

Determination of growth and maintenance coefficients by calorespirometry.

This study describes a calorespirometric method for determining the coefficients of the correlation of specific respiration and growth rates. To validate the calorespirometric method, coefficients obtained from calorespirometric data are compared with coefficients obtained from mass and elongation growth rates measured at three temperatures on oat (Avena sativa L.) shoots. Calorespirometric measurements were also made on leaf tissue of varying age from Verbascum thapsus L., Convolvulus arvensis L., and Helianthus tuberosus Nutt. Measurements on A. sativa, C. arvensis and H. tuberosus at several temperatures show maintenance coefficients generally increase with temperature, but, in disagreement with accepted theory, growth coefficients for C. arvensis and A. sativa vary with temperature. A comparison of rates expressed as intensive and extensive quantities showed that the decline in specific respiration and growth rates with age is caused by dilution-by-growth, not down-regulation of respiration rate by reduced demand. The ratio of heat rate to CO2 rate increases with leaf age, and, for fully mature leaves, exceeds the maximum possible value for carbohydrates. This shows that the catabolic substrate may vary with leaf age in immature leaves and cannot be assumed to consist only of carbohydrates in mature leaves. Dilution-by-growth, substrate variation, and inseparability of the variables in the growth-maintenance model all complicate physiological interpretation of the slope and intercept of plots of specific respiration rates v. specific growth rates.

Analysis of respiratory metabolism correlates well with the response of Eucalyptus camaldulensis seedlings to NaCl and high pH.

Growth of sand-cultured Eucalyptus camaldulensis Dehnh. (river red gum) seedlings from six wide-ranging provenances was reduced in the presence of 150 mM NaCl, a high pH of 9.5, and combined NaCl and high pH, compared with no applied NaCl and neutral pH. Effects of these stress conditions on respiration rates and substrate carbon conversion efficiencies of rapidly-expanding leaf tissue were measured with calorespirometric techniques. Growth rates were calculated from respiration parameters. Respiration rate, measured as metabolic heat production rate (q), showed no consistent trend with either NaCl or high pH, whereas the rate measured as CO2 production rate (R CO2) was generally lower with both treatments. The ratio of heat lost per mole of CO2 produced [q/(R CO2)] was consistently increased by both stresses. Stress causes a larger fraction of metabolic energy produced by aerobic metabolism to be lost as heat, relative to non-stressed controls. Consequently, a larger fraction of photosynthetic product in stressed seedlings must be metabolized to CO2 per mole of C incorporated into biomass. Our results indicate that 0.42 mol substrate C is converted to CO2 per mole C incorporated into biomass for control plants, compared with 0.96 mol for plants treated with combined NaCl and high pH. Respiratory responses to treatment varied with provenance. Specific growth rates, calculated from repiratory parameters (q and RCO2) of stressed E. camaldulensis seedlings, generally paralleled experimentally-determined reduced growth (dry weight) of these seedlings. Thus, measurements of leaf respiration allow calculation of growth inhibition caused by NaCl and high pH stress. However, we could not discriminate among provenances in this experiment with only one level of NaCl and pH.