Proteostasis is adaptive: Balancing chaperone holdases against foldases.

Because a cell must adapt to different stresses and growth rates, its proteostasis system must too. How do cells detect and adjust proteome folding to different conditions? Here, we explore a biophysical cost-benefit principle, namely that the cell should keep its proteome as folded as possible at the minimum possible energy cost. This can be achieved by differential expression of chaperones-balancing foldases (which accelerate folding) against holdases (which act as parking spots). The model captures changes in the foldase-holdase ratio observed both within organisms during aging and across organisms of varying metabolic rates. This work describes a simple biophysical mechanism by which cellular proteostasis adapts to meet the needs of a changing growth environment.

Measurement of human serum IgD levels.

This unit describes an ELISA for the quantitative measurement of IgD levels in human serum. The ELISA is highly specific and sensitive, with a minimum detectable concentration of 30 pg/ml and more than 10,000-fold specificity for IgD over all other human immunoglobulins. Linear dilution characteristics enable measurement of IgD concentrations ranging over 5 orders of magnitude. These factors are vital for the IgD assay, since IgD makes up only a small proportion of the total immunoglobulins present in normal sera, and IgD serum concentrations are known to vary widely between individuals.

A new sensitive and specific enzyme-linked immunosorbent assay for IgD.

We have developed a new highly specific ELISA for IgD, and then used it to measure levels of circulating IgD in the serum of 480 un-selected patients from the East Anglia region of UK. The assay is both extremely sensitive and specific, with a minimum detected IgD concentration of 30 pg/ml and more than 10,000-fold specificity for IgD over all other human immunoglobulins. The assay shows linear dilution characteristics with both purified IgD and human serum, and spiking of purified IgD into either purified immunoglobulins or human serum shows c. 100% recovery. Furthermore, intra-assay and inter-assay coefficients of variation for repeated measurements of the same samples are below 10% and 15% respectively. Measurement of IgD levels on the un-selected patient population showed levels to range from <300 pg/ml to over 100 microg/ml, with a geometric mean of 8 microg/ml. The distribution is approximately normal after log transformation. Levels of circulating IgD were higher in men than in women. There was a significant negative correlation between levels of IgD and age in women, but not in men. Moreover, after adjustment for age and sex, there were statistically significantly higher levels of circulating IgD in male (but not female) smokers, compared to their non-smoking counterparts. These results highlight the care that needs to be taken to control for age, sex and cigarette smoking when examining levels of circulating IgD in future studies.

Huntington disease patients and transgenic mice have similar pro-catabolic serum metabolite profiles.

There has been considerable progress recently towards developing therapeutic strategies for Huntington's disease (HD), with several compounds showing beneficial effects in transgenic mouse models. However, human trials in HD are difficult, costly and time-consuming due to the slow disease course, insidious onset and patient-to-patient variability. Identification of molecular biomarkers associated with disease progression will aid the development of effective therapies by allowing further validation of animal models and by providing hopefully more sensitive measures of disease progression. Here, we apply metabolic profiling by gas chromatography-time-of-flight-mass spectrometry to serum samples from human HD patients and a transgenic mouse model in a hypothesis-generating search for disease biomarkers. We observed clear differences in metabolic profiles between transgenic mice and wild-type littermates, with a trend for similar differences in human patients and control subjects. Thus, the metabolites responsible for distinguishing transgenic mice also comprised a metabolic signature tentatively associated with the human disease. The candidate biomarkers composing this HD-associated metabolic signature in mouse and humans are indicative of a change to a pro-catabolic phenotype in early HD preceding symptom onset, with changes in various markers of fatty acid breakdown (including glycerol and malonate) and also in certain aliphatic amino acids. Our data raise the prospect of a robust molecular definition of progression of HD prior to symptom onset, and if validated in a genuinely prospective fashion these biomarker trajectories could facilitate the development of useful therapies for this disease.

A pattern of anti-carbohydrate antibody responses present in patients with advanced atherosclerosis.

We have previously shown that an antibody pool present in normal human serum binds cytokine receptors in vitro and may therefore interfere with assays that capture cytokines using their receptors. Here we show that this antibody pool is the same as the natural antibody termed anti-gal, that binds to the alpha-galactosyl carbohydrate epitope (alpha-gal) and which is the predominant obstacle to xenotransplantation. We report that there are high levels of IgD anti alpha-gal in most volunteers, in addition to the IgG2, IgA and IgM immunoglobulin isotypes against alpha-gal previously described. To determine if anti-gal may interfere with assays that depend on capture of cytokine with its receptor, we measured levels of several anti-carbohydrate antibodies in a cohort of patients with advanced atherosclerosis that had previously been used to measure levels of active TGF-beta using such an assay. For many isotype / carbohydrate combinations, there is a large and significant difference between the levels of anti-carbohydrate antibodies in patients with atherosclerosis and controls, after adjustment for age, sex and blood group. These results are similar to the previous data obtained for active TGF-beta, and therefore we cannot discount the possibility that anti-gal contributed to the previous data. Following further adjustment for several risk factors associated with cardiovascular disease, several anti-carbohydrate antibodies were still significantly different between patients and controls. Therefore, anti-carbohydrate antibodies may represent a new class of risk factors that may be associated with presence of advanced atherosclerosis, although larger studies will be required to confirm this hypothesis.

Circulating levels of MCP-1 and eotaxin are not associated with presence of atherosclerosis or previous myocardial infarction.

The chemokines are a family of signalling proteins that participate in regulation of the immune system and have been implicated in the pathogenesis of vascular diseases. Deleting the gene encoding the chemokine MCP-1 in mouse models of atherosclerosis reduces lipid lesion formation and circulating chemokines are upregulated in man immediately following myocardial infarction (MI) or coronary angioplasty. We have therefore investigated whether circulating levels of two chemokines (MCP-1 and eotaxin) differ between subjects with and without atherosclerosis. We have used three different methods of measuring the presence and extent of atherosclerosis in human subjects: duplex ultrasonography of the carotid arteries and clinical diagnosis of coronary heart disease on individuals from the general population and coronary angiography on patients with suspected heart disease. There was no difference in the levels of circulating MCP-1 or eotaxin, measured by ELISA, between subjects with and without atherosclerosis. Furthermore, any increase in circulating MCP-1 following acute MI must be short-lived, since chemokine levels were not different in subjects who had had an MI previously compared to those who had not. We conclude that although there may be a transient increase in circulating chemokine levels following coronary angioplasty, there is no difference in the levels of circulating MCP-1 or eotaxin in subjects with and without atherosclerosis.

Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics.

Although a wide range of risk factors for coronary heart disease have been identified from population studies, these measures, singly or in combination, are insufficiently powerful to provide a reliable, noninvasive diagnosis of the presence of coronary heart disease. Here we show that pattern-recognition techniques applied to proton nuclear magnetic resonance (1H-NMR) spectra of human serum can correctly diagnose not only the presence, but also the severity, of coronary heart disease. Application of supervised partial least squares-discriminant analysis to orthogonal signal-corrected data sets allows >90% of subjects with stenosis of all three major coronary vessels to be distinguished from subjects with angiographically normal coronary arteries, with a specificity of >90%. Our studies show for the first time a technique capable of providing an accurate, noninvasive and rapid diagnosis of coronary heart disease that can be used clinically, either in population screening or to allow effective targeting of treatments such as statins.