Liquid chromatography mass spectrometry has become one of the analytical platforms of choice for metabolomics studies. However, LC-MS metabolomics data can suffer from the effects of various systematic biases. These include batch effects, day-to-day variations in instrument performance, signal intensity loss due to time-dependent effects of the LC column performance, accumulation of contaminants in the MS ion source and MS sensitivity among others. In this study we aimed to test a singular value decomposition-based method, called EigenMS, for normalization of metabolomics data. We analyzed a clinical human dataset where LC-MS serum metabolomics data and physiological measurements were collected from thirty nine healthy subjects and forty with type 2 diabetes and applied EigenMS to detect and correct for any systematic bias. EigenMS works in several stages. First, EigenMS preserves the treatment group differences in the metabolomics data by estimating treatment effects with an ANOVA model (multiple fixed effects can be estimated). Singular value decomposition of the residuals matrix is then used to determine bias trends in the data. The number of bias trends is then estimated via a permutation test and the effects of the bias trends are eliminated. EigenMS removed bias of unknown complexity from the LC-MS metabolomics data, allowing for increased sensitivity in differential analysis. Moreover, normalized samples better correlated with both other normalized samples and corresponding physiological data, such as blood glucose level, glycated haemoglobin, exercise central augmentation pressure normalized to heart rate of 75, and total cholesterol. We were able to report 2578 discriminatory metabolite peaks in the normalized data (p<0.05) as compared to only 1840 metabolite signals in the raw data. Our results support the use of singular value decomposition-based normalization for metabolomics data.

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble proteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation of tissue-type plasminogen activator. This system has all the advantages of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retaining intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.

Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS–causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF–mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress–inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix proteins. Mutations in genes for extracellular matrix proteins are generally thought to exert their pathogenic effects because of resulting defects in extracellular matrix. However, it is becoming increasingly clear that such mutations can also have significant effects inside the cell due to the induction of ER stress. Mutations in type X collagen cause a dwarfism called metaphyseal chondrodysplasia type Schmid. A gene targeted mouse model expressing mutant type X collagen exhibited an expanded hypertrophic zone of the growth plate and significant increases in cellular ER stress, as noted previously. VEGF expression was disrupted leading to decreases in the rate of vascular invasion. To directly assess the role of elevated ER stress in disease pathogenesis, transgenic mouse lines expressing an exogenous, ER stress–inducing protein (cog mutant of thyroglobulin—Tg^cog) targeted to hypertrophic chondrocytes were generated. Mice expressing Tg^cog protein showed elevated ER stress, an expanded hypertrophic zone, and reduced bone growth demonstrating that elevated ER stress and the resultant UPR is the principal pathogenic mechanism causing this cartilage pathology. It is possible that therapeutic strategies aimed at alleviating ER stress may be beneficial in this and other connective tissue diseases caused by mutant extracellular matrix genes.

Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.

Atlantic salmon production in Tasmania (Southern Australia) occurs near the upper limits of the species thermal tolerance. Summer water temperatures can average over 19 °C over several weeks and have negative effects on performance and health. Liver tissue exerts important metabolic functions in thermal adaptation. With the aim of identifying mechanisms underlying liver plasticity in response to chronic elevated temperature in Atlantic salmon, label-free shotgun proteomics was used to explore quantitative protein changes after 43 days of exposure to elevated temperature. A total of 276 proteins were differentially (adjusted p-value < 0.05) expressed between the control (15 °C) and elevated (21 °C) temperature treatments. As identified by Ingenuity Pathway Analysis (IPA), transcription and translation mechanisms, protein degradation via the proteasome, and cytoskeletal components were down-regulated at elevated temperature. In contrast, an up-regulated response was identified for NRF2-mediated oxidative stress, endoplasmic reticulum stress, and amino acid degradation. The proteome response was paralleled by reduced fish condition factor and hepato-somatic index at elevated temperature. The present study provides new evidence of the interplay among different cellular machineries in a scenario of heat-induced energy deficit and oxidative stress, and refines present understanding of how Atlantic salmon cope with chronic exposure to temperature near the upper limits of thermal tolerance.

Summary: Light underneath Antarctic sea‐ice is below detectable limits for up to 4 months of the year. The ability of Antarctic sea‐ice diatoms to survive this prolonged darkness relies on their metabolic capability. This study is the first to examine the proteome of a prominent sea‐ice diatom in response to extended darkness, focusing on the protein‐level mechanisms of dark survival. The Antarctic diatom Fragilariopsis cylindrus was grown under continuous light or darkness for 120 d. The whole cell proteome was quantitatively analysed by nano‐LC−MS/MS to investigate metabolic changes that occur during sustained darkness and during recovery under illumination. Enzymes of metabolic pathways, particularly those involved in respiratory processes, tricarboxylic acid cycle, glycolysis, the Entner−Doudoroff pathway, the urea cycle and the mitochondrial electron transport chain became more abundant in the dark. Within the plastid, carbon fixation halted while the upper sections of the glycolysis, gluconeogenesis and pentose phosphate pathways became less active. We have discovered how F. cylindrus utilises an ancient alternative metabolic mechanism that enables its capacity for long‐term dark survival. By sustaining essential metabolic processes in the dark, F. cylindrus retains the functionality of the photosynthetic apparatus, ensuring rapid recovery upon re‐illumination.