Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration.

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H[Formula: see text]O/D[Formula: see text]O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H[Formula: see text]O and D[Formula: see text]O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D[Formula: see text]O than in H[Formula: see text]O, and collagen in D[Formula: see text]O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H[Formula: see text]O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D[Formula: see text]O is less hydrated than in H[Formula: see text]O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.

[Plasma proteomic analysis of diabetes improvement by vitamin D supplementation].

OBJECTIVE: To analyze the effect of vitamin D supplementation on the improvement of diabetes mellitus based on plasma proteomics. METHODS: Five-week-old SPF spontaneously obese rats with type 2 diabetes were randomly divided into a diabetic group and a diabetic vitamin D intervention group, and the control group was Zucker lean rats. The fasting blood glucose of the rats in each group was compared with that of the diabetic vitamin D group, and the plasma proteins of the rats in each group were compared by quantitative analysis of the high-resolution mass spectrometry system iTRAQ, and KEGG signaling pathway analysis was performed. RESULTS: The fasting blood glucose of rats in the diabetic vitamin D intervention group was significantly lower than that of the diabetic group, and the proteins that were differentially expressed in the diabetic vitamin D intervention group were significantly improved. KEGG signaling pathway analysis revealed that the differential proteins in the diabetic group were mainly distributed among enzymes, exosomal proteins, and peptidases and inhibitors, and that the number of differences in these three classes of proteins was significantly reduced in the diabetic intervention group. CONCLUSION: Vitamin D supplementation can improve the differential expression of fasting glucose and plasma proteins in the diabetic rats.

[Study on the CFHR1 level and its genetic polymorphisms in type 2 diabetes mellitus patients].

OBJECTIVE: To explore the characteristics in CFHR1 concentration and the frequency of CFHR1 gene polymorphisms of patients with type 2 diabetes mellitus (T2DM) based on the high level of complement factor H (CFH) expression among such patients and the similarity between CFHR1 and CFH in terms of sequence and functions. METHODS: Fifty T2DM patients and 30 healthy controls were selected. The plasma samples were separated by pI with OFFGEL electrophoresis following solution digestion. Further separation and identification were carried out on a Nano HPLC-Chip-MS/MS system. Differentially expressed proteins were identified by comparison. Enzyme-linked immunosorbent assay (ELISA) was used to validate the result. Genomic DNA of the two groups was extracted. Polymerase chain reaction and sequencing were used to determine the single nucleotide polymorphisms in the 6 exons of the CFHR1 gene. RESULTS: The CFHR1 level in plasma of T2DM patients were significantly higher than that of the healthy controls (P=2.78× 10-11). A significant difference in allelic frequencies of rs12406079 of the fifth exon of the CFHR1 gene was found between the two groups (χ 2=5.692, P=0.017). CONCLUSION: The concentration of CFHR1 and frequencies of CFHR1 gene polymorphisms among patients with T2DM differ significantly from healthy subjects. Polymorphisms of the CFHR1 gene are associated with T2DM.

Glycation of paraoxonase 1 by high glucose instigates endoplasmic reticulum stress to induce endothelial dysfunction in vivo.

High-density lipoprotein (HDL) modulates low-density lipoprotein and cell membrane oxidation through the action of paraoxonase-1 (PON1). Endoplasmic reticulum (ER) stress has been linked to a wide range of human pathologies including diabetes, obesity, and atherosclerosis. Previous studies have reported that PON1 is glycated in diabetes. The aim of this study is to investigate whether and how PON1 glycation contributes to endothelial dysfunction in diabetes. ER stress markers were monitored by western blot. Endothelial function was determined by organ bath. Incubation of recombinant PON1 proteins with high glucose increased PON1 glycation and reduced PON1 activity. Exposure of HUVECs to glycated PON1 induced prolonged ER stress and reduced SERCA activity, which were abolished by tempol, apocynin, BAPTA, and p67 and p22 siRNAs. Chronic administration of amino guanidine or 4-PBA prevented endothelial dysfunction in STZ-injected rats. Importantly, injection of glycated PON1 but not native PON1 induced aberrant ER stress and endothelial dysfunction in rats, which were attenuated by tempol, BAPTA, and 4-PBA. In conclusion, glycation of PON1 by hyperglycemia induces endothelial dysfunction through ER stress. In perspectives, PON1 glycation is a novel risk factor of hyperglycemia-induced endothelial dysfunction. Therefore, inhibition of oxidative stress, chelating intracellular Ca2+, and ER chaperone would be considered to reduce vascular complications in diabetes.

[Proteomics study on ficolin 3 in the human plasma of type 2 diabetics].

OBJECTIVE: To explore the expression trend of ficolin 3 (FCN3) in type 2 diabetes (T2DM) plasma. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2DE) was used to separate the plasma proteins from T2DM patients and healthy control subjects. MALDI-TOF-TOF was used to identify the differential proteins. Western Blot and enzyme-linked immune response (ELISA) were used to verify the results from 2DE. RESULTS: The experiment on 2DE showed complement C1s subcomponent, complement C3, C9 and FCN3 were up-regulated in the plasma of T2DM patients. The Western Blot results showed that C9 and FCN3 increased in the plasma of T2DM patients with respect to T2DM-free subjects. The experiment results on ELISA showed that the plasma concentration of FCN3 were (50.88 ± 3.85 ) and (36.20 ± 2.75 ) µg/mL (P = 0.0062) in T2DM and T2DM-free, respectively. The plasma concentration of C9 were (26.22 ± 1.43) and (19.23 ± 1.55) µg/mL (P = 0.0022) in T2DM and T2DM-free. FCN3 and C9 were up-regulated in T2DM plasma. CONCLUSION: FCN3 over-expressed in the plasma of T2DM patients,which activated the complement system in greater degree.

[Study on the prevalence of lipid metabolism disorders and quantitative analysis of apolipoproteins in T2DM patients].

OBJECTIVE: To investigate the disorder of lipid metabolism in type 2diabetic patients. To assessed the association of cholesterol and apolipoprotein with derangement of glucose metabolism. METHODS: The levels of TC, TG, HDL-C, LDL-C, Apo A1, Apo A2, Apo AB48, Apo B100 and Apo E were all measured in 60 type 2 diabetic patients and 60 healthy controls. The differences of these clinical indicators in the two groups have been compared. Correlation analysis, multiple regression analysis have been applied in this paper. RESULTS: The levels of TC, TG, LDL-C were significantly higher( P < 0. 01) and the level of HDL-C was significantly lower( P < 0. 01) in patients with type 2 diabetes which indicating a lipid metabolic abnormalities. The levels of Apo A2, Apo B100, Apo E were significantly higher in type 2 diabetic patients than those in control group. Multiple regression analysis showed that Apo B100, Apo B48, LDL-C, TG were the main factors of FBG level. It was showed that apolipoproteins, especially Apo B, isassociated with glucose metabolism disorder. CONCLUSION: T2 DM patients are obviously associated with abnormal lipid metabolism, and the apolipoproteins may play an important role in derangement of glucose metabolism.

[Removal of high-abundance proteins in plasma of the obese by improved TCA/acetone precipitation method].

OBJECTIVE: To develop an improved trichloroacetic acid (TCA)/acetone precipitation method for removal of high-abundance proteins in plasma of the obese. METHODS: Volumes of TCA/acetone solution (1, 3, 4, 5, 6, 8, 10 and 20 times of the sample) and concentrations of TCA (10%, 30%, 50%, 60%, 70% TCA/acetone solution) have been investigated to optimize the conditions of sample preparation. SDS-PAGE were used to separate and tested proteins in the supernatant and sediment. The best concentration of the TCA/acetone solution was first determined by SDS-PAGE. The protein in precipitation from 10% TCA/acetone solution processing and the new determined concentration TCA/acetone solution processing were verified by 2-D-SDS-PAGE. And then the digested products of the protein in precipitation and supernatant by trypsin were analyzed by nano HPLC-Chip-MS/MS to verify which is the best concentration to process the plasma. RESULTS: The best volume of TCA/acetone is four times to sample, which less or more TCA/acetone would reduce the removal efficiency of high-abundance proteins. The concentration of TCA in acetone solution should be 60%, which may remove more high-abundance proteins in plasma than 10%, 30%, 50% TCA in acetone solution. If the TCA concentration is more than 60%, the reproducibility will be much poorer due to fast precipitation of proteins. The results of mass identification showed that human plasma prepared with 60% TCA/acetone (4 times sample volume) could be verified more low-abundance proteins than 10%. CONCLUSION: The most desirable conditions for removal of high-abundance proteins in plasma is 60% TCA/acetone (4 times sample volume), especially for the plasma of obesity.

[Screening of protein markers on the plasma of obese young men].

OBJECTIVE: To screen obesity-related protein biomarkers of young men using differential proteomic method and OffGel electrophoresis. METHODS: Ten male obese volunteers with the age of 18 - 44 years were selected. The control group was matched with the ratio of 1:1 considering the factors of age and gender etc. Two milliliter venous blood was collected after 8 hours fasting. Albumin and IgG were removed from the plasma samples with highly specific immune-affinity method. Then the peptide-mixed samples were separated by pI with OffGel electrophoresis after solution digestion. Further separation and identification were performed by Nano HPLC-Chip-MS/MS system. The different proteins between the two groups were compared. RESULTS: Overall, 332 and 301 proteins were identified in the obesity and control groups, respectively. There were 43 proteins with significant differences between the two groups, 17 of which were in the control group and 26 in the obesity group. Protein function annotation results showed that the level of adiponectin was lower while the level of C-reaction protein and three other phosphatases were higher in the obesity group compared with the control group. CONCLUSION: Adiponectin, C-reaction protein and three phosphatases were closely related to obesity of young men.

[Plasma proteomic research on obesity subtype and non-obesity subtype of T2DM].

OBJECTIVE: With the increasingly serious epidemic situation of diabetes, plasma proteomic method and OFFGEL electrophoresis have been applied for screening different proteins between obese and non-obese T2DM patients, which may be used to further explain the mechanism of T2DM. METHODS: Twenty male T2DM volunteers (Obesity Subtype: 10; Non-obesity Subtype: 10) with the age of 18-44 years have been selected. The control group has been matched considering the factors of age, gender, etc. Albumin and IgG were removed from the plasma samples with highly specific immune-affinity method. Then the peptide-mixed samples were separated by pI with OFFGEL electrophoresis after solution digestion. Further separation and identification were performed by Nano HPLC-Chip-MS/MS system. Comparing the three groups, the differences were obtained and annotated on functions and its mechanism. RESULTS: 391, 415 and 371 proteins have been identified in the experimental groups and control group, respectively. The different proteins in groups and their annotations showed that adiponectin was down-regulated in obesity subtype of T2DM group, while STIM1 (stromal interaction molecule 1) was up-regulated. There were six protein kinases high expression in non-obesity DM patients, such as Serine-protein kinase ATM, Serine/threonine-protein kinase WNK1. CONCLUSION: Adiponectin, STIM1 and protein kinases may act in different roles on the pathogenesis of obesity subtype and non-obesity subtype of T2DM.