Molecular interaction networks in the analyses of sequence variation and proteomics data

Protein–protein interaction networks are typically generated in standard cell lines or model organisms as it is prohibitively difficult to record large interaction datasets from specific tissues or disease models at a reasonable pace. Although the interaction data are of high confidence, they thus do not reflect in vivo relationships as such. A wealth of physiologically relevant protein information, obtained under different conditions and from different systems, is available including information on genetic variation, protein levels, and PTMs. However, these data are difficult to assess comprehensively because the relationships between the entities remain elusive from the measurements. Here, we exemplarily highlight recent studies that gained deeper insight from genetic variation, protein, and PTM measurements using interaction information pointing toward the importance and potential of interaction networks for the interpretation of sequencing and proteomics data.     

UHT milk contains multiple forms of αS1-casein that undergo degradative changes during storage

Milk proteins are susceptible to chemical changes during processing and storage. We used proteomic tools to analyse bovine α_S1-casein in UHT milk. 2-D gels of freshly processed milk α_S1-casein was presented as five or more spots due to genetic polymorphism and variable phosphorylation. MS analysis after phosphopeptide enrichment allowed discrimination between phosphorylation states and genetic variants. We identified a new alternatively-spliced isoform with a deletion of exon 17, producing a new C-terminal sequence, K¹⁶⁴SQVNSEGLHSYGL¹⁷⁷, with a novel phosphorylation site at S¹⁷⁴. Storage of UHT milk at elevated temperatures produced additional, more acidic α_S1-casein spots on the gels and decreased the resolution of minor forms. MS analysis indicated that non-enzymatic deamidation and loss of the N-terminal dipeptide were the major contributors to the changing spot pattern. These results highlight the important role of storage temperature in the stability of milk proteins and the utility of proteomic techniques for analysis of proteins in food.     

Structure of focal adhesion kinase in healthy heart versus pathological cardiac hypertrophy: A modeling and simulation study

Focal adhesion kinase (FAK) is required for signaling in the heart. S910 phosphorylated FAK is known to cause pathological cardiac hypertrophy. The switching of FAK between its inactive (-i), activated (-a) and hyperactive (-h) state is controlled by phosphorylation. FAK consists of three domains, namely: FERM, Kinase, and FAT joined by linkers L1 and L2. The structural basis of FAK phosphorylation and signaling to the downstream pathways is not understood. In this work, we carried out homology modeling and domain assembly of full length human iFAK and aFAK. 100 ns classical molecular dynamic simulations were performed using AMBER14 and effect of S910 phosphorylation on FAK was investigated. The iFAK model superposed on a small angel X-ray scattering (SAXS) derived model with RMSD of 1.18 Å for 590 Cα atoms. aFAK showed S910 phosphorylation site in L2 shielded by FERM. S910 phosphorylation in hFAK led to its exposure accompanied by a large conformational change and exposing the previously buried Grb2 interaction site responsible for causing cardiac hypertrophy. The models of FAK are in agreement with diverse experimental data and observed differences in biological action. Understanding the structure activity relationships of FAK in response to phosphorylation is important for its future therapeutic modulation.     

Proteomic analysis of the kidney filtration barrier—Problems and perspectives

Diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. The kidney filter is localized within the renal glomeruli, small microvascular units that are responsible for ultrafiltration of about 180 liters of primary urine every day. The renal filter consists of three layers, fenestrated endothelial cells, glomerular basement membrane, and the podocytes, terminally differentiated, arborized epithelial cells. This review demonstrates the use of proteomics to generate insights into the regulation of the renal filtration barrier at a molecular level. The advantages and disadvantages of different glomerular purification methods are examined, and the technical limitations that have been significantly improved by in silico or biochemical approaches are presented. We also comment on phosphoproteomic studies that have generated considerable molecular-level understanding of the physiological regulation of the kidney filter. Lastly, we conclude with an analysis of urinary exosomes as a potential filter-derived resource for the noninvasive discovery of glomerular disease mechanisms.     

丝素膜表面磷酸化研究

从蚕丝中提取丝素蛋白制备成聚乙二醇缩水甘油醚(P E G O)的交联丝素膜,用磷酸处理,在丝素膜材料表面引入了磷酸基团;用电子能谱分析该材料的表面性质,表明85%磷酸处理丝素膜2min,可以使PEGO丝素膜表面带上较多的磷酸基团,这大大提高了丝素膜的抗凝血性,防止了细胞在丝素膜上的黏附。X-衍射表明丝素膜用磷酸处理后,增加了丝素膜的结晶度。经磷酸处理后,丝素膜的断裂伸长率与断裂强度有所下降,但是PEGO丝素膜仍保留了较好的湿态机械性能。     

Recent advances in phosphorylation of food proteins: A review

Phosphorylation is a useful method for improving the functional properties of food proteins. In this article, various methods of phosphorylation are reviewed. Dry-heating phosphorylation, a method developed recently, is also introduced. Some characteristics of phosphate groups are involved, and the effects of phosphorylation on the structural changes, the functional properties, and the physiological functions in vitro of food proteins, are discussed. The types of phosphate linkages and the phosphopeptides from phosphorylated proteins are identified. The molten (partially unfolded) conformations of food proteins formed by phosphorylation are discussed. The phosphorylation of food proteins improved a number of functional properties, including heat stability, emulsifying properties, foaming properties, gelling properties, water absorption capacity, oil absorption capacity, and calcium phosphate-solubilizing ability. In vitro physiological function studies of protein (α-lactoalbumin) indicated that the digestibility (ovalbumin) was improved and the inflammatory response (α-lactoalbumin) was suppressed by phosphorylation. Experiments with animals are necessary to evaluate the toxicity and physiological functions of phosphorylated proteins.     

Xanthohumol kills B-chronic lymphocytic leukemia cells by an apoptotic mechanism

B-chronic lymphocytic leukemia (B-CLL) is an indolent lymphoid malignancy with variable prognosis. Adverse prognostic factors comprise treatment resistance, cytogenetics (11q- and 17p-), the presence of unmutated Ig genes, and the more comprehensive activation marker Zap 70. In contrast to diminished sensitivity to chemotherapy, Zap 70+ B-CLL cells retain their responsiveness to manipulation of signal transduction and monoclonals. Xanthohumol (XA) has recently been documented to have an impact on breast cancer cell growth and invasiveness in vitro. Based on these observations, lymphocytes from patients with B-CLL were cultured in the presence of XA in vitro. XA induced a dose-dependent killing of B-CLL cells at an LD(50) ((24 h)) of 24.4 +/- 6.6 microM, independent of known adverse prognostic factors including functional loss of p53. Cell death was associated with poly (ADP)-ribose polymerase cleavage and annexin V positivity, suggestive of an apoptotic mechanism. Surprisingly, p 70(S 6 K) phosphorylation was stimulated upon XA treatment. In conclusion, XA has an antitumor activity on B-CLL cells in vitro. The molecular mechanisms behind this pro-apoptotic effect deserve further investigation.     

Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.     

Effect of amphiphilic phosphate/octenylsuccinate starch on enhancing adhesion to hydrophobic polyester fibers in sizing

Amphiphilic starch at different degree of substitution (DS) was prepared by phosphorylation and octenylsuccinylation of acid-thinned starch with sodium tripolyphosphate and 2-octenylsuccinic anhydride, respectively for improving the adhesion of starch to polyester fibers. Various assessment of starch such as ¹H NMR and FTIR analysis, apparent viscosity, starch clarity, adhesion to polyester fiber, desizing efficiency and biodegradability were conducted. The ¹H NMR and FTIR spectroscopy revealed that octenylsuccinate and phosphate substituents have been attached to the molecular chain of starch. Also, the apparent viscosity, paste clarity, desizing efficiency and biodegradability of the amphiphilic starch increased as the DS of phosphate increased. It was observed that the amphiphilic starch was an effective method for stronger adhesion unto polyester fibers but reduced at the highest DS of phosphate due to excessive hydrophilicity. As compared to acid-thinned starch, the stronger adhesion of the amphiphilic starch was attributed to the steric hindrance caused by the two substituents on starch and the good dispersibility impacted by the phosphate substituents on starch. Conclusively, the strongest adhesion on polyester fibers, good paste clarity, good desizing efficiency and enhanced biodegradability of starch were attained at phosphate and octenylsuccinate DS ratio of 0.031/0.014.