Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein–protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t_1/2 = 5.1 h and t_1/2 = 4.4 h, respectively) compared to WT-AQP2 (t_1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.     

Electrophoretic analysis of protein components in extracts of five trematodes.

The protein components of Clonorchis sinensis, Paragonimus westermani, Schistosomajaponicum, Fasciolopsis buski and Fasciola hepatica were analysed by polyacrylamide gelelectrophoresis. The results showed that the number of protein bands of C. sinensis, P.     

Is C-reactive protein a marker of inflammation?

C-reactive protein (CRP) is the prototype of acute-phase protein which is secreted by the liver in response to a variety of inflammatory cytokines. Levels of CRP can increase up to 1000-fold very rapidly after the onset of inflammation and decrease just as rapidly with the resolution of aggression. CRP is a member of the ancient highly conserved pentraxin family of proteins and it is arranged in a cyclic homopentameric structure. The important role of CRP in innate immunity is largely due to its opsonizing abilities, its capability to activate human complement and to bind to immunoglobulin G receptors. CRP can bind phosphocholine largely present in bacterial membranes, cell membrane and lipoproteins, in addition CRP can recognize nuclear constituent in damaged cells. CRP can activate C3 convertase through the classical pathway but not C5 convertase resulting in generation of opsonic complement fragments. Interactions of CRP with Fc receptors lead to the generation of proinflammatory cytokines and reactive oxygen species by monocyte/macrophage while inhibit neutrophiles functions. Recently, CRP was demonstrated to play an active role in atherogenesis and it has been largely proven that a microinflammatory state as defined by a moderate increase in CRP (up to 3 mg/l), is associated with an increased risk for arterial disease. Moreover it has been postulated that CRP may be a useful tool for monitoring drug therapy.     

Computational design of a self-assembling symmetrical β-propeller protein

The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2–10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.It is generally accepted that evolution is driven by duplications of genetic material. These events allow gene copies to develop independent regulation (1) and to express new proteins that inherit the stable architecture of the parent protein but possess a novel function (2, 3). Although this process largely explains the diversity of proteins with similar folds, it cannot account for the appearance of new protein folds. However, many proteins have a modular internal structure that most probably arose from duplication and fusion of structural elements. This type of process is most clearly demonstrated by proteins consisting of conserved domains repeated in tandem, giving a highly symmetrical tertiary structure (4, 5). Although symmetry remains a common feature of proteins (6), many present-day proteins show more limited symmetry than that of the ancestral intermediate forms suggested by the duplication theory of evolution (7–9). Since the group of Wilmanns demonstrated that a (β/α)₈−barrel protein could be constructed out of two identical halves in 2000 (10), several other groups have also reported the artificial construction of symmetrical or modular proteins, providing evidence for duplication and fusion events in nature (11–15). In the case of β-trefoil proteins, a design procedure based on Rosetta proved much more efficient than directed evolution methods at producing a symmetrical structure (15). Structural plasticity and domain swapping (16, 17) allow such extended proteins to adopt novel tertiary and quaternary structures (18), but to date there is no report of a perfectly symmetrical β-propeller protein.β-propeller proteins are composed of different numbers of repeats, each made from a single β-sheet, roughly 40 residues in length, that resembles the blade of a propeller (19, 20). β-propeller proteins are good examples of how proteins may have evolved from duplication and fusion events of simple peptide motifs (21). Examples are known of 4-, 5-, 6-, 7-, 8-, and 10-bladed proteins. These proteins have diverse functions, including varied enzymatic activities and protein–protein interactions, making them a highly interesting class to redesign both for synthetic biochemistry and as nano-building blocks. Previous attempts to create stable perfectly symmetrical β-propeller proteins have failed. Yadid and Tawfik (3, 22) screened genetic libraries encoding about 100 amino acid residues from a 236-residue five-bladed propeller (tachylectin-2) in attempts to create a fivefold symmetrical propeller. The initial proteins produced were poorly stable, but subsequent directed evolution to improve expression and folding led to domain-swapped structures through strand exchange (18). An artificial WD40-based repeat protein was designed by Nikkhah et al. using computational methods, but this protein failed to fold and adopted a molten globule state (23). Similarly, Figueroa et al. have recently described a putative artificial TIM barrel structure called “octarellin VI,” but this protein proved to be poorly soluble, and NMR indicated that it is not stably folded (24). It is widely believed that proteins with a perfectly repeated sequence motif experience “folding frustration,” the absence of a single strongly preferred tertiary structure, leading to unstable folds (11, 25). In fact, a search for identical sequence repeats within the same polypeptide chain failed to find any duplicated domains containing regular secondary structure in known natural proteins (26).We have applied a novel computational approach to the problem of creating a stable, perfectly symmetrical propeller by reverse engineering the supposed evolutionary pathway. Specifically, we wanted to address the question whether we can construct from a nonsymmetrical protein a symmetrical one that could have originated from smaller protein fragments. Ancestral sequence reconstruction was used to derive likely parent sequences assuming evolution through duplication, and then these sequences were computationally evaluated for protein stability (Fig. S1). We chose a six-bladed protein, given its additional two- and threefold pseudosymmetry. To agree with the duplication and fusion theory, such a protein should be divisible into a self-assembling unit consisting of 2 or 3 domains. Additionally, we created polypeptides carrying up to 10 identical blades and showed that these molecules also fold to give stable structures.     

Clinical significance of immunohistochemica study of P53 protein in colorectal carcinoma

AIMS To determine the clinical significance of P53 protein ex-pression in colorectal carcinoma.METHODS The expression of P53 protein in 92 colorectal car-cinomas was examined using the monoclonal antibody PAb 1801.Correlation between P53 protein expression and prognosis in col-orectal carcinoma was analyzed using log-rank test.RESULTS The frequency of P53 protein expression was 57.61%,corresponding with Dukes' staging.Analysis of survivordata demonstrated that the survival rate of colorectal carcinomawith positive P53 protein group was lower than that of negativeP53 protein group.CONCLUSIONS We conclude that the expression of P53 pro-tein is correlated with poor prognosis in colorectal carcinoma.     

Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?

Free radical hypothesis which is one of the most acknowledged aging theories was developed into oxidative stress hypothesis. Protein carbonylation is by far one of the most widely used markers of protein oxidation. We studied the role of age and gender in protein carbonyl content of saliva and plasma among 273 Chinese healthy subjects (137 females and 136 males aged between 20 and 79) and discussed the correlation between protein carbonyl content of saliva and plasma. Protein carbonyl content of saliva and plasma were, respectively, 2.391 ± 0.639 and 0.838 ± 0.274 nmol/mg. Variations of saliva and plasma different age groups all reached significant differences in both male and female (all p < 0.05) while both saliva and plasma protein carbonyls were found to be significantly correlated with age (r = 0.6582 and r = 0.5176, all p < 0.001). Gender was discovered to be unrelated to saliva and plasma protein carbonyl levels (all p > 0.05). Saliva and plasma protein carbonyls were positively related (r = 0.4405, p < 0.001). Surprisingly, saliva and plasma protein carbonyls/ferric reducing ability of plasma (FRAP) ratios were proved to be significantly correlated with age (r = 0.7796 and r = 0.6938, all p < 0.001) while saliva protein carbonyls/FRAP ratio and plasma protein carbonyls/FRAP ratio were also correlated (r = 0.5573, p < 0.001). We concluded that saliva protein carbonyls seem to be an alternative biomarker of aging while the mechanisms of protein carbonylation and oxidative stress and the relationship between saliva protein carbonyls and diseases need to be further investigated.     

Analysing protein–protein interaction networks of human liver cancer cell lines with diverse metastasis potential

Purpose Human hepatocellular carcinoma (HCC) is one of the most mortal tumor. In a previous study, we had constructed glycoprotein expression profiles and glycoprotein databases of three human liver cancer cell lines with diverse metastasis potential. In order to discover vital glycoproteins related to pathogenesis and metastasis of HCC, in this study we analyzed previous data with bioinformatic approach. Methods We took previous data to draw the protein–protein interaction (PPI) networks of liver cell lines by searching IntACT database and then using Pajeck software. Further more, we compared the differences between the three PPI networks by drawing the PPI networks of differential glycoproteins and by naming differential display PPI networks. Results Large numbers of proliferation and apoptosis-relative proteins interact with the differential glycoproteins, and among the differential glycoproteins there are many interactions. Conclusions We conclude that neither single nor several proteins cause malignant proliferation of liver cells. “Molecule groups” concept should be introduced into diagnosis and metastasis prediction of the HCC.     

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori(H.pylori)is associated withthe development of chronic gastritis,peptic ulcerand gastric cancer and gastric MALTlymphoma.H.pylori has many antigens,including urease,heat shock protein and vacuolatingcytotoxin and so on,and urease is an importantfactor in the colonization of the gastric mucosa and     

What is the role of non-native intermediates of beta-lactoglobulin in protein folding?

The mechanism of alpha-->beta transition in folding of beta-lactoglobulin is discussed based on free energy landscape analysis of a long lattice model. It is found that helical propensity of beta-lactoglobulin is driven by conformational entropy and is intrinsically coded in its native structure. We propose a view on a role of folding intermediate, which is "on-pathway" but rich in non-native structures. The present results suggest that the native structure topology plays an important role in alpha-->beta transition.     

Effect of manganese on heat stress protein synthesis of new—born rats

AIM: To study the effect of manganese (Mn) on heat stressprotein 70 (HSP70) synthesis in the brain and liver of new-bom rats whose mother-rats were exposed to Mn.METHODS: 32 female rats were randomly divided into fourgroups. One group was administrated with physiologicalsaline only as control group, the other three groups wereadministrated with 7.5, 15 and 30 mg@ kg-1 manganesechloride (MnCl2) by intraperitioneal injection every two daysfor two weeks. After delivery, the mother-rats receivedMnCl2 unceasingly for a week with the same method. Thenthe contents of Mn、 Zn、 Cu and Fe in the livers of the new-bom rats were determined by atomic absorptionspectroscopy; The level of HSP70 in the brains and thelivers of the new-born rats as detected by Westsrn-dot-blotting, and the SOD activities were measuredsimultaneously.RESULTS: The contents of Mn in the livers of new-bom ratsof the experimental groups(respective 1.38 ± 0.18, 2.73 ±0.65, 3.44 ± 0.89μg @ g-1) were significantly increasedcompared with the control group(0.88 ± 0.18μg@ g-1; p ＜0.01); The contents of Fe in the livers of new-bom rats of 15and 30 mg@ kg-1 experimental groups (426 ± 125,572 ± 175μg@g-1, respectively) were significantly increased comparedwith the control group(286±42μg@g-1; P＜0.05); the levelsof Zn in the livers of the new-bom rats of three experimentalgroups( 254 ± 49, 263 ± 47, 213 ± 28μg@ g-1, respectively)were lower than those of the control group(335 ± 50μg@g-1;respective P＜0.05, P＜0.01); and the levels of Cu showedno significant difference among the four groups (threeexperimental groups: 75 ± 21, 68 ± 241 and 78 ± 18μg@g-1;control group: 83 ± 9μg@ g-1; p ＞ 0.05). There was asignificant increase in the levels of HSP70 in the brains ofnew-bom rats of the 30 mg@kg-1 group (19.5 × 103 ± 1.3 ×103A; control group: 14.3 × 103 ± 1.4 × 103A; P＜ 0.01),andthe levels of HSP70 in the livers of new-bom rats of threeexperinental groups(respective 19.6 × 103 ± 3.9 × 103A, 18.5× 103 ± 3.8 × 103A, 22.4 × 103 ± 1.9 × 103A ) also increasedthan control group(13.3 × 103 ± 1.0 × 103A; P ＜ 0.01), butthe SOD activities showed no significant difference amongbrains of the four groups (experimental groups: 5.04 ± 0.43,4.83±0.48, 4.60±0.84 ku@g-1; control group: 4.91 ± 0.37ku@g-1; P＞ 0.05). The SOD activities in the livers of 15mg@kg-1 group(5.41 ± 0.44 ku@g-1) was lower than the controlgroup(5.95±0.36 ku@g-1; P＜0.05).CONCLUSION: While mother-rats were exposed tomanganese, the metabolisms of Mn、Zn and Fe of new- bornrats in the livers were influenced and were situated in astress status, thus HSP70 syntheses is induced in the brainsand livers of new-bom rats, but the mechanism of this effectin the developmental toxicity of Mn remains to he furtherstudied.