Predicting weakly stable regions,oligomerization state,and protein–protein interfaces in transmembrane domains of outer membrane proteins

Although the structures of many β-barrel membrane proteins are available, our knowledge of the principles that govern their energetics and oligomerization states is incomplete. Here we describe a computational method to study the transmembrane (TM) domains of β-barrel membrane proteins. Our method is based on a physical interaction model, a simplified conformational space for efficient enumeration, and an empirical potential function from a detailed combinatorial analysis. Using this method, we can identify weakly stable regions in the TM domain, which are found to be important structural determinants for β-barrel membrane proteins. By calculating the melting temperatures of the TM strands, our method can also assess the stability of β-barrel membrane proteins. Predictions on membrane enzyme PagP are consistent with recent experimental NMR and mutant studies. We have also discovered that out-clamps, in-plugs, and oligomerization are 3 general mechanisms for stabilizing weakly stable TM regions. In addition, we have found that extended and contiguous weakly stable regions often signal the existence of an oligomer and that strands located in the interfaces of protein–protein interactions are considerably less stable. Based on these observations, we can predict oligomerization states and can identify the interfaces of protein–protein interactions for β-barrel membrane proteins by using either structure or sequence information. In a set of 25 nonhomologous proteins with known structures, our method successfully predicted whether a protein forms a monomer or an oligomer with 91% accuracy; in addition, our method identified with 82% accuracy the protein–protein interaction interfaces by using sequence information only when correct strands are given.     

Predictive energy landscapes for protein–protein association

We investigate protein–protein association using the associative-memory, water-mediated, structure, and energy model (AWSEM), a coarse-grained protein folding model that has been optimized using energy-landscape theory. The potential was originally parameterized by enforcing a funneled nature for a database of dimeric interfaces but was later further optimized to create funneled folding landscapes for individual monomeric proteins. The ability of the model to predict interfaces was not tested previously. The present results show that simulated annealing of the model indeed is able to predict successfully the native interfaces of eight homodimers and four heterodimers, thus amounting to a flexible docking algorithm. We go on to address the relative importance of monomer geometry, flexibility, and nonnative intermonomeric contacts in the association process for the homodimers. Monomer surface geometry is found to be important in determining the binding interface, but it is insufficient. Using a uniform binding potential rather than the water-mediated potential results in sampling of misbound structures that are geometrically preferred but are nonetheless energetically disfavored by AWSEM, as well as in nature. Depending on the stability of the unbound monomers, nonnative contacts play different roles in the association process. For unstable monomers, thermodynamic states stabilized by nonnative interactions correspond to productive, on-pathway intermediates and can, therefore, catalyze binding through a fly-casting mechanism. For stable monomers, in contrast, states stabilized by nonnative interactions generally correspond to traps that impede binding.     

Structure analysis and expressions of a novel tetratransmembrane protein, lysosoma-associated protein transmembrane 4 β associated with hepatocellular carcinoma

AIM: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosomeassociated protein transmembrane 4 β (LAPTM4B). METHODS: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. RESULTS: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. CONCLUSION: LAPTM4B gene encoded two isoforms of tetratansmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.     

Kinetics of protein–ligand unbinding: Predicting pathways,rates, and rate-limiting steps

The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate k_off is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate k_on. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design.Understanding the thermodynamics and kinetics of protein–ligand interactions is of paramount relevance in the early stages of drug discovery (1–3). So far the major emphasis has been placed on predicting the most likely binding pose as determined by the highest binding affinity (4, 5). In contrast, it has not been possible to predict the pathways for unbinding and the associated rates. However, it is by now well-recognized that one of the most pertinent factors for sustained drug efficacy and safety is not just its affinity, but possibly even more so, the mean lifetime of the protein–ligand complex (1–3). The latter property is strictly related to the time during which the ligand remains in the binding site (1, 2), and is typically expressed by its inverse, the dissociation rate k_off (2). In principle k_off should be amenable to calculations through all-atom molecular dynamics (MD) simulations. These simulations could give detailed and useful insights into the atomic interactions at work during unbinding, especially in the ephemeral but kinetically most relevant transition state ensemble (TSE) (6, 7). Such information is of great value in designing modifications of the ligand that might improve its pharmaceutical properties.However, despite the potential of MD simulations no such calculation has yet been reported. This is a consequence of the limited timescales of MD simulations. Even with the most modern purpose-built supercomputers or massive distributed computing, one can barely reach the timescale of milliseconds (3). Unfortunately most of the reported ligand–protein dissociation times far exceed this timescale (2). These timescales can be reached either by transition path sampling methods (8, 9), quasi-classical approximations (10), by the construction of Markov state models (11, 12), or through carefully designed enhanced sampling methods (8, 13–30) that make accessible the timescale of seconds and beyond in a controlled and accurate way. The enhanced sampling method we use in this work is based on metadynamics (13–15), which has been widely and successfully applied to a variety of systems including complex protein–ligand systems (25–30), and has been rigorously proven to converge to the correct free-energy surface (31, 32).Recently, we have extended the scope of metadynamics by showing that it can also be used to recover kinetic information (15). Furthermore, we showed that by using an a posteriori statistical analysis (33) one can also establish the reliability of the kinetics thus generated. The use of metadynamics for obtaining kinetic information is still in its infancy, however its usefulness has been tested by us and other groups in a range of systems (15, 33–36).In this work, we demonstrate that the scope of the method reported in ref. 15 can be extended to study protein–ligand dissociation pathways and to determine in an accurate way the ligand unbinding rates. We reach well into the hundreds of milliseconds regime and longer, maintaining at the same time full atomic resolution for protein, ligand, and solvent. Specifically, we study the unbinding of the inhibitor benzamidine from trypsin, a serine protease protein (27, 37, 38) using classical force fields (39, 40). Using our acceleration method (15, 33) we are able to harness 21 independent successful unbinding trajectories in which the ligand goes from the bound to the fully unbound state. We find that one of the most distinctive features of the unbinding process is the role played by the water molecules (41, 42). In particular, the solvent promotes unbinding by assisting in the breakage of shielded hydrogen bonds through the formation of water bridge interactions (41).From the analysis of the unbinding trajectories we find that along the unbinding pathways the ligand rests for times ranging from nanoseconds to milliseconds in a number of intermediate structures. We calculate the rates for all possible transitions between these intermediates and construct a Markov model for the unbinding process (11, 43, 44). The overall escape rate computed from this Markov model is in good agreement with the direct estimation of the mean unbinding time that comes from the metadynamics runs. Reassured by this agreement we use the Markov model to determine the dominant unbinding pathways and rate-limiting steps. To this end, starting from the metadynamics reactive trajectories, we perform a committor analysis and determine the TSE (6). Using the recently computed value of the binding affinity (27) we also estimate the binding rate constant k_on. Our calculated unbinding and binding rates compare reasonably well with the known experimental measurement (37), especially taking into account the margin of error in the experiment and the inaccuracy of the force field used in the simulations (42). Unprecedented structural features of the target are also disclosed. In particular, we find that in its apo state trypsin can exist in two forms. In the first form, loop Val207–Tyr224 (hereafter labeled loop L) oscillates around the crystallographic state. In the other form, a small distortion of this loop is stabilized. The mean lifetime of this distorted state is nearly 0.7 ms and during this time the ligand cannot reach the binding site.We believe that this metadynamics-based strategy is, to our knowledge, the first direct approach for calculating k_off from MD simulations of unbinding. Previous studies have focused on the calculation of k_on and the magnitude of k_off was only indirectly obtained (12, 38). Our strategy should be easily applicable for calculating unbinding pathways and rates for generic protein–ligand systems, thus complementing and extending the role of enhanced sampling-based simulations in drug discovery.     

Serum β2-microglobulin,sialic acid,and C-reactive protein in systemic lupus erythematosus

Summary Serum ₂-microglobulin (₂m), sialic acid and C-reactive protein (CRP) were studied in 58 patients with systemic lupus erythematosus (SLE) on 186 occasions. Serum ₂m was significantly higher in SLE patients than in control subjects. Increased serum ₂m levels were seen in 68% of the patients with only extrarenal manifestations of SLE, in 75% of the patients with renal manifestations but normal glomerular filtration rate, and in 100% of the patients with renal failure. Serum ₂m levels in 12 SLE patients with associated Sjögren's syndrome were similar to those in patients without that syndrome. Serum sialic acid was also significantly increased in the SLE patients. Sixty-one (33%) of the 186 sera were positive for CRP (5 mg/l). The CRP elevation was not accompanied by recognized intercurrent infection or other superimposed cause of tissue injury and inflammation in 37 instances (61%). Under such conditions CRP was only moderately increased.     

Allosteric switch regulates protein–protein binding through collective motion

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.Intermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2–4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5–9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11).     

血清C-反应蛋白(C-reactive protein,CRP)与糖尿病

CRP是1930年威廉姆和托马斯在对肺炎球菌性肺炎进行研究的时候发现了一种能够凝集肺炎球菌C-菌多糖的蛋白,一些流行病调查和临床研究发现心血管病变常伴随有炎性标志物的升高, 2型糖尿病它与心血管病有着共同土壤,使得CRP在2型糖尿病患者中的变化研究被受关注.近年来的一些研究发现CRP在糖尿病患者中升高,它的升高可能与动脉硬化以外的因素有关.因此,引发了糖尿病的低度炎性病变假说.本文就近年来CRP与糖尿病研究主要状况进行概述.     

The antiviral protein,viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

Lipid droplets are intracellular lipid-storage organelles that are thought to be derived from the endoplasmic reticulum (ER). Several pathogens, notably hepatitis C virus, use lipid droplets for replication. Numerous questions remain about how lipid droplets are generated and used by viruses. Here we show that the IFN-induced antiviral protein viperin, which localizes to the cytosolic face of the ER and inhibits HCV, localizes to lipid droplets. We show that the N-terminal amphipathic α-helix of viperin that is responsible for ER localization is also necessary and sufficient to localize both viperin and the fluorescent protein dsRed to lipid droplets. Point mutations in the α-helix that prevent ER association also disrupt lipid droplet association, and sequential deletion mutants indicate that the same number of helical turns are necessary for ER and lipid droplet association. Finally, we show that the N-terminal amphipathic α-helix of the hepatitis C viral protein NS5A can localize dsRed and viperin to lipid droplets. These findings indicate that the amphipathic α-helices of viperin and NS5A are lipid droplet-targeting domains and suggest that viperin inhibits HCV by localizing to lipid droplets using a domain and mechanism similar to that used by HCV itself.     

Aβ neurotoxicity depends on interactions between copper ions, prion protein, and N-methyl-D-aspartate receptors

N-methyl-d-aspartate receptors (NMDARs) mediate critical CNS functions, whereas excessive activity contributes to neuronal damage. At physiological glycine concentrations, NMDAR currents recorded from cultured rodent hippocampal neurons exhibited strong desensitization in the continued presence of NMDA, thus protecting neurons from calcium overload. Reducing copper availability by specific chelators (bathocuproine disulfonate, cuprizone) induced nondesensitizing NMDAR currents even at physiologically low glycine concentrations. This effect was mimicked by, and was not additive with, genetic ablation of cellular prion protein (PrP(C)), a key copper-binding protein in the CNS. Acute ablation of PrP(C) by enzymatically cleaving its cell-surface GPI anchor yielded similar effects. Biochemical studies and electrophysiological measurements revealed that PrP(C) interacts with the NMDAR complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor. Synthetic human Aβ(1-42) (10 nM-5 μM) produced an identical effect that could be mitigated by addition of excess copper ions or NMDAR blockers. Taken together, Aβ(1-42), copper chelators, or PrP(C) inactivation all enhance the activity of glycine at the NMDAR, giving rise to pathologically large nondesensitizing steady-state NMDAR currents and neurotoxicity. We propose a physiological role for PrP(C), one that limits excessive NMDAR activity that might otherwise promote neuronal damage. In addition, we provide a unifying molecular mechanism whereby toxic species of Aβ(1-42) might mediate neuronal and synaptic injury, at least in part, by disrupting the normal copper-mediated, PrP(C)-dependent inhibition of excessive activity of this highly calcium-permeable glutamate receptor.     

Host–rabies virus protein–protein interactions as druggable antiviral targets

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.     

Expression of GNAZ,encoding the Gαz protein,predicts survival in mantle cell lymphoma

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gα_z protein – a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gα_z contribute to the MCL pathobiology.     

H-Ras forms dimers on membrane surfaces via a protein–protein interface

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein–protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K_d is measured to be ∼1 × 10³ molecules/µm², and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.In mammalian signal transduction, Ras functions as a binary switch in fundamental processes including proliferation, differentiation, and survival (1). Ras is a network hub; various upstream signaling pathways can activate Ras-GDP to Ras-GTP, which subsequently selects between multiple downstream effectors to elicit a varied but specific biochemical response (2, 3). Signaling specificity is achieved by a combination of conformational plasticity in Ras itself (4, 5) and dynamic control of Ras spatial organization (6, 7). Isoform-specific posttranslational lipidation targets the main H-, N-, and K-Ras isoforms to different subdomains of the plasma membrane (8–10). For example, H-Ras localizes to cholesterol-sensitive membrane domains, whereas K-Ras does not (11). A common C-terminal S-farnesyl moiety operates in concert with one (N-Ras) or two (H-Ras) palmitoyl groups, or with a basic sequence of six lysines in K-Ras4B (12), to provide the primary membrane anchorage. Importantly, the G-domain (residues 1–166) and the hypervariable region (HVR) (residues 167–189) dynamically modulate the lipid anchor localization preference to switch between distinct membrane populations (13). For example, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is necessary for efficient activation of the effector Raf and GTP loading of the G-domain promotes this redistribution by a mechanism that requires the HVR (14). However, the molecular details of the coupling between lipid anchor partitioning and nucleotide-dependent protein–membrane interactions remain unclear.In addition to biochemical evidence for communication between the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion affects Ras structure and orientation (15–17). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET measurements are consistent with a reorientation of Ras with respect to the membrane upon GTP binding (19, 20). Further modeling showed that the membrane binding region and the canonical switch I and II regions communicate across the protein via long-range side-chain interactions (21) in a conformational selection mechanism (22). Whereas these allosteric modes likely contribute to Ras partitioning and reorientation in vivo, direct functional consequences on Ras protein–protein interactions are poorly understood.Members of the Ras superfamily of small GTPases are widely considered to be monomeric (23). However, several members across the Ras GTPase subfamilies are now known to dimerize (24–28), and a class of small GTPases that use dimerization instead of GTPase activating proteins (GAPs) for GTPase activity has been identified (29). Recently, semisynthetic natively lipidated N-Ras was shown to cluster on supported membranes in vitro, in a manner broadly consistent with molecular mechanics (MM) modeling of dimers (30). For Ras, dimerization could be important because Raf, which is recruited to the membrane by binding to Ras, requires dimerization for activation. Soluble Ras does not activate Raf in vitro (31), but because artificial dimerization of GST-fused H-Ras leads to Raf activation in solution, it has been hypothesized that Ras dimers exist on membranes (32). However, presumed dimers were only detected after chemical cross-linking (32), and the intrinsic oligomeric properties of Ras remain unknown.Here, we use a combination of time-resolved fluorescence spectroscopy and microscopy to characterize H-Ras(C118S, 1–181) and H-Ras(C118S, 1–184) [referred to as Ras(C181) and Ras(C181,C184) from here on] anchored to supported lipid bilayers. By tethering H-Ras to membranes at cys181 (or both at cys181 and cys184) via a membrane-miscible lipid tail, we eliminate effects of lipid anchor clustering while preserving the HVR region between the G-domain and the N-terminal palmitoylation site at cys181 (or cys184), which is predicted to undergo large conformational changes upon membrane binding and nucleotide exchange (18). Labeling is achieved through a fluorescent Atto488-linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras forms surface density-dependent clusters. Photon counting histogram (PCH) analysis and single-molecule tracking (SMT) reveal that H-Ras clusters are dimers and that no higher-order oligomers are formed. A Y64A point mutation in the loop between beta strand 3 (β3) and alpha helix 2 (α2) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either part of, or allosterically coupled to, the dimer interface. The 2D dimerization K_d is measured to be on the order of 1 × 10³ molecules/µm², within the broad range of Ras surface densities measured in vivo (10, 33–35). Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution, suggesting that a membrane-induced structural change in H-Ras leads to dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive to the details of HVR lipidation, suggesting that dimerization is a general property of H-Ras on membrane surfaces.     

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.     

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