Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca²⁺ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca²⁺, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca²⁺ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca²⁺, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.Actin is crucial to such processes as cell movement, cell division, and apoptosis, which are regulated by numerous actin-binding proteins, including gelsolin, Arp2/3, and profilin (for review, see Ref. 1). Gelsolin, the most potent actin filament-severing protein known, can bind to, sever, cap, and nucleate actin filaments in a calcium-, pH-, ATP-, and phospholipid-dependent manner (for review, see Ref. 2). Villin, found in microvilli of absorptive epithelium, is a second member of the gelsolin family of actin-binding proteins. In addition to standard gelsolin-type activities, villin is able to bundle actin filaments and is subject to regulation by tyrosine phosphorylation as well as by Ca²⁺ and phosphatidylinositol 4,5-bisphosphate (for review, see Ref. 3). Many comparisons have been made between gelsolin and villin. The two share 50% amino acid sequence identity and show similar proteolytic cleavage patterns (4). Both contain six similarly folded domains, but villin possesses a seventh domain at its C terminus, the headpiece (HP)² domain, which folds into a compact structure that introduces a second F-actin-binding site into the protein. Recent studies indicate that villin uses the HP F-actin-binding sites to achieve bundling (5). In an environment devoid of free Ca²⁺, gelsolin and villin assume inactive conformations. After binding Ca²⁺, both undergo conformational rearrangements that expose their binding sites for F-actin. In villin, this includes revealing the HP actin-binding site through a “hinge mechanism” (6).Biochemical and structural studies have revealed eight Ca²⁺-binding sites of two types in gelsolin (for review, see Ref. 7). Each of the six domains contains a complete and evolutionarily conserved site, termed type 2, whereas G1 and G4 provide partial Ca²⁺ coordination at interfaces with actin through sites termed type 1. Sequential mutagenesis of these sites in villin has identified six functional Ca²⁺-binding sites (8): two major sites, one each of type 1 and type 2, in V1, plus four type 2 sites in V2–V6. The type 1 site in V1 regulates F-actin-capping and F-actin-severing activities, whereas the lower affinity type 2 site in V1 only affects severing (9). The other four sites are involved in stabilizing villin conformation, but they do not directly influence actin-severing activity. NMR studies of a fragment of villin that consists of V6 and the HP domain have implicated V6 residues Asn⁶⁴⁷, Asp⁶⁴⁸, and Glu⁶⁷⁰ in binding Ca²⁺ (10). These experiments also revealed the first 80 residues of V6 to undergo significant conformational change as a result of Ca²⁺ binding.Nanomolar to micromolar concentrations of free Ca²⁺ govern the actin-binding activities of gelsolin. In contrast, micromolar and millimolar concentrations of calcium ions are required for villin to exhibit capping and severing, respectively. However, after tyrosine phosphorylation, villin can sever actin filaments even at nanomolar Ca²⁺ concentrations (11). Furthermore, although the actin-severing ability of the N-terminal half of villin is calcium-dependent, that by the N-terminal half of gelsolin is not. In contrast, the binding of G-actin of the C-terminal half of both villin and gelsolin requires Ca²⁺. Creation of hybrid proteins demonstrated that the domains of villin and gelsolin are not interchangeable (12).Abundant x-ray crystallographic structural information exists for gelsolin, including the calcium ion-free (Ca²⁺-free), inactive structure of the intact protein (13), the activated N- and C-terminal halves, each in a bimolecular complex with actin (7, 14), and the activated C-terminal half on its own (15, 16). Structural data for intact villin are unavailable and are limited to fragment V1 (17), solved using NMR methods, and the HP domain, solved by NMR and x-ray crystallography (18, 19). NMR experiments also indicate that HP is connected to V6 by a 40-residue disordered linker. As a result, HP has been proposed to bind actin independently of the remainder of the protein (10).In this report, we present the structure of Ca²⁺-free, isolated villin V6, which exhibits a typical gelsolin domain fold. The long helix in V6 in this structure is straight, unlike the corresponding helix in G6 of intact Ca²⁺-free gelsolin, which is bent, and only straightens on calcium activation of the intact protein. Hence, V6 appears to be in an active conformation in the absence of Ca²⁺. Molecular dynamics simulations indicate that the preferred state of the long helix is also straight for isolated G6 in the absence of Ca²⁺. Furthermore, they suggest a bistable mechanism of helix conformational change regulated by the presence of the remaining domains, by calcium ions, and by other interactants. We therefore propose a mechanism for the gelsolin family proteins whereby Ca²⁺ triggers the straightening of the domain 6 helix in the native conformation of the inactive proteins to propagate more widespread conformational changes.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mud-puddling in the yellow-spined bamboo locust, Ceracris kiangsu (Oedipodidae: Orthoptera): Does it detect and prefer salts or nitrogenous compounds from human urine?

C. kiangsu adults were observed visiting human urine, especially on hot summer days. The main chemicals in fresh human urine include inorganic salts and CO(NH₂)_2. When human urine was incubated, NH₄HCO₃ became the richest nitrogenous compound. The phagostimulants, repellents and attractants in urine were identified here. On the filter papers treated with fresh or incubated urine samples, the 5th instar nymphs and the adults started and continued gnawing around the edges, in contrast to the 3rd and the 4th instar nymphs. The consumed areas were dramatically greater on the filters treated with the urine samples incubated for 3-6 days. The feedings of both male and female adults were also stimulated by several urine-borne components such as NaCl, NaH₂PO₄, Na₂SO₄, KCl, NH₄Cl and NH₄HCO₃ but not by CO(NH₂)₂. Among them NaCl was the most powerful phagostimulant. The repelling, or attractive/arresting effects of CO(NH₂)₂ and NH₄HCO₃ were also evaluated by a two-choice test. When exposed to water- and CO(NH₂)₂ solution-immersed filters simultaneously, the adults prefer to stay on water-immersed filter. In contrast, when provided water- and NH₄HCO₃ solution-treated filters, the adults prefer to stay on NH₄HCO₃ solution-treated filter. This demonstrated that CO(NH₂)₂ acted as a repellent and NH₄HCO₃ as an attractant/arrestant. In the bamboo forest, similar feeding behavior was also elicited by NaCl, NH₄HCO₃ but not by CO(NH₂)₂. Comparing to NaCl solution, a mixed solution of NaCl and CO(NH₂)₂ (1:1) significantly decreased the consumed area of the treated filters whereas a mixed solution of NaCl and NH₄HCO₃ (1:1) dramatically increased the consumed area. These results demonstrated that the phagostimulatory effect by NaCl was reduced by CO(NH₂)₂ in fresh urine and was enhanced by NH₄HCO₃ in incubated urine.     

Glucose biosensor based on electrodeposited platinum nanoparticles and three-dimensional porous chitosan membranes

The chitosan with three-dimensional porous structure greatly increased the effective electrode surface for loading of platinum nanoparticles and promoted efficient electron transfer. The resulting biosensor had a response time (within 5 s) and a linear response from 6 μM to 4.2 mM glucose with a detection limit of 2 μM (S/N = 3). Moreover, the methodology can be applied for the immobilization of other enzymes.     

The configuration,solvatochromism and metallo‐responses of two novel cyano‐containing oligo(phenylene‐vinylene) derivatives

Two novel cyano‐containing oligo(phenylenevinylene) (OPV) derivatives have been designed and synthesized. Photophysical and sensing properties of the two compounds were studied. Such studies reveal the intramolecular charge transfer process between cyano groups and OPV core. The results showed that the alkyl difference of substituted OPV leads to the changes of molecular configuration and metallo‐response of two compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Hyper-activated pro-inflammatory CD16 monocytes correlate with the severity of liver injury and fibrosis in patients with chronic hepatitis B

Background

Extensive mononuclear cell infiltration is strongly correlated with liver damage in patients with chronic hepatitis B virus (CHB) infection. Macrophages and infiltrating monocytes also participate in the development of liver damage and fibrosis in animal models. However, little is known regarding the immunopathogenic role of peripheral blood monocytes and intrahepatic macrophages.

Methodology/Principal Findings

The frequencies, phenotypes, and functions of peripheral blood and intrahepatic monocyte/macrophage subsets were analyzed in 110 HBeAg positive CHB patients, including 32 immune tolerant (IT) carriers and 78 immune activated (IA) patients. Liver biopsies from 20 IA patients undergoing diagnosis were collected for immunohistochemical analysis. IA patients displayed significant increases in peripheral blood monocytes and intrahepatic macrophages as well as CD16⁺ subsets, which were closely associated with serum alanine aminotransferase (ALT) levels and the liver histological activity index (HAI) scores. In addition, the increased CD16⁺ monocytes/macrophages expressed higher levels of the activation marker HLA-DR compared with CD16⁻ monocytes/macrophages. Furthermore, peripheral blood CD16⁺ monocytes preferentially released inflammatory cytokines and hold higher potency in inducing the expansion of Th17 cells. Of note, hepatic neutrophils also positively correlated with HAI scores.

Conclusions

These distinct properties of monocyte/macrophage subpopulations participate in fostering the inflammatory microenvironment and liver damage in CHB patients and further represent a collaborative scenario among different cell types contributing to the pathogenesis of HBV-induced liver disease.     

A model on liquid penetration into soft material with application to needle-free jet injection

A mathematical model has been presented for a high speed liquid jet penetration into soft solid by a needle-free injection system. The model consists of a cylindrical column formed by the initial jet penetration and an expansion sphere due to continuous deposition of the liquid. By solving the equations of energy conservation and volume conservation, the penetration depth and the radius of the expansion sphere can be predicted. As an example, the calculation results were presented for a typical needle-free injection system into which a silicon rubber was injected into. The calculation results were compared with the experimental results.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.